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Li P, Ying S, Zou Y, Wang X, Zhang R, Huang C, Dai M, Xu K, Feng G, Li X, Jiang H, Li Z, Zhang Y, Li W, Zhou Q. NSun2-Mediated tsRNAs Alleviate Liver Fibrosis via FAK Dephosphorylation. Cell Prolif 2025:e70058. [PMID: 40355098 DOI: 10.1111/cpr.70058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2025] [Revised: 04/13/2025] [Accepted: 04/22/2025] [Indexed: 05/14/2025] Open
Abstract
Sinusoidal capillarization - key symptoms of liver fibrosis progression - represents potential therapeutic targets. tRNA modification-mediated tRNA-derived small RNAs (tsRNAs) play a role in angiogenesis. NSun2, an RNA methyltransferase, generates a significant number of tsRNAs. However, the role of NSun2 and its mediated tsRNAs in liver fibrosis remains unclear. In this study, NSun2 deficiency was found to inhibit sinusoidal capillarization, alleviating liver fibrosis. Furthermore, endothelial cell angiogenesis and migration were disrupted in NSun2 knockout mice. Mechanistically, reduced NSun2 expression led to alterations in the functional tsRNAs tRF-1-S25 and tRF-5-V31, which regulate sinusoidal capillarization by targeting key proteins, including DUSP1 and FAK - crucial clinical targets. Moreover, intravenous injection of tRF-1-S25 and tRF-5-V31 inhibitor rescued liver fibrosis in mice. In conclusion, tsRNAs generated by NSun2-mediated modification of tRNAs inhibit sinusoidal capillarization. Furthermore, targeting the DUSP1/FAK/p-FAK pathway offers an innovative approach to treat this disease.
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Affiliation(s)
- Pengcheng Li
- College of Life Science, Northeast Agricultural University of China, Harbin, China
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
| | - Sunyang Ying
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
| | - Yu Zou
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Xin Wang
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
- Medical School, University of Chinese Academy of Sciences, Beijing, China
| | - Runxue Zhang
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Cheng Huang
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Moyu Dai
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Kai Xu
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Guihai Feng
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Xin Li
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Haiping Jiang
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Zhikun Li
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Ying Zhang
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Wei Li
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
| | - Qi Zhou
- College of Life Science, Northeast Agricultural University of China, Harbin, China
- State key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
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2
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Guo D, Yu Q, Tong Y, Qian X, Meng Y, Ye F, Jiang X, Wu L, Yang Q, Li S, Li M, Wu Q, Xiao L, He X, Zhu R, Liu G, Nie D, Luo S, Ma L, Jin RA, Liu Z, Liang X, Yan D, Lu Z. OXCT1 succinylation and activation by SUCLA2 promotes ketolysis and liver tumor growth. Mol Cell 2025; 85:843-856.e6. [PMID: 39862868 DOI: 10.1016/j.molcel.2024.12.025] [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: 05/23/2024] [Revised: 11/11/2024] [Accepted: 12/30/2024] [Indexed: 01/27/2025]
Abstract
Ketone bodies generated in hepatocytes in the adult liver are used for nonhepatic tissues as an energy source. However, ketolysis is reactivated in hepatocellular carcinoma (HCC) cells with largely unelucidated mechanisms. Here, we demonstrate that 3-oxoacid CoA-transferase 1 (OXCT1), a rate-limiting enzyme in ketolysis, interacts with SUCLA2 upon IGF1 stimulation in HCC cells. This interaction results from ERK2-mediated SUCLA2 S124 phosphorylation and subsequent PIN1-mediated cis-trans isomerization of SUCLA2. OXCT1-associated SUCLA2 generates succinyl-CoA, which not only serves as a substrate for OXCT1 but also directly succinylates OXCT1 at K421 and activates OXCT1. SUCLA2-regulated OXCT1 activation substantially enhances ketolysis, HCC cell proliferation, and tumor growth in mice. Notably, treatment with acetohydroxamic acid, an OXCT1 inhibitor used clinically for urinary infection, inhibits liver tumor growth in mice and significantly enhances lenvatinib therapy. Our findings highlight the role of SUCLA2-coupled regulation of OXCT1 succinylation in ketolysis and unveil an unprecedented strategy for treating HCC by interrupting ketolysis.
