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1
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Matsuda A, Masuzawa R, Takahashi K, Takano K, Endo T. MEK inhibitors and DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, prevent the migration and invasion of KRAS-mutant cancer cells. Cytoskeleton (Hoboken) 2025; 82:32-44. [PMID: 38872577 DOI: 10.1002/cm.21881] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Revised: 05/08/2024] [Accepted: 05/16/2024] [Indexed: 06/15/2024]
Abstract
The Ras-induced ERK pathway (Raf-MEK-ERK signaling cascade) regulates a variety of cellular responses including cell proliferation, survival, and migration. Activating mutations in RAS genes, particularly in the KRAS gene, constitutively activate the ERK pathway, resulting in tumorigenesis, cancer cell invasion, and metastasis. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Consequently, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner and can serve as a tumor suppressor that targets mutant Ras protein-induced tumorigenesis. We show here that MEK inhibitors and DA-Raf interfere with the in vitro collective cell migration and invasion of human KRAS-mutant carcinoma cell lines, the lung adenocarcinoma A549, colorectal carcinoma HCT116, and pancreatic carcinoma MIA PaCa-2 cells. DA-Raf expression was silenced in these cancer cell lines. All these cell lines had high collective migration abilities and invasion properties in Matrigel, compared with nontumor cells. Their migration and invasion abilities were impaired by suppressing the ERK pathway with the MEK inhibitors U0126 and trametinib, an approved anticancer drug. Expression of DA-Raf in MIA PaCa-2 cells reduced the ERK activity and hindered the migration and invasion abilities. Therefore, DA-Raf may function as an invasion suppressor protein in the KRAS-mutant cancer cells by blocking the Ras-ERK pathway when DA-Raf expression is induced in invasive cancer cells.
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Affiliation(s)
- Aoi Matsuda
- Department of Biology, Graduate School of Science, Chiba University, Chiba, Chiba, Japan
| | - Ryuichi Masuzawa
- Department of Biology, Graduate School of Science, Chiba University, Chiba, Chiba, Japan
| | - Kazuya Takahashi
- Department of Biology, Graduate School of Science, Chiba University, Chiba, Chiba, Japan
| | - Kazunori Takano
- Department of Biology, Graduate School of Science, Chiba University, Chiba, Chiba, Japan
| | - Takeshi Endo
- Department of Biology, Graduate School of Science, Chiba University, Chiba, Chiba, Japan
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2
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Műzes G, Sipos F. Inflammasomes Are Influenced by Epigenetic and Autophagy Mechanisms in Colorectal Cancer Signaling. Int J Mol Sci 2024; 25:6167. [PMID: 38892354 PMCID: PMC11173330 DOI: 10.3390/ijms25116167] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2024] [Revised: 05/31/2024] [Accepted: 05/31/2024] [Indexed: 06/21/2024] Open
Abstract
Inflammasomes contribute to colorectal cancer signaling by primarily inducing inflammation in the surrounding tumor microenvironment. Its role in inflammation is receiving increasing attention, as inflammation has a protumor effect in addition to inducing tissue damage. The inflammasome's function is complex and controlled by several layers of regulation. Epigenetic processes impact the functioning or manifestation of genes that are involved in the control of inflammasomes or the subsequent signaling cascades. Researchers have intensively studied the significance of epigenetic mechanisms in regulation, as they encompass several potential therapeutic targets. The regulatory interactions between the inflammasome and autophagy are intricate, exhibiting both advantageous and harmful consequences. The regulatory aspects between the two entities also encompass several therapeutic targets. The relationship between the activation of the inflammasome, autophagy, and epigenetic alterations in CRC is complex and involves several interrelated pathways. This article provides a brief summary of the newest studies on how epigenetics and autophagy control the inflammasome, with a special focus on their role in colorectal cancer. Based on the latest findings, we also provide an overview of the latest therapeutic ideas for this complex network.
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Affiliation(s)
- Györgyi Műzes
- Immunology Division, Department of Internal Medicine and Hematology, Semmelweis University, 1088 Budapest, Hungary
| | - Ferenc Sipos
- Immunology Division, Department of Internal Medicine and Hematology, Semmelweis University, 1088 Budapest, Hungary
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3
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Bhatia V, Esmati L, Bhullar RP. Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells. Mol Cell Biochem 2024; 479:567-577. [PMID: 37131040 DOI: 10.1007/s11010-023-04725-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Accepted: 03/31/2023] [Indexed: 05/04/2023]
Abstract
Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.
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Affiliation(s)
- Vikram Bhatia
- Manitoba Chemosensory Biology Research Group and Department of Oral Biology, Dr. Gerald Niznick College of Dentistry, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada
- Children's Hospital Research Institute of Manitoba (CHRIM), Winnipeg, MB, R3E 3P4, Canada
| | - Laya Esmati
- Manitoba Chemosensory Biology Research Group and Department of Oral Biology, Dr. Gerald Niznick College of Dentistry, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada
| | - Rajinder P Bhullar
- Manitoba Chemosensory Biology Research Group and Department of Oral Biology, Dr. Gerald Niznick College of Dentistry, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada.
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, R3E 0W2, Canada.
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Tariq M, Ikeya T, Togashi N, Fairall L, Kamei S, Mayooramurugan S, Abbott LR, Hasan A, Bueno-Alejo C, Sukegawa S, Romartinez-Alonso B, Muro Campillo MA, Hudson AJ, Ito Y, Schwabe JW, Dominguez C, Tanaka K. Structural insights into the complex of oncogenic KRas4B G12V and Rgl2, a RalA/B activator. Life Sci Alliance 2024; 7:e202302080. [PMID: 37833074 PMCID: PMC10576006 DOI: 10.26508/lsa.202302080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2023] [Revised: 09/28/2023] [Accepted: 10/02/2023] [Indexed: 10/15/2023] Open
Abstract
About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.
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Affiliation(s)
- Mishal Tariq
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
| | - Teppei Ikeya
- Department of Chemistry, Tokyo Metropolitan University, Hachioji, Japan
| | - Naoyuki Togashi
- Department of Chemistry, Tokyo Metropolitan University, Hachioji, Japan
| | - Louise Fairall
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
| | - Shun Kamei
- Department of Chemistry, Tokyo Metropolitan University, Hachioji, Japan
| | | | - Lauren R Abbott
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
| | - Anab Hasan
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
| | - Carlos Bueno-Alejo
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
| | - Sakura Sukegawa
- Department of Chemistry, Tokyo Metropolitan University, Hachioji, Japan
| | - Beatriz Romartinez-Alonso
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
| | | | - Andrew J Hudson
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
- Department of Chemistry, University of Leicester, Leicester, UK
| | - Yutaka Ito
- Department of Chemistry, Tokyo Metropolitan University, Hachioji, Japan
| | - John Wr Schwabe
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
| | - Cyril Dominguez
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
- Leicester Institute of Structure and Chemical Biology, University of Leicester, Leicester, UK
| | - Kayoko Tanaka
- Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
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Andrade F, German-Cortés J, Montero S, Carcavilla P, Baranda-Martínez-Abascal D, Moltó-Abad M, Seras-Franzoso J, Díaz-Riascos ZV, Rafael D, Abasolo I. The Nanotechnology-Based Approaches against Kirsten Rat Sarcoma-Mutated Cancers. Pharmaceutics 2023; 15:1686. [PMID: 37376135 DOI: 10.3390/pharmaceutics15061686] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2023] [Revised: 05/18/2023] [Accepted: 05/27/2023] [Indexed: 06/29/2023] Open
Abstract
Kirsten rat sarcoma (KRAS) is a small GTPase which acts as a molecular switch to regulate several cell biological processes including cell survival, proliferation, and differentiation. Alterations in KRAS have been found in 25% of all human cancers, with pancreatic cancer (90%), colorectal cancer (45%), and lung cancer (35%) being the types of cancer with the highest mutation rates. KRAS oncogenic mutations are not only responsible for malignant cell transformation and tumor development but also related to poor prognosis, low survival rate, and resistance to chemotherapy. Although different strategies have been developed to specifically target this oncoprotein over the last few decades, almost all of them have failed, relying on the current therapeutic solutions to target proteins involved in the KRAS pathway using chemical or gene therapy. Nanomedicine can certainly bring a solution for the lack of specificity and effectiveness of anti-KRAS therapy. Therefore, nanoparticles of different natures are being developed to improve the therapeutic index of drugs, genetic material, and/or biomolecules and to allow their delivery specifically into the cells of interest. The present work aims to summarize the most recent advances related to the use of nanotechnology for the development of new therapeutic strategies against KRAS-mutated cancers.
