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Zhong C, Chen D, Gong D, Sheng X, Lin Y, Li R, Li Y. Transcriptomic response of overexpression ZNF32 in breast cancer cells. Sci Rep 2024; 14:28407. [PMID: 39557972 PMCID: PMC11574142 DOI: 10.1038/s41598-024-80125-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Accepted: 11/15/2024] [Indexed: 11/20/2024] Open
Abstract
Breast cancer is one of the deadliest malignancies in women worldwide. Zinc finger protein 32 (ZNF32) has been reported to be involved in autophagy and stem cell like properties of breast cancer cells. However, the effects, mechanisms, target genes and pathways of ZNF32 in breast cancer development have not been fully explored. In this study, stable ZNF32 overexpression breast cancer cell line was generated, and we used RNA-seq and RT-qPCR to quantify and verify the changes in transcription levels in breast cancer cells under ZNF32 overexpression. Transcriptome analysis showed that high expression of ZNF32 is accompanied by changes in downstream focal adhesion, ECM-receptor interaction, PI3K-AKT, HIPPO and TNF signaling pathways, which are critical for the occurrence and development of cancer. Multiple differentially expressed genes (DEGs) were significantly involved in cell proliferation, adhesion and migration, including 11 DEGs such as CA9, CRLF1 and ENPP2P with fundamental change of regulation modes. All the 11 DEGs were validated by RT-qPCR, and 9 of them contained potential transcriptional binding sequences of ZNF32 in their promoter region. This study provides a holistic perspective on the role and molecular mechanism of ZNF32 in breast cancer progression.
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Affiliation(s)
- Chaosong Zhong
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Education Ministry, Southwest Minzu University, Chengdu, China
- College of Animal and Veterinary Sciences, Southwest Minzu University, No. 16, South Section 4, First Ring Road, Chengdu, 610041, Sichuan, China
| | - Dingshuang Chen
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Education Ministry, Southwest Minzu University, Chengdu, China
- College of Animal and Veterinary Sciences, Southwest Minzu University, No. 16, South Section 4, First Ring Road, Chengdu, 610041, Sichuan, China
| | - Di Gong
- School of Basic Medical Science, Chengdu University, Chengdu, China
| | - Xueqing Sheng
- College of Animal and Veterinary Sciences, Southwest Minzu University, No. 16, South Section 4, First Ring Road, Chengdu, 610041, Sichuan, China
| | - Yaqiu Lin
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Education Ministry, Southwest Minzu University, Chengdu, China
- College of Animal and Veterinary Sciences, Southwest Minzu University, No. 16, South Section 4, First Ring Road, Chengdu, 610041, Sichuan, China
| | - Ruiwen Li
- Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China
| | - Yanyan Li
- Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Education Ministry, Southwest Minzu University, Chengdu, China.
- College of Animal and Veterinary Sciences, Southwest Minzu University, No. 16, South Section 4, First Ring Road, Chengdu, 610041, Sichuan, China.
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Bhowmick C, Rahaman M, Bhattacharya S, Mukherjee M, Chakravorty N, Dutta PK, Mahadevappa M. Identification of hub genes to determine drug-disease correlation in breast carcinomas. Med Oncol 2023; 41:36. [PMID: 38153604 DOI: 10.1007/s12032-023-02246-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2023] [Accepted: 11/11/2023] [Indexed: 12/29/2023]
Abstract
The exact molecular mechanism underlying the heterogeneous drug response against breast carcinoma remains to be fully understood. It is urgently required to identify key genes that are intricately associated with varied clinical response of standard anti-cancer drugs, clinically used to treat breast cancer patients. In the present study, the utility of transcriptomic data of breast cancer patients in discerning the clinical drug response using machine learning-based approaches were evaluated. Here, a computational framework has been developed which can be used to identify key genes that can be linked with clinical drug response and progression of cancer, offering an immense opportunity to predict potential prognostic biomarkers and therapeutic targets. The framework concerned utilizes DeSeq2, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cytoscape, and machine learning techniques to find these crucial genes. Total RNA extraction and qRT-PCR were performed to quantify relative expression of few hub genes selected from the networks. In our study, we have experimentally checked the expression of few key hub genes like APOA2, DLX5, APOC3, CAMK2B, and PAK6 that were predicted to play an immense role in breast cancer tumorigenesis and progression in response to anti-cancer drug Paclitaxel. However, further experimental validations will be required to get mechanistic insights of these genes in regulating the drug response and cancer progression which will likely to play pivotal role in cancer treatment and precision oncology.
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Affiliation(s)
- Chiranjib Bhowmick
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Motiur Rahaman
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Shatarupa Bhattacharya
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Mandrita Mukherjee
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Nishant Chakravorty
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Pranab Kumar Dutta
- Department of Electrical Engineering, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India
| | - Manjunatha Mahadevappa
- School of Medical Science and Technology, Indian Institute of Technology Kharagpur, West Medinipur, Kharagpur, West Bengal, 721302, India.
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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: 9] [Impact Index Per Article: 4.5] [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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Mitochondrial Alterations in Prostate Cancer: Roles in Pathobiology and Racial Disparities. Int J Mol Sci 2023; 24:ijms24054482. [PMID: 36901912 PMCID: PMC10003184 DOI: 10.3390/ijms24054482] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/15/2023] [Accepted: 02/19/2023] [Indexed: 03/12/2023] Open
Abstract
Prostate cancer (PCa) affects millions of men worldwide and is a major cause of cancer-related mortality. Race-associated PCa health disparities are also common and are of both social and clinical concern. Most PCa is diagnosed early due to PSA-based screening, but it fails to discern between indolent and aggressive PCa. Androgen or androgen receptor-targeted therapies are standard care of treatment for locally advanced and metastatic disease, but therapy resistance is common. Mitochondria, the powerhouse of cells, are unique subcellular organelles that have their own genome. A large majority of mitochondrial proteins are, however, nuclear-encoded and imported after cytoplasmic translation. Mitochondrial alterations are common in cancer, including PCa, leading to their altered functions. Aberrant mitochondrial function affects nuclear gene expression in retrograde signaling and promotes tumor-supportive stromal remodeling. In this article, we discuss mitochondrial alterations that have been reported in PCa and review the literature related to their roles in PCa pathobiology, therapy resistance, and racial disparities. We also discuss the translational potential of mitochondrial alterations as prognostic biomarkers and as effective targets for PCa therapy.
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CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain. Int J Mol Sci 2022; 23:ijms23168842. [PMID: 36012107 PMCID: PMC9408373 DOI: 10.3390/ijms23168842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 08/05/2022] [Accepted: 08/06/2022] [Indexed: 11/17/2022] Open
Abstract
The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.
