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Jeon S, Jeon Y, Lim JY, Kim Y, Cha B, Kim W. Emerging regulatory mechanisms and functions of biomolecular condensates: implications for therapeutic targets. Signal Transduct Target Ther 2025; 10:4. [PMID: 39757214 DOI: 10.1038/s41392-024-02070-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 10/01/2024] [Accepted: 11/06/2024] [Indexed: 01/07/2025] Open
Abstract
Cells orchestrate their processes through complex interactions, precisely organizing biomolecules in space and time. Recent discoveries have highlighted the crucial role of biomolecular condensates-membrane-less assemblies formed through the condensation of proteins, nucleic acids, and other molecules-in driving efficient and dynamic cellular processes. These condensates are integral to various physiological functions, such as gene expression and intracellular signal transduction, enabling rapid and finely tuned cellular responses. Their ability to regulate cellular signaling pathways is particularly significant, as it requires a careful balance between flexibility and precision. Disruption of this balance can lead to pathological conditions, including neurodegenerative diseases, cancer, and viral infections. Consequently, biomolecular condensates have emerged as promising therapeutic targets, with the potential to offer novel approaches to disease treatment. In this review, we present the recent insights into the regulatory mechanisms by which biomolecular condensates influence intracellular signaling pathways, their roles in health and disease, and potential strategies for modulating condensate dynamics as a therapeutic approach. Understanding these emerging principles may provide valuable directions for developing effective treatments targeting the aberrant behavior of biomolecular condensates in various diseases.
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Affiliation(s)
- Soyoung Jeon
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Yeram Jeon
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Ji-Youn Lim
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea
| | - Yujeong Kim
- Department of Life Science, University of Seoul, Seoul, South Korea
| | - Boksik Cha
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, South Korea.
| | - Wantae Kim
- Department of Life Science, University of Seoul, Seoul, South Korea.
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2
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Herrmannová A, Jelínek J, Pospíšilová K, Kerényi F, Vomastek T, Watt K, Brábek J, Mohammad MP, Wagner S, Topisirovic I, Valášek LS. Perturbations in eIF3 subunit stoichiometry alter expression of ribosomal proteins and key components of the MAPK signaling pathways. eLife 2024; 13:RP95846. [PMID: 39495207 PMCID: PMC11534336 DOI: 10.7554/elife.95846] [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] [Indexed: 11/05/2024] Open
Abstract
Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.
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Affiliation(s)
- Anna Herrmannová
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Jan Jelínek
- Laboratory of Bioinformatics, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Klára Pospíšilová
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Farkas Kerényi
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Tomáš Vomastek
- Laboratory of Cell Signaling, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Kathleen Watt
- Science for Life Laboratory, Department of Oncology-Pathology, Karolinska InstitutetSolnaSweden
| | - Jan Brábek
- Lady Davis Institute, Laboratory of Cancer Cell Invasion, Faculty of Science, Charles UniversityPragueCzech Republic
| | - Mahabub Pasha Mohammad
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Susan Wagner
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
| | - Ivan Topisirovic
- Lady Davis Institute, Gerald Bronfman Department of Oncology, Department of Biochemistry, Division of Experimental Medicine, McGill UniversityMontréalCanada
| | - Leoš Shivaya Valášek
- Laboratory of Regulation of Gene Expression, Institute of Microbiology of the Czech Academy of SciencesPragueCzech Republic
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3
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Gregory MD, Ofosu-Asante K, Lazarte JMS, Puente PE, Tawfeeq N, Belony N, Huang Y, Offringa IA, Lamango NS. Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis. PLoS One 2024; 19:e0312563. [PMID: 39436906 PMCID: PMC11495567 DOI: 10.1371/journal.pone.0312563] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Accepted: 09/11/2024] [Indexed: 10/25/2024] Open
Abstract
KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations.
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Affiliation(s)
- Matthew D. Gregory
- Institute of Public Health, Florida A&M University College of Pharmacy Pharmaceutical Sciences, Tallahassee, FL, United States of America
| | - Kweku Ofosu-Asante
- Institute of Public Health, Florida A&M University College of Pharmacy Pharmaceutical Sciences, Tallahassee, FL, United States of America
| | - Jassy Mary S. Lazarte
- Institute of Public Health, Florida A&M University College of Pharmacy Pharmaceutical Sciences, Tallahassee, FL, United States of America
| | - Pablo E. Puente
- Keck School of Medicine, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA, United States of America
| | - Nada Tawfeeq
- Institute of Public Health, Florida A&M University College of Pharmacy Pharmaceutical Sciences, Tallahassee, FL, United States of America
| | - Nadine Belony
- University of Florida Department of Mechanical and Aerospace Engineering, Gainesville, FL, United States of America
| | - Yong Huang
- University of Florida Department of Mechanical and Aerospace Engineering, Gainesville, FL, United States of America
| | - Ite A. Offringa
- Keck School of Medicine, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA, United States of America
| | - Nazarius S. Lamango
- Institute of Public Health, Florida A&M University College of Pharmacy Pharmaceutical Sciences, Tallahassee, FL, United States of America
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4
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Hossain MA. Targeting the RAS upstream and downstream signaling pathway for cancer treatment. Eur J Pharmacol 2024; 979:176727. [PMID: 38866361 DOI: 10.1016/j.ejphar.2024.176727] [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: 03/08/2024] [Revised: 06/05/2024] [Accepted: 06/06/2024] [Indexed: 06/14/2024]
Abstract
Cancer often involves the overactivation of RAS/RAF/MEK/ERK (MAPK) and PI3K-Akt-mTOR pathways due to mutations in genes like RAS, RAF, PTEN, and PIK3CA. Various strategies are employed to address the overactivation of these pathways, among which targeted therapy emerges as a promising approach. Directly targeting specific proteins, leads to encouraging results in cancer treatment. For instance, RTK inhibitors such as imatinib and afatinib selectively target these receptors, hindering ligand binding and reducing signaling initiation. These inhibitors have shown potent efficacy against Non-Small Cell Lung Cancer. Other inhibitors, like lonafarnib targeting Farnesyltransferase and GGTI 2418 targeting geranylgeranyl Transferase, disrupt post-translational modifications of proteins. Additionally, inhibition of proteins like SOS, SH2 domain, and Ras demonstrate promising anti-tumor activity both in vivo and in vitro. Targeting downstream components with RAF inhibitors such as vemurafenib, dabrafenib, and sorafenib, along with MEK inhibitors like trametinib and binimetinib, has shown promising outcomes in treating cancers with BRAF-V600E mutations, including myeloma, colorectal, and thyroid cancers. Furthermore, inhibitors of PI3K (e.g., apitolisib, copanlisib), AKT (e.g., ipatasertib, perifosine), and mTOR (e.g., sirolimus, temsirolimus) exhibit promising efficacy against various cancers such as Invasive Breast Cancer, Lymphoma, Neoplasms, and Hematological malignancies. This review offers an overview of small molecule inhibitors targeting specific proteins within the RAS upstream and downstream signaling pathways in cancer.
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Affiliation(s)
- Md Arafat Hossain
- Department of Pharmacy, Bangabandhu Sheikh Mujibur Rahman Science and Technology University, Gopalganj, 8100, Bangladesh.
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5
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Yun SD, Scott E, Chang JY, Bahramimoghaddam H, Lynn M, Lantz C, Russell DH, Laganowsky A. Capturing RAS oligomerization on a membrane. Proc Natl Acad Sci U S A 2024; 121:e2405986121. [PMID: 39145928 PMCID: PMC11348296 DOI: 10.1073/pnas.2405986121] [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: 03/22/2024] [Accepted: 07/09/2024] [Indexed: 08/16/2024] Open
Abstract
RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.
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Affiliation(s)
- Sangho D. Yun
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | - Elena Scott
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | - Jing-Yuan Chang
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | | | - Michael Lynn
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | - Carter Lantz
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | - David H. Russell
- Department of Chemistry, Texas A&M University, College Station, TX77843
| | - Arthur Laganowsky
- Department of Chemistry, Texas A&M University, College Station, TX77843
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6
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Mozzarelli AM, Simanshu DK, Castel P. Functional and structural insights into RAS effector proteins. Mol Cell 2024; 84:2807-2821. [PMID: 39025071 PMCID: PMC11316660 DOI: 10.1016/j.molcel.2024.06.027] [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: 03/08/2024] [Revised: 06/24/2024] [Accepted: 06/25/2024] [Indexed: 07/20/2024]
Abstract
RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets.
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Affiliation(s)
- Alessandro M Mozzarelli
- Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY, USA; Laura and Isaac Perlmutter NYU Cancer Center, NYU Langone Health, New York, NY, USA
| | - Dhirendra K Simanshu
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
| | - Pau Castel
- Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of Medicine, New York, NY, USA; Laura and Isaac Perlmutter NYU Cancer Center, NYU Langone Health, New York, NY, USA.
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7
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Jeon H, Tkacik E, Eck MJ. Signaling from RAS to RAF: The Molecules and Their Mechanisms. Annu Rev Biochem 2024; 93:289-316. [PMID: 38316136 DOI: 10.1146/annurev-biochem-052521-040754] [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] [Indexed: 02/07/2024]
Abstract
RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).
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Affiliation(s)
- Hyesung Jeon
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA;
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
| | - Emre Tkacik
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA;
- Systems, Synthetic, and Quantitative Biology PhD Program, Harvard Medical School, Boston, Massachusetts, USA
| | - Michael J Eck
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA;
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
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8
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Torres-Jiménez J, Espinar JB, de Cabo HB, Berjaga MZ, Esteban-Villarrubia J, Fraile JZ, Paz-Ares L. Targeting KRAS G12C in Non-Small-Cell Lung Cancer: Current Standards and Developments. Drugs 2024; 84:527-548. [PMID: 38625662 DOI: 10.1007/s40265-024-02030-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/27/2024] [Indexed: 04/17/2024]
Abstract
Among the most common molecular alterations detected in non-small-cell lung cancer (NSCLC) are mutations in Kristen Rat Sarcoma viral oncogene homolog (KRAS). KRAS mutant NSCLC is a heterogenous group of diseases, different from other oncogene-driven tumors in terms of biology and response to therapies. Despite efforts to develop drugs aimed at inhibiting KRAS or its signaling pathways, KRAS had remained undruggable for decades. The discovery of a small pocket in the binding switch II region of KRASG12C has revolutionized the treatment of KRASG12C-mutated NSCLC patients. Sotorasib and adagrasib, direct KRASG12C inhibitors, have been approved by the US Food and Drug Administration (FDA) and other regulatory agencies for patients with previously treated KRASG12C-mutated NSCLC, and these advances have become practice changing. However, first-line treatment in KRASG12C-mutated NSCLC does not differ from NSCLC without actionable driver genomic alterations. Treatment with KRASG12C inhibitors is not curative and patients develop progressive disease, so understanding associated mechanisms of drug resistance is key. New KRASG12C inhibitors and several combination therapy strategies, including with immune checkpoint inhibitors, are being studied in clinical trials. The aim of this review is to explore the clinical impact of KRAS, and outline different treatment approaches, focusing on the novel treatment of KRASG12C-mutated NSCLC.
