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Jiang T, Zhang C, Cao X, Tian Y, Cai H, Kong S, Lu J, Wang H, Lu Z. EZH1/2 plays critical roles in oocyte meiosis prophase I in mice. Biol Res 2024; 57:83. [PMID: 39511641 PMCID: PMC11545252 DOI: 10.1186/s40659-024-00564-4] [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: 06/18/2024] [Accepted: 10/30/2024] [Indexed: 11/15/2024] Open
Abstract
BACKGROUD abnormalities or defects in oocyte meiosis can result in decreased oocyte quality, reduced ovarian reserve, and female diseases. However, the mechanisms of oocyte meiosis remain largely unknown, especially epigenetic regulation. Here, we explored the role of EZH1/2 (histone methyltransferase of H3K27) in mouse oocyte meiosis by inhibiting its activity and deleting its gene. RESULTS with embryonic ovary cultured in vitro, EZH1/2 was demonstrated to be essential for oocyte development during meiosis prophase I in mice. Activity inhibition or gene knockout of EZH1/2 resulted in cell apoptosis and a reduction in oocyte numbers within embryonic ovaries. By observing the expression of some meiotic marker protein (γ-H2AX, diplotene stage marker MSY2 and synapsis complex protein SCP1), we found that function deficiency of EZH1/2 resulted in failure of DNA double-strand breaks (DSBs) repair and break of meiotic progression in fetal mouse ovaries. Moreover, Ezh1/2 deficiency led to the suppression of ATM (Ataxia Telangiectasia Mutated kinase) phosphorylation and a decrease in the expression of key DNA repair proteins Hormad1, Mre11, Rad50, and Nbs1 in fetal mouse ovaries, underscoring the enzyme's pivotal role in initiating DNA repair. RNA-seq analysis revealed that Ezh1/2-deletion induced abnormal expression of multiple genes involved into several function of oocyte development in embryonic ovaries. Knockout of Ezh1/2 in ovaries also affected the levels of H3K9me3 and H4K20me2, as well as the expression of their target genes L3mbtl4 and Fbxo44. CONCLUSIONS our study demonstrated that EZH1/2 plays a role in the DSBs repair in oocyte meiosis prophase I via multiple mechanisms and offers new insights into the physiological regulatory role of histone modification in fetal oocyte guardianship and female fertility.
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Affiliation(s)
- Ting Jiang
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian, 361005, China
| | - Chengxiu Zhang
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian, 361005, China
| | - Xinjing Cao
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian, 361005, China
| | - Yingpu Tian
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian, 361005, China
| | - Han Cai
- Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, School of Medicine, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, Fujian, 361102, China
| | - Shuangbo Kong
- Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, School of Medicine, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, Fujian, 361102, China
| | - Jinhua Lu
- Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, School of Medicine, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, Fujian, 361102, China
| | - Haibin Wang
- Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, School of Medicine, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, Fujian, 361102, China.
- Medical College of Xiamen University, Xiamen, Fujian, 361102, China.
| | - Zhongxian Lu
- School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research, Xiamen University, Xiamen, Fujian, 361005, China.
- Fujian Provincial Key Laboratory of Reproductive Health Research, Department of Obstetrics and Gynecology, School of Medicine, The First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, Fujian, 361102, China.
- School of Pharmaceutical Sciences, Xiamen University, Zhuangjin Hall, Room 363, Xiamen, Fujian, 361102, China.
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Frei K, Schecher S, Daher T, Hörner N, Richter J, Hildebrand U, Schindeldecker M, Witzel HR, Tsaur I, Porubsky S, Gaida MM, Roth W, Tagscherer KE. Inhibition of the Cyclin K-CDK12 complex induces DNA damage and increases the effect of androgen deprivation therapy in prostate cancer. Int J Cancer 2024; 154:1082-1096. [PMID: 37916780 DOI: 10.1002/ijc.34778] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Revised: 09/07/2023] [Accepted: 10/10/2023] [Indexed: 11/03/2023]
Abstract
Androgen deprivation therapy (ADT) is the mainstay of the current first-line treatment concepts for patients with advanced prostate carcinoma (PCa). However, due to treatment failure and recurrence investigation of new targeted therapeutics is urgently needed. In this study, we investigated the suitability of the Cyclin K-CDK12 complex as a novel therapeutic approach in PCa using the new covalent CDK12/13 inhibitor THZ531. Here we show that THZ531 impairs cellular proliferation, induces apoptosis, and decreases the expression of selected DNA repair genes in PCa cell lines, which is associated with an increasing extent of DNA damage. Furthermore, combination of THZ531 and ADT leads to an increase in these anti-tumoral effects in androgen-sensitive PCa cells. The anti-proliferative and pro-apoptotic activity of THZ531 in combination with ADT was validated in an ex vivo PCa tissue culture model. In a retrospective immunohistochemical analysis of 300 clinical tissue samples we show that Cyclin K (CycK) but not CDK12 expression correlates with a more aggressive type of PCa. In conclusion, this study demonstrates the clinical relevance of the CycK-CDK12 complex as a promising target for combinational therapy with ADT in PCa and its importance as a prognostic biomarker for patients with PCa.
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Affiliation(s)
- Katharina Frei
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Sabrina Schecher
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Tamas Daher
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Nina Hörner
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Jutta Richter
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Ute Hildebrand
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Mario Schindeldecker
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- Tissue Biobank of the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Hagen R Witzel
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Igor Tsaur
- Department of Urology and Pediatric Urology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Stefan Porubsky
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Matthias M Gaida
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Wilfried Roth
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Katrin E Tagscherer
- Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
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3
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Somanath PR, Chernoff J, Cummings BS, Prasad SM, Homan HD. Targeting P21-Activated Kinase-1 for Metastatic Prostate Cancer. Cancers (Basel) 2023; 15:2236. [PMID: 37190165 PMCID: PMC10137274 DOI: 10.3390/cancers15082236] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Revised: 04/06/2023] [Accepted: 04/09/2023] [Indexed: 05/17/2023] Open
Abstract
Metastatic prostate cancer (mPCa) has limited therapeutic options and a high mortality rate. The p21-activated kinase (PAK) family of proteins is important in cell survival, proliferation, and motility in physiology, and pathologies such as infectious, inflammatory, vascular, and neurological diseases as well as cancers. Group-I PAKs (PAK1, PAK2, and PAK3) are involved in the regulation of actin dynamics and thus are integral for cell morphology, adhesion to the extracellular matrix, and cell motility. They also play prominent roles in cell survival and proliferation. These properties make group-I PAKs a potentially important target for cancer therapy. In contrast to normal prostate and prostatic epithelial cells, group-I PAKs are highly expressed in mPCA and PCa tissue. Importantly, the expression of group-I PAKs is proportional to the Gleason score of the patients. While several compounds have been identified that target group-I PAKs and these are active in cells and mice, and while some inhibitors have entered human trials, as of yet, none have been FDA-approved. Probable reasons for this lack of translation include issues related to selectivity, specificity, stability, and efficacy resulting in side effects and/or lack of efficacy. In the current review, we describe the pathophysiology and current treatment guidelines of PCa, present group-I PAKs as a potential druggable target to treat mPCa patients, and discuss the various ATP-competitive and allosteric inhibitors of PAKs. We also discuss the development and testing of a nanotechnology-based therapeutic formulation of group-I PAK inhibitors and its significant potential advantages as a novel, selective, stable, and efficacious mPCa therapeutic over other PCa therapeutics in the pipeline.
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Affiliation(s)
- Payaningal R. Somanath
- Department of Clinical & Administrative Pharmacy, College of Pharmacy, University of Georgia, Augusta, GA 30912, USA
- MetasTx LLC, Basking Ridge, NJ 07920, USA
| | - Jonathan Chernoff
- MetasTx LLC, Basking Ridge, NJ 07920, USA
- Fox Chase Cancer Center, Philadelphia, PA 19111, USA
| | - Brian S. Cummings
- MetasTx LLC, Basking Ridge, NJ 07920, USA
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA
| | - Sandip M. Prasad
- Morristown Medical Center, Atlantic Health System, Morristown, NJ 07960, USA
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Manzar N, Ganguly P, Khan UK, Ateeq B. Transcription networks rewire gene repertoire to coordinate cellular reprograming in prostate cancer. Semin Cancer Biol 2023; 89:76-91. [PMID: 36702449 DOI: 10.1016/j.semcancer.2023.01.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 01/04/2023] [Accepted: 01/18/2023] [Indexed: 01/24/2023]
Abstract
Transcription factors (TFs) represent the most commonly deregulated DNA-binding class of proteins associated with multiple human cancers. They can act as transcriptional activators or repressors that rewire the cistrome, resulting in cellular reprogramming during cancer progression. Deregulation of TFs is associated with the onset and maintenance of various cancer types including prostate cancer. An emerging subset of TFs has been implicated in the regulation of multiple cancer hallmarks during tumorigenesis. Here, we discuss the role of key TFs which modulate transcriptional cicuitries involved in the development and progression of prostate cancer. We further highlight the role of TFs associated with key cancer hallmarks, including, chromatin remodeling, genome instability, DNA repair, invasion, and metastasis. We also discuss the pluripotent function of TFs in conferring lineage plasticity, that aids in disease progression to neuroendocrine prostate cancer. At the end, we summarize the current understanding and approaches employed for the therapeutic targeting of TFs and their cofactors in the clinical setups to prevent disease progression.
