Impairment of rigidity sensing caused by mutant TP53 gain of function in osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary malignant pediatric bone tumor and is characterized by high heterogeneity. Studies have revealed a wide range of phenotypic differences among OS cell lines in terms of their in vivo tumorigenicity and in vitro colony-forming abilities. However, the underlying molecular mechanism of these discrepancies remains unclear. The potential role of mechanotransduction in tumorigenicity is of particular interest. To this end, we tested the tumorigenicity and anoikis resistance of OS cell lines both in vitro and in vivo. We utilized a sphere culture model, a soft agar assay, and soft and rigid hydrogel surface culture models to investigate the function of rigidity sensing in the tumorigenicity of OS cells. Additionally, we quantified the expression of sensor proteins, including four kinases and seven cytoskeletal proteins, in OS cell lines. The upstream core transcription factors of rigidity-sensing proteins were further investigated. We detected anoikis resistance in transformed OS cells. The mechanosensing function of transformed OS cells was also impaired, with general downregulation of rigidity-sensing components. We identified toggling between normal and transformed growth based on the expression pattern of rigidity-sensing proteins in OS cells. We further uncovered a novel TP53 mutation (R156P) in transformed OS cells, which acquired gain of function to inhibit rigidity sensing, thus sustaining transformed growth. Our findings suggest a fundamental role of rigidity-sensing components in OS tumorigenicity as mechanotransduction elements through which cells can sense their physical microenvironment. In addition, the gain of function of mutant TP53 appears to serve as an executor for such malignant programs.

References

[1] Shao H, Ge M, Zhang J, Zhao T, Zhang S. Osteoclasts differential-related prognostic biomarker for osteosarcoma based on single cell, bulk cell and gene expression datasets. BMC Cancer, 2022, 22

[2] Shoaib Z, Fan TM, Irudayaraj JMK. Osteosarcoma mechanobiology and therapeutic targets. Br. J. Pharmacol., 2022, 179: 201-217

[3] Zhou Y et al. Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma. Nat. Commun., 2020, 11

[4] Lauvrak SU et al. Functional characterisation of osteosarcoma cell lines and identification of mRNAs and miRNAs associated with aggressive cancer phenotypes. Br. J. Cancer, 2013, 109: 2228-2236

[5] Wolfenson H, Yang B, Sheetz MP. Steps in mechanotransduction pathways that control cell morphology. Annu. Rev. Physiol., 2019, 81: 585-605

[6] Yang B et al. Stopping transformed cancer cell growth by rigidity sensing. Nat. Mater., 2020, 19: 239-250

[7] Cagigas ML et al. Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell-substrate adhesion in cancer cells and mechanosensitivity of cell proliferation. Nat. Mater., 2022, 21: 120-128

[8] Wolfenson H et al. Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat. Cell Biol., 2016, 18: 33-42

[9] Wang J et al. Anoikis-associated lung cancer metastasis: mechanisms and therapies. Cancers, 2022, 14: 4791

[10] Simpson CD, Anyiwe K, Schimmer AD. Anoikis resistance and tumor metastasis. Cancer Lett., 2008, 272: 177-185

[11] Díaz-Montero CM, McIntyre BW. Acquisition of anoikis resistance in human osteosarcoma cells. Eur. J. Cancer, 2003, 39: 2395-2402

[12] Marei HE et al. p53 signaling in cancer progression and therapy. Cancer Cell Int., 2021, 21

[13] Wang Z, Strasser A, Kelly GL. Should mutant TP53 be targeted for cancer therapy? Cell Death Differ., 2022, 29: 911-920

[14] Synoradzki KJ et al. TP53 in biology and treatment of osteosarcoma. Cancers, 2021, 13: 4284

[15] Diessner BJ et al. Nearly half of TP53 germline variants predicted to be pathogenic in patients with osteosarcoma are de novo: a report from the Children’s Oncology Group. JCO Precis Oncol., 2020, 20: 00087

[16] Chen Z, Guo J, Zhang K, Guo Y. TP53 mutations and survival in osteosarcoma patients: a meta-analysis of published data. Dis. Markers, 2016, 2016: 4639575

[17] Del Giudice I et al. Behind the scenes of non-nodal MCL: downmodulation of genes involved in actin cytoskeleton organization, cell projection, cell adhesion, tumour invasion, TP53 pathway and mutated status of immunoglobulin heavy chain genes. Br. J. Haematol., 2012, 156: 601-611