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Affiliation(s)
- Dong Guo
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Qiujing Yu
- Department of Health Management Center & Institute of Health Management, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, Sichuan 611731, China.
| | - Yingying Tong
- Cancer Center, Beijing Luhe Hospital, Capital Medical University, Beijing 101149, China
| | - Xu Qian
- Department of Clinical Laboratory, Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, China
| | - Ying Meng
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Fei Ye
- College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, Zhejiang 310018, China
| | - Xiaoming Jiang
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Lihui Wu
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Qingqing Yang
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Suyao Li
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Min Li
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Qingang Wu
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Liwei Xiao
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Xuxiao He
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Rongxuan Zhu
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Guijun Liu
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Dou Nie
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Shudi Luo
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China
| | - Leina Ma
- Department of Oncology, the Affiliated Hospital of Qingdao University, Qingdao Cancer Institute, Qingdao, Shandong 266071, China
| | - Ren-An Jin
- Zhejiang Key Laboratory of Multi-omics Precision Diagnosis and Treatment of Liver Diseases, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, China
| | - Zhihua Liu
- State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
| | - Xiao Liang
- Zhejiang Key Laboratory of Multi-omics Precision Diagnosis and Treatment of Liver Diseases, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310016, China.
| | - Dong Yan
- Cancer Center, Beijing Luhe Hospital, Capital Medical University, Beijing 101149, China.
| | - Zhimin Lu
- Zhejiang Provincial Key Laboratory of Pancreatic Disease, the First Affiliated Hospital, Zhejiang Key Laboratory of Frontier Medical Research on Cancer Metabolism, Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310029, China; Institute of Fundamental and Transdisciplinary Research, Cancer Center, Zhejiang University, Hangzhou, Zhejiang 310029, China.
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3
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Almazan J, Turapov T, Kircher DA, Stanley KA, Culver K, Medellin AP, Field MN, Parkman GL, Colman H, Coma S, Pachter JA, Holmen SL. Combined inhibition of focal adhesion kinase and RAF/MEK elicits synergistic inhibition of melanoma growth and reduces metastases. Cell Rep Med 2025; 6:101943. [PMID: 39922199 PMCID: PMC11866499 DOI: 10.1016/j.xcrm.2025.101943] [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: 07/12/2024] [Revised: 10/04/2024] [Accepted: 01/13/2025] [Indexed: 02/10/2025]
Abstract
This study addresses the urgent need for effective therapies for patients with brain metastases from cutaneous melanoma, a major cause of treatment failure despite recent therapeutic advances. Utilizing mouse models that mimic human melanoma brain metastases, this study investigates the necessity of focal adhesion kinase (FAK) in the development of distant metastases and its potential as a therapeutic target. Pharmacological inhibition of FAK demonstrates significant efficacy in reducing the development of brain metastases in preclinical mouse models. Importantly, the study provides insight into the crosstalk between FAK and mitogen-activated protein kinase (MAPK) pathway signaling and highlights the synergistic effects of combined inhibition of FAK, rapidly accelerated fibrosarcoma (RAF), and mitogen-activated protein kinase kinase (MEK) in cutaneous melanoma. These findings provide the rationale for clinical evaluation of the efficacy of the FAK inhibitor defactinib and the RAF/MEK inhibitor avutometinib in patients with brain metastases from cutaneous melanoma.
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Affiliation(s)
- Jared Almazan
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - Tursun Turapov
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - David A Kircher
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - Karly A Stanley
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - Katie Culver
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - A Paulina Medellin
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - MiKaela N Field
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | - Gennie L Parkman
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Zoology, Weber State University, Ogden, UT 84408, USA
| | - Howard Colman
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Neurosurgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA
| | | | | | - Sheri L Holmen
- Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA; Department of Surgery, University of Utah Health Sciences Center, Salt Lake City, UT 84112, USA.
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4
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Jain K, Kishan K, Minhaj RF, Kanchanawong P, Sheetz MP, Changede R. Immobile Integrin Signaling Transit and Relay Nodes Organize Mechanosignaling through Force-Dependent Phosphorylation in Focal Adhesions. ACS NANO 2025; 19:2070-2088. [PMID: 39760672 DOI: 10.1021/acsnano.4c03214] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2025]
Abstract
Transmembrane signaling receptors, such as integrins, organize as nanoclusters that provide several advantages, including increasing avidity, sensitivity (increasing the signal-to-noise ratio), and robustness (signaling threshold) of the signal in contrast to signaling by single receptors. Furthermore, compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, whether nanoclusters function as signaling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at subdiffraction limited resolution of ∼100 nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in an immobile population of integrin β3 organized as nanoclusters, which (ii) in turn serve to organize nanoclusters of associated key adhesome proteins-vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signaling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct protein dynamics, which is closely correlated to their function in signaling. (iv) Long-lived nanoclusters function as signaling hubs─wherein immobile integrin nanoclusters organize phosphorylated FAK to form stable nanoclusters in close proximity to them, which are disassembled in response to inactivation signal by removal of force and in turn activation of phosphatase PTPN12. (v) Signaling takes place in response to external signals such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. We term these functional nanoclusters as integrin signaling transit and relay nodes (STARnodes). Taken together, these results demonstrate that integrin STARnodes seed signaling downstream of the integrin receptors by organizing hubs of signaling proteins (FAK, paxillin, vinculin) to relay the incoming signal intracellularly and bring about robust function.