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Affiliation(s)
- Nishat Manzar
- Molecular Oncology Laboratory, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Promit Ganguly
- Molecular Oncology Laboratory, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Umar Khalid Khan
- Molecular Oncology Laboratory, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Bushra Ateeq
- Molecular Oncology Laboratory, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India; Mehta Family Center for Engineering in Medicine, Indian Institute of Technology Kanpur, Kanpur 208016, India.
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Chen Y, Yan W, Chen Y, Zhu J, Wang J, Jin H, Wu H, Zhang G, Zhan S, Xi Q, Shi T, Chen W. SLC6A14 facilitates epithelial cell ferroptosis via the C/EBPβ-PAK6 axis in ulcerative colitis. Cell Mol Life Sci 2022; 79:563. [PMID: 36272033 PMCID: PMC11802994 DOI: 10.1007/s00018-022-04594-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 09/22/2022] [Accepted: 10/08/2022] [Indexed: 11/24/2022]
Abstract
Emerging evidence suggests that ferroptosis is involved in the pathogenesis of ulcerative colitis (UC). However, the key regulator of this process remains uncertain. In this study, we aimed to explore the roles of solute carrier (SLC) family 6 member 14 (SLC6A14) in regulating ferroptosis in UC. The expression of SLC6A14 was significantly increased and positively associated with that of prostaglandin-endoperoxide synthase 2 (PTGS2) in tissue samples from patients with UC. Moreover, a series of in vitro and in vivo experiments showed that SLC6A14 knockdown markedly suppressed ferroptosis. RNA sequencing revealed that SLC6A14 inhibited the expression of P21 (RAC1)-activated kinase 6 (PAK6) and that PAK6 knockdown abolished the effects of SLC6A14 on RAS-selective lethal 3 (RSL3)-induced ferroptosis in Caco-2 cells. Furthermore, chromatin immunoprecipitation (ChIP) and Western blot analysis demonstrated that SLC6A14 negatively regulated PAK6 expression in a CCAAT enhancer binding protein beta (C/EBPβ)-dependent manner. Collectively, these findings indicate that SLC6A14 facilitates ferroptosis in UC by promoting C/EBPβ expression and binding activity to inhibit PAK6 expression, suggesting that targeting SLC6A14-C/EBPβ-PAK6 axis-mediated ferroptosis may be a promising therapeutic alternative for UC.
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Affiliation(s)
- Yanjun Chen
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Wenying Yan
- Center for Systems Biology, Department of Bioinformatics, School of Biology and Basic Medical Sciences, Soochow University, Suzhou, China
| | - Yuqi Chen
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Jinghan Zhu
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Jiayu Wang
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Haiyan Jin
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Hongya Wu
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Guangbo Zhang
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou, China
- Jiangsu Key Laboratory of Gastrointestinal Tumor Immunology, The First Affiliated Hospital of Soochow University, Suzhou, 215000, Jiangsu, China
| | - Shenghua Zhan
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China
- Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou, China
| | - Qinhua Xi
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Tongguo Shi
- Jiangsu Institute of Clinical Immunology, The First Affiliated Hospital of Soochow University, Suzhou, China.
- Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou, China.
- Jiangsu Key Laboratory of Gastrointestinal Tumor Immunology, The First Affiliated Hospital of Soochow University, Suzhou, 215000, Jiangsu, China.
| | - Weichang Chen
- Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, China.
- Jiangsu Key Laboratory of Clinical Immunology, Soochow University, Suzhou, China.
- Jiangsu Key Laboratory of Gastrointestinal Tumor Immunology, The First Affiliated Hospital of Soochow University, Suzhou, 215000, Jiangsu, China.
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p21-Activated kinases as promising therapeutic targets in hematological malignancies. Leukemia 2022; 36:315-326. [PMID: 34697424 DOI: 10.1038/s41375-021-01451-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 09/30/2021] [Accepted: 10/07/2021] [Indexed: 01/12/2023]
Abstract
The p21-Activated Kinases (PAKs) are a family of six serine/threonine kinases that were originally identified as downstream effectors of the Rho GTPases Cdc42 and Rac. Since the first PAK was discovered in 1994, studies have revealed their fundamental and biological importance in the development of physiological systems. Within the cell, PAKs also play significant roles in regulating essential cellular processes such as cytoskeletal dynamics, gene expression, cell survival, and cell cycle progression. These processes are often deregulated in numerous cancers when different PAKs are overexpressed or amplified at the chromosomal level. Furthermore, PAKs modulate multiple oncogenic signaling pathways which facilitate apoptosis escape, uncontrolled proliferation, and drug resistance. There is growing insight into the critical roles of PAKs in regulating steady-state hematopoiesis, including the properties of hematopoietic stem cells (HSC), and the initiation and progression of hematological malignancies. This review will focus on the most recent studies that provide experimental evidence showing how specific PAKs regulate the properties of leukemic stem cells (LSCs) and drug-resistant cells to initiate and maintain hematological malignancies. The current understanding of the molecular and cellular mechanisms by which the PAKs operate in specific human leukemia or lymphomas will be discussed. From a translational point of view, PAKs have been suggested to be critical therapeutic targets and potential prognosis markers; thus, this review will also discuss current therapeutic strategies against hematological malignancies using existing small-molecule PAK inhibitors, as well as promising combination treatments, to sensitize drug-resistant cells to conventional therapies. The challenges of toxicity and non-specific targeting associated with some PAK inhibitors, as well as how future approaches for PAK inhibition to overcome these limitations, will also be addressed.
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Tan S, Zhou Y, Zhao H, Wu J, Yu H, Yang Y, Yang Y, Zhao H, Li H. Comprehensive transcriptome analysis of hypothalamus reveals genes associated with disorders of sex development in pigs. J Steroid Biochem Mol Biol 2021; 210:105875. [PMID: 33746111 DOI: 10.1016/j.jsbmb.2021.105875] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 03/05/2021] [Accepted: 03/09/2021] [Indexed: 12/14/2022]
Abstract
XX sex reversal, also called XX disorders of sex development (XX-DSD), is a condition affecting the development of the gonads or genitalia, and is relatively common in pigs. However, its genetic etiology and transcriptional regulation mechanism in the hypothalamic-pituitary-gonadal axis (HPGA) remain mostly unknown. XX-DSD (SRY-negative) pigs and normal sows were selected by external genitalia observation. The hypothalamus, which is the integrated center of the HPGA was sampled for whole-transcriptome RNA-seq. The role of DEmiRNA was validated by its overexpression and knockdown in vitro. A total of 1,258 lncRNAs, 1,086 mRNAs, and 61 microRNAs differentially expressed in XX-DSD pigs compared with normal female pigs. Genes in the hormone biosynthesis and secretion pathway significantly up-regulated, and the up-regulation of GNRH1, KISS1 and AVP may associate with the abnormal secretion of GnRH. We also predicted the lncRNA-miRNA-mRNA co-expression triplets and constructed three competing endogenous RNA (ceRNA) potentially associated with XX-DSD. Functional enrichment studies suggested that TCONS_00340886, TCONS_00000204 and miR-181a related to GnRH secretion. Further, miR-181a inhibitor up-regulated GNRH1, PAK6, and CAMK4 in the GT1-7 cells. Conversely, transfection of miR-181a mimics obtained the opposite trends. The expression levels of FSHR, LHR, ESR1 and ESR2 were significantly higher in XX-DSD gondas than those in normal sows. Taken together, we proposed that the balance of endocrine had broken in XX-DSD pigs. The current study is the first to examine the transcriptomic profile in the hypothalamus of XX-DSD pigs. It provides new insight into coding and non-coding RNAs that may be associated with DSD in pigs.
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Affiliation(s)
- Shuwen Tan
- Department of Ecology, Tibetan Centre for Ecology and Conservation at WHU-TU, Hubei Key Laboratory of Cell Homeostasis, College of Life Science, Wuhan University, Wuhan 430072, China; Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Yi Zhou
- College of Basic Medicine, Zunyi Medical University, Zunyi 563006, China
| | - Haiquan Zhao
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Jinhua Wu
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Hui Yu
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Yin Yang
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Yalan Yang
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China
| | - Huabin Zhao
- Department of Ecology, Tibetan Centre for Ecology and Conservation at WHU-TU, Hubei Key Laboratory of Cell Homeostasis, College of Life Science, Wuhan University, Wuhan 430072, China; College of Science, Tibet University, Lhasa 850000, China.
| | - Hua Li
- Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan 528225, China.