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Affiliation(s)
- Javier Torres-Jiménez
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain.
| | - Javier Baena Espinar
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
| | - Helena Bote de Cabo
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
| | - María Zurera Berjaga
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
| | - Jorge Esteban-Villarrubia
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
| | - Jon Zugazagoitia Fraile
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
- Lung Cancer Group, Clinical Research Program, CNIO (Centro Nacional de Investigaciones Oncológicas) and Instituto de Investigación i+12, Madrid, Spain
| | - Luis Paz-Ares
- Medical Oncology Department, Hospital Universitario 12 de Octubre, Avda de Córdoba s/n, 28041, Madrid, Spain
- Lung Cancer Group, Clinical Research Program, CNIO (Centro Nacional de Investigaciones Oncológicas) and Instituto de Investigación i+12, Madrid, Spain
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9
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Shanderson RL, Ferguson ID, Siprashvili Z, Ducoli L, Li AM, Miao W, Srinivasan S, Velasco MG, Li Y, Ye J, Khavari PA. Mitochondrial Raf1 Regulates Glutamine Catabolism. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.08.581297. [PMID: 38496616 PMCID: PMC10942467 DOI: 10.1101/2024.03.08.581297] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2024]
Abstract
Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.
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Affiliation(s)
- Ronald L. Shanderson
- Program in Cancer Biology, Stanford University, Stanford, CA, 94305, USA
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | - Ian D. Ferguson
- Program in Cancer Biology, Stanford University, Stanford, CA, 94305, USA
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | - Zurab Siprashvili
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | - Luca Ducoli
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | - Albert M. Li
- Program in Cancer Biology, Stanford University, Stanford, CA, 94305, USA
- Department of Radiation Oncology, Stanford University, Stanford, CA, 94305, USA
| | - Weili Miao
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | - Suhas Srinivasan
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
| | | | - Yang Li
- Department of Radiation Oncology, Stanford University, Stanford, CA, 94305, USA
| | - Jiangbin Ye
- Program in Cancer Biology, Stanford University, Stanford, CA, 94305, USA
- Department of Radiation Oncology, Stanford University, Stanford, CA, 94305, USA
| | - Paul A. Khavari
- Program in Cancer Biology, Stanford University, Stanford, CA, 94305, USA
- Program in Epithelial Biology, Stanford University, Stanford, CA, 94305, USA
- Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA, 94304, USA
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10
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Riaud M, Maxwell J, Soria-Bretones I, Dankner M, Li M, Rose AAN. The role of CRAF in cancer progression: from molecular mechanisms to precision therapies. Nat Rev Cancer 2024; 24:105-122. [PMID: 38195917 DOI: 10.1038/s41568-023-00650-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 11/27/2023] [Indexed: 01/11/2024]
Abstract
The RAF family of kinases includes key activators of the pro-tumourigenic mitogen-activated protein kinase pathway. Hyperactivation of RAF proteins, particularly BRAF and CRAF, drives tumour progression and drug resistance in many types of cancer. Although BRAF is the most studied RAF protein, partially owing to its high mutation incidence in melanoma, the role of CRAF in tumourigenesis and drug resistance is becoming increasingly clinically relevant. Here, we summarize the main known regulatory mechanisms and gene alterations that contribute to CRAF activity, highlighting the different oncogenic roles of CRAF, and categorize RAF1 (CRAF) mutations according to the effect on kinase activity. Additionally, we emphasize the effect that CRAF alterations may have on drug resistance and how precision therapies could effectively target CRAF-dependent tumours. Here, we discuss preclinical and clinical findings that may lead to improved treatments for all types of oncogenic RAF1 alterations in cancer.
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Affiliation(s)
- Melody Riaud
- Gerald Bronfman Department of Oncology, McGill University, Montreal, Quebec, Canada
- Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
| | - Jennifer Maxwell
- Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
- Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
| | - Isabel Soria-Bretones
- Gerald Bronfman Department of Oncology, McGill University, Montreal, Quebec, Canada
- Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
- Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada
| | - Matthew Dankner
- Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
- Faculty of Medicine, McGill University, Montreal, Quebec, Canada
- Rosalind & Morris Goodman Cancer Institute, McGill University, Montreal, Quebec, Canada
| | - Meredith Li
- Gerald Bronfman Department of Oncology, McGill University, Montreal, Quebec, Canada
| | - April A N Rose
- Gerald Bronfman Department of Oncology, McGill University, Montreal, Quebec, Canada.
- Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada.
- Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
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11
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Bahar ME, Kim HJ, Kim DR. Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies. Signal Transduct Target Ther 2023; 8:455. [PMID: 38105263 PMCID: PMC10725898 DOI: 10.1038/s41392-023-01705-z] [Citation(s) in RCA: 83] [Impact Index Per Article: 41.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2023] [Revised: 11/03/2023] [Accepted: 11/12/2023] [Indexed: 12/19/2023] Open
Abstract
Metastatic dissemination of solid tumors, a leading cause of cancer-related mortality, underscores the urgent need for enhanced insights into the molecular and cellular mechanisms underlying metastasis, chemoresistance, and the mechanistic backgrounds of individuals whose cancers are prone to migration. The most prevalent signaling cascade governed by multi-kinase inhibitors is the mitogen-activated protein kinase (MAPK) pathway, encompassing the RAS-RAF-MAPK kinase (MEK)-extracellular signal-related kinase (ERK) pathway. RAF kinase is a primary mediator of the MAPK pathway, responsible for the sequential activation of downstream targets, such as MEK and the transcription factor ERK, which control numerous cellular and physiological processes, including organism development, cell cycle control, cell proliferation and differentiation, cell survival, and death. Defects in this signaling cascade are associated with diseases such as cancer. RAF inhibitors (RAFi) combined with MEK blockers represent an FDA-approved therapeutic strategy for numerous RAF-mutant cancers, including melanoma, non-small cell lung carcinoma, and thyroid cancer. However, the development of therapy resistance by cancer cells remains an important barrier. Autophagy, an intracellular lysosome-dependent catabolic recycling process, plays a critical role in the development of RAFi resistance in cancer. Thus, targeting RAF and autophagy could be novel treatment strategies for RAF-mutant cancers. In this review, we delve deeper into the mechanistic insights surrounding RAF kinase signaling in tumorigenesis and RAFi-resistance. Furthermore, we explore and discuss the ongoing development of next-generation RAF inhibitors with enhanced therapeutic profiles. Additionally, this review sheds light on the functional interplay between RAF-targeted therapies and autophagy in cancer.
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Affiliation(s)
- Md Entaz Bahar
- Department of Biochemistry and Convergence Medical Sciences and Institute of Medical Science, Gyeongsang National University, College of Medicine, Jinju, South Korea
| | - Hyun Joon Kim
- Department of Anatomy and Convergence Medical Sciences and Institute of Medical Science, Gyeongsang National University, College of Medicine, Jinju, South Korea
| | - Deok Ryong Kim
- Department of Biochemistry and Convergence Medical Sciences and Institute of Medical Science, Gyeongsang National University, College of Medicine, Jinju, South Korea.
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12
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Shree S, McLean MA, Stephen AG, Sligar SG. Revealing KRas4b topology on the membrane surface. Biochem Biophys Res Commun 2023; 678:122-127. [PMID: 37633182 PMCID: PMC10528110 DOI: 10.1016/j.bbrc.2023.08.035] [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: 08/04/2023] [Accepted: 08/16/2023] [Indexed: 08/28/2023]
Abstract
KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.
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Affiliation(s)
- Shweta Shree
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States
| | - Mark A McLean
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States
| | - Andrew G Stephen
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD, 21701, United States
| | - Stephen G Sligar
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States.
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13
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Park E, Rawson S, Schmoker A, Kim BW, Oh S, Song K, Jeon H, Eck MJ. Cryo-EM structure of a RAS/RAF recruitment complex. Nat Commun 2023; 14:4580. [PMID: 37516774 PMCID: PMC10387098 DOI: 10.1038/s41467-023-40299-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Accepted: 07/19/2023] [Indexed: 07/31/2023] Open
Abstract
RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.
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Affiliation(s)
- Eunyoung Park
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA
- Pfizer R&D Center, 3200 Walnut St, Boulder, CO, 80301, USA
| | - Shaun Rawson
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA
| | - Anna Schmoker
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
| | - Byeong-Won Kim
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
- New Drug Development Center, Osong Medical Innovation Foundation, Cheongju, Chungbuk, 28160, Republic of Korea
| | - Sehee Oh
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA
| | - Kangkang Song
- Department of Biochemistry & Molecular Biotechnology, University of Massachusetts Chan Medical School, 364 Plantation St, Worcester, MA, 01605, USA
| | - Hyesung Jeon
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
| | - Michael J Eck
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
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14
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Cuevas-Navarro A, Wagner M, Van R, Swain M, Mo S, Columbus J, Allison MR, Cheng A, Messing S, Turbyville TJ, Simanshu DK, Sale MJ, McCormick F, Stephen AG, Castel P. RAS-dependent RAF-MAPK hyperactivation by pathogenic RIT1 is a therapeutic target in Noonan syndrome-associated cardiac hypertrophy. SCIENCE ADVANCES 2023; 9:eadf4766. [PMID: 37450595 PMCID: PMC10348673 DOI: 10.1126/sciadv.adf4766] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Accepted: 06/14/2023] [Indexed: 07/18/2023]
Abstract
RIT1 is a RAS guanosine triphosphatase (GTPase) that regulates different aspects of signal transduction and is mutated in lung cancer, leukemia, and in the germline of individuals with Noonan syndrome. Pathogenic RIT1 proteins promote mitogen-activated protein kinase (MAPK) hyperactivation; however, this mechanism remains poorly understood. Here, we show that RAF kinases are direct effectors of membrane-bound mutant RIT1 necessary for MAPK activation. We identify critical residues in RIT1 that facilitate interaction with membrane lipids and show that these are necessary for association with RAF kinases and MAPK activation. Although mutant RIT1 binds to RAF kinases directly, it fails to activate MAPK signaling in the absence of classical RAS proteins. Consistent with aberrant RAF/MAPK activation as a driver of disease, we show that pathway inhibition alleviates cardiac hypertrophy in a mouse model of RIT1 mutant Noonan syndrome. These data shed light on the function of pathogenic RIT1 and identify avenues for therapeutic intervention.