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Felgueiras J, Lobo J, Camilo V, Carneiro I, Matos B, Henrique R, Jerónimo C, Fardilha M. PP1 catalytic isoforms are differentially expressed and regulated in human prostate cancer. Exp Cell Res 2022; 418:113282. [PMID: 35841980 DOI: 10.1016/j.yexcr.2022.113282] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2021] [Revised: 06/21/2022] [Accepted: 07/10/2022] [Indexed: 11/29/2022]
Abstract
The Ser/Thr-protein phosphatase PP1 (PP1) is a positive regulator of the androgen receptor (AR), which suggests major roles for PP1 in prostate carcinogenesis. However, studies dedicated to the characterization of PP1 in PCa are currently scarce. Here we analyzed the expression and localization of the PP1 catalytic (PP1c) isoforms in formalin-fixed, paraffin-embedded prostate tissue samples, as well as in PCa cell lines. We also analyzed well-characterized PCa cohorts to determine their transcript levels, identify genetic alterations, and assess promoter methylation of PP1c-coding genes. We found that PP-1A was upregulated and relocalized towards the nucleus in PCa and that PPP1CA was frequently amplified in PCa, particularly in advanced stages. PP-1B was downregulated in PCa but upregulated in a subset of tumors with AR amplification. PP-1G transcript levels were found to be associated with Gleason score. PP1c-coding genes were rarely mutated in PCa and were not prone to regulation by promoter methylation. Protein phosphorylation, on the other hand, might be an important regulatory mechanism of PP1c isoforms' activity. Altogether, our results suggest differential expression, localization, and regulation of PP1c isoforms in PCa and support the need for investigating isoform-specific roles in prostate carcinogenesis in future studies.
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Affiliation(s)
- Juliana Felgueiras
- Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro, Aveiro, Portugal; Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal
| | - João Lobo
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal; Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
| | - Vânia Camilo
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal
| | - Isa Carneiro
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal; Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal
| | - Bárbara Matos
- Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro, Aveiro, Portugal; Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal
| | - Rui Henrique
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal; Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal & Porto Comprehensive Cancer Center (P.CCC), Portugal; Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
| | - Margarida Fardilha
- Laboratory of Signal Transduction, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro, Aveiro, Portugal.
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Xue ST, Zheng B, Cao SQ, Ding JC, Hu GS, Liu W, Chen C. Long non-coding RNA LINC00680 functions as a ceRNA to promote esophageal squamous cell carcinoma progression through the miR-423-5p/PAK6 axis. Mol Cancer 2022; 21:69. [PMID: 35255921 PMCID: PMC8900330 DOI: 10.1186/s12943-022-01539-3] [Citation(s) in RCA: 82] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Accepted: 02/12/2022] [Indexed: 12/13/2022] Open
Abstract
Background Esophageal squamous cell carcinoma (ESCC) is a common invasive malignancy worldwide with poor clinical outcomes. Increasing amount of long non-coding RNAs (lncRNAs) have been reported to be involved in cancer development. However, lncRNAs that are functional in ESCC and the underlying molecular mechanisms remain largely unknown. Methods Transcriptomic analysis was performed to identify dysregulated lncRNAs in ESCC tissue samples. The high expression of LINC00680 in ESCC was validated by RT-qPCR, and the oncogenic functions of LINC00680 was investigated by cell proliferation, colony formation, migration and invasion assays in ESCC cells in vitro and xenografts derived from ESCC cells in mice. RNA-seq, competitive endogenous RNA (ceRNA) network analysis, and luciferase reporter assays were carried out to identify LINC00680 target genes and the microRNAs (miRNAs) bound to LINC00680. Antisense oligonucleotides (ASOs) were used for in vivo treatment. Results Transcriptome profiling revealed that a large number of lncRNAs was dysregulated in ESCC tissues. Notably, LINC00680 was highly expressed, and upregulation of LINC00680 was associated with large tumor size, advanced tumor stage, and poor prognosis. Functionally, knockdown of LINC00680 restrained ESCC cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, LINC00680 was found to act as a ceRNA by sponging miR-423-5p to regulate PAK6 (p21-activated kinase 6) expression in ESCC cells. The cell viability and motility inhibition induced by LINC00680 knockdown was significantly reversed upon PAK6 restoration and miR-423-5p inhibition. Furthermore, ASO targeting LINC00680 substantially suppressed ESCC both in vitro and in vivo. Conclusions An oncogenic lncRNA, LINC00680, was identified in ESCC, which functions as a ceRNA by sponging miR-423-5p to promote PAK6 expression and ESCC. LINC00680/miR-423-5p/PAK6 axis may serve as promising diagnostic and prognostic biomarkers and therapeutic targets for ESCC. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-022-01539-3.
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Mosaddeghzadeh N, Nouri K, Krumbach OHF, Amin E, Dvorsky R, Ahmadian MR. Selectivity Determinants of RHO GTPase Binding to IQGAPs. Int J Mol Sci 2021; 22:12596. [PMID: 34830479 PMCID: PMC8625570 DOI: 10.3390/ijms222212596] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Revised: 11/16/2021] [Accepted: 11/19/2021] [Indexed: 12/13/2022] Open
Abstract
IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.
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Affiliation(s)
- Niloufar Mosaddeghzadeh
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
| | - Kazem Nouri
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
- Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Oliver H. F. Krumbach
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
| | - Ehsan Amin
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
- Medical Faculty, Institute of Neural and Sensory Physiology, University Hospital Düsseldorf, Heinrich Heine University, 40225 Düsseldorf, Germany
| | - Radovan Dvorsky
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
| | - Mohammad R. Ahmadian
- Medical Faculty, Institute of Biochemistry and Molecular Biology II, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany; (N.M.); (K.N.); (O.H.F.K.); (E.A.); (R.D.)
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The miR-185/PAK6 axis predicts therapy response and regulates survival of drug-resistant leukemic stem cells in CML. Blood 2021; 136:596-609. [PMID: 32270193 DOI: 10.1182/blood.2019003636] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Accepted: 03/09/2020] [Indexed: 12/15/2022] Open
Abstract
Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of microRNAs (miRNAs) in regulating drug resistance and leukemic stem cell (LSC) fate, we performed global transcriptome profiling in treatment-naive chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor: its restored expression impaired survival of drug-resistant cells, sensitized them to TKIs in vitro, and markedly eliminated long-term repopulating LSCs and infiltrating blast cells, conferring a survival advantage in preclinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185, and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity, sensitizing therapy-resistant cells to TKIs. Thus, miR-185 presents as a potential predictive biomarker, and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL1 may provide a valuable strategy for overcoming drug resistance in patients.
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Gu Y, Lin X, Kapoor A, Li T, Major P, Tang D. Effective Prediction of Prostate Cancer Recurrence through the IQGAP1 Network. Cancers (Basel) 2021; 13:430. [PMID: 33498739 PMCID: PMC7865788 DOI: 10.3390/cancers13030430] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 01/11/2021] [Accepted: 01/20/2021] [Indexed: 02/07/2023] Open
Abstract
IQGAP1 expression was analyzed in: (1) primary prostate cancer, (2) xenografts produced from LNCaP, DU145, and PC3 cells, 3) tumor of PTEN-/- and TRAMP mice, and (3) castration resistant PC (CRPC) produced by LNCaP xenografts and PTEN-/- mice. IQGAP1 downregulations occurred in CRPC and advanced PCs. The downregulations were associated with rapid PC recurrence in the TCGA PanCancer (n = 492, p = 0.01) and MSKCC (n = 140, p = 4 × 10-6) cohorts. Differentially expressed genes (n = 598) relative to IQGAP1 downregulation were identified with enrichment in chemotaxis, cytokine signaling, and others along with reductions in immune responses. A novel 27-gene signature (Sig27gene) was constructed from these DEGs through random division of the TCGA cohort into a Training and Testing population. The panel was validated using an independent MSKCC cohort. Sig27gene robustly predicts PC recurrence at (hazard ratio) HR 2.72 and p < 2 × 10-16 in two independent PC cohorts. The prediction remains significant after adjusting for multiple clinical features. The novel and robust nature of Sig27gene underlie its great translational potential as a prognostic biomarker to predict PC relapse risk in patients with primary PC.
