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RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in...

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Autores principales: Pranatharthi, Annapurna, Thomas, Pavana, Udayashankar, Avinash H., Bhavani, Chandra, Suresh, Srinag Bangalore, Krishna, Sudhir, Thatte, Jayashree, Srikantia, Nirmala, Ross, Cecil R., Srivastava, Sweta
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729006/
https://www.ncbi.nlm.nih.gov/pubmed/31488179
http://dx.doi.org/10.1186/s13046-019-1385-7
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author Pranatharthi, Annapurna
Thomas, Pavana
Udayashankar, Avinash H.
Bhavani, Chandra
Suresh, Srinag Bangalore
Krishna, Sudhir
Thatte, Jayashree
Srikantia, Nirmala
Ross, Cecil R.
Srivastava, Sweta
author_facet Pranatharthi, Annapurna
Thomas, Pavana
Udayashankar, Avinash H.
Bhavani, Chandra
Suresh, Srinag Bangalore
Krishna, Sudhir
Thatte, Jayashree
Srikantia, Nirmala
Ross, Cecil R.
Srivastava, Sweta
author_sort Pranatharthi, Annapurna
collection PubMed
description BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response. METHODS: Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies. RESULTS: Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation. CONCLUSIONS: Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13046-019-1385-7) contains supplementary material, which is available to authorized users.
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spelling pubmed-67290062019-09-12 RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer Pranatharthi, Annapurna Thomas, Pavana Udayashankar, Avinash H. Bhavani, Chandra Suresh, Srinag Bangalore Krishna, Sudhir Thatte, Jayashree Srikantia, Nirmala Ross, Cecil R. Srivastava, Sweta J Exp Clin Cancer Res Research BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response. METHODS: Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies. RESULTS: Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation. CONCLUSIONS: Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13046-019-1385-7) contains supplementary material, which is available to authorized users. BioMed Central 2019-09-05 /pmc/articles/PMC6729006/ /pubmed/31488179 http://dx.doi.org/10.1186/s13046-019-1385-7 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Pranatharthi, Annapurna
Thomas, Pavana
Udayashankar, Avinash H.
Bhavani, Chandra
Suresh, Srinag Bangalore
Krishna, Sudhir
Thatte, Jayashree
Srikantia, Nirmala
Ross, Cecil R.
Srivastava, Sweta
RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title_full RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title_fullStr RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title_full_unstemmed RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title_short RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
title_sort rhoc regulates radioresistance via crosstalk of rock2 with the dna repair machinery in cervical cancer
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729006/
https://www.ncbi.nlm.nih.gov/pubmed/31488179
http://dx.doi.org/10.1186/s13046-019-1385-7
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