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Affiliation(s)
- Antonio Cuevas-Navarro
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Morgan Wagner
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Richard Van
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Monalisa Swain
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Stephanie Mo
- Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA
| | - John Columbus
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Madeline R. Allison
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Alice Cheng
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Simon Messing
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Thomas J. Turbyville
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Dhirendra K. Simanshu
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Matthew J. Sale
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Frank McCormick
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Andrew G. Stephen
- NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick, MD 21702, USA
| | - Pau Castel
- Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY 10016, USA
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15
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Sadeghi Shaker M, Rokni M, Mahmoudi M, Farhadi E. Ras family signaling pathway in immunopathogenesis of inflammatory rheumatic diseases. Front Immunol 2023; 14:1151246. [PMID: 37256120 PMCID: PMC10225558 DOI: 10.3389/fimmu.2023.1151246] [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: 01/25/2023] [Accepted: 04/26/2023] [Indexed: 06/01/2023] Open
Abstract
The Ras (rat sarcoma virus) is a GTP-binding protein that is considered one of the important members of the Ras-GTPase superfamily. The Ras involves several pathways in the cell that include proliferation, migration, survival, differentiation, and fibrosis. Abnormalities in the expression level and activation of the Ras family signaling pathway and its downstream kinases such as Raf/MEK/ERK1-2 contribute to the pathogenic mechanisms of rheumatic diseases including immune system dysregulation, inflammation, and fibrosis in systemic sclerosis (SSc); destruction and inflammation of synovial tissue in rheumatoid arthritis (RA); and autoantibody production and immune complexes formation in systemic lupus erythematosus (SLE); and enhance osteoblast differentiation and ossification during skeletal formation in ankylosing spondylitis (AS). In this review, the basic biology, signaling of Ras, and abnormalities in this pathway in rheumatic diseases including SSc, RA, AS, and SLE will be discussed.
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Affiliation(s)
- Mina Sadeghi Shaker
- Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Mohsen Rokni
- Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Department of Immunology, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran
| | - Mahdi Mahmoudi
- Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran
- Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Elham Farhadi
- Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran
- Inflammation Research Center, Tehran University of Medical Sciences, Tehran, Iran
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16
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Leone GM, Candido S, Lavoro A, Vivarelli S, Gattuso G, Calina D, Libra M, Falzone L. Clinical Relevance of Targeted Therapy and Immune-Checkpoint Inhibition in Lung Cancer. Pharmaceutics 2023; 15:1252. [PMID: 37111737 PMCID: PMC10142433 DOI: 10.3390/pharmaceutics15041252] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 04/12/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
Lung cancer (LC) represents the second most diagnosed tumor and the malignancy with the highest mortality rate. In recent years, tremendous progress has been made in the treatment of this tumor thanks to the discovery, testing, and clinical approval of novel therapeutic approaches. Firstly, targeted therapies aimed at inhibiting specific mutated tyrosine kinases or downstream factors were approved in clinical practice. Secondly, immunotherapy inducing the reactivation of the immune system to efficiently eliminate LC cells has been approved. This review describes in depth both current and ongoing clinical studies, which allowed the approval of targeted therapies and immune-checkpoint inhibitors as standard of care for LC. Moreover, the present advantages and pitfalls of new therapeutic approaches will be discussed. Finally, the acquired importance of human microbiota as a novel source of LC biomarkers, as well as therapeutic targets to improve the efficacy of available therapies, was analyzed. Therapy against LC is increasingly becoming holistic, taking into consideration not only the genetic landscape of the tumor, but also the immune background and other individual variables, such as patient-specific gut microbial composition. On these bases, in the future, the research milestones reached will allow clinicians to treat LC patients with tailored approaches.
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Affiliation(s)
- Gian Marco Leone
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123 Catania, Italy
| | - Saverio Candido
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123 Catania, Italy
- Research Center for Prevention, Diagnosis and Treatment of Cancer, University of Catania, 95123 Catania, Italy
| | - Alessandro Lavoro
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123 Catania, Italy
| | - Silvia Vivarelli
- Department of Biomedical and Dental Sciences, Morphological and Functional Imaging, Section of Occupational Medicine, University of Messina, 98125 Messina, Italy
| | - Giuseppe Gattuso
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123 Catania, Italy
| | - Daniela Calina
- Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania
| | - Massimo Libra
- Department of Biomedical and Biotechnological Sciences, University of Catania, 95123 Catania, Italy
- Research Center for Prevention, Diagnosis and Treatment of Cancer, University of Catania, 95123 Catania, Italy
| | - Luca Falzone
- Epidemiology and Biostatistics Unit, Istituto Nazionale Tumori IRCCS “Fondazione G. Pascale”, 80131 Naples, Italy;
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17
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Baicalin Inhibits Airway Smooth Muscle Cells Proliferation through the RAS Signaling Pathway in Murine Asthmatic Airway Remodeling Model. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2023; 2023:4144138. [PMID: 36814956 PMCID: PMC9940961 DOI: 10.1155/2023/4144138] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 12/31/2022] [Accepted: 01/12/2023] [Indexed: 02/17/2023]
Abstract
Background Studies that looked at asthma airway remodeling pathogenesis and prevention have led to the discovery of the rat sarcoma viral oncogene (RAS) signaling pathway as a key mechanism that controls airway smooth muscle cell (ASMC) proliferation. Baicalin has great anti-inflammatory, proliferation-inhibited, and respiratory disease-relieving properties. However, the inhibitory effects and mechanisms of baicalin on ASMC-mediated airway remodeling in mice are still poorly understood. Methods After establishing the asthmatic mice model by ovalbumin (OVA) and interfering with baicalin, airway remodeling characteristics such as airway resistance, mRNA, and protein expression levels of remodeling-related cytokines were measured by histopathological assessment, quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and western blot. Further efforts on detailed mechanisms were used antibody arrays to compare the expression and activation of proteins involved in the RAS signaling pathway. In addition, validation experiments were performed in ASMC proliferation model and low-expression cells of the target gene by using shRNA. Results In OVA-induced asthmatic mice model, baicalin significantly reduced the infiltration of inflammatory cells in lung tissue, attenuated airway resistance, and decreased mRNA and protein expression levels of remodeling-related cytokines such as interleukin-13 (IL-13), vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), matrix metallopeptidase 9 (MMP9), and tissue inhibitor of metalloproteinase 1 (TIMP1). The results of antibody arrays involved in RAS signaling pathway revealed that OVA and baicalin administration altered the activation of protein kinase C alpha type (PKC-α), A-rapidly accelerated fibrosarcoma (A-RAF), mitogen-activated protein kinase 2 (MEK2), extracellular regulated MAP kinase (ERK), MAPK interacting serine/threonine kinase 1 (MNK1), and ETS transcription factor 1 (ELK1). The above results were further verified in the ASMC proliferation model. A-RAF silencing (shA-RAF) could promote ASMC proliferation and downregulate p-MEK2, p-ERK, p-MNK1, and p-ELK1 expression. Conclusion The effects of baicalin against airway remodeling and ASMC proliferation might partially be achieved by suppressing the RAS signaling pathway. Baicalin may be a new therapeutic option for managing airway remodeling in asthma patients.
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18
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Spencer-Smith R, Terrell EM, Insinna C, Agamasu C, Wagner ME, Ritt DA, Stauffer J, Stephen AG, Morrison DK. RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation. Mol Cell 2022; 82:4262-4276.e5. [PMID: 36347258 PMCID: PMC9677513 DOI: 10.1016/j.molcel.2022.10.016] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Revised: 07/16/2022] [Accepted: 10/14/2022] [Indexed: 11/09/2022]
Abstract
BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.
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Affiliation(s)
- Russell Spencer-Smith
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA
| | - Elizabeth M Terrell
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA
| | - Christine Insinna
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA
| | - Constance Agamasu
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702 USA
| | - Morgan E Wagner
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702 USA
| | - Daniel A Ritt
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA
| | - Jim Stauffer
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA
| | - Andrew G Stephen
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702 USA
| | - Deborah K Morrison
- Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702 USA.
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19
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Novel natural inhibitors targeting KRAS G12C by computational study. Anticancer Drugs 2022; 33:779-788. [PMID: 35980001 DOI: 10.1097/cad.0000000000001317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Ideal leading and nominee compounds with inhibiting effects on KRAS G12C were selected from the ZINC database, laying a cornerstone for the progress of anticancer drugs. A variety of computational virtual screening methods were utilized to screen possible inhibitors of KRAS G12C. LibDock was utilized to estimate 17 930 compounds and the top 20 were nominated for additional study, which was absorption, distribution, metabolism, and excretion and harmfulness prediction. Molecule docking was employed to prove the binding connection between certain ligands and KRAS G12C. Natural novel compounds ZINC000012494057 and ZINC000003789195 were selected to bind stably with KRAS G12C. In addition, they had lower scores in Ames mutagenicity, rodent carcinogenicity, cytochrome P450 2D6(CYP2D6) tolerance, and non-developmental toxicity potential. Molecular dynamic simulations demonstrate that the combination of ZINC000012494057 and ZINC000003789195 with KRAS G12C has more favorable potential energy, which provides conditions for their stable existence in the natural environment. Natural compounds ZINC000012494057 and ZINC000003789195 were identified as KRAS G12C potential inhibitors. These two compounds have been verified to have enormous importance for the progress of anticancer medicines.