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Affiliation(s)
- Kashish Jain
- Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
| | - Kishan Kishan
- Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
- Neurobit Inc., New York, New York 10036, United States
| | - Rida F Minhaj
- Department of Biomedical Engineering, National University of Singapore, Singapore 117583, Singapore
| | - Pakorn Kanchanawong
- Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
- Department of Biomedical Engineering, National University of Singapore, Singapore 117583, Singapore
| | - Michael P Sheetz
- Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
- Molecular Mechanomedicine Program, Biochemistry and Molecular Biology Department, University of Texas Medical Branch, Galveston, Texas 77555, United States
| | - Rishita Changede
- Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore
- Teora Pte. Ltd, Singapore 139955, Singapore
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5
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Yang J, Wang P, Zhang Y, Zhang M, Sun Q, Chen H, Dong L, Chu Z, Xue B, Hoff WD, Zhao C, Wang W, Wei Q, Cao Y. Photo-tunable hydrogels reveal cellular sensing of rapid rigidity changes through the accumulation of mechanical signaling molecules. Cell Stem Cell 2025; 32:121-136.e6. [PMID: 39437791 DOI: 10.1016/j.stem.2024.09.016] [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: 02/25/2024] [Revised: 07/08/2024] [Accepted: 09/23/2024] [Indexed: 10/25/2024]
Abstract
Cells use traction forces to sense mechanical cues in their environment. While the molecular clutch model effectively explains how cells exert more forces on stiffer substrates, it falls short in addressing their adaptation to dynamic mechanical fluctuations prevalent in tissues and organs. Here, using hydrogel with photo-responsive rigidity, we show that cells' response to rigidity changes is frequency dependent. Strikingly, at certain frequencies, cellular traction forces exceed those on static substrates 4-fold stiffer, challenging the established molecular clutch model. We discover that the discrepancy between the rapid adaptation of traction forces and the slower deactivation of mechanotransduction signaling proteins results in their accumulation, thereby enhancing long-term cellular traction in dynamic settings. Consequently, we propose a new model that melds immediate mechanosensing with extended mechanical signaling. Our study underscores the significance of dynamic rigidity in the development of synthetic biomaterials, emphasizing the importance of considering both immediate and prolonged cellular responses.
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Affiliation(s)
- Jiapeng Yang
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Peng Wang
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Yu Zhang
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Man Zhang
- College of Biomedical Engineering, Sichuan University, Chengdu 610065, China
| | - Qian Sun
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Huiyan Chen
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Liang Dong
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Zhiqin Chu
- Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong 999077, China; Joint Appointment with School of Biomedical Sciences, The University of Hong Kong, Hong Kong 999077, China
| | - Bin Xue
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Wouter David Hoff
- Department of Physics, Oklahoma State University, Stillwater, OK 74078, USA
| | - Changsheng Zhao
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China
| | - Wei Wang
- Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China
| | - Qiang Wei
- College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials and Engineering, Sichuan University, Chengdu 610065, China.
| | - Yi Cao
- Jinan Microecological Biomedicine Shandong Laboratory, Jinan 250021, China; Chemistry and Biomedicine Innovation Center (ChemBIC), ChemBioMed Interdisciplinary Research Center at Nanjing University, Department of Physics, Nanjing University, Nanjing 210093, China; Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou 325001, China.
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Muneer G, Chen CS, Lee TT, Chen BY, Chen YJ. A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics. J Proteome Res 2024; 23:3294-3309. [PMID: 39038167 DOI: 10.1021/acs.jproteome.3c00862] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/24/2024]
Abstract
Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl β-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 μg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.
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Affiliation(s)
- Gul Muneer
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
- Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
- Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan
| | - Ciao-Syuan Chen
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | - Tzu-Tsung Lee
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | - Bo-Yu Chen
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | - Yu-Ju Chen
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
- Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan
- Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
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7
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Lu KP, Zhou XZ. Pin1-catalyzed conformational regulation after phosphorylation: A distinct checkpoint in cell signaling and drug discovery. Sci Signal 2024; 17:eadi8743. [PMID: 38889227 PMCID: PMC11409840 DOI: 10.1126/scisignal.adi8743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Accepted: 05/30/2024] [Indexed: 06/20/2024]
Abstract
Protein phosphorylation is one of the most common mechanisms regulating cellular signaling pathways, and many kinases and phosphatases are proven drug targets. Upon phosphorylation, protein functions can be further regulated by the distinct isomerase Pin1 through cis-trans isomerization. Numerous protein targets and many important roles have now been elucidated for Pin1. However, no tools are available to detect or target cis and trans conformation events in cells. The development of Pin1 inhibitors and stereo- and phospho-specific antibodies has revealed that cis and trans conformations have distinct and often opposing cellular functions. Aberrant conformational changes due to the dysregulation of Pin1 can drive pathogenesis but can be effectively targeted in age-related diseases, including cancers and neurodegenerative disorders. Here, we review advances in understanding the roles of Pin1 signaling in health and disease and highlight conformational regulation as a distinct signal transduction checkpoint in disease development and treatment.