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Bantle CM, French CT, Cummings JE, Sadasivan S, Tran K, Slayden RA, Smeyne RJ, Tjalkens RB. Manganese exposure in juvenile C57BL/6 mice increases glial inflammatory responses in the substantia nigra following infection with H1N1 influenza virus. PLoS One 2021; 16:e0245171. [PMID: 33493177 PMCID: PMC7833173 DOI: 10.1371/journal.pone.0245171] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Accepted: 12/22/2020] [Indexed: 01/22/2023] Open
Abstract
Infection with Influenza A virus can lead to the development of encephalitis and subsequent neurological deficits ranging from headaches to neurodegeneration. Post-encephalitic parkinsonism has been reported in surviving patients of H1N1 infections, but not all cases of encephalitic H1N1 infection present with these neurological symptoms, suggesting that interactions with an environmental neurotoxin could promote more severe neurological damage. The heavy metal, manganese (Mn), is a potential interacting factor with H1N1 because excessive exposure early in life can induce long-lasting effects on neurological function through inflammatory activation of glial cells. In the current study, we used a two-hit model of neurotoxin-pathogen exposure to examine whether exposure to Mn during juvenile development would induce a more severe neuropathological response following infection with H1N1 in adulthood. To test this hypothesis, C57BL/6 mice were exposed to MnCl2 in drinking water (50 mg/kg/day) for 30 days from days 21–51 postnatal, then infected intranasally with H1N1 three weeks later. Analyses of dopaminergic neurons, microglia and astrocytes in basal ganglia indicated that although there was no significant loss of dopaminergic neurons within the substantia nigra pars compacta, there was more pronounced activation of microglia and astrocytes in animals sequentially exposed to Mn and H1N1, as well as altered patterns of histone acetylation. Whole transcriptome Next Generation Sequencing (RNASeq) analysis was performed on the substantia nigra and revealed unique patterns of gene expression in the dual-exposed group, including genes involved in antioxidant activation, mitophagy and neurodegeneration. Taken together, these results suggest that exposure to elevated levels of Mn during juvenile development could sensitize glial cells to more severe neuro-immune responses to influenza infection later in life through persistent epigenetic changes.
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Affiliation(s)
- Collin M. Bantle
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - C. Tenley French
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Jason E. Cummings
- Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America
| | - Shankar Sadasivan
- Department of Neuroscience, Vickie & Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
| | - Kevin Tran
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Richard A. Slayden
- Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America
| | - Richard J. Smeyne
- Department of Neuroscience, Vickie & Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
| | - Ronald B. Tjalkens
- Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, United States of America
- * E-mail:
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Zheng J, Zhang C, Li Y, Jiang Y, Xing B, Du X. p21-activated kinase 6 controls mitosis and hepatocellular carcinoma progression by regulating Eg5. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2020; 1868:118888. [PMID: 33098954 DOI: 10.1016/j.bbamcr.2020.118888] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 10/07/2020] [Accepted: 10/09/2020] [Indexed: 02/07/2023]
Abstract
P21-activated kinases 6 (PAK6) associated with many fundamental cellular processes in cancer including cell-cell adhesion, migration and apoptosis. Here, we report a novel function of PAK6 in mitosis. Expression of PAK6 peaks in the M phase. Knockdown of PAK6 increases cell number in G2/M and promotes cell proliferation. PAK6 specifically colocalizes with Eg5 in the centrosome. Depletion of PAK6 results in multipolar spindle and a simultaneous upregulation of Eg5. Further, the PAK6 depletion-induced multiple spindle and cell cycle progression is reversed by knockdown of Eg5. These data suggest that PAK6 regulates spindle formation and cell cycle by regulating Eg5 expression. Additionally, expression of PAK6 is upregulated when Eg5 is downregulated or inhibited. Thus, PAK6 and Eg5 negatively inter-regulate each other. Significantly, the effect of PAK6 expression on the outcome of the HCC patients is controlled by Eg5 expression. Inhibition of Eg5 reverses PAK6 depletion-promoted cell invasion. Collectively, our data indicate that the inter-regulation between PAK6 and Eg5 might promote the progression of HCC.
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Affiliation(s)
- Jiaojiao Zheng
- Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China
| | - Chunfeng Zhang
- Department of Medical Genetics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China
| | - Yuan Li
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Hepatopancreatobiliary Surgery Department I, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Yang Jiang
- Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China
| | - Baocai Xing
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Hepatopancreatobiliary Surgery Department I, Peking University Cancer Hospital & Institute, Beijing 100142, China.
| | - Xiaojuan Du
- Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100083, China.
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10
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Venkadakrishnan VB, Ben-Salem S, Heemers HV. AR-dependent phosphorylation and phospho-proteome targets in prostate cancer. Endocr Relat Cancer 2020; 27:R193-R210. [PMID: 32276264 PMCID: PMC7583603 DOI: 10.1530/erc-20-0048] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Accepted: 04/06/2020] [Indexed: 12/17/2022]
Abstract
Prostate cancer (CaP) is the second leading cause of cancer-related deaths in Western men. Because androgens drive CaP by activating the androgen receptor (AR), blocking AR's ligand activation, known as androgen deprivation therapy (ADT), is the default treatment for metastatic CaP. Despite an initial remission, CaP eventually develops resistance to ADT and progresses to castration-recurrent CaP (CRPC). CRPC continues to rely on aberrantly activated AR that is no longer inhibited effectively by available therapeutics. Interference with signaling pathways downstream of activated AR that mediate aggressive CRPC behavior may lead to alternative CaP treatments. Developing such therapeutic strategies requires a thorough mechanistic understanding of the most clinically relevant and druggable AR-dependent signaling events. Recent proteomics analyses of CRPC clinical specimens indicate a shift in the phosphoproteome during CaP progression. Kinases and phosphatases represent druggable entities, for which clinically tested inhibitors are available, some of which are incorporated already in treatment plans for other human malignancies. Here, we reviewed the AR-associated transcriptome and translational regulon, and AR interactome involved in CaP phosphorylation events. Novel and for the most part mutually exclusive AR-dependent transcriptional and post-transcriptional control over kinase and phosphatase expression was found, with yet other phospho-regulators interacting with AR. The multiple mechanisms by which AR can shape and fine-tune the CaP phosphoproteome were reflected in diverse aspects of CaP biology such as cell cycle progression and cell migration. Furthermore, we examined the potential, limitations and challenges of interfering with AR-mediated phosphorylation events as alternative strategy to block AR function during CaP progression.
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Affiliation(s)
- Varadha Balaji Venkadakrishnan
- Department of Cancer Biology, Cleveland Clinic, Cleveland, Ohio, USA
- Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio, USA
| | - Salma Ben-Salem
- Department of Cancer Biology, Cleveland Clinic, Cleveland, Ohio, USA
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11
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Su VL, Simon B, Draheim KM, Calderwood DA. Serine phosphorylation of the small phosphoprotein ICAP1 inhibits its nuclear accumulation. J Biol Chem 2020; 295:3269-3284. [PMID: 32005669 DOI: 10.1074/jbc.ra119.009794] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2019] [Revised: 01/29/2020] [Indexed: 02/06/2023] Open
Abstract
Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development.
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Affiliation(s)
- Valerie L Su
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
| | - Bertrand Simon
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
| | - Kyle M Draheim
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
| | - David A Calderwood
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520.
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12
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Gong CC, Li TT, Pei DS. PAK6: a potential anti-cancer target. BRAZ J PHARM SCI 2020. [DOI: 10.1590/s2175-97902019000318315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Affiliation(s)
| | | | - Dong-Sheng Pei
- Xuzhou Medical University, China; Xuzhou Medical University, China
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13
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Sun X, Su VL, Calderwood DA. The subcellular localization of type I p21-activated kinases is controlled by the disordered variable region and polybasic sequences. J Biol Chem 2019; 294:14319-14332. [PMID: 31391252 DOI: 10.1074/jbc.ra119.007692] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2019] [Revised: 07/30/2019] [Indexed: 12/20/2022] Open
Abstract
p21-activated kinases (PAKs) are serine/threonine kinase effectors of the small GTPases Rac and Cdc42 and major participants in cell adhesion, motility, and survival. Type II PAKs (PAK4, -5, and -6) are recruited to cell-cell boundaries, where they regulate adhesion dynamics and colony escape. In contrast, the type I PAK, PAK1, does not localize to cell-cell contacts. We have now found that the other type I PAKs (PAK2 and PAK3) also fail to target to cell-cell junctions. PAKs contain extensive similarities in sequence and domain organization; therefore, focusing on PAK1 and PAK6, we used chimeras and truncation mutants to investigate their differences in localization. We observed that a weakly conserved sequence region (the variable region), located between the Cdc42-binding CRIB domain and the kinase domain, inhibits PAK1 targeting to cell-cell junctions. Accordingly, substitution of the PAK1 variable region with that from PAK6 or removal of this region of PAK1 resulted in its localization to cell-cell contacts. We further show that Cdc42 binding is required, but not sufficient, to direct PAKs to cell-cell contacts and that an N-terminal polybasic sequence is necessary for PAK1 recruitment to cell-cell contacts, but only if the variable region-mediated inhibition is released. We propose that all PAKs contain cell-cell boundary-targeting motifs but that the variable region prevents type I PAK accumulation at junctions. This highlights the importance of this poorly conserved, largely disordered region in PAK regulation and raises the possibility that variable region inhibition may be released by cellular signals.
