Dynamic transitions of tumour cells along the epithelial–mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance.
In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation.
Our study demonstrates a prominent hybrid epithelial–mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial–mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion.
This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.
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Stanta, G. & Bonin, S. Overview on clinical relevance of intra-tumour heterogeneity. Front. Med. 5, 85 (2018).
Seoane, J., De & Mattos-Arruda, L. The challenge of intratumour heterogeneity in precision medicine. J. Intern. Med. 276, 41–51 (2014).
Marusyk, A., Almendro, V. & Polyak, K. Intra-tumour heterogeneity: a looking glass for cancer? Nat. Rev. Cancer 12, 323–34 (2012).
Lyons, J. G., Lobo, E., Martorana, A. M. & Myerscough, M. R. Clonal diversity in carcinomas: its implications for tumour progression and the contribution made to it by epithelial-mesenchymal transitions. Clin. Exp. Metastasis. 25, 665–77 (2008).
Jia, D., Jolly, M. K., Kulkarni, P. & Levine, H. Phenotypic plasticity and cell fate decisions in cancer: insights from dynamical systems theory. Cancers 9, 70 (2017).
Pastushenko, I. & Blanpain, C. EMT transition states during tumour progression and metastasis. Trends Cell Biol. 29, 212–26 (2019).
Chaffer, C. L., San Juan, B. P., Lim, E. & Weinberg, R. A. EMT, cell plasticity and metastasis. Cancer Metastasis Rev. 35, 645–54 (2016).
Martorana, A. M., Zheng, G., Crowe, T. C., O’Grady, R. L. & Lyons, J. G. Epithelial cells up-regulate matrix metalloproteinases in cells within the same mammary carcinoma that have undergone an epithelial-mesenchymal transition. Cancer Res. 58, 4970–9 (1998).
Lamouille, S., Xu, J. & Derynck, R. Molecular mechanisms of epithelial-mesenchymal transition. Nat. Rev. Mol. Cell. Biol. 15, 178–96 (2014).
Pastushenko, I., Brisebarre, A., Sifrim, A., Fioramonti, M., Revenco, T., Boumahdi, S. et al. Identification of the tumour transition states occurring during EMT. Nature 556, 463 (2018).
Asli, N. S. & Harvey, R. P. Epithelial to mesenchymal transition as a portal to stem cell characters embedded in gene networks. Bioessays 35, 191–200 (2013).
Jolly, M. K., Somarelli, J. A., Sheth, M., Biddle, A., Tripathi, S. C., Armstrong, A. J. et al. Hybrid epithelial/mesenchymal phenotypes promote metastasis and therapy resistance across carcinomas. Pharm. Ther. 194, 161–84 (2019).
Sha, Y., Haensel, D., Gutierrez, G., Du, H., Dai, X. & Nie, Q. Intermediate cell states in epithelial-to-mesenchymal transition. Phys. Biol. 16, 021001 (2019).
Saitoh, M. Involvement of partial EMT in cancer progression. J. Biochem. 256-264 (2018).
Liao, T. T. & Yang, M. H. Revisiting epithelial-mesenchymal transition in cancer metastasis: the connection between epithelial plasticity and stemness. Mol. Oncol. 11, 792–804 (2017).
George, J. T., Jolly, M. K., Xu, S., Somarelli, J. A. & Levine, H. Survival outcomes in cancer patients predicted by a partial EMT gene expression scoring metric. Cancer Res. 77, 6415–28 (2017).
Nankivell, P. & Mehanna, H. Oral dysplasia: biomarkers, treatment, and follow-up. Curr. Oncol. Rep. 13, 145–52 (2011).
Mehanna, H. M., Rattay, T., Smith, J. & McConkey, C. C. Treatment and follow-up of oral dysplasia - a systematic review and meta-analysis. Head Neck 31, 1600–9 (2009).
Wong, T. & Wiesenfeld, D. Oral cancer. Aust. Dent. J. 63, S91–S9 (2018).
Elkashty, O. A., Ashry, R. & Tran, S. D. Head and neck cancer management and cancer stem cells implication. Saudi Dent. J. 31, 395–416 (2019).
Lee, S. Y., Jeong, E. K., Ju, M. K., Jeon, H. M., Kim, M. Y., Kim, C. H. et al. Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionising radiation. Mol. Cancer 16, 10 (2017).
Esmatabadi, M. J., Bakhshinejad, B., Motlagh, F. M., Babashah, S. & Sadeghizadeh, M. Therapeutic resistance and cancer recurrence mechanisms: unfolding the story of tumour coming back. J. Biosci. 41, 497–506 (2016).
Willert, K. & Nusse, R. Beta-catenin: a key mediator of Wnt signaling. Curr. Opin. Genet. Dev. 8, 95–102 (1998).