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Affiliation(s)
- Fernanda Andrade
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona (UB), 08028 Barcelona, Spain
| | - Júlia German-Cortés
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
| | - Sara Montero
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
| | - Pilar Carcavilla
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
| | - Diego Baranda-Martínez-Abascal
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
| | - Marc Moltó-Abad
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Functional Validation & Preclinical Research (FVPR)/U20 ICTS Nanbiosis, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain
| | - Joaquín Seras-Franzoso
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain
| | - Zamira Vanessa Díaz-Riascos
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Functional Validation & Preclinical Research (FVPR)/U20 ICTS Nanbiosis, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain
| | - Diana Rafael
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Functional Validation & Preclinical Research (FVPR)/U20 ICTS Nanbiosis, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain
| | - Ibane Abasolo
- Clinical Biochemistry, Drug Delivery and Therapy Group (CB-DDT), Vall d'Hebron Institut of Research (VHIR), Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
- Centro de Investigación Biomédica en Red de Bioingenería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto De Salud Carlos III, 08035 Barcelona, Spain
- Functional Validation & Preclinical Research (FVPR)/U20 ICTS Nanbiosis, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain
- Clinical Biochemistry Service, Vall d'Hebron University Hospital, Vall d'Hebron Barcelona Hospital Campus, 08035 Barcelona, Spain
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6
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Hostallero DE, Wei L, Wang L, Cairns J, Emad A. Preclinical-to-clinical Anti-cancer Drug Response Prediction and Biomarker Identification Using TINDL. GENOMICS, PROTEOMICS & BIOINFORMATICS 2023; 21:535-550. [PMID: 36775056 PMCID: PMC10787192 DOI: 10.1016/j.gpb.2023.01.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/03/2021] [Revised: 11/28/2022] [Accepted: 01/31/2023] [Indexed: 02/12/2023]
Abstract
Prediction of the response of cancer patients to different treatments and identification of biomarkers of drug response are two major goals of individualized medicine. Here, we developed a deep learning framework called TINDL, completely trained on preclinical cancer cell lines (CCLs), to predict the response of cancer patients to different treatments. TINDL utilizes a tissue-informed normalization to account for the tissue type and cancer type of the tumors and to reduce the statistical discrepancies between CCLs and patient tumors. Moreover, by making the deep learning black box interpretable, this model identifies a small set of genes whose expression levels are predictive of drug response in the trained model, enabling identification of biomarkers of drug response. Using data from two large databases of CCLs and cancer tumors, we showed that this model can distinguish between sensitive and resistant tumors for 10 (out of 14) drugs, outperforming various other machine learning models. In addition, our small interfering RNA (siRNA) knockdown experiments on 10 genes identified by this model for one of the drugs (tamoxifen) confirmed that tamoxifen sensitivity is substantially influenced by all of these genes in MCF7 cells, and seven of these genes in T47D cells. Furthermore, genes implicated for multiple drugs pointed to shared mechanism of action among drugs and suggested several important signaling pathways. In summary, this study provides a powerful deep learning framework for prediction of drug response and identification of biomarkers of drug response in cancer. The code can be accessed at https://github.com/ddhostallero/tindl.
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Affiliation(s)
- David Earl Hostallero
- Department of Electrical and Computer Engineering, McGill University, Montreal, QC H3A, Canada; Mila - Quebec Artificial Intelligence Institute, Montreal, QC H2S, Canada
| | - Lixuan Wei
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA
| | - Liewei Wang
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA
| | - Junmei Cairns
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
| | - Amin Emad
- Department of Electrical and Computer Engineering, McGill University, Montreal, QC H3A, Canada; Mila - Quebec Artificial Intelligence Institute, Montreal, QC H2S, Canada; The Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A, Canada.
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7
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Báez-Flores J, Rodríguez-Martín M, Lacal J. The therapeutic potential of neurofibromin signaling pathways and binding partners. Commun Biol 2023; 6:436. [PMID: 37081086 PMCID: PMC10119308 DOI: 10.1038/s42003-023-04815-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 04/05/2023] [Indexed: 04/22/2023] Open
Abstract
Neurofibromin controls many cell processes, such as growth, learning, and memory. If neurofibromin is not working properly, it can lead to health problems, including issues with the nervous, skeletal, and cardiovascular systems and cancer. This review examines neurofibromin's binding partners, signaling pathways and potential therapeutic targets. In addition, it summarizes the different post-translational modifications that can affect neurofibromin's interactions with other molecules. It is essential to investigate the molecular mechanisms that underlie neurofibromin variants in order to provide with functional connections between neurofibromin and its associated proteins for possible therapeutic targets based on its biological function.
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Affiliation(s)
- Juan Báez-Flores
- Laboratory of Functional Genetics of Rare Diseases, Department of Microbiology and Genetics, University of Salamanca (USAL), 37007, Salamanca, Spain
- Institute of Biomedical Research of Salamanca (IBSAL), 37007, Salamanca, Spain
| | - Mario Rodríguez-Martín
- Laboratory of Functional Genetics of Rare Diseases, Department of Microbiology and Genetics, University of Salamanca (USAL), 37007, Salamanca, Spain
- Institute of Biomedical Research of Salamanca (IBSAL), 37007, Salamanca, Spain
| | - Jesus Lacal
- Laboratory of Functional Genetics of Rare Diseases, Department of Microbiology and Genetics, University of Salamanca (USAL), 37007, Salamanca, Spain.
- Institute of Biomedical Research of Salamanca (IBSAL), 37007, Salamanca, Spain.
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8
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Thuya WL, Kong LR, Syn NL, Ding LW, Cheow ESH, Wong RTX, Wang T, Goh RMWJ, Song H, Jayasinghe MK, Le MT, Hu JC, Yong WP, Lee SC, Wong ALA, Sethi G, Hung HT, Ho PCL, Thiery JP, Sze SK, Guo T, Soo RA, Yang H, Lim YC, Wang L, Goh BC. FAM3C in circulating tumor-derived extracellular vesicles promotes non-small cell lung cancer growth in secondary sites. Theranostics 2023; 13:621-638. [PMID: 36632230 PMCID: PMC9830426 DOI: 10.7150/thno.72297] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Accepted: 09/07/2022] [Indexed: 01/04/2023] Open
Abstract
Rationale: Metastasis is a complex process with a molecular underpinning that remains unclear. We hypothesize that cargo proteins conducted by extracellular vesicles (EVs) released from tumors may confer growth and metastasis potential on recipient cells. Here, we report that a cytokine-like secreted protein, FAM3C, contributes to late-stage lung tumor progression. Methods: EV protein profiling was conducted with an unbiased proteomic mass spectrometry analysis on non-small cell lung cancer (NSCLC) and normal lung fibroblast cell lines. Expression of FAM3C was confirmed in a panel of NSCLC cell lines, and correlated to the invasive and metastatic potentials. Functional phenotype of endogenous FAM3C and tumor-derived EVs (TDEs) were further investigated using various biological approaches in RNA and protein levels. Metastasis potential of TDEs secreted by FAM3C-overexpressing carcinoma cells was validated in mouse models. Results: Transcriptomic meta-analysis of pan-cancer datasets confirmed the overexpression of FAM3C - a gene encoding for interleukin-like EMT inducer (ILEI) - in NSCLC tumors, with strong association with poor patient prognosis and cancer metastasis. Aberrant expression of FAM3C in lung carcinoma cells enhances cellular transformation and promotes distant lung tumor colonization. In addition, higher FAM3C concentrations were detected in EVs extracted from plasma samples of NSCLC patients compared to those of healthy subjects. More importantly, we defined a hitherto-unknown mode of microenvironmental crosstalk involving FAM3C in EVs, whereby the delivery and uptake of FAM3C via TDEs enhances oncogenic signaling - in recipient cells that phenocopies the cell-endogenous overexpression of FAM3C. The oncogenicity transduced by FAM3C is executed via a novel interaction with the Ras-related protein RalA, triggering the downstream activation of the Src/Stat3 signaling cascade. Conclusions: Our study describes a novel mechanism for FAM3C-driven carcinogenesis and shed light on EV FAM3C as a driver for metastatic lung tumors that could be exploited for cancer therapeutics.
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Affiliation(s)
- Win Lwin Thuya
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Li Ren Kong
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Nicholas L Syn
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, China
| | - Ling-Wen Ding
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Esther Sok Hwee Cheow
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Regina Tong Xin Wong
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Tingting Wang
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | | | - Hongyan Song
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Migara K Jayasinghe
- Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Minh Tn Le
- Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Jian Cheng Hu
- NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Wei-Peng Yong
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore
| | - Soo-Chin Lee
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore
| | - Andrea Li-Ann Wong
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore
| | - Gautam Sethi
- Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Huynh The Hung
- Division of Cellular and Molecular Research, National Cancer Centre, Singapore
| | - Paul Chi-Lui Ho
- Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore
| | - Jean-Paul Thiery
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,INSERM Unit 1186, Comprehensive Cancer Center, Institut Gustave Roussy, Villejuif, France
| | - Siu Kwan Sze
- Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
| | - Tiannan Guo
- Zhejiang Provincial Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, China
| | - Ross A Soo
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore
| | - Henry Yang
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Yaw Chyn Lim
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599
| | - Lingzhi Wang
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Boon-Cher Goh
- Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599.,Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,NUS Centre for Cancer Research (N2CR), Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Department of Haematology-Oncology, National University Cancer Institute, National University Health System, Singapore.,Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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9
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Ral GTPases are critical regulators of spinal cord myelination and homeostasis. Cell Rep 2022; 40:111413. [PMID: 36170840 DOI: 10.1016/j.celrep.2022.111413] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 07/12/2022] [Accepted: 09/01/2022] [Indexed: 11/20/2022] Open
Abstract
Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.
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10
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Roman M, Hwang E, Sweet-Cordero EA. Synthetic Vulnerabilities in the KRAS Pathway. Cancers (Basel) 2022; 14:cancers14122837. [PMID: 35740503 PMCID: PMC9221492 DOI: 10.3390/cancers14122837] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Revised: 06/03/2022] [Accepted: 06/05/2022] [Indexed: 02/06/2023] Open
Abstract
Mutations in Kristen Rat Sarcoma viral oncogene (KRAS) are among the most frequent gain-of-function genetic alterations in human cancer. Most KRAS-driven cancers depend on its sustained expression and signaling. Despite spectacular recent success in the development of inhibitors targeting specific KRAS alleles, the discovery and utilization of effective directed therapies for KRAS-mutant cancers remains a major unmet need. One potential approach is the identification of KRAS-specific synthetic lethal vulnerabilities. For example, while KRAS-driven oncogenesis requires the activation of a number of signaling pathways, it also triggers stress response pathways in cancer cells that could potentially be targeted for therapeutic benefit. This review will discuss how the latest advances in functional genomics and the development of more refined models have demonstrated the existence of molecular pathways that can be exploited to uncover synthetic lethal interactions with a promising future as potential clinical treatments in KRAS-mutant cancers.