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Affiliation(s)
- Xiaowen Sun
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
| | - Valerie L Su
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520
| | - David A Calderwood
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520.,Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520
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14
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Goyette SR, Schott E, Uwimana A, Nelson DW, Boganski J. Detection of the steroid receptor interacting protein, PAK6, in a neuronal cell line. Heliyon 2019; 5:e01294. [PMID: 30923762 PMCID: PMC6423815 DOI: 10.1016/j.heliyon.2019.e01294] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Revised: 01/18/2019] [Accepted: 02/26/2019] [Indexed: 01/31/2023] Open
Abstract
PAK6 is a Group II p21 activated kinase that unlike traditional signal transduction proteins interacts with multiple binding partners including sex-steroid receptors. PAK6 acts as a nodal checkpoint integrating multiple cellular inputs to promote distinct cellular outcomes, some of which are associated with cytoskeletal remodeling. Despite the possibility that PAK6 may couple sex-specific neuronal function and therefore serve as a valuable research, diagnostic and therapeutic target, there is currently no standardized protocol for assessing PAK6 activity in a neuronal cell line. Here, we present a protocol for assessing PAK6 levels in a commonly used neuronal cell line, PC-12. In comparison with other methodology, this approach (1) does not require ex-planted tissue to identify PAK6 in neurons and (2) unlike other protocols which require steroid depleted media for detection of PAK6 in non-neuronal cell lines, such as prostate cancer cell lines, we were easily able to detect PAK6 in PC-12 cells grown in complete, steroid-containing media. Thus the present protocol allows for the efficient detection of native PAK6 in PC-12 cells to expedite targeted basic research of the emerging importance of PAK6 function in the brain as well as to accelerate the identification and isolation of potential therapeutic targets not only in cancerous but brain disease states as well.
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Affiliation(s)
| | - Eric Schott
- Shields Science Center, Stonehill College, Easton MA, 02357, USA
| | | | - David W Nelson
- Shields Science Center, Stonehill College, Easton MA, 02357, USA
| | - Jacob Boganski
- Shields Science Center, Stonehill College, Easton MA, 02357, USA
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15
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Castillo J, Knol JC, Korver CM, Piersma SR, Pham TV, de Goeij-de Haas RR, van Pelt AMM, Jimenez CR, Jansen BJH. Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer. Mol Cell Proteomics 2019; 18:S132-S144. [PMID: 30683686 PMCID: PMC6427237 DOI: 10.1074/mcp.ra118.001278] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Indexed: 12/25/2022] Open
Abstract
Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico, protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.
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Affiliation(s)
- Judit Castillo
- Lead Pharma BV, Pivot Park, Kloosterstraat 9, 5349 AB Oss, The Netherlands;.
| | - Jaco C Knol
- OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands
| | - Cindy M Korver
- Center for Reproductive Medicine, Research Institute Amsterdam Reproduction and Development, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
| | - Sander R Piersma
- OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands
| | - Thang V Pham
- OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands
| | - Richard R de Goeij-de Haas
- OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands
| | - Ans M M van Pelt
- Center for Reproductive Medicine, Research Institute Amsterdam Reproduction and Development, Amsterdam UMC, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands
| | - Connie R Jimenez
- OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands
| | - Bastiaan J H Jansen
- Lead Pharma BV, Pivot Park, Kloosterstraat 9, 5349 AB Oss, The Netherlands;.
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16
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Wang K, Baldwin GS, Nikfarjam M, He H. p21-activated kinase signalling in pancreatic cancer: New insights into tumour biology and immune modulation. World J Gastroenterol 2018; 24:3709-3723. [PMID: 30197477 PMCID: PMC6127653 DOI: 10.3748/wjg.v24.i33.3709] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 06/22/2018] [Accepted: 06/27/2018] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer is one of the most aggressive and lethal malignancies worldwide, with a very poor prognosis and a five-year survival rate less than 8%. This dismal outcome is largely due to delayed diagnosis, early distant dissemination and resistance to conventional chemo-therapies. Kras mutation is a well-defined hallmark of pancreatic cancer, with over 95% of cases harbouring Kras mutations that give rise to constitutively active forms of Kras. As important down-stream effectors of Kras, p21-activated kinases (PAKs) are involved in regulating cell proliferation, apoptosis, invasion/migration and chemo-resistance. Immunotherapy is now emerging as a promising treatment modality in the era of personalized anti-cancer therapeutics. In this review, basic knowledge of PAK structure and regulation is briefly summarised and the pivotal role of PAKs in Kras-driven pancreatic cancer is highlighted in terms of tumour biology and chemo-resistance. Finally, the involvement of PAKs in immune modulation in the tumour microenvironment is discussed and the potential advantages of targeting PAKs are explored.
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Affiliation(s)
- Kai Wang
- Department of Surgery, University of Melbourne, Melbourne 3084, Australia
| | - Graham S Baldwin
- Department of Surgery, University of Melbourne, Melbourne 3084, Australia
| | - Mehrdad Nikfarjam
- Department of Surgery, University of Melbourne, Melbourne 3084, Australia
| | - Hong He
- Department of Surgery, University of Melbourne, Melbourne 3084, Australia
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17
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Insights into Male Androgenetic Alopecia: Differential Gene Expression Profiling of Plucked Hair Follicles and Integration with Genetic Data. J Invest Dermatol 2018; 139:235-238. [PMID: 30009830 DOI: 10.1016/j.jid.2018.06.182] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Revised: 06/07/2018] [Accepted: 06/25/2018] [Indexed: 12/13/2022]
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18
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Maßberg D, Jovancevic N, Offermann A, Simon A, Baniahmad A, Perner S, Pungsrinont T, Luko K, Philippou S, Ubrig B, Heiland M, Weber L, Altmüller J, Becker C, Gisselmann G, Gelis L, Hatt H. The activation of OR51E1 causes growth suppression of human prostate cancer cells. Oncotarget 2018; 7:48231-48249. [PMID: 27374083 PMCID: PMC5217014 DOI: 10.18632/oncotarget.10197] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2015] [Accepted: 06/06/2016] [Indexed: 01/23/2023] Open
Abstract
The development of prostate cancer (PCa) is regulated by the androgen-dependent activity of the androgen receptor (AR). Androgen-deprivation therapy (ADT) is therefore the gold standard treatment to suppress malignant progression of PCa. Nevertheless, due to the development of castration resistance, recurrence of disease after initial response to ADT is a major obstacle to successful treatment. As G-protein coupled receptors play a fundamental role in PCa physiology, they might represent promising alternative or combinatorial targets for advanced diseases. Here, we verified gene expression of the olfactory receptors (ORs) OR51E1 [prostate-specific G-protein coupled receptor 2 (PSGR2)] and OR51E2 (PSGR) in human PCa tissue by RNA-Seq analysis and RT-PCR and elucidated the subcellular localization of both receptor proteins in human prostate tissue. The OR51E1 agonist nonanoic acid (NA) leads to the phosphorylation of various protein kinases and growth suppression of the PCa cell line LNCaP. Furthermore, treatment with NA causes reduction of androgen-mediated AR target gene expression. Interestingly, NA induces cellular senescence, which coincides with reduced E2F1 mRNA levels. In contrast, treatment with the structurally related compound 1-nonanol or the OR2AG1 agonist amyl butyrate, neither of which activates OR51E1, did not lead to reduced cell growth or an induction of cellular senescence. However, decanoic acid, another OR51E1 agonist, also induces cellular senescence. Thus, our results suggest the involvement of OR51E1 in growth processes of PCa cells and its impact on AR-mediated signaling. These findings provide novel evidences to support the functional importance of ORs in PCa pathogenesis.
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Affiliation(s)
- Désirée Maßberg
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
| | | | - Anne Offermann
- Pathology of the University Hospital of Luebeck and the Leibniz Research Center Borstel, Luebeck and Borstel, Germany
| | - Annika Simon
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
| | - Aria Baniahmad
- Institute of Human Genetics, Jena University Hospital, Jena, Germany
| | - Sven Perner
- Pathology of the University Hospital of Luebeck and the Leibniz Research Center Borstel, Luebeck and Borstel, Germany
| | | | - Katarina Luko
- Institute of Human Genetics, Jena University Hospital, Jena, Germany
| | - Stathis Philippou
- Institute for Pathology und Cytology, Augusta-Kranken-Anstalt gGmbH Bochum, Bochum, Germany
| | - Burkhard Ubrig
- Clinic for Urology, Augusta-Kranken-Anstalt gGmbH Bochum, Bochum, Germany
| | - Markus Heiland
- Clinic for Urology, Augusta-Kranken-Anstalt gGmbH Bochum, Bochum, Germany
| | - Lea Weber
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
| | | | | | - Günter Gisselmann
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
| | - Lian Gelis
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany.,Present address: Global Drug Discovery - Clinical Sciences, Bayer Pharma AG, Wuppertal, Germany
| | - Hanns Hatt
- Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
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19
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Civiero L, Cogo S, Kiekens A, Morganti C, Tessari I, Lobbestael E, Baekelandt V, Taymans JM, Chartier-Harlin MC, Franchin C, Arrigoni G, Lewis PA, Piccoli G, Bubacco L, Cookson MR, Pinton P, Greggio E. PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2. Front Mol Neurosci 2017; 10:417. [PMID: 29311810 PMCID: PMC5735978 DOI: 10.3389/fnmol.2017.00417] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2017] [Accepted: 11/30/2017] [Indexed: 12/28/2022] Open
Abstract
Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.