Polakis, P. Wnt signaling and cancer. Genes Dev. 14, 1837–51 (2000).
Sedgwick, A. E. & D’Souza-Schorey, C. Wnt signaling in cell motility and invasion: drawing parallels between development and cancer. Cancers 8, 80 (2016).
Zhan, T., Rindtorff, N. & Boutros, M. Wnt signaling in cancer. Oncogene 36, 1461–73 (2017).
Martin-Orozco, E., Sanchez-Fernandez, A., Ortiz-Parra, I. & Ayala-San Nicolas, M. WNT signaling in tumours: the way to evade drugs and immunity. Front. Immunol. 10, 2854 (2019).
Mirabelli, C. K., Nusse, R., Tuveson, D. A. & Williams, B. O. Perspectives on the role of Wnt biology in cancer. Sci. Signal. 12, 589 (2019).
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–5 (2012).
Pfaffl, M. W., Tichopad, A., Prgomet, C. & Neuvians, T. P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–15 (2004).
Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–9 (2006).
Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).
Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: Precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).
Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–9 (2000).
Grigore, A. D., Jolly, M. K., Jia, D., Farach-Carson, M. C. & Levine, H. Tumour budding: the name is EMT. Partial EMT. J. Clin. Med. 5, 51 (2016).
Berx, G., Staes, K., van Hengel, J., Molemans, F., Bussemakers, M. J., van Bokhoven, A. et al. Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1). Genomics 26, 281–9 (1995).
Liu, C. Y., Lin, H. H., Tang, M. J. & Wang, Y. K. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation. Oncotarget 6, 15966–83 (2015).
Weiswald, L. B., Bellet, D. & Dangles-Marie, V. Spherical cancer models in tumour biology. Neoplasia 17, 1–15 (2015).
Bosch, F. X., Andl, C., Abel, U. & Kartenbeck, J. E-cadherin is a selective and strongly dominant prognostic factor in squamous cell carcinoma: a comparison of E-cadherin with desmosomal components. Int. J. Cancer 114, 779–90 (2005).
Ren, X., Wang, J., Lin, X. & Wang, X. E-cadherin expression and prognosis of head and neck squamous cell carcinoma: evidence from 19 published investigations. OncoTargets Ther. 9, 2447–53 (2016).
Nagaraja, S. S. & Nagarajan, D. Radiation-induced pulmonary epithelial-mesenchymal transition: a review on targeting molecular pathways and mediators. Curr. Drug Targets 19, 1191–204 (2018).
Zhang, X., Li, X., Zhang, N., Yang, Q. & Moran, M. S. Low doses ionising radiation enhances the invasiveness of breast cancer cells by inducing epithelial-mesenchymal transition. Biochem. Biophys. Res. Commun. 412, 188–92 (2011).
Lu, J., Zhong, Y., Chen, J., Lin, X., Lin, Z., Wang, N. et al. Radiation enhances the epithelial- mesenchymal transition of A549 cells via miR3591-5p/USP33/PPM1A. Cell. Physiol. Biochem. 50, 721–33 (2018).
Nieman, M. T., Prudoff, R. S., Johnson, K. R. & Wheelock, M. J. N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression. J. Cell Biol. 147, 631–44 (1999).
Kuo, L. J. & Yang, L. X. Gamma-H2AX - a novel biomarker for DNA double-strand breaks. Vivo 22, 305–9 (2008).
Albini, A. & Benelli, R. The chemoinvasion assay: a method to assess tumour and endothelial cell invasion and its modulation. Nat. Protoc. 2, 504–11 (2007).
Steinhart, Z. & Angers, S. Wnt signaling in development and tissue homeostasis. Development 145, 11 (2018).
Chen, B., Dodge, M. E., Tang, W., Lu, J., Ma, Z., Fan, C. W. et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5, 100–7 (2009).
Moncharmont, C., Levy, A., Gilormini, M., Bertrand, G., Chargari, C., Alphonse, G. et al. Targeting a cornerstone of radiation resistance: cancer stem cell. Cancer Lett. 322, 139–47 (2012).
Mandal, M., Ghosh, B., Anura, A., Mitra, P., Pathak, T., & Chatterjee, J. Modeling continuum of epithelial mesenchymal transition plasticity. Integr. Biol. 8, 167–76 (2016).
Jolly, M. K., Preca, B. T., Tripathi, S. C., Jia, D., George, J. T., Hanash, S. M. et al. Interconnected feedback loops among ESRP1, HAS2, and CD44 regulate epithelial-mesenchymal plasticity in cancer. APL Bioeng. 2, 031908 (2018).