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11
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Eves BJ, Gebregiworgis T, Gasmi-Seabrook GM, Kuntz DA, Privé GG, Marshall CB, Ikura M. Structures of RGL1 RAS-Association domain in complex with KRAS and the oncogenic G12V mutant. J Mol Biol 2022; 434:167527. [DOI: 10.1016/j.jmb.2022.167527] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 02/24/2022] [Accepted: 03/01/2022] [Indexed: 11/28/2022]
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12
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Hou P, Wang YA. Conquering oncogenic KRAS and its bypass mechanisms. Theranostics 2022; 12:5691-5709. [PMID: 35966590 PMCID: PMC9373815 DOI: 10.7150/thno.71260] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Accepted: 05/05/2022] [Indexed: 11/19/2022] Open
Abstract
Aberrant activation of KRAS signaling is common in cancer, which has catalyzed heroic drug development efforts to target KRAS directly or its downstream signaling effectors. Recent works have yielded novel small molecule drugs with promising preclinical and clinical activities. Yet, no matter how a cancer is addicted to a specific target - cancer's genetic and biological plasticity fashions a variety of resistance mechanisms as a fait accompli, limiting clinical benefit of targeted interventions. Knowledge of these mechanisms may inform combination strategies to attack both oncogenic KRAS and subsequent bypass mechanisms.
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Affiliation(s)
- Pingping Hou
- Center for Cell Signaling, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA.,Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA.,Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08903, USA.,Lead contact
| | - Y Alan Wang
- Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
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13
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Ottaiano A, Santorsola M, Caraglia M, Circelli L, Gigantino V, Botti G, Nasti G. Genetic regressive trajectories in colorectal cancer: A new hallmark of oligo-metastatic disease? Transl Oncol 2021; 14:101131. [PMID: 34034007 PMCID: PMC8144733 DOI: 10.1016/j.tranon.2021.101131] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 05/17/2021] [Accepted: 05/19/2021] [Indexed: 02/07/2023] Open
Abstract
Colorectal cancer (CRC) originates as consequence of multiple genetic alterations. Some of the involved genes have been extensively studied (APC, TP53, KRAS, SMAD4, PIK3CA, MMR genes) in highly heterogeneous and poly-metastatic cohorts. However, about 10% of metastatic CRC patients presents with an indolent oligo-metastatic disease differently from other patients with poly-metastatic and aggressive clinical course. Which are the genetic dynamics underlying the differences between oligo- and poly-metastatic CRC? The understanding of the genetic trajectories (primary→metastatic) of CRC, in patients selected to represent homogenous clinical models, is crucial to make genotype/phenotype correlations and to identify the molecular events pushing the disease towards an increasing malignant phenotype. This information is crucial to plan innovative therapeutic strategies aimed to reverse or inhibit these phenomena. In the present study, we review the genetic evolution of CRC with the intent to give a developmental perspective on the border line between oligo- and poly-metastatic diseases.
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Affiliation(s)
- Alessandro Ottaiano
- Istituto Nazionale Tumori di Napoli, IRCCS "G. Pascale", Via M. Semmola, 80131, Naples, Italy.
| | - Mariachiara Santorsola
- Istituto Nazionale Tumori di Napoli, IRCCS "G. Pascale", Via M. Semmola, 80131, Naples, Italy
| | - Michele Caraglia
- Department of Precision Medicine, University of Campania "L. Vanvitelli", Via L. De Crecchio, 7 80138, Naples, Italy; Biogem Scarl, Institute of Genetic Research, Laboratory of Precision and Molecular Oncology, 83031, Ariano Irpino, Italy
| | - Luisa Circelli
- AMES-Centro Polidiagnostico Strumentale, 80013, Casalnuovo di Napoli, Italy
| | - Valerio Gigantino
- Innovalab scarl, Molecular Biology, Centro Direzionale, isola A2, 80143, Naples, Italy
| | - Gerardo Botti
- Istituto Nazionale Tumori di Napoli, IRCCS "G. Pascale", Via M. Semmola, 80131, Naples, Italy
| | - Guglielmo Nasti
- Istituto Nazionale Tumori di Napoli, IRCCS "G. Pascale", Via M. Semmola, 80131, Naples, Italy
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14
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Rio-Vilariño A, del Puerto-Nevado L, García-Foncillas J, Cebrián A. Ras Family of Small GTPases in CRC: New Perspectives for Overcoming Drug Resistance. Cancers (Basel) 2021; 13:3757. [PMID: 34359657 PMCID: PMC8345156 DOI: 10.3390/cancers13153757] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Revised: 07/20/2021] [Accepted: 07/23/2021] [Indexed: 12/11/2022] Open
Abstract
Colorectal cancer remains among the cancers with the highest incidence, prevalence, and mortality worldwide. Although the development of targeted therapies against the EGFR and VEGFR membrane receptors has considerably improved survival in these patients, the appearance of resistance means that their success is still limited. Overactivation of several members of the Ras-GTPase family is one of the main actors in both tumour progression and the lack of response to cytotoxic and targeted therapies. This fact has led many resources to be devoted over the last decades to the development of targeted therapies against these proteins. However, they have not been as successful as expected in their move to the clinic so far. In this review, we will analyse the role of these Ras-GTPases in the emergence and development of colorectal cancer and their relationship with resistance to targeted therapies, as well as the status and new advances in the design of targeted therapies against these proteins and their possible clinical implications.
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Affiliation(s)
| | | | - Jesús García-Foncillas
- Translational Oncology Division, Hospital Universitario Fundación Jimenez Diaz, 28040 Madrid, Spain; (A.R.-V.); (L.d.P.-N.)
| | - Arancha Cebrián
- Translational Oncology Division, Hospital Universitario Fundación Jimenez Diaz, 28040 Madrid, Spain; (A.R.-V.); (L.d.P.-N.)
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15
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Thies KA, Cole MW, Schafer RE, Spehar JM, Richardson DS, Steck SA, Das M, Lian AW, Ray A, Shakya R, Knoblaugh SE, Timmers CD, Ostrowski MC, Chakravarti A, Sizemore GM, Sizemore ST. The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer. Breast Cancer Res 2021; 23:65. [PMID: 34118960 PMCID: PMC8196523 DOI: 10.1186/s13058-021-01438-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Accepted: 05/13/2021] [Indexed: 02/01/2023] Open
Abstract
Background Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. Methods The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. Results Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. Conclusions Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC. Supplementary Information The online version contains supplementary material available at 10.1186/s13058-021-01438-3.
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Affiliation(s)
- Katie A Thies
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Matthew W Cole
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Rachel E Schafer
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Jonathan M Spehar
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Dillon S Richardson
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Sarah A Steck
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Manjusri Das
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Arthur W Lian
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Alo Ray
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Reena Shakya
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Target Validation Shared Resource, The Ohio State University, Columbus, OH, 43210, USA
| | - Sue E Knoblaugh
- Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, 43210, USA
| | - Cynthia D Timmers
- The Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA.,Division of Hematology and Oncology, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Michael C Ostrowski
- The Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA.,Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Arnab Chakravarti
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Gina M Sizemore
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA
| | - Steven T Sizemore
- Arthur G. James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA. .,Department of Radiation Oncology, The Ohio State University, 646A TMRF, 420 W. 12th Avenue, Columbus, OH, 43210, USA.
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16
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Nászai M, Bellec K, Yu Y, Román-Fernández A, Sandilands E, Johansson J, Campbell AD, Norman JC, Sansom OJ, Bryant DM, Cordero JB. RAL GTPases mediate EGFR-driven intestinal stem cell proliferation and tumourigenesis. eLife 2021; 10:e63807. [PMID: 34096503 PMCID: PMC8216719 DOI: 10.7554/elife.63807] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2020] [Accepted: 06/03/2021] [Indexed: 02/07/2023] Open
Abstract
RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine and the mechanisms of how they contribute to tumourigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine, via induction of EGFR internalisation. Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to influencing stem cell proliferation during damage-induced intestinal regeneration, this role of RAL GTPases impacts on EGFR-dependent tumourigenic growth in the intestine and in human mammary epithelium. However, the effect of oncogenic RAS in the intestine is independent from RAL function. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of adult tissue homeostasis and malignant transformation.
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MESH Headings
- Animals
- Animals, Genetically Modified
- Breast Neoplasms/enzymology
- Breast Neoplasms/genetics
- Breast Neoplasms/pathology
- Cell Line, Tumor
- Cell Proliferation
- Cell Transformation, Neoplastic/genetics
- Cell Transformation, Neoplastic/metabolism
- Cell Transformation, Neoplastic/pathology
- Drosophila Proteins/genetics
- Drosophila Proteins/metabolism
- Drosophila melanogaster/enzymology
- Drosophila melanogaster/genetics
- Endocytosis
- ErbB Receptors/genetics
- ErbB Receptors/metabolism
- Female
- Humans
- Hyperplasia
- Intestinal Mucosa/metabolism
- Intestinal Mucosa/pathology
- Lung Neoplasms/enzymology
- Lung Neoplasms/genetics
- Lung Neoplasms/pathology
- Mammary Glands, Human/enzymology
- Mammary Glands, Human/pathology
- Mice, Inbred C57BL
- Mitogen-Activated Protein Kinases/metabolism
- Monomeric GTP-Binding Proteins/genetics
- Monomeric GTP-Binding Proteins/metabolism
- Receptors, Invertebrate Peptide/genetics
- Receptors, Invertebrate Peptide/metabolism
- Signal Transduction
- Stem Cells/metabolism
- Stem Cells/pathology
- ral GTP-Binding Proteins/genetics
- ral GTP-Binding Proteins/metabolism
- Mice
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Affiliation(s)
- Máté Nászai
- Wolfson Wohl Cancer Research CentreGlasgowUnited Kingdom
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
| | - Karen Bellec
- Wolfson Wohl Cancer Research CentreGlasgowUnited Kingdom
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
| | - Yachuan Yu
- Wolfson Wohl Cancer Research CentreGlasgowUnited Kingdom
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - Alvaro Román-Fernández
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - Emma Sandilands
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - Joel Johansson
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | | | - Jim C Norman
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - Owen J Sansom
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - David M Bryant
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
| | - Julia B Cordero
- Wolfson Wohl Cancer Research CentreGlasgowUnited Kingdom
- Institute of Cancer Sciences, University of GlasgowGlasgowUnited Kingdom
- Cancer Research UK Beatson InstituteGlasgowUnited Kingdom
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17
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Rajendran R, Sudha D, Chidambaram S, Nagarajan H, Vetrivel U, Arunachalam JP. Retinoschisis and Norrie disease: a missing link. BMC Res Notes 2021; 14:204. [PMID: 34039417 PMCID: PMC8157631 DOI: 10.1186/s13104-021-05617-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Accepted: 05/15/2021] [Indexed: 11/10/2022] Open
Abstract
OBJECTIVE Retinoschisis and Norrie disease are X-linked recessive retinal disorders caused by mutations in RS1 and NDP genes respectively. Both are likely to be monogenic and no locus heterogeneity has been reported. However, there are reports showing overlapping features of Norrie disease and retinoschisis in a NDP knock-out mouse model and also the involvement of both the genes in retinoschisis patients. Yet, the exact molecular relationships between the two disorders have still not been understood. The study investigated the association between retinoschisin (RS1) and norrin (NDP) using in vitro and in silico approaches. Specific protein-protein interaction between RS1 and NDP was analyzed in human retina by co-immunoprecipitation assay and MALDI-TOF mass spectrometry. STRING database was used to explore the functional relationship. RESULT Co-immunoprecipitation demonstrated lack of a direct interaction between RS1 and NDP and was further substantiated by mass spectrometry. However, STRING revealed a potential indirect functional association between the two proteins. Progressively, our analyses indicate that FZD4 protein interactome via PLIN2 as well as the MAP kinase signaling pathway to be a likely link bridging the functional relationship between retinoschisis and Norrie disease.