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Affiliation(s)
- Yan Gu
- Department of Medicine, McMaster University, Hamilton, ON L8S 4L8, Canada; (Y.G.); (X.L.)
- Urological Cancer Center for Research and Innovation (UCCRI), St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada;
- The Research Institute of St Joe’s Hamilton, St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada
| | - Xiaozeng Lin
- Department of Medicine, McMaster University, Hamilton, ON L8S 4L8, Canada; (Y.G.); (X.L.)
- Urological Cancer Center for Research and Innovation (UCCRI), St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada;
- The Research Institute of St Joe’s Hamilton, St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada
| | - Anil Kapoor
- Urological Cancer Center for Research and Innovation (UCCRI), St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada;
- The Research Institute of St Joe’s Hamilton, St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada
- Department of Surgery, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Taosha Li
- Life-Tech Industry Alliance, Shenzhen 518000, China;
| | - Pierre Major
- Department of Oncology, McMaster University, Hamilton, ON L8S 4L8, Canada;
| | - Damu Tang
- Department of Medicine, McMaster University, Hamilton, ON L8S 4L8, Canada; (Y.G.); (X.L.)
- Urological Cancer Center for Research and Innovation (UCCRI), St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada;
- The Research Institute of St Joe’s Hamilton, St Joseph’s Hospital, Hamilton, ON L8N 4A6, Canada
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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: 14] [Impact Index Per Article: 2.8] [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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12
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Protein phosphatase 1 in tumorigenesis: is it worth a closer look? Biochim Biophys Acta Rev Cancer 2020; 1874:188433. [PMID: 32956763 DOI: 10.1016/j.bbcan.2020.188433] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2020] [Revised: 08/26/2020] [Accepted: 09/12/2020] [Indexed: 02/06/2023]
Abstract
Cancer cells take advantage of signaling cascades to meet their requirements for sustained growth and survival. Cell signaling is tightly controlled by reversible protein phosphorylation mechanisms, which require the counterbalanced action of protein kinases and protein phosphatases. Imbalances on this system are associated with cancer development and progression. Protein phosphatase 1 (PP1) is one of the most relevant protein phosphatases in eukaryotic cells. Despite the widely recognized involvement of PP1 in key biological processes, both in health and disease, its relevance in cancer has been largely neglected. Here, we provide compelling evidence that support major roles for PP1 in tumorigenesis.
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13
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Verma A, Artham S, Alwhaibi A, Adil MS, Cummings BS, Somanath PR. PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling. Biochem Pharmacol 2020; 177:113943. [PMID: 32240651 DOI: 10.1016/j.bcp.2020.113943] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Accepted: 03/26/2020] [Indexed: 12/12/2022]
Abstract
Metastatic prostate cancer (PCa) has high mortality and a poor 5-year survival rate primarily due to the lack of effective treatments. Bone is the primary site of PCa metastasis in humans and the development of reliable therapeutic options for bone metastatic PCa will make a huge impact in reducing the mortality among these patients. Although P21 activated kinases (PAKs) have been studied in the past for their role in cancer, the efficacy of targeting PAKs to treat lung and bone metastatic PCa has not been tested yet. In the current study, we report that targeting PAK1 using IPA-3, an allosteric inhibitor of PAK1 kinase activity, significantly inhibits the murine metastatic PCa (RM1) cell proliferation and motility in vitro, and metastasis to the lungs in vivo. More importantly, we demonstrate for the first time that treatment with IPA-3 can blunt metastatic PCa-induced bone remodeling in vivo as analyzed by the 3-dimensional microcomputer tomography analysis. Our study has identified IPA-3 as a potential drug to treat bone metastatic PCa.
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Affiliation(s)
- Arti Verma
- Clinical and Experimental Therapeutics, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States
| | - Sandeep Artham
- Clinical and Experimental Therapeutics, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States
| | - Abdulrahman Alwhaibi
- Clinical and Experimental Therapeutics, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States
| | - Mir S Adil
- Clinical and Experimental Therapeutics, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States
| | - Brian S Cummings
- Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia Athens, GA, United States; Interdisciplinary Toxicology Program, University of Georgia, United States
| | - Payaningal R Somanath
- Clinical and Experimental Therapeutics, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA, United States; Department of Medicine and Vascular Biology Center, Augusta University, Augusta, GA, United States.
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14
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Li T, Li Y, Liu T, Hu B, Li J, Liu C, Liu T, Li F. Mitochondrial PAK6 inhibits prostate cancer cell apoptosis via the PAK6-SIRT4-ANT2 complex. Theranostics 2020; 10:2571-2586. [PMID: 32194820 PMCID: PMC7052886 DOI: 10.7150/thno.42874] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Accepted: 01/11/2020] [Indexed: 12/18/2022] Open
Abstract
Rationale: P21-activated kinase 6 (PAK6) is a member of the class II PAKs family, which is a conserved family of serine/threonine kinases. Although the effects of PAK6 on many malignancies, especially in prostate cancer, have been studied for a long time, the role of PAK6 in mitochondria remains unknown. Methods: The expression of PAK6, SIRT4 and ANT2 in prostate cancer and adjacent non-tumor tissues was detected by immunohistochemistry. Immunofuorescence and immunoelectron microscopy were used to determine the subcellular localization of PAK6. Immunoprecipitation, immunofuorescence and ubiquitination assays were performed to determine how PAK6 regulates SIRT4, how SIRT4 regulates ANT2, and how PAK6 regulates ANT2. Flow cytometry detection and xenograft models were used to evaluate the impact of ANT2 mutant expression on the prostate cancer cell cycle and apoptosis regulation. Results: The present study revealed that the PAK6-SIRT4-ANT2 complex is involved in mitochondrial apoptosis in prostate cancer cells. It was found that PAK6 is mainly located in the mitochondrial inner membrane, in which PAK6 promotes SIRT4 ubiquitin-mediated proteolysis. Furthermore, SIRT4 deprives the ANT2 acetylation at K105 to promote its ubiquitination degradation. Hence, PAK6 adjusts the acetylation level of ANT2 through the PAK6-SIRT4-ANT2 pathway, in order to regulate the stability of ANT2. Meanwhile, PAK6 directly phosphorylates ANT2 atT107 to inhibit the apoptosis of prostate cancer cells. Therefore, the phosphorylation and deacetylation modifications of ANT2 are mutually regulated, leading to tumor growth in vivo. Consistently, these clinical prostate cancer tissue evaluations reveal that PAK6 is positively correlated with ANT2 expression, but negatively correlated with SIRT4. Conclusion: These present findings suggest the pivotal role of the PAK6-SIRT4-ANT2 complex in the apoptosis of prostate cancer. This complex could be a potential biomarker for the treatment and prognosis of prostate cancer.