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20
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Tortosa E, Sengupta Ghosh A, Li Q, Wong WR, Hinkle T, Sandoval W, Rose CM, Hoogenraad CC. Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration. EMBO J 2022; 41:e110155. [PMID: 35611591 PMCID: PMC9289706 DOI: 10.15252/embj.2021110155] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 04/21/2022] [Accepted: 04/22/2022] [Indexed: 11/09/2022] Open
Abstract
Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.
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Affiliation(s)
- Elena Tortosa
- Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA
| | | | - Qingling Li
- Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA
| | - Weng Ruh Wong
- Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA
| | - Trent Hinkle
- Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA
| | - Wendy Sandoval
- Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA
| | - Christopher M Rose
- Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA
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21
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Andreadelis I, Kiriakidi S, Lamprakis C, Theodoropoulou A, Doerr S, Chatzigoulas A, Manchester J, Velez-Vega C, Duca JS, Cournia Z. Membrane Composition and Raf[CRD]-Membrane Attachment Are Driving Forces for K-Ras4B Dimer Stability. J Phys Chem B 2022; 126:1504-1519. [PMID: 35142524 DOI: 10.1021/acs.jpcb.1c01184] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf[CRD] domain. We conclude that the presence of effectors with the Raf[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.
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Affiliation(s)
- Ioannis Andreadelis
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
| | - Sofia Kiriakidi
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
| | - Christos Lamprakis
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
| | | | - Stefan Doerr
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
| | - Alexios Chatzigoulas
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
| | - John Manchester
- Computer-Aided Drug Discovery, Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States
| | - Camilo Velez-Vega
- Computer-Aided Drug Discovery, Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States
| | - José S Duca
- Computer-Aided Drug Discovery, Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States
| | - Zoe Cournia
- Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527 Athens, Greece
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22
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Asimgil H, Ertetik U, Çevik NC, Ekizce M, Doğruöz A, Gökalp M, Arık-Sever E, Istvanffy R, Friess H, Ceyhan GO, Demir IE. Targeting the undruggable oncogenic KRAS: the dawn of hope. JCI Insight 2022; 7:e153688. [PMID: 35014625 PMCID: PMC8765045 DOI: 10.1172/jci.insight.153688] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
KRAS mutations are the drivers of various cancers, including non-small cell lung cancer, colon cancer, and pancreatic cancer. Over the last 30 years, immense efforts have been made to inhibit KRAS mutants and oncogenic KRAS signaling using inhibitors. Recently, specific targeting of KRAS mutants with small molecules revived the hopes for successful therapies for lung, pancreatic, and colorectal cancer patients. Moreover, advances in gene editing, protein engineering, and drug delivery formulations have revolutionized cancer therapy regimens. New therapies aim to improve immune surveillance and enhance antitumor immunity by precisely targeting cancer cells harboring oncogenic KRAS. Here, we review recent KRAS-targeting strategies, their therapeutic potential, and remaining challenges to overcome. We also highlight the potential synergistic effects of various combinatorial therapies in preclinical and clinical trials.
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Affiliation(s)
- Hande Asimgil
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Utku Ertetik
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Nedim Can Çevik
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Menar Ekizce
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Alper Doğruöz
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Muazzez Gökalp
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Elif Arık-Sever
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Rouzanna Istvanffy
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany
- German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany
- SFB/Collaborative Research Centre 1321 Modelling and Targeting Pancreatic Cancer, Munich, Germany
| | - Helmut Friess
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany
- German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany
- SFB/Collaborative Research Centre 1321 Modelling and Targeting Pancreatic Cancer, Munich, Germany
| | - Güralp Onur Ceyhan
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
| | - Ihsan Ekin Demir
- Department of Surgery, Klinikum rechts der Isar, Technical University of Munich, School of Medicine, Munich, Germany
- Department of General Surgery, Hepatopancreatobiliary-Unit, School of Medicine, Kerem Aydınlar Campus at Acıbadem University, Istanbul, Turkey
- German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany
- SFB/Collaborative Research Centre 1321 Modelling and Targeting Pancreatic Cancer, Munich, Germany
- Else Kröner Clinician Scientist Professor for Translational Pancreatic Surgery, Munich, Germany
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23
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He W, Cao X, Kong K, Rong K, Han S, Qin A. Ceritinib (LDK378) prevents bone loss via suppressing Akt and NF-κB-induced osteoclast formation. Front Endocrinol (Lausanne) 2022; 13:939959. [PMID: 36425467 PMCID: PMC9679281 DOI: 10.3389/fendo.2022.939959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Accepted: 10/10/2022] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND Ceritinib is used for the treatment of patients with anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC), who are at the risk of developing bone metastasis. During bone metastasis, tumor cells release factors that induce osteoclast formation, resulting in osteolysis. However, the effect of ceritinib on osteoclast formation remains unclear. METHODS Osteoclastogenesis was induced to assess the effect of ceritinib on osteoclast formation and osteoclast-specific gene expression. Western blotting was used to examine the molecular mechanisms underlying the effect of ceritinib on osteoclast differentiation. An in vivo ovariectomized mouse model was established to validate the effect of ceritinib in suppressing osteoclast formation and preventing bone loss. RESULTS The differentiation of osteoclasts and the expression of osteoclast-specific genes were inhibited upon ceritinib stimulation. Ceritinib suppressed Akt and p65 phosphorylation during the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The administration of ceritinib to ovariectomized mice ameliorated trabecular bone loss by inhibiting osteoclast formation. CONCLUSIONS Ceritinib is beneficial in preventing bone loss by suppressing osteoclastic Akt and nuclear factor κB (NF-κB) signaling.
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Affiliation(s)
- Wenxin He
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Centre National de la Recherche Scientifique–Laboratoire International Associé (CNRS-LIA) Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xiankun Cao
- Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Keyu Kong
- Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Kewei Rong
- Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Shuai Han
- Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Nanning, Guangxi, China
| | - An Qin
- Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- *Correspondence: An Qin,
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24
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Li W, Li F, Zhang X, Lin HK, Xu C. Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduct Target Ther 2021; 6:422. [PMID: 34924561 PMCID: PMC8685280 DOI: 10.1038/s41392-021-00825-8] [Citation(s) in RCA: 74] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 11/02/2021] [Accepted: 11/05/2021] [Indexed: 12/11/2022] Open
Abstract
More and more in-depth studies have revealed that the occurrence and development of tumors depend on gene mutation and tumor heterogeneity. The most important manifestation of tumor heterogeneity is the dynamic change of tumor microenvironment (TME) heterogeneity. This depends not only on the tumor cells themselves in the microenvironment where the infiltrating immune cells and matrix together forming an antitumor and/or pro-tumor network. TME has resulted in novel therapeutic interventions as a place beyond tumor beds. The malignant cancer cells, tumor infiltrate immune cells, angiogenic vascular cells, lymphatic endothelial cells, cancer-associated fibroblastic cells, and the released factors including intracellular metabolites, hormonal signals and inflammatory mediators all contribute actively to cancer progression. Protein post-translational modification (PTM) is often regarded as a degradative mechanism in protein destruction or turnover to maintain physiological homeostasis. Advances in quantitative transcriptomics, proteomics, and nuclease-based gene editing are now paving the global ways for exploring PTMs. In this review, we focus on recent developments in the PTM area and speculate on their importance as a critical functional readout for the regulation of TME. A wealth of information has been emerging to prove useful in the search for conventional therapies and the development of global therapeutic strategies.
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Affiliation(s)
- Wen Li
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China
| | - Feifei Li
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China
- Guangxi Collaborative Innovation Center for Biomedicine (Guangxi-ASEAN Collaborative Innovation Center for Major Disease Prevention and Treatment), Guangxi Medical University, 530021, Nanning, Guangxi, China
| | - Xia Zhang
- Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), 400038, Chongqing, China
| | - Hui-Kuan Lin
- Department of Cancer Biology, Wake Forest Baptist Medical Center, Wake Forest University, Winston Salem, NC, 27101, USA
| | - Chuan Xu
- Integrative Cancer Center & Cancer Clinical Research Center, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, School of Medicine, University of Electronic Science and Technology of China, 610042, Chengdu, P. R. China.
- Department of Cancer Biology, Wake Forest Baptist Medical Center, Wake Forest University, Winston Salem, NC, 27101, USA.
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25
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Seegobin K, Majeed U, Wiest N, Manochakian R, Lou Y, Zhao Y. Immunotherapy in Non-Small Cell Lung Cancer With Actionable Mutations Other Than EGFR. Front Oncol 2021; 11:750657. [PMID: 34926258 PMCID: PMC8671626 DOI: 10.3389/fonc.2021.750657] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Accepted: 11/10/2021] [Indexed: 12/11/2022] Open
Abstract
While first line targeted therapies are the current standard of care treatment for non-small cell lung cancer (NSCLC) with actionable mutations, the cancer cells inevitably acquire resistance to these agents over time. Immune check-point inhibitors (ICIs) have improved the outcomes of metastatic NSCLC, however, its efficacy in those with targetable drivers is largely unknown. In this manuscript, we reviewed the published data on ICI therapies in NSCLC with ALK, ROS1, BRAF, c-MET, RET, NTRK, KRAS, and HER2 (ERBB2) alterations. We found that the objective response rates (ORRs) associated with ICI treatments in lung cancers harboring the BRAF (0-54%), c-MET (12-49%), and KRAS (18.7-66.7%) alterations were comparable to non-mutant NSCLC, whereas the ORRs in RET fusion NSCLC (less than10% in all studies but one) and ALK fusion NSCLC (0%) were relatively low. The ORRs reported in small numbers of patients and studies of ROS1 fusion, NTRK fusion, and HER 2 mutant NSCLC were 0-17%, 50% and 7-23%, respectively, making the efficacy of ICIs in these groups of patients less clear. In most studies, no significant correlation between treatment outcome and PD-L1 expression or tumor mutation burden (TMB) was identified, and how to select patients with NSCLC harboring actionable mutations who will likely benefit from ICI treatment remains unknown.