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Affiliation(s)
- Rahini Rajendran
- Central Inter-Disciplinary Research Facility (CIDRF), Sri Balaji Vidyapeeth (Deemed To Be University), Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry, 607402, India
| | - Dhandayuthapani Sudha
- SN ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Nungambakkam, Chennai, 600006, India
| | - Subbulakshmi Chidambaram
- Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, 605014, India
| | - Hemavathy Nagarajan
- Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, 600006, India
| | - Umashankar Vetrivel
- National Institute of Traditional Medicine, Indian Council of Medical Research, Belagavi, 590010, India
| | - Jayamuruga Pandian Arunachalam
- Central Inter-Disciplinary Research Facility (CIDRF), Sri Balaji Vidyapeeth (Deemed To Be University), Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry, 607402, India. .,SN ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Nungambakkam, Chennai, 600006, India.
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18
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Cornish J, Owen D, Mott HR. RLIP76: A Structural and Functional Triumvirate. Cancers (Basel) 2021; 13:cancers13092206. [PMID: 34064388 PMCID: PMC8124665 DOI: 10.3390/cancers13092206] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2021] [Revised: 04/12/2021] [Accepted: 04/29/2021] [Indexed: 11/16/2022] Open
Abstract
RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications.
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19
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Liu X, Li Z, Wang Y. Advances in Targeted Therapy and Immunotherapy for Pancreatic Cancer. Adv Biol (Weinh) 2021; 5:e1900236. [PMID: 33729700 DOI: 10.1002/adbi.201900236] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 08/19/2020] [Indexed: 12/24/2022]
Abstract
Pancreatic cancer is a highly aggressive malignancy with an overall 5-year survival rate of <6% due to therapeutic resistance and late-stage diagnosis. These statistics have not changed despite 50 years of research and therapeutic development. Pancreatic cancer is predicted to become the second leading cause of cancer mortality by the year 2030. Currently, the treatment options for pancreatic cancer are limited. This disease is usually diagnosed at a late stage, which prevents curative surgical resection. Chemotherapy is the most frequently used approach for pancreatic cancer treatment and has limited effects. In many other cancer types, targeted therapy and immunotherapy have made great progress and have been shown to be very promising prospects; these treatments also provide hope for pancreatic cancer. The need for research on targeted therapy and immunotherapy is pressing due to the poor prognosis of pancreatic cancer, and in recent years, there have been some breakthroughs for targeted therapy and immunotherapy in pancreatic cancer. This review summarizes the current preclinical and clinical studies of targeted therapy and immunotherapy for pancreatic cancer and ends by describing the challenges and outlook.
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Affiliation(s)
- Xiaoxiao Liu
- CAS Key Laboratory of Tissue Microenvironment and Tumor, SINH - Changzheng Hospital Joint Center for Translational Medicine, Institutes for Translational Medicine (CAS-SMMU), Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Zhang Li
- CAS Key Laboratory of Tissue Microenvironment and Tumor, SINH - Changzheng Hospital Joint Center for Translational Medicine, Institutes for Translational Medicine (CAS-SMMU), Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
| | - Yuexiang Wang
- CAS Key Laboratory of Tissue Microenvironment and Tumor, SINH - Changzheng Hospital Joint Center for Translational Medicine, Institutes for Translational Medicine (CAS-SMMU), Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
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20
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Hurd CA, Brear P, Revell J, Ross S, Mott HR, Owen D. Affinity maturation of the RLIP76 Ral binding domain to inform the design of stapled peptides targeting the Ral GTPases. J Biol Chem 2021; 296:100101. [PMID: 33214225 PMCID: PMC7949049 DOI: 10.1074/jbc.ra120.015735] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Revised: 11/10/2020] [Accepted: 11/19/2020] [Indexed: 12/18/2022] Open
Abstract
Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction.
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Affiliation(s)
- Catherine A Hurd
- Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Paul Brear
- Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Jefferson Revell
- AstraZeneca, Sir Aaron Klug Building, Granta Park, Cambridge, UK
| | - Sarah Ross
- Research and Early Development, Oncology R&D, AstraZeneca, Cambridge, UK
| | - Helen R Mott
- Department of Biochemistry, University of Cambridge, Cambridge, UK.
| | - Darerca Owen
- Department of Biochemistry, University of Cambridge, Cambridge, UK.
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21
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Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives. Biomolecules 2020; 10:biom10111535. [PMID: 33182807 PMCID: PMC7698260 DOI: 10.3390/biom10111535] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2020] [Revised: 11/03/2020] [Accepted: 11/08/2020] [Indexed: 02/07/2023] Open
Abstract
RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.
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22
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Kelly MR, Kostyrko K, Han K, Mooney NA, Jeng EE, Spees K, Dinh PT, Abbott KL, Gwinn DM, Sweet-Cordero EA, Bassik MC, Jackson PK. Combined Proteomic and Genetic Interaction Mapping Reveals New RAS Effector Pathways and Susceptibilities. Cancer Discov 2020; 10:1950-1967. [PMID: 32727735 PMCID: PMC7710624 DOI: 10.1158/2159-8290.cd-19-1274] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 06/08/2020] [Accepted: 07/24/2020] [Indexed: 11/16/2022]
Abstract
Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.This article is highlighted in the In This Issue feature, p. 1775.
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Affiliation(s)
- Marcus R Kelly
- Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California.,Program in Cancer Biology, Stanford University School of Medicine, Stanford, California
| | - Kaja Kostyrko
- Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California
| | - Kyuho Han
- Department of Genetics, Stanford University School of Medicine, Stanford, California
| | - Nancie A Mooney
- Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California
| | - Edwin E Jeng
- Department of Genetics, Stanford University School of Medicine, Stanford, California
| | - Kaitlyn Spees
- Department of Genetics, Stanford University School of Medicine, Stanford, California
| | - Phuong T Dinh
- Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California
| | - Keene L Abbott
- Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California
| | - Dana M Gwinn
- Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California
| | - E Alejandro Sweet-Cordero
- Division of Hematology and Oncology, Department of Pediatrics, University of California, San Francisco, San Francisco, California.
| | - Michael C Bassik
- Department of Genetics, Stanford University School of Medicine, Stanford, California. .,Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California
| | - Peter K Jackson
- Baxter Laboratory, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California. .,Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California.,Department of Pathology, Stanford University School of Medicine, Stanford, California
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23
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Jang H, Zhang M, Nussinov R. The quaternary assembly of KRas4B with Raf-1 at the membrane. Comput Struct Biotechnol J 2020; 18:737-748. [PMID: 32257057 PMCID: PMC7125320 DOI: 10.1016/j.csbj.2020.03.018] [Citation(s) in RCA: 45] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2020] [Revised: 03/17/2020] [Accepted: 03/19/2020] [Indexed: 02/07/2023] Open
Abstract
Proximally located in the membrane, oncogenic Ras dimers (or nanoclusters) can recruit and promote Raf dimerization and MAPK (Raf/MEK/ERK) signaling. Among Ras isoforms, KRas4B is the most frequently mutated. Recent data on the binary KRas4B–Raf-1 complex suggested that Raf-1 CRD not only executes membrane anchorage, but also supports the high-affinity interaction of Raf-1 RBD with KRas4B catalytic domain. For a detailed mechanistic picture of Raf activation at the membrane, we employ explicit MD simulations of the quaternary KRas4B–Raf-1 complex. The complex contains two active GTP-bound KRas4B proteins forming a dimer through the allosteric lobe interface and two tandem RBD-CRD segments of Raf-1 interacting with the effector lobes at both ends of the KRas4B dimer. We show that Raf-1 RBD-CRD supports stable KRas4B dimer at preferred interface and orientation at the membrane, thereby cooperatively enhancing the affinity of the KRas4B–Raf-1 interaction. We propose that a Ras dimer at the membrane can increase the population of proximal Raf kinase domains, promoting kinase domain dimerization in the cytoplasm. Collectively, the dynamic Ras–Raf assembly promotes Raf activation not by allostery; instead, Ras activates Raf by shifting its ensemble toward kinase domain-accessible states through enhanced affinity at the membrane.