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Affiliation(s)
- Laura Civiero
- Department of Biology, University of Padova, Padova, Italy
| | - Susanna Cogo
- Department of Biology, University of Padova, Padova, Italy
- School of Pharmacy, University of Reading, Reading, United Kingdom
| | | | - Claudia Morganti
- Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | | | - Evy Lobbestael
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Veerle Baekelandt
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Jean-Marc Taymans
- Université de Lille, Institut National de la Santé et de la Recherche Médicale, CHU Lille, UMR-S1172, JPArc, Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Lille, France
- Institut National de la Santé et de la Recherche Médicale, UMR-S 1172, Team “Early Stages of Parkinson's Disease”, Lille, France
| | - Marie-Christine Chartier-Harlin
- Université de Lille, Institut National de la Santé et de la Recherche Médicale, CHU Lille, UMR-S1172, JPArc, Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Lille, France
- Institut National de la Santé et de la Recherche Médicale, UMR-S 1172, Team “Early Stages of Parkinson's Disease”, Lille, France
| | - Cinzia Franchin
- Department of Biomedical Sciences, University of Padova, Padova, Italy
- Proteomics Center, University of Padova and Azienda Ospedaliera di Padova, Padova, Italy
| | - Giorgio Arrigoni
- Department of Biomedical Sciences, University of Padova, Padova, Italy
- Proteomics Center, University of Padova and Azienda Ospedaliera di Padova, Padova, Italy
| | - Patrick A. Lewis
- School of Pharmacy, University of Reading, Reading, United Kingdom
| | - Giovanni Piccoli
- Center for Integrative Biology, University of Trento, Trento, Italy
| | - Luigi Bubacco
- Department of Biology, University of Padova, Padova, Italy
| | - Mark R. Cookson
- Laboratory of Neurogenetics, National Institute on Aging/NIH, Bethesda, MD, United States
| | - Paolo Pinton
- Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | - Elisa Greggio
- Department of Biology, University of Padova, Padova, Italy
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20
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Civiero L, Greggio E. PAKs in the brain: Function and dysfunction. Biochim Biophys Acta Mol Basis Dis 2017; 1864:444-453. [PMID: 29129728 DOI: 10.1016/j.bbadis.2017.11.005] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 10/31/2017] [Accepted: 11/06/2017] [Indexed: 12/17/2022]
Abstract
p21-Activated kinases (PAKs) comprise a family of proteins covering a central role in signal transduction. They are downstream effectors of Rho GTPases and can affect a variety of processes in different cell types and tissues by remodeling the cytoskeleton and by promoting gene transcription and cell survival. Given the relevance of cytoskeletal organization in neuronal development as well as synaptic function and the importance of pro-survival signals in controlling neuronal cell fate, accumulating studies investigated the role of PAKs in the nervous system. In this review, we provide a critical overview of the role of PAKs in the nervous system, both in neuronal and non-neuronal cells, and discuss their potential link with neurodegenerative diseases.
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21
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Liposome-mediated delivery of the p21 activated kinase-1 (PAK-1) inhibitor IPA-3 limits prostate tumor growth in vivo. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2016; 12:1231-1239. [PMID: 26949163 DOI: 10.1016/j.nano.2016.01.003] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/19/2015] [Revised: 12/26/2015] [Accepted: 01/12/2016] [Indexed: 11/21/2022]
Abstract
P21 activated kinases-1 (PAK-1) is implicated in various diseases. It is inhibited by the small molecule 'inhibitor targeting PAK1 activation-3' (IPA-3), which is highly specific but metabolically unstable. To address this limitation we encapsulated IPA-3 in sterically stabilized liposomes (SSL). SSL-IPA-3 averaged 139nm in diameter, polydispersity index (PDI) of 0.05, and a zeta potential of -28.1, neither of which changed over 14days; however, the PDI increased to 0.139. Analysis of liposomal IPA-3 levels demonstrated good stability, with 70% of IPA-3 remaining after 7days. SSL-IPA-3 inhibited prostate cancer cell growth in vitro with comparable efficacy to free IPA-3. Excitingly, only a 2day/week dose of SSL-IPA-3 was needed to inhibit the growth of prostate xenografts in vivo, while a similar dose of free IPA-3 was ineffective. These data demonstrate the development and clinical utility of a novel liposomal formulation for the treatment of prostate cancer.
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22
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Chen J, Lu H, Yan D, Cui F, Wang X, Yu F, Xue Y, Feng X, Wang J, Wang X, Jiang T, Zhang M, Zhao S, Yu Y, Tang H, Peng Z. PAK6 increase chemoresistance and is a prognostic marker for stage II and III colon cancer patients undergoing 5-FU based chemotherapy. Oncotarget 2016; 6:355-67. [PMID: 25426562 PMCID: PMC4381600 DOI: 10.18632/oncotarget.2803] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2014] [Accepted: 11/07/2014] [Indexed: 12/20/2022] Open
Abstract
p21-Activated kinase 6 (PAK6) has been implicated in radiotherapy and docetaxel resistance. We have further evaluated PAK6 as a predictor of 5-fluorouracil (5-FU) treatment response in colon cancer. Here we report that in colon cancer PAK6 promotes tumor progression and chemoresistance both in vitro and in vivo. In the clinical analysis, PAK6 was overexpressed in 104 of 147 (70.75%) stage II and III patients who received 5-FU based chemotherapy after surgery. Multivariate Cox regression analysis indicated that PAK6 was an independent prognostic factor for overall survival (P < 0.001) and disease-free survival (P < 0.001). Colon cancer cell lines showed increased PAK6 expression upon 5-FU treatment. In PAK6-knockdown cells treated with 5-FU, cell viability and phosphorylation of BAD decreased, and the number of apoptotic cells, levels of cleaved caspase 3 and PARP increased compared to control cells. The opposite was observed in PAK6 overexpressing cells. Short hairpin RNA knockdown of PAK6 blocked cells in G2-M phase. Furthermore, Animal experiments results in vivo are consistent with outcomes in vitro. This study demonstrates that PAK6 is an independent prognostic factor for adjuvant 5-FU-based chemotherapy in patients with stage II and stage III colon cancer.
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Affiliation(s)
- Jian Chen
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Huijun Lu
- Department of Pathology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Dongwang Yan
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Feifei Cui
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Xiaoliang Wang
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Fudong Yu
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Yingming Xue
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Xiaodong Feng
- Basic Medical College, Taishan Medical University, Tai'an, People's Republic of China
| | - Jingtao Wang
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Xiao Wang
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Tao Jiang
- Department of Anal-Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People's Republic of China
| | - Meng Zhang
- Department of Pathology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Senlin Zhao
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Yang Yu
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Huamei Tang
- Department of Pathology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
| | - Zhihai Peng
- Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China
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Morse EM, Sun X, Olberding JR, Ha BH, Boggon TJ, Calderwood DA. PAK6 targets to cell-cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape. J Cell Sci 2015; 129:380-93. [PMID: 26598554 DOI: 10.1242/jcs.177493] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2015] [Accepted: 11/18/2015] [Indexed: 12/26/2022] Open
Abstract
The six serine/threonine kinases in the p21-activated kinase (PAK) family are important regulators of cell adhesion, motility and survival. PAK6, which is overexpressed in prostate cancer, was recently reported to localize to cell-cell adhesions and to drive epithelial cell colony escape. Here we report that PAK6 targeting to cell-cell adhesions occurs through its N-terminus, requiring both its Cdc42/Rac interactive binding (CRIB) domain and an adjacent polybasic region for maximal targeting efficiency. We find PAK6 localization to cell-cell adhesions is Cdc42-dependent, as Cdc42 knockdown inhibits PAK6 targeting to cell-cell adhesions. We further find the ability of PAK6 to drive epithelial cell colony escape requires kinase activity and is disrupted by mutations that perturb PAK6 cell-cell adhesion targeting. Finally, we demonstrate that all type II PAKs (PAK4, PAK5 and PAK6) target to cell-cell adhesions, albeit to differing extents, but PAK1 (a type I PAK) does not. Notably, the ability of a PAK isoform to drive epithelial colony escape correlates with its targeting to cell-cell adhesions. We conclude that PAKs have a broader role in the regulation of cell-cell adhesions than previously appreciated.
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Affiliation(s)
- Elizabeth M Morse
- Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Xiaowen Sun
- Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Jordan R Olberding
- Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Byung Hak Ha
- Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Titus J Boggon
- Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA
| | - David A Calderwood
- Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA
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24
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Civiero L, Cirnaru MD, Beilina A, Rodella U, Russo I, Belluzzi E, Lobbestael E, Reyniers L, Hondhamuni G, Lewis PA, Van den Haute C, Baekelandt V, Bandopadhyay R, Bubacco L, Piccoli G, Cookson MR, Taymans JM, Greggio E. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain. J Neurochem 2015; 135:1242-56. [PMID: 26375402 PMCID: PMC4715492 DOI: 10.1111/jnc.13369] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2015] [Revised: 08/01/2015] [Accepted: 09/04/2015] [Indexed: 12/23/2022]
Abstract
Leucine‐rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21‐activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21‐activated kinases are serine‐threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post‐mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock‐out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2‐mediated pathophysiology. ![]()
We propose p21‐activated kinase 6 (PAK6) as a novel interactor of leucine‐rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2‐linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.