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Affiliation(s)
- Tingting Li
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
| | - Yang Li
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
| | - Tong Liu
- Medical Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
| | - Bingtao Hu
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
| | - Jiabin Li
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
| | - Chen Liu
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
| | - Tao Liu
- Department of Urology, the First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, China
| | - Feng Li
- Department of Cell Biology, Key Laboratory of Cell Biology, National Health Commission of the PRC, and Key Laboratory of Medical Cell Biology, Ministry of Education of the PRC, Shenyang 110122, Liaoning, China
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15
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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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16
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Najahi‐Missaoui W, Quach ND, Jenkins A, Dabke I, Somanath PR, Cummings BS. Effect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasion. Pharmacol Res Perspect 2019; 7:e00518. [PMID: 31516713 PMCID: PMC6728842 DOI: 10.1002/prp2.518] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Accepted: 07/26/2019] [Indexed: 02/06/2023] Open
Abstract
P21-activated kinase-1 (PAK-1) is a serine/threonine kinase involved in multiple signaling pathways that mediate cellular functions such as cytoskeletal motility, cell proliferation, and survival. PAK-1 expression is altered in various cancers, including prostate and breast. Our recent studies showed that prostate cancer cells expressing higher levels of PAK-1 were resistant to the cytotoxic effects of the PAK-1 inhibitor, inhibitor targeting PAK-1 activation-3 (IPA-3), compared to those with lower expression. This study expanded these findings to other cancers (breast and melanoma) by testing the hypothesis that genetic and pharmacological inhibition of PAK-1 alters cell growth, migration, and invasion in prostate, breast, and skin cancer cell lines. We also tested the specificity of IPA-3 for PAK-1 and the hypothesis that gene silencing of PAK-1 altered the efficacy of sterically stabilized liposomes (SSL) containing IPA-3 (SSL-IPA-3). PAK-1 expression was identified in four different breast cancer cell lines, and in a melanoma cell line. The expression of PAK-1 correlated to the IC50 of IPA-3 as measured by MTT staining. PAK-1 inhibition using shRNA correlated with decreased cell migration and invasion in prostate cancer DU-145 and breast cancer MCF-7 cells. Decreased migration and invasion also correlated to decreased expression of E-cadherin and alterations in C-X-C Chemokine Receptor type 4 and Homing Cell Adhesion Molecule expression. PAK-1 inhibition increased the cytotoxicity of IPA-3, and the cytotoxicity of SSL-IPA-3 to levels comparable to that of free drug. These data demonstrate that both pharmacological and molecular inhibition of PAK-1 decreased growth in prostate, breast, and melanoma cancer cell lines, and increased the toxicity of IPA-3 and its liposomal formulation. These data also show the specificity of IPA-3 for PAK-1, are some of the first data suggesting that IPA-3 is a therapeutic treatment for breast cancer and melanoma, and demonstrate the efficacy of liposome-encapsulated IPA-3 in breast cancer cells.
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Affiliation(s)
- Wided Najahi‐Missaoui
- Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensGAUSA
| | - Nhat D. Quach
- Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensGAUSA
- Present address:
Department of Molecular Pharmacology, Physiology, & BiotechnologyBrown UniversityProvidenceRIUSA
| | - Amber Jenkins
- Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensGAUSA
- Present address:
Cancer Center of Middle GeorgiaDublinGAUSA
| | - Isha Dabke
- Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensGAUSA
- Present address:
Medical College of GeorgiaAugustaGAUSA
| | - Payaningal R. Somanath
- Clinical and Experimental Therapeutics, College of PharmacyUniversity of Georgia and Charlie Norwood VA Medical CenterAugustaGAUSA
- Department of Medicine, Vascular Biology Center and Cancer CenterGeorgia Regents UniversityAugustaGAUSA
| | - Brian S. Cummings
- Department of Pharmaceutical and Biomedical Sciences, College of PharmacyUniversity of GeorgiaAthensGAUSA
- Interdisciplinary Toxicology ProgramUniversity of GeorgiaAthensGAUSA
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17
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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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18
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Zhu L, Shu Z, Sun X. Bioinformatic analysis of four miRNAs relevant to metastasis-regulated processes in endometrial carcinoma. Cancer Manag Res 2018; 10:2337-2346. [PMID: 30122983 PMCID: PMC6078085 DOI: 10.2147/cmar.s168594] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
BACKGROUND The purpose of this study was to investigate the expression of different miRNAs in nonmetastatic and metastatic endometrial cancer Existing evidence indicates that there are many factors affecting the metastasis of endometrial cancer, and miRNAs play an unique role in many processes of endometiral cancer. MATERIALS AND METHODS miRNA sequences were downloaded from The Cancer Genome Atlas Project database, and Bioinformatics technique was used to deal with those data. RESULTS We elucidated the relation between differentially expressed miRNAs and clinical information for a total of 260 tumor tissues and 22 tumor tissues that had metastasized. We used the threshold of P <0.05| log 2 FC | >1.2 to identify potential miRNAs. Four differentially expressed miRNAs were identified in nonmetastatic and metastatic endometrial cancers. Further differential analysis of metastatic tissue revealed that miR-1247 is associated with metastasis of endometrial cancer to the lung, and miR-3200 is associated with the clinical stage of endometrial cancer. A functional enrichment analysis showed that the four miRNAs may be involved in multiple pathways of cancer, including the Wnt, NOTCH, and TGF-β signaling pathways and signaling pathways regulating pluripotency of stem cells. Protein-protein interaction analysis showed that PAK6, SNAP25, MAN1A1, MYB, ZBTB4, UST, ALDH1A3, and NRP2 are hub genes of relevant miRNAs in endometrial cancers. CONCLUSION The current study indicates that these four miRNAs may be related to molecular markers of metastasis of endometrial cancer.
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Affiliation(s)
- Lingping Zhu
- Department of General Practice, Shenzhen Longhua District Central Hospital, Shenzhen, People's Republic of China
- Department of General Practice, Zhongshan Hospital, Fudan University, Shanghai, People's Republic of China,
| | - Zhiqun Shu
- Pudong Institute for Health Development, Shanghai, People's Republic of China,
| | - Xiaoming Sun
- Department of General Practice, Zhongshan Hospital, Fudan University, Shanghai, People's Republic of China,
- Pudong Institute for Health Development, Shanghai, People's Republic of China,
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19
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LaPak KM, Vroom DC, Garg AA, Guan X, Hays JL, Song JW, Burd CE. Melanoma-associated mutants within the serine-rich domain of PAK5 direct kinase activity to mitogenic pathways. Oncotarget 2018; 9:25386-25401. [PMID: 29875996 PMCID: PMC5986637 DOI: 10.18632/oncotarget.25356] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Accepted: 04/26/2018] [Indexed: 02/07/2023] Open
Abstract
The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.
