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RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes

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Abstract

Emerging evidence illustrates that RhoC has divergent roles in cervical cancer progression where it controls epithelial to mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response. Cancer stem cells (CSCs) are the primary cause of recurrence and metastasis and exhibit all of the above phenotypes. It, therefore, becomes imperative to understand if RhoC regulates CSCs in cervical cancer. In this study, cell lines and clinical specimen-based findings demonstrate that RhoC regulates tumor phenotypes such as clonogenicity and anoikis resistance. Accordingly, inhibition of RhoC abrogated these phenotypes. RNA-seq analysis revealed that RhoC over-expression resulted in up-regulation of 27% of the transcriptome. Further, the Infinium MethylationEPIC array showed that RhoC over-expressing cells had a demethylated genome. Studies divulged that RhoC via TET2 signaling regulated the demethylation of the genome. Further investigations comprising ChIP-seq, reporter assays, and mass spectrometry revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes such as Nanog. Interestingly, clinical specimen-based investigations revealed the existence of a subset of tumor cells marked by RhoC+/Nanog+ expression. Finally, combinatorial inhibition (in vitro) of RhoC and its partners (WDR5 and TET2) resulted in increased sensitization of clinical specimen-derived cells to radiation. These findings collectively reveal a novel role for nuclear RhoC in the epigenetic regulation of Nanog and identify RhoC as a regulator of CSCs. The study nominates RhoC and associated signaling pathways as therapeutic targets.

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The datasets during and/or analyzed during the current study are available on Annotare 2.0.

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Acknowledgements

We would like to thank the Department of Science and Technology (DST), India for funding. Pavana Thomas has been awarded a fellowship by the Council for Scientific and Industrial Research (CSIR), India for the course of her PhD. Dr Sweta Srivastava was awarded a travel grant by the Indian Council for Medical Research (ICMR). We would like to thank the Central Imaging and Flow Facility (CIFF) at National Centre for Biological Sciences (NCBS), Bangalore for help with selected flow experiments.

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Authors and Affiliations

  1. Translational and Molecular Biology Laboratory (TMBL), Division of Molecular Biology and Genetics, St. John’s Research Institute (SJRI), St. John’s Medical College, Bangalore, 560034, India

    Pavana Thomas, Samyuktha Damodaran & Srinag Bangalore Suresh

  2. School of Integrative Health Sciences, The University of Trans-Disciplinary Health Sciences and Technology (TDU), Bangalore, 560064, India

    Pavana Thomas

  3. Translational and Molecular Biology Laboratory (TMBL), Division of Molecular Biology and Genetics, St. John’s Medical College Hospital, Bangalore, 560034, India

    Sweta Srivastava & Lokendra Yadav

  4. Department of Radiation Oncology, St John’s Medical College Hospital, Bangalore, 560034, India

    Avinash H. Udayashankara & Nirmala Srikantia

  5. Clinical Proteomics Unit, Division of Molecular Medicine, St. John’s Research Institute (SJRI), St. John’s Medical College, Bangalore, 560034, India

    Boby Mathew & Amit Kumar Mandal

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  1. Pavana Thomas
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Contributions

PT: concept and design, data acquisition, analysis and interpretation, drafting of the manuscript, final approval. SS: concept and design, data acquisition, analysis and interpretation, drafting of the manuscript, final approval. AHU: sample collection and clinical data analysis, revision of the manuscript, final approval. SD: data acquisition, analysis. LY: data acquisition, analysis. BM: acquisition and interpretation of 2D gel data, final approval. SBS: acquisition and analysis of flow cytometry data. AKM: acquisition and interpretation of 2D gel data. NS: sample collection and clinical data acquisition. All authors have read and approved the final manuscript.

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Correspondence to Sweta Srivastava.

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The study was approved by the Institutional Ethics Committee (IEC) at St. John’s Medical College. The samples were collected with patient consent and all experiments were carried out as per the guidelines.

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Thomas, P., Srivastava, S., Udayashankara, A.H. et al. RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes. Cell. Mol. Life Sci. 80, 1 (2023). https://doi.org/10.1007/s00018-022-04645-z

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