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Affiliation(s)
- Karan Seegobin
- Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States
| | - Umair Majeed
- Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States
| | - Nathaniel Wiest
- Department of Medicine, Mayo Clinic, Jacksonville, FL, United States
| | - Rami Manochakian
- Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States
| | - Yanyan Lou
- Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States
| | - Yujie Zhao
- Division of Hematology and Oncology, Mayo Clinic, Jacksonville, FL, United States
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26
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Kessler L, Malik S, Leoni M, Burrows F. Potential of Farnesyl Transferase Inhibitors in Combination Regimens in Squamous Cell Carcinomas. Cancers (Basel) 2021; 13:cancers13215310. [PMID: 34771475 PMCID: PMC8582567 DOI: 10.3390/cancers13215310] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 10/14/2021] [Accepted: 10/19/2021] [Indexed: 11/16/2022] Open
Abstract
Current therapies for recurrent and metastatic SCC are associated with poor outcomes, and options for later lines of treatment are limited. Insights into potential therapeutic targets, as well as mechanisms of resistance to available therapies, have begun to be elucidated, creating the basis for exploration of combination approaches to drive better patient outcomes. Tipifarnib, a farnesyl transferase inhibitor (FTI), is a small molecule drug that has demonstrated encouraging clinical activity in a genetically-defined subset of head and neck squamous cell carcinoma (HNSCC)-specifically, tumors that express a mutation in the HRAS protooncogene. More recently, bioinformatic analyses and results from patient-derived xenograft modeling indicate that HRAS pathway dependency may extend to a broader subpopulation of SCCs beyond HRAS mutants in the context of combination with agents such as cisplatin, cetuximab, or alpelisib. In addition, tipifarnib can also inactivate additional farnesylated proteins implicated in resistance to approved therapies, including immunotherapies, through a variety of distinct mechanisms, suggesting that tipifarnib could serve as an anchor for combination regimens in SCCs and other tumor types.
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27
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Tago K, Ohta S, Aoki-Ohmura C, Funakoshi-Tago M, Sashikawa M, Matsui T, Miyamoto Y, Wada T, Oshio T, Komine M, Matsugi J, Furukawa Y, Ohtsuki M, Yamauchi J, Yanagisawa K. K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors. Sci Rep 2021; 11:20658. [PMID: 34667224 PMCID: PMC8526694 DOI: 10.1038/s41598-021-00200-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Accepted: 10/01/2021] [Indexed: 12/17/2022] Open
Abstract
NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.
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Affiliation(s)
- Kenji Tago
- Division of Structural Biochemistry, Department of Biochemistry, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan.
| | - Satoshi Ohta
- Division of Structural Biochemistry, Department of Biochemistry, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Chihiro Aoki-Ohmura
- Division of Structural Biochemistry, Department of Biochemistry, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Megumi Funakoshi-Tago
- Division of Hygienic Chemistry, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, 105-8512, Japan
| | - Miho Sashikawa
- Department of Dermatology, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Takeshi Matsui
- Laboratory for Evolutionary Cell Biology of the Skin, School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo, 192-0982, Japan
| | - Yuki Miyamoto
- Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo, 157-8535, Japan
| | - Taeko Wada
- Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Tomoyuki Oshio
- Department of Dermatology, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Mayumi Komine
- Department of Dermatology, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Jitsuhiro Matsugi
- Division of Structural Biochemistry, Department of Biochemistry, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Yusuke Furukawa
- Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Mamitaro Ohtsuki
- Department of Dermatology, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
| | - Junji Yamauchi
- Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo, 157-8535, Japan.,Laboratory of Molecular Neuroscience and Neurology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan
| | - Ken Yanagisawa
- Division of Structural Biochemistry, Department of Biochemistry, School of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi, 329-0498, Japan
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28
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Najar MA, Aravind A, Dagamajalu S, Sidransky D, Ashktorab H, Smoot DT, Gowda H, Prasad TSK, Modi PK, Chatterjee A. Hyperactivation of MEK/ERK pathway by Ca 2+ /calmodulin-dependent protein kinase kinase 2 promotes cellular proliferation by activating cyclin-dependent kinases and minichromosome maintenance protein in gastric cancer cells. Mol Carcinog 2021; 60:769-783. [PMID: 34437731 DOI: 10.1002/mc.23343] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 08/10/2021] [Accepted: 08/12/2021] [Indexed: 01/04/2023]
Abstract
Although CAMKK2 is overexpressed in several cancers, its role and relevant downstream signaling pathways in gastric cancer (GC) are poorly understood. Treatment of AGS GC cells with a CAMKK2 inhibitor, STO-609, resulted in decreased cell proliferation, cell migration, invasion, colony-forming ability, and G1/S-phase arrest. Quantitative phosphoproteomics in AGS cells with the CAMKK2 inhibitor led to the identification of 9603 unique phosphosites mapping to 3120 proteins. We observed decreased phosphorylation of 1101 phosphopeptides (1.5-fold) corresponding to 752 proteins upon CAMKK2 inhibition. Bioinformatics analysis of hypo-phosphorylated proteins revealed enrichment of MAPK1/MAPK3 signaling. Kinase enrichment analysis of hypo-phosphorylated proteins using the X2K Web tool identified ERK1, cyclin-dependant kinase 1 (CDK1), and CDK2 as downstream substrates of CAMKK2. Moreover, inhibition of CAMKK2 and MEK1 resulted in decreased phosphorylation of ERK1, CDK1, MCM2, and MCM3. Immunofluorescence results were in concordance with our mass spectroscopy data and Western blot analysis results. Taken together, our data reveal the essential role of CAMKK2 in the pathobiology of GC through the activation of the MEK/ERK1 signaling cascade.
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Affiliation(s)
- Mohd A Najar
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Anjana Aravind
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Shobha Dagamajalu
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - David Sidransky
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Hassan Ashktorab
- Department of Medicine, Howard University, Washington, District of Columbia, USA
| | - Duane T Smoot
- Department of Medicine, Meharry Medical Center, Nashville, Tennessee, USA
| | - Harsha Gowda
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India.,Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka, India.,Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India
| | - T S Keshava Prasad
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Prashant K Modi
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India
| | - Aditi Chatterjee
- Center for Systems Biology and Molecular Medicine, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India.,Institute of Bioinformatics, International Technology Park, Bangalore, Karnataka, India.,Manipal Academy of Higher Education (MAHE), Manipal, Karnataka, India
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29
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Heppner DE, Eck MJ. A structural perspective on targeting the RTK/Ras/MAP kinase pathway in cancer. Protein Sci 2021; 30:1535-1553. [PMID: 34008902 PMCID: PMC8284588 DOI: 10.1002/pro.4125] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 05/13/2021] [Accepted: 05/14/2021] [Indexed: 02/06/2023]
Abstract
Precision oncology is premised on identifying and drugging proteins and pathways that drive tumorigenesis or are required for survival of tumor cells. Across diverse cancer types, the signaling pathway emanating from receptor tyrosine kinases on the cell surface to RAS and the MAP kinase pathway is the most frequent target of oncogenic mutations, and key proteins in this signaling axis including EGFR, SHP2, RAS, BRAF, and MEK have long been a focus in cancer drug discovery. In this review, we provide an overview of historical and recent efforts to develop inhibitors targeting these nodes with an emphasis on the role that an understanding of protein structure and regulation has played in inhibitor discovery and characterization. Beyond its well-established role in structure-based drug design, structural biology has revealed mechanisms of allosteric regulation, distinct effects of activating oncogenic mutations, and other vulnerabilities that have opened new avenues in precision cancer drug discovery.
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Affiliation(s)
- David E. Heppner
- Department of ChemistryUniversity at Buffalo, State University of New YorkBuffaloNew YorkUSA
- Department of Pharmacology and TherapeuticsRoswell Park Comprehensive Cancer CenterBuffaloNew YorkUSA
| | - Michael J. Eck
- Department of Cancer BiologyDana‐Farber Cancer InstituteBostonMassachusettsUSA
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonMassachusettsUSA
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30
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Zhou Y, Gorfe AA, Hancock JF. RAS Nanoclusters Selectively Sort Distinct Lipid Headgroups and Acyl Chains. Front Mol Biosci 2021; 8:686338. [PMID: 34222339 PMCID: PMC8245699 DOI: 10.3389/fmolb.2021.686338] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 05/13/2021] [Indexed: 12/12/2022] Open
Abstract
RAS proteins are lipid-anchored small GTPases that switch between the GTP-bound active and GDP-bound inactive states. RAS isoforms, including HRAS, NRAS and splice variants KRAS4A and KRAS4B, are some of the most frequently mutated proteins in cancer. In particular, constitutively active mutants of KRAS comprise ∼80% of all RAS oncogenic mutations and are found in 98% of pancreatic, 45% of colorectal and 31% of lung tumors. Plasma membrane (PM) is the primary location of RAS signaling in biology and pathology. Thus, a better understanding of how RAS proteins localize to and distribute on the PM is critical to better comprehend RAS biology and to develop new strategies to treat RAS pathology. In this review, we discuss recent findings on how RAS proteins sort lipids as they undergo macromolecular assembly on the PM. We also discuss how RAS/lipid nanoclusters serve as signaling platforms for the efficient recruitment of effectors and signal transduction, and how perturbing the PM biophysical properties affect the spatial distribution of RAS isoforms and their functions.
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Affiliation(s)
- Yong Zhou
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, United States
| | - Alemayehu A. Gorfe
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, United States
| | - John F. Hancock
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, TX, United States
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31
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Davis AP, Cooper WA, Boyer M, Lee JH, Pavlakis N, Kao SC. Efficacy of immunotherapy in KRAS-mutant non-small-cell lung cancer with comutations. Immunotherapy 2021; 13:941-952. [PMID: 34114474 DOI: 10.2217/imt-2021-0090] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
KRAS-mutant non-small-cell lung cancer is the most common molecular driver of lung adenocarcinoma in western populations. No KRAS specific therapy has been approved by the US FDA until 2021. Despite significant heterogeneity in comutations, patients typically receive single-agent immunotherapy or chemoimmunotherapy as standard first-line therapy. It is unclear whether KRAS mutations predict outcomes with immunotherapy; however, there is emerging data suggesting improved outcomes in patients with a TP53 comutation and worse outcomes in patients with a STK11/LKB1 or KEAP1 comutation.