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Affiliation(s)
- Kyle M LaPak
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
| | - Dennis C Vroom
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
| | - Ayush A Garg
- Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA
| | - Xiangnan Guan
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA
| | - John L Hays
- Division of Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
| | - Jonathan W Song
- Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA
| | - Christin E Burd
- Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA.,Department of Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA
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20
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21
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Raja R, Sahasrabuddhe NA, Radhakrishnan A, Syed N, Solanki HS, Puttamallesh VN, Balaji SA, Nanjappa V, Datta KK, Babu N, Renuse S, Patil AH, Izumchenko E, Prasad TSK, Chang X, Rangarajan A, Sidransky D, Pandey A, Gowda H, Chatterjee A. Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells. Oncotarget 2018; 7:61229-61245. [PMID: 27542207 PMCID: PMC5308647 DOI: 10.18632/oncotarget.11310] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Accepted: 08/08/2016] [Indexed: 12/21/2022] Open
Abstract
Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.
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Affiliation(s)
- Remya Raja
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India
| | | | - Aneesha Radhakrishnan
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, 605014, India
| | - Nazia Syed
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, 605014, India
| | - Hitendra S Solanki
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India
| | - Vinuth N Puttamallesh
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India
| | - Sai A Balaji
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India
| | - Vishalakshi Nanjappa
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India
| | - Keshava K Datta
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India
| | - Niraj Babu
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India
| | - Santosh Renuse
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India
| | - Arun H Patil
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,School of Biotechnology, KIIT University, Bhubaneswar, Odisha, 751024, India
| | - Evgeny Izumchenko
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21231, USA
| | - T S Keshava Prasad
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,Amrita School of Biotechnology, Amrita University, Kollam, 690 525, India.,YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, 575018, India.,NIMHANS-IOB Proteomics and Bioinformatics Laboratory, Neurobiology Research Centre, National Institute of Mental Health and Neurosciences, Bangalore, 560029, India
| | - Xiaofei Chang
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21231, USA
| | - Annapoorni Rangarajan
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India
| | - David Sidransky
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21231, USA
| | - Akhilesh Pandey
- McKusick-Nathans Institute of Genetic Medicine, Baltimore, Maryland, 21205, USA.,Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.,Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.,Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA
| | - Harsha Gowda
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, 575018, India
| | - Aditi Chatterjee
- Institute of Bioinformatics, International Tech Park, Bangalore, 560 066, India.,YU-IOB Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, 575018, India
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22
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Jiang Y, Liu W, Li T, Hu Y, Chen S, Xi S, Wen Y, Huang L, Zhao L, Xiao C, Huang X, Han Z, Liu H, Qi X, Yang Y, Yu J, Cai S, Li G. Prognostic and Predictive Value of p21-activated Kinase 6 Associated Support Vector Machine Classifier in Gastric Cancer Treated by 5-fluorouracil/Oxaliplatin Chemotherapy. EBioMedicine 2017; 22:78-88. [PMID: 28687498 PMCID: PMC5552213 DOI: 10.1016/j.ebiom.2017.06.028] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2017] [Revised: 06/26/2017] [Accepted: 06/30/2017] [Indexed: 12/23/2022] Open
Abstract
To determine whether p21-activated Kinase (PAK) 6 is a prognostic and predictive marker in gastric cancer (GC) and to construct a classifier that can identify a subset of patients who are highly sensitive to 5-fluorouracil/oxaliplatin chemotherapy. We retrospectively analyzed the expression levels of PAK6, cyclooxygenase 2, p21WAF1, Ki-67, excision repair cross-complementing gene 1, and thymidylate synthase in 242 paraffin-embedded GC specimens of the training cohort by immunohistochemistry. Then, we used support vector machine (SVM)-based methods to develop a predictive classifier for chemotherapy (chemotherapy score - CS-SVM classifier). Further validation was performed in an independent cohort of 279 patients. High PAK6 expression was associated with poor prognosis and increased chemoresistance to 5-FU/oxaliplatin chemotherapy. The CS-SVM classifier distinguished patients with stage II and III GC into low- and high-CS-SVM groups, with significant differences in the 5-year disease-free survival (DFS) and overall survival (OS) in chemotherapy patients. Moreover, chemotherapy significantly prolonged the DFS and OS of the high CS-SVM patients in the training and validation cohorts. In conclusion, PAK6 was an independent prognostic factor and increased chemoresistance. The CS-SVM classifier distinguished a subgroup of stage II and III patients who would highly benefit from chemotherapy, thus facilitating patient counseling and individualizing the management.
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Affiliation(s)
- Yuming Jiang
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China; Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
| | - Wei Liu
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Hepatic Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Tuanjie Li
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Yanfeng Hu
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Sile Chen
- Department of Gastrointestinal Surgery of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, Guangdong, China
| | - Sujuan Xi
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Infectious disease, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Yajia Wen
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Infectious disease, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Lei Huang
- German Cancer Research Center (DKFZ), Heidelberg 69120, Germany
| | - Liying Zhao
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Cuicui Xiao
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Hepatic Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Xiaohui Huang
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
| | - Zhen Han
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Hao Liu
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Xiaolong Qi
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China
| | - Yang Yang
- Guangdong Key Laboratory of Liver Disease Research, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China; Department of Hepatic Surgery, The 3rd Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Jiang Yu
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China.
| | - Shirong Cai
- Department of Gastrointestinal Surgery of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, Guangdong, China.
| | - Guoxin Li
- Department of General Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, China.
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Kumar R, Sanawar R, Li X, Li F. Structure, biochemistry, and biology of PAK kinases. Gene 2016; 605:20-31. [PMID: 28007610 DOI: 10.1016/j.gene.2016.12.014] [Citation(s) in RCA: 169] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Revised: 11/24/2016] [Accepted: 12/14/2016] [Indexed: 02/07/2023]
Abstract
PAKs, p21-activated kinases, play central roles and act as converging junctions for discrete signals elicited on the cell surface and for a number of intracellular signaling cascades. PAKs phosphorylate a vast number of substrates and act by remodeling cytoskeleton, employing scaffolding, and relocating to distinct subcellular compartments. PAKs affect wide range of processes that are crucial to the cell from regulation of cell motility, survival, redox, metabolism, cell cycle, proliferation, transformation, stress, inflammation, to gene expression. Understandably, their dysregulation disrupts cellular homeostasis and severely impacts key cell functions, and many of those are implicated in a number of human diseases including cancers, neurological disorders, and cardiac disorders. Here we provide an overview of the members of the PAK family and their current status. We give special emphasis to PAK1 and PAK4, the prototypes of groups I and II, for their profound roles in cancer, the nervous system, and the heart. We also highlight other family members. We provide our perspective on the current advancements, their growing importance as strategic therapeutic targets, and our vision on the future of PAKs.
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Affiliation(s)
- Rakesh Kumar
- Department of Biochemistry and Molecular Medicine, School of Medicine and Health Sciences, George Washington University, Washington, DC 20037, USA; Cancer Biology Program, Rajiv Gandhi Center of Biotechnology, Thiruvananthapuram 695014, India.
| | - Rahul Sanawar
- Cancer Biology Program, Rajiv Gandhi Center of Biotechnology, Thiruvananthapuram 695014, India
| | - Xiaodong Li
- Department of Cell Biology, Key Laboratory of Medical Cell Biology, Chinese Ministry of Education, China Medical University, Shenyang 110122, China
| | - Feng Li
- Department of Cell Biology, Key Laboratory of Medical Cell Biology, Chinese Ministry of Education, China Medical University, Shenyang 110122, China.