Garg, M. Epithelial, mesenchymal and hybrid epithelial/mesenchymal phenotypes and their clinical relevance in cancer metastasis. Expert Rev. Mol. Med. 19, e3 (2017).
Puram, S. V., Tirosh, I., Parikh, A. S., Patel, A. P., Yizhak, K., Gillespie, S. et al. Single-cell transcriptomic analysis of primary and metastatic tumour ecosystems in head and neck cancer. Cell 171, 1611–24.e24 (2017).
Bhatia, S., Monkman, J., Blick, T., Pinto, C., Waltham, M., Nagaraj, S. H. et al. Interrogation of phenotypic plasticity between epithelial and mesenchymal states in breast cancer. J. Clin. Med. 8, 893 (2019).
Prieto-Garcia, E., Diaz-Garcia, C. V., Garcia-Ruiz, I. & Agullo-Ortuno, M. T. Epithelial-to-mesenchymal transition in tumour progression. Med Oncol. 34, 122 (2017).
Guo, W., Keckesova, Z., Donaher, J. L., Shibue, T., Tischler, V., Reinhardt, F. et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015–28 (2012).
Gupta, P. B., Pastushenko, I., Skibinski, A., Blanpain, C. & Kuperwasser, C. Phenotypic plasticity: driver of cancer initiation, progression, and therapy resistance. Cell Stem Cell 24, 65–78 (2019).
Zhao, C., Li, X., Su, C., Li, J., Cheng, N., Ren, S. et al. High expression of E-cadherin in pleural effusion cells predicts better prognosis in lung adenocarcinoma patients. Int. J. Clin. Exp. Pathol. 8, 3104–9 (2015).
Gabbert, H. E., Mueller, W., Schneiders, A., Meier, S., Moll, R., Birchmeier, W. et al. Prognostic value of E-cadherin expression in 413 gastric carcinomas. Int. J. Cancer 69, 184–9 (1996).
Li, Z., Yin, S., Zhang, L., Liu, W. & Chen, B. Prognostic value of reduced E-cadherin expression in breast cancer: a meta-analysis. Oncotarget 8, 16445–55 (2017).
Yang, L., Wang, X. W., Zhu, L. P., Wang, H. L., Wang, B., Zhao, Q. et al. Significance and prognosis of epithelial-cadherin expression in invasive breast carcinoma. Oncol. Lett. 16, 1659–65 (2018).
Noh, M. G., Oh, S. J., Ahn, E. J., Kim, Y. J., Jung, T. Y., Jung, S. et al. Prognostic significance of E-cadherin and N-cadherin expression in gliomas. BMC Cancer 17, 583 (2017).
Querzoli, P., Coradini, D., Pedriali, M., Boracchi, P., Ambrogi, F., Raimondi, E. et al. An immunohistochemically positive E-cadherin status is not always predictive for a good prognosis in human breast cancer. Br. J. Cancer 103, 1835–9 (2010).
De Cecco, L., Nicolau, M., Giannoccaro, M., Daidone, M. G., Bossi, P., Locati, L. et al. Head and neck cancer subtypes with biological and clinical relevance: Meta-analysis of gene-expression data. Oncotarget 6, 9627–42 (2015).
Zheng, X., Carstens, J. L., Kim, J., Scheible, M., Kaye, J., Sugimoto, H. et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525–30 (2015).
Kroger, C., Afeyan, A., Mraz, J., Eaton, E. N., Reinhardt, F., Khodor, Y. L. et al. Acquisition of a hybrid E/M state is essential for tumourigenicity of basal breast cancer cells. Proc. Natl Acad. Sci. USA 116, 7353–62 (2019).
Richard, G., Dalle, S., Monet, M. A., Ligier, M., Boespflug, A., Pommier, R. M. et al. ZEB1-mediated melanoma cell plasticity enhances resistance to MAPK inhibitors. EMBO Mol. Med. 8, 1143–61 (2016).
Hashimoto, A., Hashimoto, S., Sugino, H., Yoshikawa, A., Onodera, Y., Handa, H. et al. ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance. Oncogenesis 5, e259 (2016).
Jia, W., Deshmukh, A., Mani, S. A., Jolly, M. K. & Levine, H. A possible role for epigenetic feedback regulation in the dynamics of the epithelial-mesenchymal transition (EMT). Phys. Biol. 16, 066004 (2019).
Chung, V. Y., Tan, T. Z., Ye, J., Huang, R. L., Lai, H. C., Kappei, D. et al. The role of GRHL2 and epigenetic remodeling in epithelial-mesenchymal plasticity in ovarian cancer cells. Commun. Biol 2, 272 (2019).
Maier, P., Hartmann, L., Wenz, F. & Herskind, C. Cellular pathways in response to ionising radiation and their targetability for tumour radiosensitization. Int. J. Mol. Sci. 17, 102 (2016).