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Affiliation(s)
- Alexander P Davis
- Department of Medical Oncology, Chris O'Brien Lifehouse, 119-143 Missenden Road, Camperdown, NSW 2050, Australia
| | - Wendy A Cooper
- Tissue Pathology & Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, NSW 2050, Australia.,Sydney Medical School, University of Sydney, Sydney, NSW 2050, Australia.,School of Medicine, Western Sydney University, Sydney, NSW 2571, Australia
| | - Michael Boyer
- Department of Medical Oncology, Chris O'Brien Lifehouse, 119-143 Missenden Road, Camperdown, NSW 2050, Australia.,Sydney Medical School, University of Sydney, NSW 2006, Australia
| | - Jenny H Lee
- Department of Medical Oncology, Chris O'Brien Lifehouse, 119-143 Missenden Road, Camperdown, NSW 2050, Australia.,Faculty of Medicine & Health, Macquarie University, NSW 2109, Australia
| | - Nick Pavlakis
- Sydney Medical School, University of Sydney, NSW 2006, Australia.,Department of Medical Oncology, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.,Genesis Care St Leonards, St Leonards, NSW 2065, Australia
| | - Steven C Kao
- Department of Medical Oncology, Chris O'Brien Lifehouse, 119-143 Missenden Road, Camperdown, NSW 2050, Australia.,Sydney Medical School, University of Sydney, NSW 2006, Australia.,Asbestos Disease Research Institute, Concord, NSW 2139, Australia
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32
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Reck M, Carbone DP, Garassino M, Barlesi F. Targeting KRAS in non-small-cell lung cancer: recent progress and new approaches. Ann Oncol 2021; 32:1101-1110. [PMID: 34089836 DOI: 10.1016/j.annonc.2021.06.001] [Citation(s) in RCA: 155] [Impact Index Per Article: 38.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Revised: 05/18/2021] [Accepted: 06/01/2021] [Indexed: 02/07/2023] Open
Abstract
Rat sarcoma (RAS) is the most frequently mutated oncogene in human cancer, with Kirsten rat sarcoma (KRAS) being the most commonly mutated RAS isoform. Overall, KRAS accounts for 85% of RAS mutations observed in human cancers and is present in 35% of lung adenocarcinomas (LUADs). While the use of targeted therapies and immune checkpoint inhibitors (CPIs) has drastically changed the treatment landscape of advanced non-small-cell lung cancer (NSCLC) in recent years, historic attempts to target KRAS (both direct and indirect approaches) have had little success, and no KRAS-specific targeted therapies have been approved to date for patients in this molecular subset of NSCLC. With the discovery by Ostrem, Shokat, and colleagues of the switch II pocket on the surface of the active and inactive forms of KRAS, we now have an improved understanding of the complex interactions involved in the RAS family of signaling proteins which has led to the development of a number of promising direct KRASG12C inhibitors, such as sotorasib and adagrasib. In previously treated patients with KRASG12C-mutant NSCLC, clinical activity has been shown for both sotorasib and adagrasib monotherapy; these data suggest promising new treatment options are on the horizon. With the stage now set for a new era in the treatment of KRASG12C-mutated NSCLC, many questions remain to be answered in order to further elucidate the mechanisms of resistance, how best to use combination strategies, and if KRASG12C inhibitors will have suitable activity in earlier lines of therapy for patients with advanced/metastatic NSCLC.
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Affiliation(s)
- M Reck
- Department of Thoracic Oncology, Lung Clinic Grosshansdorf, Airway Research Center North, German Center for Lung Research, Grosshansdorf, Germany.
| | - D P Carbone
- James Thoracic Oncology Center, The Ohio State University, Columbus, USA
| | - M Garassino
- Department of Medicine, Section Hematology Oncology; The University of Chicago, Chicago, USA
| | - F Barlesi
- Aix Marseille University, Marseille, France; Gustave Roussy Cancer Campus, Villejuif, France
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Nailwal NP, Doshi GM. Role of intracellular signaling pathways and their inhibitors in the treatment of inflammation. Inflammopharmacology 2021; 29:617-640. [PMID: 34002330 DOI: 10.1007/s10787-021-00813-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Accepted: 04/24/2021] [Indexed: 12/11/2022]
Abstract
Inflammation is not only a defense mechanism of the innate immune system against invaders, but it is also involved in the pathogenesis of many diseases such as atherosclerosis, thrombosis, diabetes, epilepsy, and many neurodegenerative disorders. The World Health Organization (WHO) reports worldwide estimates of people (9.6% in males and 18.0% in females) aged over 60 years, suffering from symptomatic osteoarthritis, and around 339 million suffering from asthma. Other chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease are also highly prevalent. The existing anti-inflammatory agents, both non-steroidal and steroidal, are highly effective; however, their prolonged use is marred by the severity of associated side effects. A holistic approach to ensure patient compliance requires understanding the pathophysiology of inflammation and exploring new targets for drug development. In this regard, various intracellular cell signaling pathways and their signaling molecules have been identified to be associated with inflammation. Therefore, chemical inhibitors of these pathways may be potential candidates for novel anti-inflammatory drug approaches. This review focuses on the anti-inflammatory effect of these inhibitors (for JAK/STAT, MAPK, and mTOR pathways) describing their mechanism of action through literature search, current patents, and molecules under clinical trials.
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Affiliation(s)
- Namrata P Nailwal
- Department of Pharmacology, SVKM's Dr. Bhanuben Nanavati College of Pharmacy, Mithibai Campus, Vile Parle (W), V. M. Road, 400056, Mumbai, India
| | - Gaurav M Doshi
- Department of Pharmacology, SVKM's Dr. Bhanuben Nanavati College of Pharmacy, Mithibai Campus, Vile Parle (W), V. M. Road, 400056, Mumbai, India.
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Ullah R, Yin Q, Snell AH, Wan L. RAF-MEK-ERK pathway in cancer evolution and treatment. Semin Cancer Biol 2021; 85:123-154. [PMID: 33992782 DOI: 10.1016/j.semcancer.2021.05.010] [Citation(s) in RCA: 198] [Impact Index Per Article: 49.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 05/03/2021] [Accepted: 05/06/2021] [Indexed: 12/13/2022]
Abstract
The RAF-MEK-ERK signaling cascade is a well-characterized MAPK pathway involved in cell proliferation and survival. The three-layered MAPK signaling cascade is initiated upon RTK and RAS activation. Three RAF isoforms ARAF, BRAF and CRAF, and their downstream MEK1/2 and ERK1/2 kinases constitute a coherently orchestrated signaling module that directs a range of physiological functions. Genetic alterations in this pathway are among the most prevalent in human cancers, which consist of numerous hot-spot mutations such as BRAFV600E. Oncogenic mutations in this pathway often override otherwise tightly regulated checkpoints to open the door for uncontrolled cell growth and neoplasia. The crosstalk between the RAF-MEK-ERK axis and other signaling pathways further extends the proliferative potential of this pathway in human cancers. In this review, we summarize the molecular architecture and physiological functions of the RAF-MEK-ERK pathway with emphasis on its dysregulations in human cancers, as well as the efforts made to target the RAF-MEK-ERK module using small molecule inhibitors.
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Affiliation(s)
- Rahim Ullah
- Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA
| | - Qing Yin
- Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA
| | - Aidan H Snell
- Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA
| | - Lixin Wan
- Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA; Department of Cutaneous Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.
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Zhang T, Jia C, Dong Z, Li C, Lu W. A novel mutation in NF1 gene of patient with Neurofibromatosis type 1: A case report and functional study. Mol Genet Genomic Med 2021; 9:e1643. [PMID: 33764694 PMCID: PMC8172195 DOI: 10.1002/mgg3.1643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 01/17/2021] [Accepted: 02/10/2021] [Indexed: 11/17/2022] Open
Abstract
BACKGROUND Neurofibromatosis type 1 is an autosomal dominant inherited disease and caused by NF1 gene mutation. Its clinical manifestations include multiple cafe´-au lait (CAL) spots, skinfold freckling, neurofibroma, bone dysplasia, learning disabilities, and an increased risk of malignancy. METHODS AND RESULTS Here, we reported a Chinese patient bearing with a novel NF1 mutation (c.2064delGGATGCAGCGG/p.Gly672AsnfsTer24) and complaining mainly about bone phenotype. Functional studies found that this novel mutation caused the damage of NF1 mRNA and protein levels, and lost the inhibition on Ras/Erk signaling. CONCLUSION A novel mutation in NF1 gene was identified and in vitro functional studies were performed, which provided a potential molecular mechanism to explain the bone maldevelopment of patients with neurofibromatosis type 1.
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Affiliation(s)
- Tingting Zhang
- Department of PediatricsRuijin Hospital Affiliated to Shanghai Jiao Tong UniversityShanghaiChina
| | - Caiwei Jia
- Cancer CenterShanghai Tenth People’s HospitalSchool of MedicineTongji UniversityShanghaiChina
| | - Zhiya Dong
- Department of PediatricsRuijin Hospital Affiliated to Shanghai Jiao Tong UniversityShanghaiChina
| | - Chuanyin Li
- Cancer CenterShanghai Tenth People’s HospitalSchool of MedicineTongji UniversityShanghaiChina
| | - Wenli Lu
- Department of PediatricsRuijin Hospital Affiliated to Shanghai Jiao Tong UniversityShanghaiChina
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Chen JJ, Fan Y, Boehning D. Regulation of Dynamic Protein S-Acylation. Front Mol Biosci 2021; 8:656440. [PMID: 33981723 PMCID: PMC8107437 DOI: 10.3389/fmolb.2021.656440] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Accepted: 02/16/2021] [Indexed: 12/20/2022] Open
Abstract
Protein S-acylation is the reversible addition of fatty acids to the cysteine residues of target proteins. It regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interactions, protein stability, and protein conformation. This process is regulated by palmitoyl acyltransferases that have the conserved amino acid sequence DHHC at their active site. Although they have conserved catalytic cores, DHHC enzymes vary in their protein substrate selection, lipid substrate preference, and regulatory mechanisms. Alterations in DHHC enzyme function are associated with many human diseases, including cancers and neurological conditions. The removal of fatty acids from acylated cysteine residues is catalyzed by acyl protein thioesterases. Notably, S-acylation is now known to be a highly dynamic process, and plays crucial roles in signaling transduction in various cell types. In this review, we will explore the recent findings on protein S-acylation, the enzymatic regulation of this process, and discuss examples of dynamic S-acylation.