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24
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P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate. PLoS One 2016; 11:e0153312. [PMID: 27071060 PMCID: PMC4829229 DOI: 10.1371/journal.pone.0153312] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2015] [Accepted: 03/28/2016] [Indexed: 12/25/2022] Open
Abstract
Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients' adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1-10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.
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25
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Kumar R, Li DQ. PAKs in Human Cancer Progression: From Inception to Cancer Therapeutic to Future Oncobiology. Adv Cancer Res 2016; 130:137-209. [PMID: 27037753 DOI: 10.1016/bs.acr.2016.01.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Since the initial recognition of a mechanistic role of p21-activated kinase 1 (PAK1) in breast cancer invasion, PAK1 has emerged as one of the widely overexpressed or hyperactivated kinases in human cancer at-large, allowing the PAK family to make in-roads in cancer biology, tumorigenesis, and cancer therapeutics. Much of our current understanding of the PAK family in cancer progression relates to a central role of the PAK family in the integration of cancer-promoting signals from cell membrane receptors as well as function as a key nexus-modifier of complex, cytoplasmic signaling network. Another core aspect of PAK signaling that highlights its importance in cancer progression is through PAK's central role in the cross talk with signaling and interacting proteins, as well as PAK's position as a key player in the phosphorylation of effector substrates to engage downstream components that ultimately leads to the development cancerous phenotypes. Here we provide a comprehensive review of the recent advances in PAK cancer research and its downstream substrates in the context of invasion, nuclear signaling and localization, gene expression, and DNA damage response. We discuss how a deeper understanding of PAK1's pathobiology over the years has widened research interest to the PAK family and human cancer, and positioning the PAK family as a promising cancer therapeutic target either alone or in combination with other therapies. With many landmark findings and leaps in the progress of PAK cancer research since the infancy of this field nearly 20 years ago, we also discuss postulated advances in the coming decade as the PAK family continues to shape the future of oncobiology.
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Affiliation(s)
- R Kumar
- School of Medicine and Health Sciences, George Washington University, Washington, DC, United States; Rajiv Gandhi Center of Biotechnology, Thiruvananthapuram, India.
| | - D-Q Li
- Fudan University Shanghai Cancer Center and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China; Key Laboratory of Breast Cancer in Shanghai, Shanghai Medical College, Fudan University, Shanghai, China; Key Laboratory of Epigenetics in Shanghai, Shanghai Medical College, Fudan University, Shanghai, China.
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26
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Shao YG, Ning K, Li F. Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer. World J Gastroenterol 2016; 22:1224-1235. [PMID: 26811660 PMCID: PMC4716033 DOI: 10.3748/wjg.v22.i3.1224] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Revised: 09/17/2015] [Accepted: 11/19/2015] [Indexed: 02/06/2023] Open
Abstract
P21-activated kinases (PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group I (PAK1-3) and group II (PAK4-6). Focus is currently shifting from group I PAKs to group II PAKs. Group II PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group II PAKs have become popular potential drug target candidates. However, few group II PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and “drug-like” properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group II PAKs, the importance of group II PAKs in the development and progression of gastrointestinal cancer, and small-molecule inhibitors of group II PAKs for the treatment of cancer.
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27
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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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28
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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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29
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Prencipe M, O’Neill A, O’Hurley G, Nguyen LK, Fabre A, Bjartell A, Gallagher WM, Morrissey C, Kay EW, Watson RW. Relationship between serum response factor and androgen receptor in prostate cancer. Prostate 2015; 75:1704-17. [PMID: 26250344 PMCID: PMC4579008 DOI: 10.1002/pros.23051] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 06/29/2015] [Indexed: 12/13/2022]
Abstract
BACKGROUND Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. MATERIALS AND METHODS Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. RESULTS To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. CONCLUSION Our data support SRF as a promising therapeutic target in combination with current treatments.
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Affiliation(s)
- Maria Prencipe
- UCD School of Medicine and Medical Science, Conway Institute of
Biomolecular and Biomedical Research, University College Dublin, Belfield, D4,
Dublin, Ireland
- To whom correspondence should be addressed. Tel:
+353 1716 6913,
| | - Amanda O’Neill
- UCD School of Medicine and Medical Science, Conway Institute of
Biomolecular and Biomedical Research, University College Dublin, Belfield, D4,
Dublin, Ireland
| | - Gillian O’Hurley
- OncoMark Limited, NovaUCD, Belfield Innovation Park, Belfield, D4,
Dublin, Ireland
| | - Lan K. Nguyen
- Systems Biology Ireland, University College Dublin, Belfield, D4,
Dublin, Ireland
| | - Aurelie Fabre
- Department of Pathology, St Vincent’s University Hospital,
Dublin, Ireland
| | - Anders Bjartell
- Department of Urology, Skåne University Hospital,
Malmö, Sweden
| | - William M. Gallagher
- OncoMark Limited, NovaUCD, Belfield Innovation Park, Belfield, D4,
Dublin, Ireland
- UCD School of Biomolecular and Biomedical Science, Conway Institute
of Biomolecular and Biomedical Research, University College Dublin, Belfield, D4,
Dublin, Ireland
| | - Colm Morrissey
- Department of Urology, University of Washington, Seattle,
Washington, USA
| | - Elaine W Kay
- Department of Pathology, RCSI Education and Research Centre;
Beaumont Hospital, Dublin, Ireland
| | - R William Watson
- UCD School of Medicine and Medical Science, Conway Institute of
Biomolecular and Biomedical Research, University College Dublin, Belfield, D4,
Dublin, Ireland
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30
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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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31
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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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32
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Ha BH, Morse EM, Turk BE, Boggon TJ. Signaling, Regulation, and Specificity of the Type II p21-activated Kinases. J Biol Chem 2015; 290:12975-83. [PMID: 25855792 DOI: 10.1074/jbc.r115.650416] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The p21-activated kinases (PAKs) are a family of six serine/threonine kinases that act as key effectors of RHO family GTPases in mammalian cells. PAKs are subdivided into two groups: type I PAKs (PAK1, PAK2, and PAK3) and type II PAKs (PAK4, PAK5, and PAK6). Although these groups are involved in common signaling pathways, recent work indicates that the two groups have distinct modes of regulation and have both unique and common substrates. Here, we review recent insights into the molecular level details that govern regulation of type II PAK signaling. We also consider mechanisms by which signal transduction is regulated at the level of substrate specificity. Finally, we discuss the implications of these studies for clinical targeting of these kinases.