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Affiliation(s)
- Laura Civiero
- Department of Biology, University of Padova, Padova, Italy
| | | | - Alexandra Beilina
- Laboratory of Neurogenetics, National Institute on Aging/NIH, Bethesda, Maryland, USA
| | - Umberto Rodella
- Department of Biology, University of Padova, Padova, Italy.,Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Isabella Russo
- Department of Biology, University of Padova, Padova, Italy
| | - Elisa Belluzzi
- Department of Biology, University of Padova, Padova, Italy
| | - Evy Lobbestael
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Lauran Reyniers
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Geshanthi Hondhamuni
- Department of Molecular Neuroscience UCL, Reta Lila Weston Institute of Neurological Studies, Institute of Neurology, London, UK
| | - Patrick A Lewis
- School of Pharmacy, University of Reading, Reading, UK.,Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, UK
| | - Chris Van den Haute
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium.,Leuven Viral Vector Core, KU Leuven, Leuven, Belgium
| | - Veerle Baekelandt
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Rina Bandopadhyay
- Department of Molecular Neuroscience UCL, Reta Lila Weston Institute of Neurological Studies, Institute of Neurology, London, UK
| | - Luigi Bubacco
- Department of Biology, University of Padova, Padova, Italy
| | - Giovanni Piccoli
- San Raffaele Science Park and Università Vita-Salute San Raffaele, Milano, Italy
| | - Mark R Cookson
- Laboratory of Neurogenetics, National Institute on Aging/NIH, Bethesda, Maryland, USA
| | - Jean-Marc Taymans
- Laboratory for Neurobiology and Gene Therapy, KU Leuven, Leuven, Belgium
| | - Elisa Greggio
- Department of Biology, University of Padova, Padova, Italy
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25
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Liu W, Liu Y, Liu H, Zhang W, Fu Q, Xu J, Gu J. Tumor Suppressive Function of p21-activated Kinase 6 in Hepatocellular Carcinoma. J Biol Chem 2015; 290:28489-28501. [PMID: 26442588 DOI: 10.1074/jbc.m115.658237] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2015] [Indexed: 01/16/2023] Open
Abstract
Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict a favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with tumor node metastasis stage progression and unfavorable overall survival in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal deletion mutants of PAK6 experiments indicated the tumor suppressive function of PAK6 was partially dependent on its kinase activity and nuclear translocation. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components, including EZH2, SUZ12, and EED, elucidated epigenetic control of H3K27me3-arbitrated PAK6 down-regulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.
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Affiliation(s)
- Weisi Liu
- Departments of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032
| | - Yidong Liu
- Departments of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032
| | - Haiou Liu
- Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai 200011, China
| | - Weijuan Zhang
- Departments of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai 200032
| | - Qiang Fu
- Departments of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032
| | - Jiejie Xu
- Departments of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032.
| | - Jianxin Gu
- Departments of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai 200032
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26
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Liu C, Zhang L, Huang Y, Lu K, Tao T, Chen S, Zhang X, Guan H, Chen M, Xu B. MicroRNA‑328 directly targets p21‑activated protein kinase 6 inhibiting prostate cancer proliferation and enhancing docetaxel sensitivity. Mol Med Rep 2015; 12:7389-95. [PMID: 26459798 PMCID: PMC4626198 DOI: 10.3892/mmr.2015.4390] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2015] [Accepted: 09/01/2015] [Indexed: 01/19/2023] Open
Abstract
Prostate cancer (Pca) has one of the highest mortality rates for malignant cancers worldwide. Previous research has demonstrated that numerous genes are aberrantly expressed during Pca onset and development. p21-activated protein kinase 6 (PAK6) is known to be overexpressed in primary and metastatic Pca, however the mechanism of this aberrant expression remains unknown. In the present study, immunohistochemistry demonstrated that PAK6 is overexpressed in castration-resistant Pca (CRPC). Furthermore, PAK6 overexpression was regulated by microRNA (miR)-328. Luciferase reporter assay and western blot analysis indicated that PAK6 was directly targeted by miR-328. Forced expression of miR-328 enhanced docetaxel sensitivity, inhibited cell proliferation and promoted cell apoptosis without affecting the cell cycle. This indicates that miR-328 performs important functions in CRPC progression via PAK6 regulation. This mechanism may be used to enhance the effect of docetaxel.
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Affiliation(s)
- Chunhui Liu
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Lei Zhang
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Yeqing Huang
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Kai Lu
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Tao Tao
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Shuqiu Chen
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Xiaowen Zhang
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Han Guan
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Ming Chen
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
| | - Bin Xu
- Department of Urology, Affiliated Zhongda Hospital of Southeast University, Nanjing, Jiangsu 210009, P.R. China
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Abstract
INTRODUCTION Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression, and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed "undruggable" because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells. AREAS COVERED This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, that is, RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases, and posttranslational modifications at a molecular level. EXPERT OPINION To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small-molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents.
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Affiliation(s)
- Yuan Lin
- Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio 45229, USA
| | - Yi Zheng
- Division of Experimental Hematology and Cancer Biology, Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio 45229, USA
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Al-Azayzih A, Gao F, Somanath PR. P21 activated kinase-1 mediates transforming growth factor β1-induced prostate cancer cell epithelial to mesenchymal transition. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:1229-39. [PMID: 25746720 DOI: 10.1016/j.bbamcr.2015.02.023] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 02/25/2015] [Accepted: 02/28/2015] [Indexed: 12/11/2022]
Abstract
Transforming growth factor beta (TGFβ) is believed to play a dual role in prostate cancer. Molecular mechanism by which TGFβ1 suppresses early prostate tumor growth and induces epithelial-to-mesenchymal transition (EMT) in advanced stages is not known. We determined if P21-activated kinase1 (Pak1), which mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38-MAPK and caspase-3, 9. Long-term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N-cadherin through tumor necrosis factor receptor-associated factor-6 (TRAF6)-mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1-induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38-MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1-Pak1 pathway for advanced-stage prostate cancer.
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Affiliation(s)
- Ahmad Al-Azayzih
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States; College of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan
| | - Fei Gao
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States
| | - Payaningal R Somanath
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States; Department of Medicine, Vascular Biology Center and Cancer Center, Georgia Regents University, Augusta, GA, United States.
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29
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Felgueiras J, Fardilha M. Phosphoprotein phosphatase 1-interacting proteins as therapeutic targets in prostate cancer. World J Pharmacol 2014; 3:120-139. [DOI: 10.5497/wjp.v3.i4.120] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2014] [Revised: 09/01/2014] [Accepted: 09/24/2014] [Indexed: 02/06/2023] Open
Abstract
Prostate cancer is a major public health concern worldwide, being one of the most prevalent cancers in men. Great improvements have been made both in terms of early diagnosis and therapeutics. However, there is still an urgent need for reliable biomarkers that could overcome the lack of cancer-specificity of prostate-specific antigen, as well as alternative therapeutic targets for advanced metastatic cases. Reversible phosphorylation of proteins is a post-translational modification critical to the regulation of numerous cellular processes. Phosphoprotein phosphatase 1 (PPP1) is a major serine/threonine phosphatase, whose specificity is determined by its interacting proteins. These interactors can be PPP1 substrates, regulators, or even both. Deregulation of this protein-protein interaction network alters cell dynamics and underlies the development of several cancer hallmarks. Therefore, the identification of PPP1 interactome in specific cellular context is of crucial importance. The knowledge on PPP1 complexes in prostate cancer remains scarce, with only 4 holoenzymes characterized in human prostate cancer models. However, an increasing number of PPP1 interactors have been identified as expressed in human prostate tissue, including the tumor suppressors TP53 and RB1. Efforts should be made in order to identify the role of such proteins in prostate carcinogenesis, since only 26 have yet well-recognized roles. Here, we revise literature and human protein databases to provide an in-depth knowledge on the biological significance of PPP1 complexes in human prostate carcinogenesis and their potential use as therapeutic targets for the development of new therapies for prostate cancer.
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30
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Abstract
PAKs 4, 5 and 6 are members of the group B family of p21-activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions.
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Affiliation(s)
- Audrey Minden
- Susan Lehman Cullman Laboratory for Cancer Research; Department of Chemical Biology; Ernest Mario School of Pharmacy; Rutgers, The State University of New Jersey; Piscataway, NJ USA
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31
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Zhao ZS, Manser E. PAK family kinases: Physiological roles and regulation. CELLULAR LOGISTICS 2014; 2:59-68. [PMID: 23162738 PMCID: PMC3490964 DOI: 10.4161/cl.21912] [Citation(s) in RCA: 80] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1-6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4-6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies.