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Affiliation(s)
- Kun Ping Lu
- Departments of Biochemistry and Oncology, Schulich School of Medicine & Dentistry
- Robarts Research Institute, Schulich School of Medicine & Dentistry
| | - Xiao Zhen Zhou
- Departments of Biochemistry and Oncology, Schulich School of Medicine & Dentistry
- Department of Pathology and Laboratory Medicine, Schulich School of Medicine & Dentistry
- Lawson Health Research Institute, Western University, London, ON N6G 2V4, Canada
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Liu XL, Run-Hua Z, Pan JX, Li ZJ, Yu L, Li YL. Emerging therapeutic strategies for metastatic uveal melanoma: Targeting driver mutations. Pigment Cell Melanoma Res 2024; 37:411-425. [PMID: 38411373 DOI: 10.1111/pcmr.13161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2023] [Revised: 12/29/2023] [Accepted: 01/18/2024] [Indexed: 02/28/2024]
Abstract
Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults. Although primary UM can be effectively controlled, a significant proportion of cases (40% or more) eventually develop distant metastases, commonly in the liver. Metastatic UM remains a lethal disease with limited treatment options. The initiation of UM is typically attributed to activating mutations in GNAQ or GNA11. The elucidation of the downstream pathways such as PKC/MAPK, PI3K/AKT/mTOR, and Hippo-YAP have provided potential therapeutic targets. Concurrent mutations in BRCA1 associated protein 1 (BAP1) or splicing factor 3b subunit 1 (SF3B1) are considered crucial for the acquisition of malignant potential. Furthermore, in preclinical studies, actionable targets associated with BAP1 loss or oncogenic mutant SF3B1 have been identified, offering promising avenues for UM treatment. This review aims to summarize the emerging targeted and epigenetic therapeutic strategies for metastatic UM carrying specific driver mutations and the potential of combining these approaches with immunotherapy, with particular focus on those in upcoming or ongoing clinical trials.
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Affiliation(s)
- Xiao-Lian Liu
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China
- School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China
| | - Zhou Run-Hua
- School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China
| | - Jing-Xuan Pan
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
| | - Zhi-Jie Li
- Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China
| | - Le Yu
- School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China
| | - Yi-Lei Li
- Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, China
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9
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Baranyi M, Molnár E, Hegedűs L, Gábriel Z, Petényi FG, Bordás F, Léner V, Ranđelović I, Cserepes M, Tóvári J, Hegedűs B, Tímár J. Farnesyl-transferase inhibitors show synergistic anticancer effects in combination with novel KRAS-G12C inhibitors. Br J Cancer 2024; 130:1059-1072. [PMID: 38278976 PMCID: PMC10951297 DOI: 10.1038/s41416-024-02586-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 01/10/2024] [Accepted: 01/15/2024] [Indexed: 01/28/2024] Open
Abstract
BACKGROUND Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.
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Affiliation(s)
- Marcell Baranyi
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
- KINETO Lab Ltd, H-1037, Budapest, Hungary
| | - Eszter Molnár
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
| | - Luca Hegedűs
- Department of Thoracic Surgery, University Medicine Essen - Ruhrlandklinik, University Duisburg-Essen, D-45239, Essen, Germany
| | - Zsófia Gábriel
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
- Pázmány Péter Catholic University Faculty of Information Technology and Bionics, H-1083, Budapest, Hungary
| | - Flóra Gréta Petényi
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
- Pázmány Péter Catholic University Faculty of Information Technology and Bionics, H-1083, Budapest, Hungary
| | - Fanni Bordás
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
- Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, H-1117, Budapest, Hungary
| | | | - Ivan Ranđelović
- KINETO Lab Ltd, H-1037, Budapest, Hungary
- Department of Experimental Pharmacology and the National Tumor Biology Laboratory, National Institute of Oncology, H-1122, Budapest, Hungary
| | - Mihály Cserepes
- KINETO Lab Ltd, H-1037, Budapest, Hungary
- Department of Experimental Pharmacology and the National Tumor Biology Laboratory, National Institute of Oncology, H-1122, Budapest, Hungary
| | - József Tóvári
- Department of Experimental Pharmacology and the National Tumor Biology Laboratory, National Institute of Oncology, H-1122, Budapest, Hungary
| | - Balázs Hegedűs
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary.