[18] Muller PA et al. Mutant p53 drives invasion by promoting integrin recycling. Cell., 2009, 139: 1327-1341

[19] Frisch SM, Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 1994, 124: 619-626

[20] Zhu Z et al. Anoikis and metastatic potential of cloudman S91 melanoma cells. Cancer Res., 2001, 61: 1707-1716

[21] Ko SY, Naora H. HOXA9 promotes homotypic and heterotypic cell interactions that facilitate ovarian cancer dissemination via its induction of P-cadherin. Mol. Cancer, 2014, 13

[22] Weiswald LB et al. A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation. Br. J. Cancer, 2013, 108: 1720-1731

[23] Moya IM, Halder G. Hippo-YAP/TAZ signalling in organ regeneration and regenerative medicine. Nat. Rev. Mol. Cell Biol., 2019, 20: 211-226

[24] Tang Q, Su Z, Gu W, Rustgi AK. Mutant p53 on the path to metastasis. Trends Cancer, 2020, 6: 62-73

[25] Yamamoto S, Iwakuma T. Regulators of oncogenic mutant TP53 gain of function. Cancers, 2018, 11: 4

[26] Sun T et al. Anoikis resistant mediated by FASN promoted growth and metastasis of osteosarcoma. Cell Death Dis., 2019, 10

[27] Müller DA, Silvan U. On the biomechanical properties of osteosarcoma cells and their environment. Int. J. Dev. Biol., 2019, 63: 1-8

[28] Jiang T et al. Impact of hydrogel elasticity and adherence on osteosarcoma cells and osteoblasts. Adv. Healthc. Mater., 2019, 8: e1801587

[29] Yang YA et al. Local contractions regulate E-cadherin rigidity sensing. Sci. Adv., 2022, 8: eabk0387

[30] Mitchell CB et al. Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments. J. Neurooncol., 2019, 141: 303-313

[31] Yiqi Z, Ziyun L, Qin F, Xingli W, Liyu Y. Identification of 9-gene epithelial-mesenchymal transition related signature of osteosarcoma by integrating multi cohorts. Technol. Cancer Res. Treat., 2020, 19: 1533033820980769

[32] Helfman DM, Flynn P, Khan P, Saeed A. Tropomyosin as a regulator of cancer cell transformation. Adv. Exp. Med. Biol., 2008, 644: 124-131

[33] Manstein DJ, Meiring JCM, Hardeman EC, Gunning PW. Actin-tropomyosin distribution in non-muscle cells. J. Muscle Res. Cell Motil., 2020, 41: 11-22

[34] Olive KP et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell, 2004, 119: 847-860

[35] Di Fiore R et al. Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells. Bone, 2014, 60: 198-212

[36] Wu X, Cai ZD, Lou LM, Zhu YB. Expressions of p53, c-MYC, BCL-2 and apoptotic index in human osteosarcoma and their correlations with prognosis of patients. Cancer Epidemiol., 2012, 36: 212-216

[37] Levantini E, Maroni G, Del Re M, Tenen DG. EGFR signaling pathway as therapeutic target in human cancers. Semin. Cancer Biol., 2022, 85: 253-275

[38] Cooper J, Giancotti FG. Integrin signaling in cancer: mechanotransduction, stemness, epithelial plasticity, and therapeutic resistance. Cancer Cell, 2019, 35: 347-367

[39] Zhang C et al. Mechanics-driven nuclear localization of YAP can be reversed by N-cadherin ligation in mesenchymal stem cells. Nat. Commun., 2021, 12

[40] Pappireddi N, Martin L, Wühr M. A review on quantitative multiplexed proteomics. ChemBiochem, 2019, 20: 1210-1224

[41] Franklin JM, Ghosh RP, Shi Q, Reddick MP, Liphardt JT. Concerted localization-resets precede YAP-dependent transcription. Nat. Commun., 2020, 11

[42] Adzhubei IA et al. A method and server for predicting damaging missense mutations. Nat. Methods, 2010, 7: 248-249

[43] Jumper J et al. Highly accurate protein structure prediction with AlphaFold. Nature, 2021, 596: 583-589

[44] Keenan AB et al. ChEA3: transcription factor enrichment analysis by orthogonal omics integration. Nucleic Acids Res., 2019, 47: W212-w224

[45] Tate JG et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res., 2019, 47: D941-d947

Funding

National Natural Science Foundation of China (National Science Foundation of China)(82202767)