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Affiliation(s)
- Zhuo-Shen Zhao
- sGSK Group; Astar Neuroscience Research Partnership; Singapore
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32
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Tanneeru K, Balla AR, Guruprasad L. In silico3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase. J Biomol Struct Dyn 2014; 33:1710-9. [DOI: 10.1080/07391102.2014.968622] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
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Discovery and the structural basis of a novel p21-activated kinase 4 inhibitor. Cancer Lett 2014; 349:45-50. [PMID: 24704155 DOI: 10.1016/j.canlet.2014.03.024] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2013] [Revised: 03/19/2014] [Accepted: 03/23/2014] [Indexed: 12/15/2022]
Abstract
Functional versatility and elevated expression in cancers have endowed p21-activated kinase 4 (PAK4) as one of the first-in-class anti-cancer drug target. In this study, a novel PAK4 inhibitor, KY-04031 (N(2)-(2-(1H-indol-3-yl)ethyl)-N(4)-(1H-indazol-5-yl)-6-methoxy-1,3,5-triazine-2,4-diamine), was discovered using a high-throughput screening. Analysis of the complex crystal structure illustrated that both indole and indazole of KY-04031 are responsible for PAK4 hinge interaction. Moreover, the molecule's triazine core was found to mimic the ribose of the natural ATP substrate. The cell-based anti-cancer potency of KY-04031 was less effective than the pyrroloaminopyrazoles; however, the unique molecular feature of KY-04031 can be exploited in designing new PAK4 inhibitors.
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34
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Abstract
The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer.
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Affiliation(s)
- Chetan K Rane
- Susan Lehman Cullman Laboratory for Cancer Research; Department of Chemical Biology; Ernest Mario School of Pharmacy; Rutgers The State University of New Jersey; Piscataway, NJ USA
| | - Audrey Minden
- Susan Lehman Cullman Laboratory for Cancer Research; Department of Chemical Biology; Ernest Mario School of Pharmacy; Rutgers The State University of New Jersey; Piscataway, NJ USA
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35
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Abstract
p21-Activated kinases (PAKs) are positioned at the nexus of several oncogenic signalling pathways. Overexpression or mutational activation of PAK isoforms frequently occurs in various human tumours, and recent data suggest that excessive PAK activity drives many of the cellular processes that are the hallmarks of cancer. In this Review, we discuss the mechanisms of PAK activation in cancer, the key substrates that mediate the developmental and oncogenic effects of this family of kinases, and how small-molecule inhibitors of these enzymes might be best developed and deployed for the treatment of cancer.
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Affiliation(s)
- Maria Radu
- Cancer Biology Program; Fox Chase Cancer Center; Philadelphia, PA, USA
| | - Galina Semenova
- Cancer Biology Program; Fox Chase Cancer Center; Philadelphia, PA, USA
| | - Rachelle Kosoff
- Cancer Biology Program; Fox Chase Cancer Center; Philadelphia, PA, USA
- Cancer Biology program, University of Pennsylvania, Philadelphia, PA, USA
| | - Jonathan Chernoff
- Cancer Biology Program; Fox Chase Cancer Center; Philadelphia, PA, USA
- To whom correspondence should be addressed: Jonathan Chernoff, Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Ave, Philadelphia, PA 19111, USA, Tel.: (215) 728 5319; Fax: (215) 728 3616;
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36
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Role of p-21-activated kinases in cancer progression. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2014; 309:347-87. [PMID: 24529727 DOI: 10.1016/b978-0-12-800255-1.00007-7] [Citation(s) in RCA: 77] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The p-21-activated kinases (PAKs) are downstream effectors of Rho GTPases Rac and Cdc42. The PAK family consists of six members which are segregated into two subgroups (Group I and Group II) based on sequence homology. Group I PAKs (PAK1-3) are the most extensively studied but there is increasing interest in the functionality of Group II PAKs (PAK4-6). The PAK family proteins are thought to play an important role in many different cellular processes, some of which have particular significance in the context of cancer progression. This review explores established and more recent data, linking the PAK family kinases to cancer progression including expression profiles, evasion of apoptosis, promotion of cell survival, and regulation of cell invasion. Finally, we discuss attempts to therapeutically target the PAK family and outline the major obstacles that still need to be overcome.
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Fram S, King H, Sacks DB, Wells CM. A PAK6-IQGAP1 complex promotes disassembly of cell-cell adhesions. Cell Mol Life Sci 2013; 71:2759-73. [PMID: 24352566 PMCID: PMC4059965 DOI: 10.1007/s00018-013-1528-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2013] [Revised: 10/30/2013] [Accepted: 11/18/2013] [Indexed: 12/04/2022]
Abstract
p-21 activated 6 (PAK6), first identified as interacting with the androgen receptor (AR), is over-expressed in multiple cancer tissues and has been linked to the progression of prostate cancer, however little is known about PAK6 function in the absence of AR signaling. We report here that PAK6 is specifically required for carcinoma cell–cell dissociation downstream of hepatocyte growth factor (HGF) for both DU145 prostate cancer and HT29 colon cancer cells. Moreover, PAK6 overexpression can drive cells to escape from adhesive colonies in the absence of stimulation. We have localized PAK6 to cell–cell junctions and have detected a direct interaction between the kinase domain of PAK6 and the junctional protein IQGAP1. Co-expression of IQGAP1 and PAK6 increases cell colony escape and leads to elevated PAK6 activation. Further studies have identified a PAK6/E-cadherin/IQGAP1 complex downstream of HGF. Moreover, we find that β-catenin is also localized with PAK6 in cell–cell junctions and is a novel PAK6 substrate. We propose a unique role for PAK6, independent of AR signaling, where PAK6 drives junction disassembly during HGF-driven cell–cell dissociation via an IQGAP1/E-cadherin complex that leads to the phosphorylation of β-catenin and the disruption of cell–cell adhesions.
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Affiliation(s)
- Sally Fram
- Division of Cancer Studies, King's College London, New Hunts House, Guys Campus, London, SE1 1UL, UK
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Shoni M, Nagymanyoki Z, Vitonis AF, Jimenez C, Ng SW, Quade BJ, Berkowitz RS. p-21-Activated kinase-1, -4 and -6 and estrogen receptor expression pattern in normal placenta and gestational trophoblastic diseases. Gynecol Oncol 2013; 131:759-63. [DOI: 10.1016/j.ygyno.2013.09.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2013] [Revised: 09/11/2013] [Accepted: 09/11/2013] [Indexed: 11/29/2022]
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Furnari MA, Jobes ML, Nekrasova T, Minden A, Wagner GC. Differential sensitivity of Pak5, Pak6, and Pak5/Pak6 double-knockout mice to the stimulant effects of amphetamine and exercise-induced alterations in body weight. Nutr Neurosci 2013; 17:109-15. [PMID: 23710594 DOI: 10.1179/1476830513y.0000000072] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
OBJECTIVES PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. METHODS Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. RESULTS While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. DISCUSSION Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.
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Gao J, Ha BH, Lou HJ, Morse EM, Zhang R, Calderwood DA, Turk BE, Boggon TJ. Substrate and inhibitor specificity of the type II p21-activated kinase, PAK6. PLoS One 2013; 8:e77818. [PMID: 24204982 PMCID: PMC3810134 DOI: 10.1371/journal.pone.0077818] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 09/04/2013] [Indexed: 01/07/2023] Open
Abstract
The p21-activated kinases (PAKs) are important effectors of Rho-family small GTPases. The PAK family consists of two groups, type I and type II, which have different modes of regulation and signaling. PAK6, a type II PAK, influences behavior and locomotor function in mice and has an ascribed role in androgen receptor signaling. Here we show that PAK6 has a peptide substrate specificity very similar to the other type II PAKs, PAK4 and PAK5 (PAK7). We find that PAK6 catalytic activity is inhibited by a peptide corresponding to its N-terminal pseudosubstrate. Introduction of a melanoma-associated mutation, P52L, into this peptide reduces pseudosubstrate autoinhibition of PAK6, and increases phosphorylation of its substrate PACSIN1 (Syndapin I) in cells. Finally we determine two co-crystal structures of PAK6 catalytic domain in complex with ATP-competitive inhibitors. We determined the 1.4 Å co-crystal structure of PAK6 with the type II PAK inhibitor PF-3758309, and the 1.95 Å co-crystal structure of PAK6 with sunitinib. These findings provide new insights into the structure-function relationships of PAK6 and may facilitate development of PAK6 targeted therapies.
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Affiliation(s)
- Jia Gao
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-biosciences, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Byung Hak Ha
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Hua Jane Lou
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Elizabeth M. Morse
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Rong Zhang
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - David A. Calderwood
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Benjamin E. Turk
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, United States of America
| | - Titus J. Boggon
- Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
- Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, United States of America
- * E-mail:
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Rudov A, Rocchi MBL, Accorsi A, Spada G, Procopio AD, Olivieri F, Rippo MR, Albertini MC. Putative miRNAs for the diagnosis of dyslexia, dyspraxia, and specific language impairment. Epigenetics 2013; 8:1023-9. [PMID: 23949389 DOI: 10.4161/epi.26026] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Disorders of human communication abilities can be classified into speech and language disorders. Speech disorders (e.g., dyspraxia) affect the sound generation and sequencing, while language disorders (e.g., dyslexia and specific language impairment, or SLI) are deficits in the encoding and decoding of language according to its rules (reading, spelling, grammar). The diagnosis of such disorders is often complicated, especially when a patient presents more than one disorder at the same time. The present review focuses on these challenges. We have combined data available from the literature with an in silico approach in an attempt to identify putative miRNAs that may have a key role in dyspraxia, dyslexia and SLI. We suggest the use of new miRNAs, which could have an important impact on the three diseases. Further, we relate those miRNAs to the axon guidance pathway and discuss possible interactions and the role of likely deregulated proteins. In addition, we describe potential differences in expressional deregulation and its role in the improvement of diagnosis. We encourage experimental investigations to test the data obtained in silico.