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Affiliation(s)
- Hyunbum Jang
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
| | - Mingzhen Zhang
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
| | - Ruth Nussinov
- Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.,Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
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24
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Chen S, Li F, Xu D, Hou K, Fang W, Li Y. The Function of RAS Mutation in Cancer and Advances in its Drug Research. Curr Pharm Des 2020; 25:1105-1114. [PMID: 31057104 DOI: 10.2174/1381612825666190506122228] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Accepted: 04/18/2019] [Indexed: 12/12/2022]
Abstract
RAS (H-ras, K-ras, and N-ras), as the second largest mutated gene driver in various human cancers, has long been a vital research target for cancer. Its function is to transform the extracellular environment into a cascade of intracellular signal transduction. RAS mutant protein regulates tumor cell proliferation, apoptosis, metabolism and angiogenesis through downstream MAPK, PI3K and other signaling pathways. In KRAS or other RAS-driven cancers, current treatments include direct inhibitors and upstream/downstream signaling pathway inhibitors. However, the research on these inhibitors has been largely restricted due to their escape inhibition and off-target toxicity. In this paper, we started with the role of normal and mutant RAS genes in cancer, elucidated the relevant RAS regulating pathways, and highlighted the important research advancements in RAS inhibitor research. We concluded that for the crosstalk between RAS pathways, the effect of single regulation may be limited, and the multi-target drug combined compensation mechanism is becoming a research hotspot.
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Affiliation(s)
- Shijie Chen
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
| | - Fengyang Li
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
| | - Dan Xu
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
| | - Kai Hou
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
| | - Weirong Fang
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
| | - Yunman Li
- State Key Laboratory of Natural Medicines, Department of Physiology, China Phar maceutical University, Nanjing 210009, China
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25
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Arner EN, Du W, Brekken RA. Behind the Wheel of Epithelial Plasticity in KRAS-Driven Cancers. Front Oncol 2019; 9:1049. [PMID: 31681587 PMCID: PMC6798880 DOI: 10.3389/fonc.2019.01049] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Accepted: 09/26/2019] [Indexed: 12/15/2022] Open
Abstract
Cellular plasticity, a feature associated with epithelial-to-mesenchymal transition (EMT), contributes to tumor cell survival, migration, invasion, and therapy resistance. Phenotypic plasticity of the epithelium is a critical feature in multiple phases of human cancer in an oncogene- and tissue-specific context. Many factors can drive epithelial plasticity, including activating mutations in KRAS, which are found in an estimated 30% of all cancers. In this review, we will introduce cellular plasticity and its effect on cancer progression and therapy resistance and then summarize the drivers of EMT with an emphasis on KRAS effector signaling. Lastly, we will discuss the contribution of cellular plasticity to metastasis and its potential clinical implications. Understanding oncogenic KRAS cellular reprogramming has the potential to reveal novel strategies to control metastasis in KRAS-driven cancers.
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Affiliation(s)
- Emily N Arner
- Cancer Biology Graduate Program, Department of Surgery and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Wenting Du
- Cancer Biology Graduate Program, Department of Surgery and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Rolf A Brekken
- Cancer Biology Graduate Program, Department of Surgery and the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX, United States.,Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, United States
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26
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Spiegelman NA, Zhang X, Jing H, Cao J, Kotliar IB, Aramsangtienchai P, Wang M, Tong Z, Rosch KM, Lin H. SIRT2 and Lysine Fatty Acylation Regulate the Activity of RalB and Cell Migration. ACS Chem Biol 2019; 14:2014-2023. [PMID: 31433161 DOI: 10.1021/acschembio.9b00492] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Protein lysine fatty acylation is increasingly recognized as a prevalent and important protein post-translation modification. Recently, it has been shown that K-Ras4a, R-Ras2, and Rac1 are regulated by lysine fatty acylation. Here, we investigated whether other members of the Ras superfamily could also be regulated by lysine fatty acylation. Several small GTPases exhibit hydroxylamine resistant fatty acylation, suggesting they may also have protein lysine fatty acylation. We further characterized one of these GTPases, RalB. We show that RalB has C-terminal lysine fatty acylation, with the predominant modification site being Lys200. The lysine acylation of RalB is regulated by SIRT2, a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacylases. Lysine fatty acylated RalB exhibited enhanced plasma membrane localization and recruited its known effectors Sec5 and Exo84, members of the exocyst complex, to the plasma membrane. RalB lysine fatty acylation did not affect the proliferation or anchorage-independent growth but did affect the trans-well migration of A549 lung cancer cells. This study thus identified an additional function for protein lysine fatty acylation and the deacylase SIRT2.
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Affiliation(s)
- Nicole A. Spiegelman
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Xiaoyu Zhang
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Hui Jing
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Ji Cao
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Ilana B. Kotliar
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
- Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Ave., New York, New York 10065, United States
| | - Pornpun Aramsangtienchai
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Miao Wang
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Zhen Tong
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Kelly M. Rosch
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
| | - Hening Lin
- Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
- Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
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27
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Singh MK, Martin APJ, Joffre C, Zago G, Camonis J, Coppey M, Parrini MC. Localization of RalB signaling at endomembrane compartments and its modulation by autophagy. Sci Rep 2019; 9:8910. [PMID: 31222145 PMCID: PMC6586930 DOI: 10.1038/s41598-019-45443-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Accepted: 05/30/2019] [Indexed: 02/02/2023] Open
Abstract
The monomeric GTPase RalB controls crucial physiological processes, including autophagy and invasion, but it still remains unclear how this multi-functionality is achieved. Previously, we reported that the RalGEF (Guanine nucleotide Exchange Factor) RGL2 binds and activates RalB to promote invasion. Here we show that RGL2, a major activator of RalB, is also required for autophagy. Using a novel automated image analysis method, Endomapper, we quantified the endogenous localization of the RGL2 activator and its substrate RalB at different endomembrane compartments, in an isogenic normal and Ras-transformed cell model. In both normal and Ras-transformed cells, we observed that RGL2 and RalB substantially localize at early and recycling endosomes, and to lesser extent at autophagosomes, but not at trans-Golgi. Interestingly the use of a FRET-based RalB biosensor indicated that RalB signaling is active at these endomembrane compartments at basal level in rich medium. Furthermore, induction of autophagy by nutrient starvation led to a considerable reduction of early and recycling endosomes, in contrast to the expected increase of autophagosomes, in both normal and Ras-transformed cells. However, autophagy mildly affected relative abundances of both RGL2 and RalB at early and recycling endosomes, and at autophagosomes. Interestingly, RalB activity increased at autophagosomes upon starvation in normal cells. These results suggest that the contribution of endosome membranes (carrying RGL2 and RalB molecules) increases total pool of RGL2-RalB at autophagosome forming compartments and might contribute to amplify RalB signaling to support autophagy.
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Affiliation(s)
- Manish Kumar Singh
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France
- ART group, Inserm U830, 75005, Paris, France
| | - Alexandre P J Martin
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France
- ART group, Inserm U830, 75005, Paris, France
| | - Carine Joffre
- Centre de Recherches en Cancérologie de Toulouse (CRCT), Inserm UMR1037, Toulouse, France
| | - Giulia Zago
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France
- ART group, Inserm U830, 75005, Paris, France
| | - Jacques Camonis
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France
- ART group, Inserm U830, 75005, Paris, France
| | - Mathieu Coppey
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France
- LOCCO group, UMR168, 75005, Paris, France
| | - Maria Carla Parrini
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, 75005, Paris, France.
- ART group, Inserm U830, 75005, Paris, France.
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28
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Galino J, Cervellini I, Zhu N, Stöberl N, Hütte M, Fricker FR, Lee G, McDermott L, Lalli G, Bennett DLH. RalGTPases contribute to Schwann cell repair after nerve injury via regulation of process formation. J Cell Biol 2019; 218:2370-2387. [PMID: 31201266 PMCID: PMC6605803 DOI: 10.1083/jcb.201811002] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2018] [Revised: 04/10/2019] [Accepted: 05/15/2019] [Indexed: 12/02/2022] Open
Abstract
RalA and RalB are involved in cell migration and membrane dynamics. This study finds that ablation of RalGTPases impairs nerve regeneration and alters Schwann cell process formation; conversely, activation of RalGTPases enhancea Schwann cell process formation, migration, and axon myelination. RalA and RalB are small GTPases that are involved in cell migration and membrane dynamics. We used transgenic mice in which one or both GTPases were genetically ablated to investigate the role of RalGTPases in the Schwann cell (SC) response to nerve injury and repair. RalGTPases were dispensable for SC function in the naive uninjured state. Ablation of both RalA and RalB (but not individually) in SCs resulted in impaired axon remyelination and target reinnervation following nerve injury, which resulted in slowed recovery of motor function. Ral GTPases were localized to the leading lamellipodia in SCs and were required for the formation and extension of both axial and radial processes of SCs. These effects were dependent on interaction with the exocyst complex and impacted on the rate of SC migration and myelination. Our results show that RalGTPases are required for efficient nerve repair by regulating SC process formation, migration, and myelination, therefore uncovering a novel role for these GTPases.
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Affiliation(s)
- Jorge Galino
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Ilaria Cervellini
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Ning Zhu
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Nina Stöberl
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Meike Hütte
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Florence R Fricker
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Garrett Lee
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Lucy McDermott
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
| | - Giovanna Lalli
- Wolfson Centre for Age-Related Diseases, King's College London, Guy's Campus, London, UK
| | - David L H Bennett
- The Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
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29
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Johansson J, Naszai M, Hodder MC, Pickering KA, Miller BW, Ridgway RA, Yu Y, Peschard P, Brachmann S, Campbell AD, Cordero JB, Sansom OJ. RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes. Cell Stem Cell 2019; 24:592-607.e7. [PMID: 30853556 PMCID: PMC6459002 DOI: 10.1016/j.stem.2019.02.002] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2018] [Revised: 12/24/2018] [Accepted: 02/05/2019] [Indexed: 01/05/2023]
Abstract
Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
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Affiliation(s)
- Joel Johansson
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK
| | - Mate Naszai
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK
| | | | | | - Bryan W Miller
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK
| | | | - Yachuan Yu
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK
| | | | | | | | - Julia B Cordero
- Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
| | - Owen J Sansom
- Cancer Research UK Beatson Institute, Glasgow G61 1BD, UK; Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UK.