- Department of Thoracic Surgery, University Medicine Essen - Ruhrlandklinik, University Duisburg-Essen, D-45239, Essen, Germany.
| | - József Tímár
- Department of Pathology, Forensic and Insurance Medicine, Semmelweis University, H-1091, Budapest, Hungary
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10
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Jain K, Minhaj RF, Kanchanawong P, Sheetz MP, Changede R. Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.02.25.581925. [PMID: 38464288 PMCID: PMC10925161 DOI: 10.1101/2024.02.25.581925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/12/2024]
Abstract
Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).
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Affiliation(s)
- Kashish Jain
- Mechanobiology Institute, National University of Singapore, Singapore, Singapore
| | - Rida F Minhaj
- Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore
| | - Pakorn Kanchanawong
- Mechanobiology Institute, National University of Singapore, Singapore, Singapore
- Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore
| | - Michael P Sheetz
- Mechanobiology Institute, National University of Singapore, Singapore, Singapore
- Molecular Mechanomedicine Program, Biochemistry and Molecular Biology Department, University of Texas Medical Branch, Galveston, TX, USA
| | - Rishita Changede
- Mechanobiology Institute, National University of Singapore, Singapore, Singapore
- TeOra Pte. Ltd, Singapore, Singapore
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11
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Hu HH, Wang SQ, Shang HL, Lv HF, Chen BB, Gao SG, Chen XB. Roles and inhibitors of FAK in cancer: current advances and future directions. Front Pharmacol 2024; 15:1274209. [PMID: 38410129 PMCID: PMC10895298 DOI: 10.3389/fphar.2024.1274209] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Accepted: 01/30/2024] [Indexed: 02/28/2024] Open
Abstract
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that exhibits high expression in various tumors and is associated with a poor prognosis. FAK activation promotes tumor growth, invasion, metastasis, and angiogenesis via both kinase-dependent and kinase-independent pathways. Moreover, FAK is crucial for sustaining the tumor microenvironment. The inhibition of FAK impedes tumorigenesis, metastasis, and drug resistance in cancer. Therefore, developing targeted inhibitors against FAK presents a promising therapeutic strategy. To date, numerous FAK inhibitors, including IN10018, defactinib, GSK2256098, conteltinib, and APG-2449, have been developed, which have demonstrated positive anti-tumor effects in preclinical studies and are undergoing clinical trials for several types of tumors. Moreover, many novel FAK inhibitors are currently in preclinical studies to advance targeted therapy for tumors with aberrantly activated FAK. The benefits of FAK degraders, especially in terms of their scaffold function, are increasingly evident, holding promising potential for future clinical exploration and breakthroughs. This review aims to clarify FAK's role in cancer, offering a comprehensive overview of the current status and future prospects of FAK-targeted therapy and combination approaches. The goal is to provide valuable insights for advancing anti-cancer treatment strategies.
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Affiliation(s)
- Hui-Hui Hu
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
| | - Sai-Qi Wang
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
- State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou University, Zhengzhou, China
| | - Hai-Li Shang
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
| | - Hui-Fang Lv
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
| | - Bei-Bei Chen
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
- State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou University, Zhengzhou, China
| | - She-Gan Gao
- Henan Key Laboratory of Microbiome and Esophageal Cancer Prevention and Treatment, Henan Key Laboratory of Cancer Epigenetics, Cancer Hospital, The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China
| | - Xiao-Bing Chen
- Department of Oncology, The Affiliated Cancer Hospital of Zhengzhou University and Henan Cancer Hospital, Henan Engineering Research Center of Precision Therapy of Gastrointestinal Cancer and Zhengzhou Key Laboratory for Precision Therapy of Gastrointestinal Cancer, Zhengzhou, China
- State Key Laboratory of Esophageal Cancer Prevention & Treatment, Zhengzhou University, Zhengzhou, China
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12
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Li X, Combs JD, Salaita K, Shu X. Polarized focal adhesion kinase activity within a focal adhesion during cell migration. Nat Chem Biol 2023; 19:1458-1468. [PMID: 37349581 PMCID: PMC10732478 DOI: 10.1038/s41589-023-01353-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Accepted: 05/03/2023] [Indexed: 06/24/2023]
Abstract
Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.
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Affiliation(s)
- Xiaoquan Li
- Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA
- Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA
| | | | - Khalid Salaita
- Department of Chemistry, Emory University, Atlanta, GA, USA
| | - Xiaokun Shu
- Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
- Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA.
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13
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Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas. Cancers (Basel) 2023; 15:cancers15051587. [PMID: 36900379 PMCID: PMC10000455 DOI: 10.3390/cancers15051587] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 02/27/2023] [Accepted: 02/28/2023] [Indexed: 03/08/2023] Open
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. METHODS A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. RESULTS High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. CONCLUSIONS Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.