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Ishitsuka Y, Hanaoka Y, Tanemura A, Fujimoto M. Cutaneous Squamous Cell Carcinoma in the Age of Immunotherapy. Cancers (Basel) 2021; 13:1148. [PMID: 33800195 PMCID: PMC7962464 DOI: 10.3390/cancers13051148] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Revised: 03/02/2021] [Accepted: 03/03/2021] [Indexed: 12/11/2022] Open
Abstract
Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent skin cancer globally. Because most cSCC cases are manageable by local excision/radiotherapy and hardly become life-threatening, they are often excluded from cancer registries in most countries. Compared with cutaneous melanoma that originates from the melanin-producing, neural crest-derived epidermal resident, keratinocyte (KC)-derived cancers are influenced by the immune system with regards to their pathogenetic behaviour. Congenital or acquired immunosurveillance impairments compromise tumoricidal activity and raises cSCC incidence rates. Intriguingly, expanded applications of programmed death-1 (PD-1) blockade therapies have revealed cSCC to be one of the most amenable targets, particularly when compared with the mucosal counterparts arisen in the esophagus or the cervix. The clinical observation reminds us that cutaneous tissue has a peculiarly high immunogenicity that can evoke tumoricidal recall responses topically. Here we attempt to redefine cSCC biology and review current knowledge about cSCC from multiple viewpoints that involve epidemiology, clinicopathology, molecular genetics, molecular immunology, and developmental biology. This synthesis not only underscores the primal importance of the immune system, rather than just a mere accumulation of ultraviolet-induced mutations but also reinforces the following hypothesis: PD-1 blockade effectively restores the immunity specially allowed to exist within the fully cornified squamous epithelium, that is, the epidermis.
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Affiliation(s)
- Yosuke Ishitsuka
- Department of Dermatology Integrated Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; (Y.H.); (A.T.); (M.F.)
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Van QN, Prakash P, Shrestha R, Balius TE, Turbyville TJ, Stephen AG. RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention. Biomolecules 2021; 11:377. [PMID: 33802474 PMCID: PMC8000715 DOI: 10.3390/biom11030377] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Revised: 02/24/2021] [Accepted: 02/24/2021] [Indexed: 01/18/2023] Open
Abstract
RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein-protein and protein-lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.
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Affiliation(s)
| | | | | | | | | | - Andrew G. Stephen
- Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, National Cancer Institute RAS Initiative, Inc., Frederick, MD 21702, USA; (Q.N.V.); (P.P.); (R.S.); (T.E.B.); (T.J.T.)
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Van QN, López CA, Tonelli M, Taylor T, Niu B, Stanley CB, Bhowmik D, Tran TH, Frank PH, Messing S, Alexander P, Scott D, Ye X, Drew M, Chertov O, Lösche M, Ramanathan A, Gross ML, Hengartner NW, Westler WM, Markley JL, Simanshu DK, Nissley DV, Gillette WK, Esposito D, McCormick F, Gnanakaran S, Heinrich F, Stephen AG. Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane. Proc Natl Acad Sci U S A 2020; 117:24258-24268. [PMID: 32913056 PMCID: PMC7533834 DOI: 10.1073/pnas.2006504117] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
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Affiliation(s)
- Que N Van
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Cesar A López
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545
| | - Marco Tonelli
- National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706
| | - Troy Taylor
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Ben Niu
- National Mass Spectrometry Resource, Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130
| | - Christopher B Stanley
- Computational Sciences and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831
| | - Debsindhu Bhowmik
- Computational Sciences and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831
| | - Timothy H Tran
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Peter H Frank
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Simon Messing
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Patrick Alexander
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Daniel Scott
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213
| | - Xiaoying Ye
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Matt Drew
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Oleg Chertov
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Mathias Lösche
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213
- Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899
| | - Arvind Ramanathan
- Data Science and Learning Division, Argonne National Laboratory, Lemont, IL 60439
| | - Michael L Gross
- National Mass Spectrometry Resource, Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130
| | - Nicolas W Hengartner
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545
| | - William M Westler
- National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706
| | - John L Markley
- National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706
| | - Dhirendra K Simanshu
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Dwight V Nissley
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - William K Gillette
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Dominic Esposito
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702
| | - Frank McCormick
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702;
| | - S Gnanakaran
- Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545
| | - Frank Heinrich
- Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213
- Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899
| | - Andrew G Stephen
- National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD 21702;
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Uprety D, Adjei AA. KRAS: From undruggable to a druggable Cancer Target. Cancer Treat Rev 2020; 89:102070. [DOI: 10.1016/j.ctrv.2020.102070] [Citation(s) in RCA: 58] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Revised: 07/04/2020] [Accepted: 07/06/2020] [Indexed: 02/07/2023]
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Ngo VA, Sarkar S, Neale C, Garcia AE. How Anionic Lipids Affect Spatiotemporal Properties of KRAS4B on Model Membranes. J Phys Chem B 2020; 124:5434-5453. [PMID: 32438809 DOI: 10.1021/acs.jpcb.0c02642] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
RAS proteins are small membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the spatiotemporal properties of RAS through dimerization/clustering and signaling fidelity. To understand the effects of anionic lipids on key spatiotemporal properties of RAS, we dissected 1 ms of data from all-atom molecular dynamics simulations for KRAS4B on two model anionic lipid membranes that have 30% of POPS mixed with neutral POPC and 8% of PIP2 mixed with POPC. We unveiled the orientation space of KRAS4B, whose kinetics were slower and more distinguishable on the membrane containing PIP2 than the membrane containing POPS. Particularly, the PIP2-mixed membrane can differentiate a third kinetic orientation state from the other two known orientation states. We observed that each orientation state may yield different binding modes with an RAF kinase, which is required for activating the MAPK/ERK signaling pathway. However, an overall occluded probability, for which RAF kinases cannot bind KRAS4B, remains unchanged on the two different membranes. We identified rare fast diffusion modes of KRAS4B that appear coupled with orientations exposed to cytosolic RAF. Particularly, on the membrane having PIP2, we found nonlinear correlations between the orientation states and the conformations of the cationic farnesylated hypervariable region, which acts as an anchor in the membrane. Using diffusion coefficients estimated from the all-atom simulations, we quantified the effect of PIP2 and POPS on the KRAS4B dimerization via Green's function reaction dynamics simulations, in which the averaged dimerization rate is 12.5% slower on PIP2-mixed membranes.
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Affiliation(s)
- Van A Ngo
- Center for Nonlinear Studies (CNLS), Los Alamos National Lab, Los Alamos, New Mexico 87545, United States
| | - Sumantra Sarkar
- Center for Nonlinear Studies (CNLS), Los Alamos National Lab, Los Alamos, New Mexico 87545, United States
| | - Chris Neale
- Theoretical Biology and Biophysics Group, T-6, Los Alamos National Lab, Los Alamos, New Mexico 87545, United States
| | - Angel E Garcia
- Center for Nonlinear Studies (CNLS), Los Alamos National Lab, Los Alamos, New Mexico 87545, United States
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Nakamura A, Oki C, Sawada S, Yoshii T, Kuwata K, Rudd AK, Devaraj NK, Noma K, Tsukiji S. Designer Palmitoylation Motif-Based Self-Localizing Ligand for Sustained Control of Protein Localization in Living Cells and Caenorhabditis elegans. ACS Chem Biol 2020; 15:837-843. [PMID: 32182034 DOI: 10.1021/acschembio.0c00014] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Inducing protein translocation to the plasma membrane (PM) is an important approach for manipulating diverse signaling molecules/pathways in living cells. We previously devised a new chemogenetic system, in which a protein fused to Escherichia coli dihydrofolate reductase (eDHFR) can be rapidly translocated from the cytoplasm to the PM using a trimethoprim (TMP)-based self-localizing ligand (SL), mgcTMP. However, mgcTMP-induced protein translocation turned out to be transient and spontaneously reversed within 1 h, limiting its application. Here, we first demonstrated that the spontaneous reverse translocation was caused by cellular degradation of mgcTMP, presumably by proteases. To address this problem, we newly developed a proteolysis-resistant SL, mDcTMP. This mDcTMP now allows sustained PM localization of eDHFR-fusion proteins (over several hours to a day), and it was applicable to inducing prolonged signal activation and cell differentiation. mDcTMP also worked in live nematodes, making it an attractive new tool for probing and controlling living systems.
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Affiliation(s)
- Akinobu Nakamura
- Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
| | - Choji Oki
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
| | - Shunsuke Sawada
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
| | - Tatsuyuki Yoshii
- Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
- PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
| | - Keiko Kuwata
- Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Andrew K. Rudd
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States
| | - Neal K. Devaraj
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States
| | - Kentaro Noma
- PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
- Group of Nutritional Neuroscience, Neuroscience Institute, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan
| | - Shinya Tsukiji
- Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
- Department of Nanopharmaceutical Sciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
- Frontier Research Institute for Materials Science (FRIMS), Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
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Kundlacz C, Pourcelot M, Fablet A, Amaral Da Silva Moraes R, Léger T, Morlet B, Viarouge C, Sailleau C, Turpaud M, Gorlier A, Breard E, Lecollinet S, van Rijn PA, Zientara S, Vitour D, Caignard G. Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway. J Virol 2019; 93:e00336-19. [PMID: 31167915 PMCID: PMC6675888 DOI: 10.1128/jvi.00336-19] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Accepted: 05/22/2019] [Indexed: 12/22/2022] Open
Abstract
Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection.IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.
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Affiliation(s)
- Cindy Kundlacz
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Marie Pourcelot
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Aurore Fablet
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | | | - Thibaut Léger
- Mass Spectrometry and Proteomics Facility, Jacques Monod Institute, UMR 7592, Paris Diderot University, CNRS, Paris Cedex 13, France
| | - Bastien Morlet
- Mass Spectrometry and Proteomics Facility, Jacques Monod Institute, UMR 7592, Paris Diderot University, CNRS, Paris Cedex 13, France
| | - Cyril Viarouge
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Corinne Sailleau
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Mathilde Turpaud
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Axel Gorlier
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Emmanuel Breard
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Sylvie Lecollinet
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Piet A van Rijn
- Department of Virology, Wageningen Bioveterinary Research, Lelystad, The Netherlands
- Department of Biochemistry, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa
| | - Stephan Zientara
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Damien Vitour
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
| | - Grégory Caignard
- UMR Virologie, INRA, École Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France
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Boned Del Río I, Young LC, Sari S, Jones GG, Ringham-Terry B, Hartig N, Rejnowicz E, Lei W, Bhamra A, Surinova S, Rodriguez-Viciana P. SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics. Proc Natl Acad Sci U S A 2019; 116:13330-13339. [PMID: 31213532 PMCID: PMC6613145 DOI: 10.1073/pnas.1902658116] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.