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30
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Cruz VH, Arner EN, Du W, Bremauntz AE, Brekken RA. Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer. JCI Insight 2019; 5:126117. [PMID: 30938713 PMCID: PMC6538328 DOI: 10.1172/jci.insight.126117] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 03/27/2019] [Indexed: 01/11/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB-dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl-TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease.
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Affiliation(s)
- Victoria H. Cruz
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | - Emily N. Arner
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | - Wenting Du
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
| | | | - Rolf A. Brekken
- Division of Surgical Oncology, Department of Surgery, and the Hamon Center for Therapeutic Oncology Research
- Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
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31
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Gong S, Chen Y, Meng F, Zhang Y, Wu H, Li C, Zhang G. RCC2, a regulator of the RalA signaling pathway, is identified as a novel therapeutic target in cisplatin-resistant ovarian cancer. FASEB J 2019; 33:5350-5365. [PMID: 30768358 DOI: 10.1096/fj.201801529rr] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Currently, cisplatin (DDP) is the first-line chemotherapeutic agent used for treatment of ovarian cancer, but gradually acquired drug resistance minimizes its therapeutic outcomes. We aimed to identify crucial genes associated with DDP resistance in ovarian cancer and uncover potential mechanisms. Two sets of gene expression data were downloaded from Gene Expression Omnibus, and bioinformatics analysis was conducted. In our study, the differentially expressed genes between DDP-sensitive and DDP-resistant ovarian cancer were screened in GSE15709 and GSE51373 database, and chromosome condensation 2 regulator (RCC2) and nucleoporin 160 were identified as 2 genes that significantly up-regulated in DDP-resistant ovarian cancer cell lines compared with DDP-sensitive cell lines. Moreover, RCC2, Ral small GTPase (RalA), and Ral binding protein-1 (RalBP1) expression was found to be significantly higher in DDP-resistant ovarian cancer tissues than in DDP-sensitive tissues. RCC2 plays a positive role in cell proliferation, apoptosis, and migration in DDP-resistant ovarian cancer cell lines in vitro and in vivo. Furthermore, RCC2 could interact with RalA, thus promoting its downstream effector RalBP1. RalA knockdown could reverse the effects of RCC2 overexpression on DDP-resistant ovarian cancer cell proliferation, apoptosis, and migration. Similarly, RalA overexpression could alleviate the effects of RCC2 knockdown in DDP-resistant ovarian cancer cells. Taken together, RCC2 may function as an oncogene, regulating the RalA signaling pathway, and intervention of RCC2 expression might be a promising therapeutic strategy for DDP-resistant ovarian cancer.-Gong, S., Chen, Y., Meng, F., Zhang, Y., Wu, H., Li, C., Zhang, G. RCC2, a regulator of the RalA signaling pathway, is identified as a novel therapeutic target in cisplatin-resistant ovarian cancer.
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Affiliation(s)
- Shipeng Gong
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yongning Chen
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Fanliang Meng
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Yadi Zhang
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Huan Wu
- Department of Obstetrics and Gynecology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China; and
| | - Chanyuan Li
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Guangping Zhang
- Department of Gynecology, People's Hospital of Huadu District, Guangzhou, China
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Erickson KE, Rukhlenko OS, Posner RG, Hlavacek WS, Kholodenko BN. New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling. Semin Cancer Biol 2019; 54:162-173. [PMID: 29518522 PMCID: PMC6123307 DOI: 10.1016/j.semcancer.2018.02.008] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Revised: 02/13/2018] [Accepted: 02/22/2018] [Indexed: 01/04/2023]
Abstract
RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.
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Affiliation(s)
- Keesha E Erickson
- Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM, USA
| | - Oleksii S Rukhlenko
- Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland
| | - Richard G Posner
- Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA
| | - William S Hlavacek
- Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM, USA; University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA
| | - Boris N Kholodenko
- Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland; Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Ireland; School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland.
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Abstract
Our understanding of fundamental biological processes within platelets is continually evolving. A critical feature of platelet biology relates to the intricate uptake, packaging and release of bioactive cargo from storage vesicles, essential in mediating a range of classical (haemostasis/thrombosis) and non-classical (regeneration/inflammation/metastasis) roles platelets assume. Pivotal to the molecular control of these vesicle trafficking events are the small GTPases of the Ras superfamily, which function as spatially distinct, molecular switches controlling essential cellular processes. Herein, we specifically focus on members of the Rab, Arf and Ras subfamilies, which comprise over 130 members and platelet proteomic datasets suggest that more than half of these are expressed in human platelets. We provide an update of current literature relating to trafficking roles for these GTPases in platelets, particularly regarding endocytic and exocytic events, but also vesicle biogenesis and provide speculative argument for roles that other related GTPases and regulatory proteins may adopt in platelets. Advances in our understanding of small GTPase function in the anucleate platelet has been hampered by the lack of specific molecular tools, but it is anticipated that this will be greatly accelerated in the years ahead and will be crucial to the identification of novel therapeutic targets controlling different platelet processes.
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Affiliation(s)
- Tony G Walsh
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Yong Li
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Andreas Wersäll
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
| | - Alastair W Poole
- a From the School of Physiology, Pharmacology and Neuroscience, Biomedical Sciences Building , University of Bristol , Bristol , UK
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Zago G, Veith I, Singh MK, Fuhrmann L, De Beco S, Remorino A, Takaoka S, Palmeri M, Berger F, Brandon N, El Marjou A, Vincent-Salomon A, Camonis J, Coppey M, Parrini MC. RalB directly triggers invasion downstream Ras by mobilizing the Wave complex. eLife 2018; 7:40474. [PMID: 30320548 PMCID: PMC6226288 DOI: 10.7554/elife.40474] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 10/14/2018] [Indexed: 12/27/2022] Open
Abstract
The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies. Cancers develop when cells in the body divide rapidly in an uncontroled manner. It is generally possible to cure cancers that remain contained within a small area. However, if the tumor cells start to move, the cancer may spread in the body and become life threatening. Currently, most of the anti-cancer treatments act to reduce the multiplication of these cells, but not their ability to migrate. A signal protein called Ras stimulates human cells to grow and move around. In healthy cells, the activity of Ras is tightly controled to ensure cells only divide and migrate at particular times, but in roughly 30% of all human cancers, Ras is abnormally active. Ras switches on another protein, named RalB, which is also involved in inappropriate cell migration. Yet, it is not clear how RalB is capable to help Ras trigger the migration of cells. Zago et al. used an approach called optogenetics to specifically activate the RalB protein in human cells using a laser that produces blue light. When activated, the light-controlled RalB started abnormal cell migration; this was used to dissect which molecules and mechanisms were involved in the process. Taken together, the experiments showed that, first, Ras ‘turns on’ RalB by changing the location of two proteins that control RalB. Then, the activated RalB regulates the exocyst, a group of proteins that travel within the cell. In turn, the exocyst recruits another group of proteins, named the Wave complex, which is part of the molecular motor required for cells to migrate. Zago et al. also found that, in patients, the RalB protein was present at abnormally high levels in samples of breast cancer cells that had migrated to another part of the body. Overall, these findings indicate that the role of RalB protein in human cancers is larger than previously thought, and they highlight a new pathway that could be a target for new anti-cancer drugs.
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Affiliation(s)
- Giulia Zago
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Irina Veith
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Manish Kumar Singh
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Laetitia Fuhrmann
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,Department of Pathology, Institut Curie, Paris, France
| | - Simon De Beco
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,LOCCO Group, UMR168, Paris, France
| | - Amanda Remorino
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,LOCCO Group, UMR168, Paris, France
| | - Saori Takaoka
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Marjorie Palmeri
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Frédérique Berger
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,Department of Biostatistics, Institut Curie, Paris, France
| | - Nathalie Brandon
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Ahmed El Marjou
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,Protein Expression and Purification Core Facility, Paris, France
| | - Anne Vincent-Salomon
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,Department of Pathology, Institut Curie, Paris, France
| | - Jacques Camonis
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
| | - Mathieu Coppey
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,LOCCO Group, UMR168, Paris, France
| | - Maria Carla Parrini
- Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France.,ART Group, Inserm U830, Paris, France
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Bisht S, Feldmann G. Novel Targets in Pancreatic Cancer Therapy - Current Status and Ongoing Translational Efforts. Oncol Res Treat 2018; 41:596-602. [PMID: 30269126 DOI: 10.1159/000493437] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2018] [Accepted: 09/03/2018] [Indexed: 12/11/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC, pancreatic cancer) carries one of the poorest overall prognoses of all human malignancies known to date. Despite the introduction of novel therapeutic regimens, the outcome has not markedly improved over the past decades, the incidence rates are almost identical to the mortality rates, and PDAC is projected to soon become the second most common cause of cancer-related mortality in Western countries. Despite this clear medical need to develop novel therapeutic strategies against this dire malady, this need has so far not been addressed with sufficient institutional attention and support in terms of research funding and strategical programs. Given the still growing life expectancy and projected demographic changes with a growing proportion of senior citizens in many European societies, this discrepancy is likely to become even more pressing in the future. This article provides a brief overview of ongoing preclinical efforts to identify novel targets and, based on this, to develop novel strategies to treat advanced pancreatic cancer and improve survival and the quality of life of patients suffering from this malignancy.