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14
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Honda R, Tempaku Y, Sulidan K, Palmer HEF, Mashima K. Phosphorylation/dephosphorylation of PTP-PEST at Serine 39 is crucial for cell migration. J Biochem 2023; 173:73-84. [PMID: 36250939 DOI: 10.1093/jb/mvac084] [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: 07/20/2022] [Revised: 10/11/2022] [Accepted: 10/13/2022] [Indexed: 02/07/2023] Open
Abstract
We investigated the molecular details of the role of protein tyrosine phosphatase (PTP)-PEST in cell migration. PTP-PEST knockout mouse embryonic fibroblasts (KO MEFs) and MEF cells expressing a dominant-negative mutant of PTP-PEST showed significant suppression of cell migration compared to MEF cells expressing wild-type PTP-PEST (WT MEFs). Moreover, MEF cells harbouring a constitutively active mutant of PTP-PEST (S39A MEFs) showed a marked decrease in cell migration. In addition, MEF cells with no PTP-PEST or little PTP activity rapidly adhered to fibronectin and made many focal adhesions compared to WT MEF cells. In contrast, S39A MEF cells showed weak adhesion to fibronectin and formed a few focal adhesions. Furthermore, investigating the subcellular localization showed that Ser39-phosphorylated PTP-PEST was favourably situated in the adherent area of the pseudopodia. Therefore, we propose that suppression of PTP-PEST enzyme activity due to Ser39-phosphorylation in pseudopodia and at the leading edge of migrating cells induces rapid and good adherence to the extracellular matrix. Thus, suppression of PTP activity by Ser39-phosphorylation is critical for cell migration. Three amino acid substitutions in human PTP-PEST have been previously reported to alter PTP activity. These amino acid substitutions in mouse PTP-PEST altered the migration of MEF cells in a positive correlation.
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Affiliation(s)
- Reika Honda
- Department of Life Science, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
| | - Yasuko Tempaku
- Department of Life Science, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
| | - Kaidiliayi Sulidan
- Department of Life Science, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
| | - Helen E F Palmer
- Department of Life Science, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
| | - Keisuke Mashima
- Department of Life Science, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan.,Life Science Research Center, Rikkyo (St. Paul's) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
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15
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Chen XR, Igumenova TI. Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase. Adv Biol Regul 2023; 87:100938. [PMID: 36496344 PMCID: PMC9992314 DOI: 10.1016/j.jbior.2022.100938] [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: 11/23/2022] [Accepted: 11/29/2022] [Indexed: 12/03/2022]
Abstract
The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.
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Affiliation(s)
- Xiao-Ru Chen
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, 77843, USA
| | - Tatyana I Igumenova
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, 77843, USA.
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16
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Carrasco-Ceballos JM, Barrera-Hernández D, Locia-Espinosa J, Sampieri CL, Lara-Reyes JA, Hernández-Aguilar ME, Aranda-Abreu GE, Toledo-Cárdenas MR, Chi-Castañeda LD, Pérez-Estudillo CA, Rojas-Durán F. Involvement of the PRL-PAK1 Pathway in Cancer Cell Migration. CANCER DIAGNOSIS & PROGNOSIS 2023; 3:17-25. [PMID: 36632591 PMCID: PMC9801455 DOI: 10.21873/cdp.10174] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Accepted: 10/06/2022] [Indexed: 12/31/2022]
Abstract
Prolactin (PRL) is a polypeptide hormone synthesized in the lactotrophs of the adenohypophysis and in extrahypophyseal glands (such as the prostate and breasts) where it promotes their development. PRL is also involved in cancer development in these glands. It has been shown to stimulate cancer cell migration, suggesting its possible involvement in metastasis, in which cell migration plays an essential role. However, the role of PRL in cell migration is still unclear. Moreover, the intracellular mechanisms activated by PRL to carry out cell migration are less well understood. PRL exerts its effects via the PRL receptor (PRLR), which leads intracellularly to phosphorylation of Janus protein kinase 2 (JAK2), which in turn phosphorylates p21-activated protein kinase (PAK1), leading to an increase in cell migration. Although several studies have described the involvement of the PRL-PAK1 pathway in breast cancer cell migration, the molecular mechanisms have not been fully elucidated and there is no integration of these into signaling pathways. This study was conducted based on literature search of review articles and original research in the PubMed database, using the following keywords: PRL, cell migration, PRL and cell migration, PAK1 and signaling pathways. The aim of this review article was to describe the major signaling pathways controlled by PRL-PAK1 and propose a comprehensive model of the signaling pathways associated with PRL-PAK1.