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Affiliation(s)
| | - Elizabeth M Morse
- Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520
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33
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Hedman AC, Smith JM, Sacks DB. The biology of IQGAP proteins: beyond the cytoskeleton. EMBO Rep 2015; 16:427-46. [PMID: 25722290 DOI: 10.15252/embr.201439834] [Citation(s) in RCA: 172] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2014] [Accepted: 01/07/2015] [Indexed: 01/02/2023] Open
Abstract
IQGAP scaffold proteins are evolutionarily conserved in eukaryotes and facilitate the formation of complexes that regulate cytoskeletal dynamics, intracellular signaling, and intercellular interactions. Fungal and mammalian IQGAPs are implicated in cytokinesis. IQGAP1, IQGAP2, and IQGAP3 have diverse roles in vertebrate physiology, operating in the kidney, nervous system, cardio-vascular system, pancreas, and lung. The functions of IQGAPs can be corrupted during oncogenesis and are usurped by microbial pathogens. Therefore, IQGAPs represent intriguing candidates for novel therapeutic agents. While modulation of the cytoskeletal architecture was initially thought to be the primary function of IQGAPs, it is now clear that they have roles beyond the cytoskeleton. This review describes contributions of IQGAPs to physiology at the organism level.
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Affiliation(s)
- Andrew C Hedman
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
| | - Jessica M Smith
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
| | - David B Sacks
- Department of Laboratory Medicine, National Institutes of Health, Bethesda, MD, USA
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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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Abstract
Transformation of a normal cell to a cancer cell is caused by mutations in genes that regulate proliferation, apoptosis, and invasion. Small GTPases such as Ras, Rho, Rac and Cdc42 orchestrate many of the signals that are required for malignant transformation. The p21-activated kinases (PAKs) are effectors of Rac and Cdc42. PAKs are a family of serine/threonine protein kinases comprised of six isoforms (PAK1–6), and they play important roles in cytoskeletal dynamics, cell survival and proliferation. They act as key signal transducers in several cancer signaling pathways, including Ras, Raf, NFκB, Akt, Bad and p53. Although PAKs are not mutated in cancers, they are overexpressed, hyperactivated or amplified in several human tumors and their role in cell transformation make them attractive therapeutic targets. This review discusses the evidence that PAK is important for cell transformation and some key signaling pathways it regulates. This review primarily discusses Group I PAKs (PAK1, PAK2 and PAK3) as Group II PAKs (PAK4, PAK5 and PAK6) are discussed elsewhere in this issue (by Minden).
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Affiliation(s)
- Diana Zi Ye
- Department of Pharmacology; Perelman School of Medicine; University of Pennsylvania; Philadelphia, PA USA
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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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Hodgson MC, Deryugina EI, Suarez E, Lopez SM, Lin D, Xue H, Gorlov IP, Wang Y, Agoulnik IU. INPP4B suppresses prostate cancer cell invasion. Cell Commun Signal 2014. [PMID: 25248616 DOI: 10.1186/preaccept-2663637391256502] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer. RESULTS We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B. CONCLUSION Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors.
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Abstract
Background INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer. Results We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B. Conclusion Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12964-014-0061-y) contains supplementary material, which is available to authorized users.
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Tse EYT, Ching YP. The role of p21-activated kinases in hepatocellular carcinoma metastasis. J Mol Signal 2014; 9:7. [PMID: 25093037 PMCID: PMC4121300 DOI: 10.1186/1750-2187-9-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2014] [Accepted: 07/18/2014] [Indexed: 01/06/2023] Open
Abstract
The p21-activated kinases (PAKs) are downstream effectors of the Rho family small GTPases as well as a wide variety of mitogenic factors and have been implicated in cancer formation, development and metastasis. PAKs phosphorylate a wide spectrum of substrates to mediate extracellular signals and regulate cytoskeletal remodeling, cell motility and survival. In this review, we aim to summarize the findings regarding the oncogenic role and the underlying mechanisms of PAKs signaling in various cancers, and in particular highlight the prime importance of PAKs in hepatocellular carcinoma (HCC) progression and metastasis. Recent studies exploring the potential therapeutic application of PAK inhibitors will also be discussed.
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Affiliation(s)
- Edith Yuk Ting Tse
- Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
| | - Yick Pang Ching
- Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China ; State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China
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Zapatero A, Morente M, Nieto S, Martín de Vidales C, Lopez C, Adrados M, Arellano R, Artiga MJ, Garcia-Vicente F, Herranz LM, Leaman O. Predictive value of PAK6 and PSMB4 expression in patients with localized prostate cancer treated with dose-escalation radiation therapy and androgen deprivation therapy. Urol Oncol 2014; 32:1327-32. [PMID: 24946957 DOI: 10.1016/j.urolonc.2014.05.004] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Revised: 05/12/2014] [Accepted: 05/13/2014] [Indexed: 10/25/2022]
Abstract
PURPOSE The present study analyzed the expression by immunochemistry of the novel markers P21-activated protein kinase 6 (PAK6) and proteasome beta-4 subunit (PSMB4) in men with localized prostate cancer (PC) who were treated with dose-escalation radiotherapy (RT) and androgen deprivation therapy. MATERIALS AND METHODS Between 1996 and 2004, a cohort of 129 patients with PC who underwent diagnostic biopsies pretreatment and 24 to 36 months following RT were enrolled in this study. Suitable archival diagnostic tissue was obtained from 89 patients. Median follow-up was 129 months (48-198). Correlation analysis was done to assess association between PAK6 and PSMB4 expression and clinical outcome. RESULTS PAK6 and PSMB4 were expressed in the cytoplasm in 62% and 96.7% of diagnostic biopsies, respectively. Increased staining for PAK6 was significantly (P = 0.04) correlated with higher Gleason scores. In the multivariate analysis, the intensity of PSMB4 staining was an independent predictor of local relapse (hazard ratio = 8.6, P = 0.04). CONCLUSIONS To our knowledge, this is the first description of PAK6 and PSMB4 expression in the diagnostic specimens of men with PC who were treated with RT. If confirmed by further studies, increased expression of these genes could be used to identify patients at a high risk of developing local failure following high-dose RT, thus better tailoring treatments for the individual patient.
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Affiliation(s)
- Almudena Zapatero
- Department of Radiation Oncology, La Princesa University Hospital, Madrid, Spain.