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Affiliation(s)
- Alexander Rudov
- Department of Biomolecular Sciences; Urbino University ''Carlo Bo''; Urbino, Italy
| | | | - Augusto Accorsi
- Department of Biomolecular Sciences; Urbino University ''Carlo Bo''; Urbino, Italy
| | - Giorgio Spada
- Dipartimento di Scienze di Base e Fondamenti; Urbino University ''Carlo Bo''; Urbino, Italy
| | | | - Fabiola Olivieri
- Department of Clinical and Molecular Sciences; Università Politecnica delle Marche; Ancona, Italy
| | - Maria Rita Rippo
- Department of Clinical and Molecular Sciences; Università Politecnica delle Marche; Ancona, Italy
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Goc A, Al-Azayzih A, Abdalla M, Al-Husein B, Kavuri S, Lee J, Moses K, Somanath PR. P21 activated kinase-1 (Pak1) promotes prostate tumor growth and microinvasion via inhibition of transforming growth factor β expression and enhanced matrix metalloproteinase 9 secretion. J Biol Chem 2012; 288:3025-35. [PMID: 23258534 DOI: 10.1074/jbc.m112.424770] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
P21-activated kinases (Paks) are major effectors downstream of the small Rho family of GTPases. Among the six isoforms, Pak1 is the most ubiquitous and the best characterized member. Previous studies have shown that inhibition of Pak6, which is predominantly present in the prostate compared with other tissues, inhibits prostate tumor growth in vivo. Even though Pak1 has been identified in normal prostatic epithelial cells and cancer cells, its specific role in the development of prostate cancer remains unclear. We report here that highly invasive prostate cancer cells express significantly higher levels of Pak1 protein compared with non-invasive prostate cancer cells. Furthermore, prostate tumor tissues and prostate cancer metastasized to lungs showed a higher expression of Pak1 compared with normal tissues. Interestingly, Pak6 protein expression levels did not change with the invasive/metastatic potential of the cancer cells or tumors. Although inhibition of Pak1, and not Pak6, resulted in impaired PC3 cell migration, the effects of Pak1 knockdown on transendothelial migration (microinvasion), tumor growth, and tumor angiogenesis was higher compared with Pak6 knockdown. Finally, gene array data revealed reduced expression of matrix metalloproteinase 9 with the ablation of either Pak1 or Pak6 gene expression in PC3 cells, whereas protein levels of TGFβ was elevated significantly with specific modulation of Pak1 activity or ablation of the Pak1 gene. Our observations suggest that although some level of functional redundancy exists between Pak1 and Pak6 in prostate cancer cells, targeting Pak1 is a potential option for the management of prostate tumor growth, microinvasion, and metastasis.
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Affiliation(s)
- Anna Goc
- Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, Georgia
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Liu T, Li Y, Gu H, Zhu G, Li J, Cao L, Li F. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2). J Biol Chem 2012; 288:3359-69. [PMID: 23132866 DOI: 10.1074/jbc.m112.384289] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.
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Affiliation(s)
- Tong Liu
- Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, Ministry of Education, China Medical University, Shenyang 110001, China
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Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate. Proc Natl Acad Sci U S A 2012; 109:16107-12. [PMID: 22988085 DOI: 10.1073/pnas.1214447109] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The type II p21-activated kinases (PAKs) are key effectors of RHO-family GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-X(L) antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation.
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Sampson N, Ruiz C, Zenzmaier C, Bubendorf L, Berger P. PAGE4 positivity is associated with attenuated AR signaling and predicts patient survival in hormone-naive prostate cancer. THE AMERICAN JOURNAL OF PATHOLOGY 2012; 181:1443-54. [PMID: 22885105 DOI: 10.1016/j.ajpath.2012.06.040] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2012] [Revised: 05/30/2012] [Accepted: 06/26/2012] [Indexed: 11/27/2022]
Abstract
Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.
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Affiliation(s)
- Natalie Sampson
- Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria.
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Shepelev MV, Korobko IV. Pak6 protein kinase is a novel effector of an atypical Rho family GTPase Chp/RhoV. BIOCHEMISTRY (MOSCOW) 2012; 77:26-32. [PMID: 22339630 DOI: 10.1134/s0006297912010038] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Chp/RhoV is an atypical Rho GTPase whose functions are far from being fully understood. To date several effector proteins of Chp have been identified, including p21-activated kinases Pak1, Pak2, and Pak4. Using a yeast two-hybrid system and co-immunoprecipitation, here we show that another p21-activated kinase, Pak6, is a novel Chp-binding protein. Interaction between Chp and Pak6 depends on the activation state of the GTPase, suggesting that Pak6 is an effector protein for Chp. Point mutations in the effector domain of Chp or in the CRIB motif of Pak6 significantly impair the interaction between Chp and Pak6 upon co-immunoprecipitation, suggesting that the binding interface involves the effector domain of Chp and the CRIB motif in Pak6. We found that Chp does not affect the phosphorylation status of the S560 residue in the catalytic domain of Pak6 when Chp and Pak6 are co-expressed in HEK293 cells. Therefore, similarly to Cdc42, Chp is not likely to activate Pak6. In NCI-H1299 cells, Chp co-localizes with Pak6 on vesicular structures in activation state-dependent manner. Taking the data together, we report here the identification of p21-activated kinase Pak6 as a novel effector of the atypical Rho GTPase Chp. Our data suggest further directions in elucidating biological functions of these proteins.
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Affiliation(s)
- M V Shepelev
- Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia.
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Liu J, Youn H, Yang J, Du N, Liu J, Liu H, Li B. G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells. Prostate 2011; 71:1276-86. [PMID: 21308712 PMCID: PMC3143312 DOI: 10.1002/pros.21345] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2010] [Accepted: 12/21/2010] [Indexed: 11/10/2022]
Abstract
BACKGROUND The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. METHODS Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA-binding activity was determined using chromatin-immunoprecipitation (ChIP) assay. RESULTS First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β-cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gα(s) and Gα(12) genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gα(s) or Gα(12) active mutants enhanced androgen-induced AR transactivation, of which Gα(s) active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gα(s) but not Gα(12) subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, ChIP analysis revealed that both Gα(s) or Gα(12) subunits are involved in androgen-induced AR interaction with the AR target gene PSA promoter region. CONCLUSION These data suggest that caveolae-associated G-protein alpha subunits are involved in AR transactivation by modulating the activities of different PI3K isoforms.
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Affiliation(s)
- Jianjun Liu
- Department of Urology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China
| | - Hyewon Youn
- Institute of Radiation Medicine and Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul 110-799, Korea
- Departments of Urology, the University of Kansas Medical Center, Kansas City, Kansas 66160
| | - Jun Yang
- Departments of Urology, the University of Kansas Medical Center, Kansas City, Kansas 66160
- Department of Urology, Tongji Hospital, Huazhong University of Science & Technology, Wuhan, Hubei 430030, China
| | - Ningchao Du
- Department of Urology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China
| | - Jihong Liu
- Departments of Urology, the University of Kansas Medical Center, Kansas City, Kansas 66160
| | - Hongwei Liu
- Department of Urology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China
| | - Benyi Li
- Department of Urology, the Affiliated Hospital of Guangdong Medical College, Zhanjiang, Guangdong 524001, China
- Departments of Urology, the University of Kansas Medical Center, Kansas City, Kansas 66160
- Corresponding Author: Benyi Li, MD/PhD, KUMC Urology, 3901 Rainbow Blvd, Kansas City, KS 66160. Tel: 913-588-4773; Fax: 913-588-4756;
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Upregulation of p21-activated Kinase 6 in rat brain cortex after traumatic brain injury. J Mol Histol 2011; 42:195-203. [DOI: 10.1007/s10735-011-9324-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2011] [Accepted: 03/28/2011] [Indexed: 12/30/2022]
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Gene expression analysis reveals a different transcriptomic landscape in female and male breast cancer. Breast Cancer Res Treat 2010; 127:601-10. [PMID: 20625818 DOI: 10.1007/s10549-010-1015-8] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2010] [Accepted: 06/23/2010] [Indexed: 10/19/2022]
Abstract
Male breast cancer (MBC) is a poorly characterized disease because of its rarity. Clinical management is based on results obtained from randomized trials conducted in women notwithstanding data in the literature suggesting relevant gender-associated differences in terms of biological and clinical behavior. However, a genome-wide characterization of MBC on a transcriptional level is lacking. In this study, gene expression profiles of 37 estrogen receptor positive (ER+) MBC specimens were compared to that of 53 ER+ Female Breast Cancer (FBC) samples similar for clinical and patho-biological features. Almost 1000 genes were found differentially expressed (FDR < 1%) between female and male patients and biological interpretation highlighted a gender-associated modulation of key biological processes ranging from energy metabolism to regulation of translation and matrix remodeling as well as immune system recruitment. Moreover, an analysis of genes correlated to steroid receptors and ERBB2 suggested a prominent role for the androgen receptor in MBC with a minor relevance for progesterone receptor and ERBB2, although, similarly to FBC, a genomic amplification could be observed. Our findings support the idea that breast cancer is a quite different disease in male and female patients and the underlying gender-related biological differences are likely to have clinical implications connected with different susceptibility to treatment.
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