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Zhang Y, Luu BE, Storey KB. FoxO4 activity is regulated by phosphorylation and the cellular environment during dehydration in the African clawed frog, Xenopus laevis. Biochim Biophys Acta Gen Subj 2018; 1862:1721-1728. [DOI: 10.1016/j.bbagen.2018.05.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2018] [Revised: 04/07/2018] [Accepted: 05/04/2018] [Indexed: 01/10/2023]
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Inchanalkar S, Deshpande NU, Kasherwal V, Jayakannan M, Balasubramanian N. Polymer Nanovesicle-Mediated Delivery of MLN8237 Preferentially Inhibits Aurora Kinase A To Target RalA and Anchorage-Independent Growth in Breast Cancer Cells. Mol Pharm 2018; 15:3046-3059. [PMID: 29863884 DOI: 10.1021/acs.molpharmaceut.8b00163] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The small GTPase RalA is a known mediator of anchorage-independent growth in cancers and is differentially regulated by adhesion and aurora kinase A (AURKA). Hence, inhibiting AURKA offers a means of specifically targeting RalA (over RalB) in cancer cells. MLN8237 (alisertib) is a known inhibitor of aurora kinases; its specificity for AURKA, however, is compromised by its poor solubility and transport across the cell membrane. A polymer nanovesicle platform is used for the first time to deliver and differentially inhibit AURKA in cancer cells. For this purpose, polysaccharide nanovesicles made from amphiphilic dextran were used as nanocarriers to successfully administer MLN8237 (VMLN) in cancer cells in 2D and 3D microenvironments. These nanovesicles (<200 nm) carry the drug in their intermembrane space with up to 85% of it released by the action of esterase enzyme(s). Lysotracker experiments reveal the polymer nanovesicles localize in the lysosomal compartment of the cell, where they are enzymatically targeted and MLN released in a controlled manner. Rhodamine B fluorophore trapped in the nanovesicles hydrophilic core (VMLN+RhB) allows us to visualize its uptake and localization in cells in a 2D and 3D microenvironment. In breast cancer, MCF-7 cells VMLN inhibits AURKA significantly better than the free drug at low concentrations (0.02-0.04 μM). This ensures that the drug in VMLN at these concentrations can specifically inhibit up to 94% of endogenous AURKA without affecting AURKB. This targeting of AURKA causes the downstream differential inhibition of active RalA (but not RalB). Free MLN8237 at similar concentrations and conditions failed to affect RalA activation. VMLN-mediated inhibition of RalA, in turn, disrupts the anchorage-independent growth of MCF-7 cells supporting a role for the AURKA-RalA crosstalk in mediating the same. These studies not only identify the polysaccharide nanovesicle to be an improved way to efficiently deliver low concentrations of MLN8237 to inhibit AURKA but, in doing so, also help reveal a role for AURKA and its crosstalk with RalA in anchorage-independent growth of MCF-7 cells.
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Sphingomyelin Metabolism Is a Regulator of K-Ras Function. Mol Cell Biol 2018; 38:MCB.00373-17. [PMID: 29158292 DOI: 10.1128/mcb.00373-17] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 11/08/2017] [Indexed: 01/07/2023] Open
Abstract
K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans, as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.
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Moghadam AR, Patrad E, Tafsiri E, Peng W, Fangman B, Pluard TJ, Accurso A, Salacz M, Shah K, Ricke B, Bi D, Kimura K, Graves L, Najad MK, Dolatkhah R, Sanaat Z, Yazdi M, Tavakolinia N, Mazani M, Amani M, Ghavami S, Gartell R, Reilly C, Naima Z, Esfandyari T, Farassati F. Ral signaling pathway in health and cancer. Cancer Med 2017; 6:2998-3013. [PMID: 29047224 PMCID: PMC5727330 DOI: 10.1002/cam4.1105] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2016] [Revised: 04/10/2017] [Accepted: 04/14/2017] [Indexed: 12/12/2022] Open
Abstract
The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors.
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Affiliation(s)
- Adel Rezaei Moghadam
- Department of Human Anatomy and Cell ScienceUniversity of ManitobaWinnipegCanada
| | - Elham Patrad
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Elham Tafsiri
- Department of Pediatrics, Columbia Presbyterian Medical CenterNew YorkNew York
| | - Warner Peng
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Benjamin Fangman
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Timothy J Pluard
- Saint Luke's HospitalUniversity of Missouri at Kansas CityKansas CityMissouri
| | - Anthony Accurso
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Michael Salacz
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Kushal Shah
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Brandon Ricke
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Danse Bi
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Kyle Kimura
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Leland Graves
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Marzieh Khajoie Najad
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Roya Dolatkhah
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Zohreh Sanaat
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Mina Yazdi
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Naeimeh Tavakolinia
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Mohammad Mazani
- Pasteur Institute of IranTehranIran
- Ardabil University of Medical Sciences, BiochemistryArdabilIran
| | - Mojtaba Amani
- Pasteur Institute of IranTehranIran
- Ardabil University of Medical Sciences, BiochemistryArdabilIran
| | - Saeid Ghavami
- Department of Human Anatomy and Cell ScienceUniversity of ManitobaWinnipegCanada
| | - Robyn Gartell
- Department of Pediatrics, Columbia Presbyterian Medical CenterNew YorkNew York
| | - Colleen Reilly
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Zaid Naima
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Tuba Esfandyari
- Department of Medicine, Molecular Medicine LaboratoryThe University of Kansas Medical SchoolKansas CityKansas
| | - Faris Farassati
- Research Service (151)Kansas City Veteran Affairs Medical Center & Midwest Biomedical Research Foundation4801 E Linwood BlvdKansas CityMissouri64128‐2226
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Gulyás G, Radvánszki G, Matuska R, Balla A, Hunyady L, Balla T, Várnai P. Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins. J Biol Chem 2017; 292:18862-18877. [PMID: 28939768 DOI: 10.1074/jbc.m117.806679] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2017] [Revised: 09/11/2017] [Indexed: 11/06/2022] Open
Abstract
Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C β or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2 but not PtdIns(3,4,5)P3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras-mediated signals.
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Affiliation(s)
- Gergő Gulyás
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary
| | - Glória Radvánszki
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary
| | - Rita Matuska
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary
| | - András Balla
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary.,MTA-SE Laboratory of Molecular Physiology, Hungarian Academy of Sciences and Semmelweis University, Budapest 1094, Hungary, and
| | - László Hunyady
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary.,MTA-SE Laboratory of Molecular Physiology, Hungarian Academy of Sciences and Semmelweis University, Budapest 1094, Hungary, and
| | - Tamas Balla
- Section on Molecular Signal Transduction, Eunice Kennedy Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892
| | - Péter Várnai
- From the Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest 1094, Hungary,
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Miao Y, Wu J, Abraham SN. Ubiquitination of Innate Immune Regulator TRAF3 Orchestrates Expulsion of Intracellular Bacteria by Exocyst Complex. Immunity 2017; 45:94-105. [PMID: 27438768 DOI: 10.1016/j.immuni.2016.06.023] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2016] [Revised: 04/26/2016] [Accepted: 06/21/2016] [Indexed: 12/27/2022]
Abstract
Although the intracellular trafficking system is integral to most physiologic activities, its role in mediating immune responses to infection has remained elusive. Here, we report that infected bladder epithelial cells (BECs) mobilized the exocyst complex, a powerful exporter of subcellular vesicles, to rapidly expel intracellular bacteria back for clearance. Toll-like receptor (TLR) 4 signals emanating from bacteria-containing vesicles (BCVs) were found to trigger K33-linked polyubiquitination of TRAF3 at Lys168, which was then detected by RalGDS, a guanine nucleotide exchange factor (GEF) that precipitated the assembly of the exocyst complex. Although this distinct modification of TRAF3 served to connect innate immune signaling to the cellular trafficking apparatus, it crucially ensured temporal and spatial accuracy in determining which among the many subcellular vesicles was recognized and selected for expulsion in response to innate immune signaling.
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Affiliation(s)
- Yuxuan Miao
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
| | - Jianxuan Wu
- Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA
| | - Soman N Abraham
- Department of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27710, USA; Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA; Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA; Program in Emerging Infectious Diseases, Duke-National University of Singapore, Singapore 169857, Singapore
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42
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Integrin-Dependent Regulation of Small GTPases: Role in Cell Migration. J Indian Inst Sci 2017. [DOI: 10.1007/s41745-016-0010-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Ganapathy S, Fagman JB, Shen L, Yu T, Zhou X, Dai W, Makriyannis A, Chen C. Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C. Oncotarget 2016; 7:84326-84337. [PMID: 27741517 PMCID: PMC5356664 DOI: 10.18632/oncotarget.12607] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Accepted: 10/04/2016] [Indexed: 01/08/2023] Open
Abstract
Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.
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Affiliation(s)
| | - Johan B Fagman
- The Institute of Clinic Sciences, Sahlgrenska Academy, Gothenburg, SE
| | - Ling Shen
- Center for Drug Discovery, Northeastern University, Boston, MA, USA
| | - Tianqi Yu
- Center for Drug Discovery, Northeastern University, Boston, MA, USA
| | - Xiaodong Zhou
- Center for Drug Discovery, Northeastern University, Boston, MA, USA
- The First Affiliated Hospital of Nanchang University, Nanchang, China
| | - Wei Dai
- Department of Environmental Medicine, New York University, Tuxedo, NY, USA
| | | | - Changyan Chen
- Center for Drug Discovery, Northeastern University, Boston, MA, USA
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Ahmad DAJ, Negm OH, Alabdullah ML, Mirza S, Hamed MR, Band V, Green AR, Ellis IO, Rakha EA. Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers. Breast Cancer Res Treat 2016; 159:457-67. [PMID: 27592113 PMCID: PMC5021722 DOI: 10.1007/s10549-016-3967-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2016] [Accepted: 08/26/2016] [Indexed: 12/21/2022]
Abstract
BACKGROUND Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. METHODS Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. RESULTS MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER+ subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER+ and ER- cell lines. CONCLUSIONS MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathway.