| | - Manuel Morente
- Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Santiago Nieto
- Department of Pathology, University Hospital Henares, Madrid, Spain
| | | | - Consuelo Lopez
- Department of Pathology, La Princesa University Hospital, Madrid, Spain
| | - Magdalena Adrados
- Department of Pathology, La Princesa University Hospital, Madrid, Spain
| | - Ramón Arellano
- Department of Urology, La Princesa University Hospital, Madrid, Spain
| | | | | | | | - Olwen Leaman
- Department of Radiation Oncology, La Princesa University Hospital, Madrid, Spain
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Liu W, Liu H, Liu Y, Xu L, Zhang W, Zhu Y, Xu J, Gu J. Prognostic Significance of p21-activated Kinase 6 Expression in Patients with Clear Cell Renal Cell Carcinoma. Ann Surg Oncol 2014; 21 Suppl 4:S575-83. [DOI: 10.1245/s10434-014-3680-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2014] [Indexed: 02/06/2023]
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AR variant ARv567es induces carcinogenesis in a novel transgenic mouse model of prostate cancer. Neoplasia 2014; 15:1009-17. [PMID: 24027426 DOI: 10.1593/neo.13784] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2013] [Revised: 06/17/2013] [Accepted: 06/24/2013] [Indexed: 02/02/2023] Open
Abstract
Androgen deprivation therapy remains the primary treatment modality for patients with metastatic prostate cancer but is uniformly marked by progression to castration-resistant prostate cancer (CRPC) after a period of regression. Continued activation of androgen receptor (AR) signaling is attributed as one of the most important mechanisms underlying failure of therapy. Recently, the discovery of constitutively active AR splice variants (AR-Vs) adds more credence to this idea. Expression of AR-Vs in metastases portends a rapid progression of the tumor. However, the precise role of the AR-Vs in CRPC still remains unknown. ARv567es is one of the two AR variants frequently found in human CRPC xenografts and metastases. Herein, we developed a probasin (Pb) promoter-driven ARv567es transgenic mouse, Pb-ARv567es, to evaluate the role of ARv567es in both autonomous prostate growth and progression to CRPC. We found that expression of ARv567es in the prostate results in epithelial hyperplasia by 16 weeks and invasive adenocarcinoma is evident by 1 year of age. The underlying genetic cellular events involved a cell cycle-related transcriptome and differential expression of a spectrum of genes that are critical for tumor initiation and progression. These findings indicate that ARv567es could induce tumorigenesis de novo and signifies the critical role of AR-Vs in CRPC. Thus, the Pb-ARv567es mouse could provide a novel model in which the role of AR variants in prostate cancer progression can be examined.
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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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Chen H, Miao J, Li H, Wang C, Li J, Zhu Y, Wang J, Wu X, Qiao H. Expression and prognostic significance of p21-activated kinase 6 in hepatocellular carcinoma. J Surg Res 2014; 189:81-8. [PMID: 24576777 DOI: 10.1016/j.jss.2014.01.049] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2013] [Revised: 01/15/2014] [Accepted: 01/24/2014] [Indexed: 12/12/2022]
Abstract
BACKGROUND p21-activated protein kinase (PAK) 6 is a serine-threonine kinase belonging to the PAK family. Previous studies have indicated that abnormal expressions of PAK1, PAK2, and PAK5 played critical roles in hepatocellular carcinoma (HCC). Recent studies suggested that deregulation of PAK6 expression played an important role in oncogenesis. To explore the potential roles of PAK6 in HCC, expression of PAK6 was detected in human HCC specimens. METHODS Immunohistochemistry and Western blot analysis were performed for PAK6 in 121 HCC samples. The data were correlated with clinicopathologic features. The univariate and multivariate survival analyses were also performed to determine their clinical prognostic significance. RESULTS PAK6 was overexpressed in HCC as compared with the adjacent noncancerous liver tissues. High expression of PAK6 was associated with Edmondson-Steiner grade (P = 0.006) and number of tumor nodules (P < 0.001), and PAK6 was positively correlated with proliferation marker Ki-67 (P < 0.01). Univariate analysis suggested that PAK6 expression was associated with poor prognosis (P < 0.001). Multivariate analysis indicated that PAK6 and Ki-67 protein expressions were independent prognostic markers for HCC (P = 0.0245 and 0.0331, respectively). CONCLUSIONS Our results suggest that PAK6 overexpression is involved in the pathogenesis of HCC; it may be an independent poor prognostic factor for HCC.
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Affiliation(s)
- Hongwei Chen
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China.
| | - Jinlin Miao
- Department of Magnetic Resonance Imaging, The Fourth People's Hospital of Taizhou, Taizhou, China
| | - Hongchen Li
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Chunhua Wang
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Junliang Li
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Yong Zhu
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Jianxin Wang
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Xia Wu
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
| | - Hongying Qiao
- Department of Clinical Laboratory, The First Hospital of Qinhuangdao, Qinhuangdao, China
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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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P21-activated kinase 5 plays essential roles in the proliferation and tumorigenicity of human hepatocellular carcinoma. Acta Pharmacol Sin 2014; 35:82-8. [PMID: 23685956 DOI: 10.1038/aps.2013.31] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 03/08/2013] [Indexed: 12/15/2022]
Abstract
AIM To investigate the roles of P21-activated kinase 5 (PAK5) in proliferation and tumorigenicity of human hepatocellular carcinoma (HCC). METHODS HCC and matched paraneoplastictis tissue samples were obtained from 30 patients. Human HCC cell lines SMMC7721, HepG2, Hep3B, SK-HEP-1, Huh-7, and liver cell line HL-7702 were examined. The expression of PAK5 gene was studied using real-time qPCR and Western blotting. Cell proliferation was quantified with the MTT assay. Cell cycle was analyzed with flow cytometry. The tumorigenicity of Lv-shRNA-transfected HepG2 cells was evaluated in BALB/cA nude mice. RESULTS The mRNA level of PAK5 was significantly higher in 25 out of 30 HCC samples compared to the matched paraneoplastic tissues. The HCC cell lines showed varying expression of PAK5 protein, and the highest level was found in the HepG2 cells. PAK5 gene silencing in HepG2 cells markedly reduced the cell proliferation and colony formation, and induced cell cycle arrest in the G1 phase. Furthermore, PAK5 gene silencing suppressed the tumor formation in nude mice, and significantly decreased the expression of HCC-related genes Cyclin D1 and beta-catenin. CONCLUSION PAK5 may play essential roles in the initiation and progression of human HCC. Thus, it may be an effective therapeutic target or perhaps serve as a clinical diagnostic or prognostic marker in human HCC.
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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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Abstract
Coordination of the activity of multiple small GTPases is required for the regulation of many physiological processes, including cell migration. There are now several examples of functional interplay between small GTPase pairs, but the mechanisms that control GTPase activity in time and space are only partially understood. Here, we build on the hypothesis that small GTPases are part of a large, integrated network and propose that key proteins within this network integrate multiple signaling events and coordinate multiple small GTPase activities. Specifically, we identify the scaffolding protein IQGAP1 as a master regulator of multiple small GTPases, including Cdc42, Rac1, Rap1, and RhoA. In addition, we demonstrate that IQGAP1 promotes Arf6 activation downstream of β1 integrin engagement. Furthermore, following literature-curated searches and recent mass spectrometric analysis of IQGAP1-binding partners, we report that IQGAP1 recruits other small GTPases, including RhoC, Rac2, M-Ras, RhoQ, Rab10, and Rab5, small GTPase regulators, including Tiam1, RacGAP1, srGAP2 and HERC1, and small GTPase effectors, including PAK6, N-WASP, several sub-units of the Arp2/3 complex and the formin mDia1. Therefore, we propose that IQGAP1 acts as a small GTPase scaffolding platform within the small GTPase network, and recruits and/or regulates small GTPases, small GTPase regulators and effectors to orchestrate cell behavior. Finally, to identify other putative key regulators of small GTPase crosstalk, we have assembled a small GTPase network using protein-protein interaction databases.
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Affiliation(s)
- Guillaume Jacquemet
- Wellcome Trust Centre for Cell-Matrix Research; Faculty of Life Sciences; University of Manchester; Manchester, UK
| | - Martin J Humphries
- Wellcome Trust Centre for Cell-Matrix Research; Faculty of Life Sciences; University of Manchester; Manchester, 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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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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