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Affiliation(s)
| | - David Barrera-Hernández
- Departamento de Biología de la Reproducción "Dr. Carlos Gual Castro", Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, México
| | - José Locia-Espinosa
- Facultad de Química Farmacéutica Biológica, Universidad Veracruzana, Xalapa, México
| | | | | | | | | | | | | | | | - Fausto Rojas-Durán
- Instituto de Investigaciones Cerebrales, Universidad Veracruzana, Xalapa, México
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17
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The role of RAS oncogenes in controlling epithelial mechanics. Trends Cell Biol 2023; 33:60-69. [PMID: 36175301 PMCID: PMC9850021 DOI: 10.1016/j.tcb.2022.09.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Revised: 09/01/2022] [Accepted: 09/05/2022] [Indexed: 01/27/2023]
Abstract
Mutations in RAS are key oncogenic drivers and therapeutic targets. Oncogenic Ras proteins activate a network of downstream signalling pathways, including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), promoting cell proliferation and survival. However, there is increasing evidence that RAS oncogenes also alter the mechanical properties of both individual malignant cells and transformed tissues. Here we discuss the role of oncogenic RAS in controlling mechanical cell phenotypes and how these mechanical changes promote oncogenic transformation in single cells and tissues. RAS activation alters actin organisation and actomyosin contractility. These changes alter cell rheology and impact mechanosensing through changes in substrate adhesion and YAP/TAZ-dependent mechanotransduction. We then discuss how these changes play out in cell collectives and epithelial tissues by driving large-scale tissue deformations and the expansion of malignant cells. Uncovering how RAS oncogenes alter cell mechanics will lead to a better understanding of the morphogenetic processes that underlie tumour formation in RAS-mutant cancers.
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18
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Zhevlakova I, Xiong L, Liu H, Dudiki T, Ciocea A, Podrez E, Byzova TV. Opposite roles of Kindlin orthologs in cell survival and proliferation. Cell Prolif 2022; 55:e13280. [PMID: 35860876 PMCID: PMC9436913 DOI: 10.1111/cpr.13280] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 05/13/2022] [Accepted: 05/19/2022] [Indexed: 11/30/2022] Open
Abstract
OBJECTIVE It is unclear why adhesion-dependent cells such as epithelium undergo anoikis without anchorage, while adhesion-independent blood cells thrive in suspension. The adhesive machinery of these cells is similar, with the exception of Kindlin orthologs, Kindlin 2 (K2) and Kindlin 3 (K3). Here we address how Kindlins control cell survival and proliferation in anchorage-dependent and independent cells. MATERIAL AND METHODS To demonstrate the opposite roles of Kindlin's in cell survival we utilized in vivo and in vitro models and K3 and K2 knockdown and knockin cells. We used human lymphocytes from the K3 deficient patients in tumour model, K3 knockout and knockin macrophages and K2 knockout and knockin MEF cells for experiments in under conditions of adhesion and in suspension. RESULTS Depletion of K3 promotes cell proliferation and survival of anchorage-independent cells regardless of cell attachment. In contrast, the absence of K2 in anchorage-dependent cells accelerates apoptosis and limits proliferation. K3 deficiency promotes human lymphoma growth and survival in vivo. Kindlins' interaction with paxillin, is critical for their differential roles in cell anchorage. While disruption of K2-paxillin binding leads to increased apoptosis, the lack of K3-paxillin binding has an opposite effect in adhesion-independent cells. CONCLUSION Kindlin ortologs and their interaction to cytoskeletal protein paxillin define the mechanisms of anchorage dependence. Our study identifies the key elements of the cell adhesion machinery in cell survival and tumour metastasis, proposing possible targets for tumour treatment.
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Affiliation(s)
- Irina Zhevlakova
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
| | - Luyang Xiong
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
| | - Huan Liu
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
- Present address:
CVRC, Simiches Research CenterMassachusetts General Hospital, Harvard Medical SchoolBostonMassachusettsUSA
| | - Tejasvi Dudiki
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
| | - Alieta Ciocea
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
- Present address:
Hondros College of NursingWestervilleOhioUSA
| | - Eugene Podrez
- Department of Inflammation and ImmunityLerner Research Institute, Cleveland ClinicClevelandOhioUSA
| | - Tatiana V. Byzova
- Department of NeurosciencesLerner Research Institute, Cleveland ClinicClevelandOhioUSA
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Han KJ, Mikalayeva V, Gerber SA, Kettenbach AN, Skeberdis VA, Prekeris R. Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation. Life Sci Alliance 2022; 5:5/9/e202101346. [PMID: 35512830 PMCID: PMC9070665 DOI: 10.26508/lsa.202101346] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 04/26/2022] [Accepted: 04/26/2022] [Indexed: 11/24/2022] Open
Abstract
Rab40c is a SOCS box-containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5-mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesion's number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.