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Affiliation(s)
- Isabel Boned Del Río
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Lucy C Young
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Sibel Sari
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Greg G Jones
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Benjamin Ringham-Terry
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Nicole Hartig
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Ewa Rejnowicz
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Winnie Lei
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms
| | - Amandeep Bhamra
- Proteomics Research Core Facility, University College London Cancer Institute, WC1E 6DD London, United Kingdom
| | - Silvia Surinova
- Proteomics Research Core Facility, University College London Cancer Institute, WC1E 6DD London, United Kingdom
| | - Pablo Rodriguez-Viciana
- University College London Cancer Institute, University College London, WC1E 6DD London, United Kingdoms;
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Murugan AK, Grieco M, Tsuchida N. RAS mutations in human cancers: Roles in precision medicine. Semin Cancer Biol 2019; 59:23-35. [PMID: 31255772 DOI: 10.1016/j.semcancer.2019.06.007] [Citation(s) in RCA: 87] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2018] [Revised: 05/13/2019] [Accepted: 06/07/2019] [Indexed: 02/07/2023]
Abstract
Ras proteins play a crucial role as a central component of the cellular networks controlling a variety of signaling pathways that regulate growth, proliferation, survival, differentiation, adhesion, cytoskeletal rearrangements and motility of a cell. Almost, 4 decades passed since Ras research was started and ras genes were originally discovered as retroviral oncogenes. Later on, mutations of the human RAS genes were linked to tumorigenesis. Genetic analyses found that RAS is one of the most deregulated oncogenes in human cancers. In this review, we summarize the pioneering works which allowed the discovery of RAS oncogenes, the finding of frequent mutations of RAS in various human cancers, the role of these mutations in tumorigenesis and mutation-activated signaling networks. We further describe the importance of RAS mutations in personalized or precision medicine particularly in molecular targeted therapy, as well as their use as diagnostic and prognostic markers as therapeutic determinants in human cancers.
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Affiliation(s)
- Avaniyapuram Kannan Murugan
- Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 Japan.
| | - Michele Grieco
- DiSTABiF, Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, via Vivaldi 43, Caserta 81100 Italy
| | - Nobuo Tsuchida
- Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 Japan.
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Cao Z, Liao Q, Su M, Huang K, Jin J, Cao D. AKT and ERK dual inhibitors: The way forward? Cancer Lett 2019; 459:30-40. [PMID: 31128213 DOI: 10.1016/j.canlet.2019.05.025] [Citation(s) in RCA: 147] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 05/03/2019] [Accepted: 05/20/2019] [Indexed: 02/07/2023]
Abstract
Phosphatidylinositol 3-kinase (PI3K)/AKT pathway regulates cell growth, proliferation, survival, mobility and invasion. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is also an important mitogenic signaling pathway involved in various cellular progresses. AKT, also named protein kinase B (PKB), is a primary mediator of the PI3K signaling pathway; and ERK at the end of MAPK signaling is the unique substrate and downstream effector of mitogen-activated protein/extracellular signal-regulated kinase (MEK). The AKT and ERK signaling are both aberrantly activated in a wide range of human cancers and have long been targeted for cancer therapy, but the clinical benefits of these targeted therapies have been limited due to complex cross-talk. Novel strategies, such as AKT/ERK dual inhibitors, may be needed.
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Affiliation(s)
- Zhe Cao
- Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. 283 Tongzipo Road, Changsha, 410013, Hunan, China
| | - Qianjin Liao
- Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. 283 Tongzipo Road, Changsha, 410013, Hunan, China
| | - Min Su
- Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. 283 Tongzipo Road, Changsha, 410013, Hunan, China
| | - Kai Huang
- Laboratory of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Guilin Medical University, 15 Lequn Road, Guilin, 541001, Guangxi, China
| | - Junfei Jin
- Laboratory of Hepatobiliary and Pancreatic Surgery, Affiliated Hospital of Guilin Medical University, 15 Lequn Road, Guilin, 541001, Guangxi, China
| | - Deliang Cao
- Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University. 283 Tongzipo Road, Changsha, 410013, Hunan, China; Department of Medical Microbiology, Immunology & Cell Biology, Simmons Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL, 62794, USA.
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47
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Surve SV, Myers PJ, Clayton SA, Watkins SC, Lazzara MJ, Sorkin A. Localization dynamics of endogenous fluorescently labeled RAF1 in EGF-stimulated cells. Mol Biol Cell 2019; 30:506-523. [PMID: 30586319 PMCID: PMC6594441 DOI: 10.1091/mbc.e18-08-0512] [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] [Indexed: 12/19/2022] Open
Abstract
Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.
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Affiliation(s)
- Sachin V Surve
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Paul J Myers
- Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904
| | - Samantha A Clayton
- Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22904
| | - Simon C Watkins
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
| | - Matthew J Lazzara
- Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904.,Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22904
| | - Alexander Sorkin
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
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48
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Three distinct regions of cRaf kinase domain interact with membrane. Sci Rep 2019; 9:2057. [PMID: 30765804 PMCID: PMC6375958 DOI: 10.1038/s41598-019-38770-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 12/19/2018] [Indexed: 12/18/2022] Open
Abstract
Raf kinases are downstream effectors of small GTPase Ras. Mutations in Ras and Raf are associated with a variety of cancers and genetic disorders. Of the three Raf isoforms, cRaf is most frequently involved in tumor initiation by Ras. Cytosolic Raf is auto-inhibited and becomes active upon recruitment to the plasma membrane. Since the catalytic domain of Raf is its kinase domain, we ask the following: does the kinase domain of Raf has potential to interact with membrane and if yes, what role does the membrane interaction play? We present a model of cRaf kinase domain in complex with a heterogeneous membrane bilayer using atomistic molecular dynamics simulation. We show that the kinase domain of cRaf has three distinct membrane-interacting regions: a polybasic motif (R.RKTR) from the regulatory αC-helix, an aromatic/hydrophobic cluster from the N-terminal acidic region (NtA) and positively charged/aromatic cluster from the activation segment (AS). We show that residues from these regions form an extended membrane-interacting surface that resembles the membrane-interacting residues from known membrane-binding domains. Activating phosphorylatable regions (NtA and AS), make direct contact with the membrane whereas R.RKTR forms specific multivalent salt bridges with PA. PA lipids dwell for longer times around the R.RKTR motif. Our results suggest that membrane interaction of monomeric cRaf kinase domain likely orchestrates the Raf activation process and modulates its function. We show that R.RKTR is a hotspot that interacts with membrane when cRaf is monomeric and becomes part of the interface upon Raf dimerization. We propose that in terms of utilizing a specific hotspot to form membrane interaction and dimer formation, both Raf and its upstream binding partner KRas, are similar.
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Terrell EM, Morrison DK. Ras-Mediated Activation of the Raf Family Kinases. Cold Spring Harb Perspect Med 2019; 9:cshperspect.a033746. [PMID: 29358316 DOI: 10.1101/cshperspect.a033746] [Citation(s) in RCA: 114] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The extracellular signal-regulated kinase (ERK) cascade comprised of the Raf, MEK, and ERK protein kinases constitutes a key effector cascade used by the Ras GTPases to relay signals regulating cell growth, survival, proliferation, and differentiation. Of the ERK cascade components, the regulation of the Raf kinases is by far the most complex, involving changes in subcellular localization, protein and lipid interactions, as well as alterations in the Raf phosphorylation state. The Raf kinases interact directly with active, membrane-localized Ras, and this interaction is often the first step in the Raf activation process, which ultimately results in ERK activation and the downstream phosphorylation of cellular targets that will specify a particular biological response. Here, we will examine our current understanding of how Ras promotes Raf activation, focusing on the molecular mechanisms that contribute to the Raf activation/inactivation cycle.
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Affiliation(s)
- Elizabeth M Terrell
- Laboratory of Cell and Developmental Signaling, NCI-Frederick, Frederick, Maryland 21702
| | - Deborah K Morrison
- Laboratory of Cell and Developmental Signaling, NCI-Frederick, Frederick, Maryland 21702
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50
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Molosh AI, Shekhar A. Neurofibromatosis type 1 as a model system to study molecular mechanisms of autism spectrum disorder symptoms. PROGRESS IN BRAIN RESEARCH 2018; 241:37-62. [PMID: 30447756 DOI: 10.1016/bs.pbr.2018.09.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Neurofibromatosis type 1 (NF1) is monogenic neurodevelopmental disorder caused by mutation of NF1 gene, which leads to increased susceptibility to various tumors formations. Additionally, majority of patients with NF1 are experience high incidence of cognitive deficits. Particularly, we review the growing number of reports demonstrated a higher incidence of autism spectrum disorder (ASD) in individuals with NF1. In this review we also discuss face validity of preclinical Nf1 mouse models. Then we describe discoveries from these animal models that have uncovered the deficiencies in the regulation of Ras and other intracellular pathways as critical mechanisms underlying the Nf1 cognitive problems. We also summarize and interpret recent preclinical and clinical studies that point toward potential pharmacological therapies for NF1 patients.
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Affiliation(s)
- Andrei I Molosh
- Department of Psychiatry, Institute of Psychiatric Research, IU School of Medicine, Indianapolis, IN, United States; Stark Neurosciences Research Institute, IU School of Medicine, Indianapolis, IN, United States.
| | - Anantha Shekhar
- Department of Psychiatry, Institute of Psychiatric Research, IU School of Medicine, Indianapolis, IN, United States; Stark Neurosciences Research Institute, IU School of Medicine, Indianapolis, IN, United States; Department of Pharmacology & Toxicology, IU School of Medicine, Indianapolis, IN, United States; Indiana Clinical and Translational Institute, IU School of Medicine, Indianapolis, IN, United States
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