Sia, J., Szmyd, R., Hau, E. & Gee, H. E. Molecular mechanisms of radiation-induced cancer cell death: a primer. Front. Cell Dev. Biol. 8, 41 (2020).
Eriksson, D. & Stigbrand, T. Radiation-induced cell death mechanisms. Tumour Biol. 31, 363–72 (2010).
Kang, Y. P., Yoon, J. H., Long, N. P., Koo, G. B., Noh, H. J., Oh, S. J. et al. Spheroid-induced epithelial-mesenchymal transition provokes global alterations of breast cancer lipidome: a multi-layered omics analysis. Front. Oncol. 9, 145 (2019).
Melissaridou, S., Wiechec, E., Magan, M., Jain, M. V., Chung, M. K., Farnebo, L. et al. The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer. Cancer Cell Int. 19, 16 (2019).
McKelvey, K. J., Hudson, A. L., Back, M., Eade, T. & Diakos, C. I. Radiation, inflammation and the immune response in cancer. Mamm. Genome 29, 843–65 (2018).
Rodriguez, J. A. HLA-mediated tumour escape mechanisms that may impair immunotherapy clinical outcomes via T-cell activation. Oncol. Lett. 14, 4415–27 (2017).
Zhao, Z., Wang, S., Lin, Y., Miao, Y., Zeng, Y., Nie, Y. et al. Epithelial-mesenchymal transition in cancer: role of the IL-8/IL-8R axis. Oncol. Lett. 13, 4577–84 (2017).
Thomson, S., Petti, F., Sujka-Kwok, I., Mercado, P., Bean, J., Monaghan, M. et al. A systems view of epithelial-mesenchymal transition signaling states. Clin. Exp. Metastasis 28, 137–55 (2011).
Gonzalez, D. M. & Medici, D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci. Signal. 7, re8 (2014).
McFaline-Figueroa, J. L., Hill, A. J., Qiu, X., Jackson, D., Shendure, J. & Trapnell, C. A pooled single-cell genetic screen identifies regulatory checkpoints in the continuum of the epithelial-to-mesenchymal transition. Nat. Genet. 51, 1389–98 (2019).
Barker, H. E., Paget, J. T., Khan, A. A. & Harrington, K. J. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat. Rev. Cancer 15, 409–25 (2015).
Zhao, Y., Yi, J., Tao, L., Huang, G., Chu, X., Song, H. et al. Wnt signaling induces radioresistance through upregulating HMGB1 in esophageal squamous cell carcinoma. Cell Death Dis. 9, 433 (2018).
Chang, H. W., Roh, J. L., Jeong, E. J., Lee, S. W., Kim, S. W., Choi, S. H. et al. Wnt signaling controls radiosensitivity via cyclooxygenase-2-mediated Ku expression in head and neck cancer. Int. J. Cancer 122, 100–7 (2008).
Santiago, L., Daniels, G., Wang, D., Deng, F. M. & Lee, P. Wnt signaling pathway protein LEF1 in cancer, as a biomarker for prognosis and a target for treatment. Am. J. Cancer Res. 7, 1389–406 (2017).
Xu, L., Zhang, L., Hu, C., Liang, S., Fei, X., Yan, N. et al. WNT pathway inhibitor pyrvinium pamoate inhibits the self-renewal and metastasis of breast cancer stem cells. Int. J. Oncol. 48, 1175–86 (2016).
Microscopy analysis, genomics, flow cytometry and irradiation experiments were performed at the Westmead Scientific Platforms, which are supported by the Westmead research hub and Westmead Institute for Medical Research, the Cancer Institute New South Wales and the National Health and Medical Research Council.
Ethics approval and consent to participate
The ethics protocol for the human OSCC tumour samples was approved by the University of Western Australia human research ethics committee (Protocol #RA/4/1/8562) and written consent from the patient was obtained in accordance with the Declaration of Helsinki.
A copy of raw data containing fastq files has been deposited on SRA under BioProject PRJNA611666.
The authors declare no competing interests.
This study was supported by the University of Sydney COMPACT research seed grant, Sydney Dental School, University of Sydney, research support and the Dr. Poyner award from Australian Dental Research Foundation (ADRF). F.Z. is supported by the University of Sydney international scholarship. G.J. is funded via a Sydney West Translational Cancer Research Centre (SW-TCRC) Ph.D. Scholarship. Cancer Institute NSW funds SW-TCRC.
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Zolghadr, F., Tse, N., Loka, D. et al. A Wnt-mediated phenotype switch along the epithelial–mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma. Br J Cancer 124, 1921–1933 (2021). https://doi.org/10.1038/s41416-021-01352-7