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Affiliation(s)
- Ke-Jun Han
- Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Valeryia Mikalayeva
- Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania
| | - Scott A Gerber
- Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.,Norris Cotton Cancer Center, Lebanon, NH, USA
| | - Arminja N Kettenbach
- Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.,Norris Cotton Cancer Center, Lebanon, NH, USA
| | - Vytenis A Skeberdis
- Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania
| | - Rytis Prekeris
- Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
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Multiphoton Microscopy Reveals DAPK1-Dependent Extracellular Matrix Remodeling in a Chorioallantoic Membrane (CAM) Model. Cancers (Basel) 2022; 14:cancers14102364. [PMID: 35625969 PMCID: PMC9139596 DOI: 10.3390/cancers14102364] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2022] [Revised: 05/06/2022] [Accepted: 05/07/2022] [Indexed: 12/12/2022] Open
Abstract
Simple Summary The formation of metastasis is not only intricately orchestrated by cancer cells but is also affected by the surrounding extracellular matrix (ECM). The barrier function of the ECM represents an obstacle that cancer cells have to overcome to disseminate from the primary tumor to form metastasis in distant organs. Here, we demonstrate an approach to studying the remodeling of a collagen-rich ECM by colorectal tumor cells using multiphoton microscopy (MPM). This approach allows the analysis of the invasion front of tumors grown on the CAM in 3D. MPM is superior to conventional histology, which is limited to 2D analysis and needs extensive tissue preparation. Abstract Cancer cells facilitate tumor growth by creating favorable tumor micro-environments (TME), altering homeostasis and immune response in the extracellular matrix (ECM) of surrounding tissue. A potential factor that contributes to TME generation and ECM remodeling is the cytoskeleton-associated human death-associated protein kinase 1 (DAPK1). Increased tumor cell motility and de-adhesion (thus, promoting metastasis), as well as upregulated plasminogen-signaling, are shown when functionally analyzing the DAPK1 ko-related proteome. However, the systematic investigation of how tumor cells actively modulate the ECM at the tissue level is experimentally challenging since animal models do not allow direct experimental access while artificial in vitro scaffolds cannot simulate the entire complexity of tissue systems. Here, we used the chorioallantoic membrane (CAM) assay as a natural, collagen-rich tissue model in combination with all-optical experimental access by multiphoton microscopy (MPM) to study the ECM remodeling potential of colorectal tumor cells with and without DAPK1 in situ and even in vivo. This approach demonstrates the suitability of the CAM assay in combination with multiphoton microscopy for studying collagen remodeling during tumor growth. Our results indicate the high ECM remodeling potential of DAPK1 ko tumor cells at the tissue level and support our findings from proteomics.
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MacNeil IA, Khan SA, Sen A, Soltani SM, Burns DJ, Sullivan BF, Laing LG. Functional signaling test identifies HER2 negative breast cancer patients who may benefit from c-Met and pan-HER combination therapy. Cell Commun Signal 2022; 20:4. [PMID: 34998412 PMCID: PMC8742957 DOI: 10.1186/s12964-021-00798-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2021] [Accepted: 11/01/2021] [Indexed: 11/29/2022] Open
Abstract
Background Research is revealing the complex coordination between cell signaling systems as they adapt to genetic and epigenetic changes. Tools to uncover these highly complex functional linkages will play an important role in advancing more efficacious disease treatments. Current tumor cell signal transduction research is identifying coordination between receptor types, receptor families, and transduction pathways to maintain tumor cell viability despite challenging tumor microenvironment conditions. Methods In this report, coactivated abnormal levels of signaling activity for c-Met and HER family receptors in live tumor cells were measured by a new clinical test to identify a subpopulation of breast cancer patients that could be responsive to combined targeted therapies. The CELsignia Multi-Pathway Signaling Function (CELsignia) Test uses an impedance biosensor to quantify an individual patient’s ex vivo live tumor cell signaling response in real-time to specific HER family and c-Met co-stimulation and targeted therapies. Results The test identified breast tumors with hyperactive HER1, HER2, HER3/4, and c-Met coordinated signaling that express otherwise normal amounts of these receptors. The supporting data of the pre-clinical verification of this test included analyses of 79 breast cancer patients’ cell response to HER and c-Met agonists. The signaling results were confirmed using clinically approved matching targeted drugs, and combinations of targeted drugs in addition to correlative mouse xenograft tumor response to HER and c-Met targeted therapies. Conclusions The results of this study demonstrated the potential benefit of a functional test for identifying a subpopulation of breast cancer patients with coordinated abnormal HER and c-Met signaling for a clinical trial testing combination targeted therapy.
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