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Affiliation(s)
- Dena A J Ahmad
- Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK.,Department of Pathology, Mosul Medical School, University of Mosul, Mosul, Iraq
| | - Ola H Negm
- School of Medicine, Queen's Medical Hospital, University of Nottingham, Derby Road, Nottingham, NG7 2UH, UK. .,Faculty of Medicine, Medical Microbiology and Immunology Department, Mansoura University, Mansoura, Egypt.
| | - M Layth Alabdullah
- Academic Unit of Clinical Oncology, School of Medicine, Nottingham City Hospital, University of Nottingham, Nottingham, UK
| | - Sameer Mirza
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska, Omaha, USA
| | - Mohamed R Hamed
- School of Medicine, Queen's Medical Hospital, University of Nottingham, Derby Road, Nottingham, NG7 2UH, UK.,Faculty of Medicine, Medical Microbiology and Immunology Department, Mansoura University, Mansoura, Egypt
| | - Vimla Band
- Department of Genetics, Cell Biology and Anatomy, University of Nebraska, Omaha, USA
| | - Andrew R Green
- Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK
| | - Ian O Ellis
- Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK
| | - Emad A Rakha
- Division of Cancer and Stem Cells, Department of Histopathology, School of Medicine, Nottingham City Hospital, The University of Nottingham and Nottingham University Hospitals NHS Trust, Nottingham, UK
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45
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Inhibition of skeletal muscle atrophy during torpor in ground squirrels occurs through downregulation of MyoG and inactivation of Foxo4. Cryobiology 2016; 73:112-9. [PMID: 27593478 DOI: 10.1016/j.cryobiol.2016.08.013] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Revised: 08/15/2016] [Accepted: 08/31/2016] [Indexed: 12/20/2022]
Abstract
Foxo4 and MyoG proteins regulate the transcription of numerous genes, including the E3 ubiquitin ligases MAFbx and MuRF1, which are activated in skeletal muscle under atrophy-inducing conditions. In the thirteen-lined ground squirrel, there is little muscle wasting that occurs during hibernation, a process characterized by bouts of torpor and arousal, despite virtual inactivity. Consequently, we were interested in studying the regulatory role of Foxo4 and MyoG on ubiquitin ligases throughout torpor-arousal cycles. Findings indicate that MAFbx and MuRF1 decreased during early torpor (ET) by 42% and 40%, respectively, relative to euthermic control (EC), although MuRF1 expression subsequently increased at late torpor (LT). The expression pattern of MyoG most closely resembled that of MAFbx, with levels decreasing during LT. In addition, the phosphorylation of Foxo4 at Thr-451 showed an initial increase during EN, followed by a decline throughout the remainder of the torpor-arousal cycle, suggesting Foxo4 inhibition. This trend was mirrored by inhibition of the Ras-Ral pathway, as the Ras and Ral proteins were decreased by 77% and 41% respectively, at ET. Foxo4 phosphorylation at S197 was depressed during entrance and torpor, suggesting Foxo4 nuclear localization, and possibly regulating the increase in MuRF1 levels at LT. These findings indicate that signaling pathways involved in regulating muscle atrophy, such as MyoG and Foxo4 through the Ras-Ral pathway, contribute to important muscle-specific changes during hibernation. Therefore, this data provides novel insight into the molecular mechanisms regulating muscle remodeling in a hibernator model.
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Coyle SM. Reverse engineering GTPase programming languages with reconstituted signaling networks. Small GTPases 2016; 7:168-72. [PMID: 27128855 PMCID: PMC5003541 DOI: 10.1080/21541248.2016.1178367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2016] [Revised: 04/08/2016] [Accepted: 04/11/2016] [Indexed: 10/21/2022] Open
Abstract
The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems.
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Affiliation(s)
- Scott M. Coyle
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA
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Thomas JC, Cooper JM, Clayton NS, Wang C, White MA, Abell C, Owen D, Mott HR. Inhibition of Ral GTPases Using a Stapled Peptide Approach. J Biol Chem 2016; 291:18310-25. [PMID: 27334922 DOI: 10.1074/jbc.m116.720243] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2016] [Indexed: 01/31/2023] Open
Abstract
Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.
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Affiliation(s)
- Jemima C Thomas
- From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom
| | - Jonathan M Cooper
- Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9039
| | - Natasha S Clayton
- From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom
| | - Chensu Wang
- Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9039
| | - Michael A White
- Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas 75390-9039
| | - Chris Abell
- Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom
| | - Darerca Owen
- From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom,
| | - Helen R Mott
- From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom,
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Jang H, Muratcioglu S, Gursoy A, Keskin O, Nussinov R. Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers. Biochem J 2016; 473:1719-32. [PMID: 27057007 PMCID: PMC7830773 DOI: 10.1042/bcj20160031] [Citation(s) in RCA: 81] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2016] [Accepted: 04/07/2016] [Indexed: 02/07/2023]
Abstract
Are the dimer structures of active Ras isoforms similar? This question is significant since Ras can activate its effectors as a monomer; however, as a dimer, it promotes Raf's activation and MAPK (mitogen-activated protein kinase) cell signalling. In the present study, we model possible catalytic domain dimer interfaces of membrane-anchored GTP-bound K-Ras4B and H-Ras, and compare their conformations. The active helical dimers formed by the allosteric lobe are isoform-specific: K-Ras4B-GTP favours the α3 and α4 interface; H-Ras-GTP favours α4 and α5. Both isoforms also populate a stable β-sheet dimer interface formed by the effector lobe; a less stable β-sandwich interface is sustained by salt bridges of the β-sheet side chains. Raf's high-affinity β-sheet interaction is promoted by the active helical interface. Collectively, Ras isoforms' dimer conformations are not uniform; instead, the isoform-specific dimers reflect the favoured interactions of the HVRs (hypervariable regions) with cell membrane microdomains, biasing the effector-binding site orientations, thus isoform binding selectivity.
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Affiliation(s)
- Hyunbum Jang
- Cancer and Inflammation Program, National Cancer Institute at Frederick, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, U.S.A
| | - Serena Muratcioglu
- Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
| | - Attila Gursoy
- Department of Computer Engineering, Koc University, Istanbul, Turkey
| | - Ozlem Keskin
- Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey
| | - Ruth Nussinov
- Cancer and Inflammation Program, National Cancer Institute at Frederick, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, U.S.A. Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
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Pawar A, Meier JA, Dasgupta A, Diwanji N, Deshpande N, Saxena K, Buwa N, Inchanalkar S, Schwartz MA, Balasubramanian N. Ral-Arf6 crosstalk regulates Ral dependent exocyst trafficking and anchorage independent growth signalling. Cell Signal 2016; 28:1225-1236. [PMID: 27269287 PMCID: PMC4973806 DOI: 10.1016/j.cellsig.2016.05.023] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2016] [Revised: 05/30/2016] [Accepted: 05/31/2016] [Indexed: 12/01/2022]
Abstract
Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells.
Ral mediates Arf6 activation downstream of integrins and oncogenic Ras. Arf6 mediates Ral-exocyst dependent delivery of raft microdomains. Active Ral supports anchorage independent Arf6 activation in bladder cancer T24 cells. Ral-Arf6 crosstalk in T24 cells regulates anchorage independent Erk signalling. A Ral-RalBP1-ARNO-Arf6 pathway mediates the Ral-Arf6 crosstalk.
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Affiliation(s)
- Archana Pawar
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Jeremy A Meier
- Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, United States
| | - Anwesha Dasgupta
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Neha Diwanji
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Neha Deshpande
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Kritika Saxena
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Natasha Buwa
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Siddhi Inchanalkar
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India
| | - Martin Alexander Schwartz
- Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, United States; Yale Cardiovascular Research Center, 300 George Street, 7th Floor, New Haven, CT 06511, United States
| | - Nagaraj Balasubramanian
- Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India.
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50
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RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases. PLoS One 2016; 11:e0154840. [PMID: 27149377 PMCID: PMC4858283 DOI: 10.1371/journal.pone.0154840] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2016] [Accepted: 04/20/2016] [Indexed: 11/19/2022] Open
Abstract
The human genome contains six genes coding for proteins validated in vitro as specific activators of the small GTPases “Ras-related protein Ral-A” and “Ras-related protein Ral-B”, generically named Ral-guanine nucleotide exchange factors (RalGEF). Ral proteins are important contributors to Ras oncogenic signaling, and RAS oncogenes are important in human Non-Small Cell Lung Carcinoma (NSCLC). Therefore in this work, RalGEF contribution to oncogenic and non-oncogenic features of human NSCLC cell lines, as anchorage-dependent and independent growth, cell survival, and proliferation, was investigated. Among all human RalGEF, silencing of RGL1 and RALGPS1 had no detectable effect. However, silencing of either RGL2, RGL3, RALGDS or, to a larger extent, RALGPS2 inhibited cell population growth in anchorage dependent and independent conditions (up to 90 and 80%, respectively). RALGPS2 silencing also caused an increase in the number of apoptotic cells, up to 45% of the cell population in transformed bronchial BZR cells. In H1299 and A549, two NSCLC cell lines, RALGPS2 silencing caused an arrest of cells in the G0/G1-phase of cell cycle. Furthermore, it was associated with the modulation of important cell cycle regulators: the E3 Ubiquitin Protein Ligase S-phase kinase-associated protein 2 (Skp2) was strongly down-regulated (both at mRNA and protein levels), and its targets, the cell cycle inhibitors p27 and p21, were up-regulated. These molecular effects were not mimicked by silencing RALA, RALB, or both. However, RALB silencing caused a modest inhibition of cell cycle progression, which in H1299 cells was associated with Cyclin D1 regulation. In conclusion, RALGPS2 is implicated in the control of cell cycle progression and survival in the in vitro growth of NSCLC cell lines. This function is largely independent of Ral GTPases and associated with modulation of Skp2, p27 and p21 cell cycle regulators.
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