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

Abstract

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.

RhoC modulates metabolic networks in cervical cancer by transcriptionally regulating the expression of genes involved in metabolism

“…The protein RhoC has been shown to be involved in multiple tumor phenotypes in cervical cancer including that of proliferation, invasion, migration, anoikis-resistance and resistance to radiation therapy (Pranatharthi et al, 2019; Srivastava et al, 2010). Transcriptional profiling of SiHa cells (a cervical carcinoma cell line) over-expressing RhoC (SiHa-R) in comparison to SiHa cells containing the background vector alone (SiHa-N), indicates global up-regulation of genes in SiHa-R cells (Pranatharthi et al, 2019). Further analysis of this data divulges the significantly enriched metabolic status of SiHa-R cells.…”

“…RhoC is a protein that has been shown to be involved in varied tumor phenotypes including metastasis, EMT, anoikis resistance and radioresistance to name a few (Pranatharthi et al, 2019; Srivastava et al, 2010). To explore the role of RhoC in oncometabolism, a transcriptomic profile of cervical cancer cell lines-one over-expressing RhoC and its control, was performed.…”

“…To explore the role of RhoC in oncometabolism, a transcriptomic profile of cervical cancer cell lines-one over-expressing RhoC and its control, was performed. Based on the transcriptomic data, it was seen that RhoC regulates many processes like DNA doublestranded break repair and cell cycle progression (Pranatharthi et al, 2019).…”

“…Beginning from tumor formation till metastasis, RhoC is required for all the intervening steps, which include invasion, anoikis resistance, intravasation, extravasation and homing (Reymond et al, 2015; Srivastava et al, 2010; Thomas, Pranatharthi, Ross, & Srivastava, 2019). We have recently observed that RhoC provides radioprotection to tumor cells, a hallmark of cancer stem cells (Pranatharthi et al, 2019). The regulation of migration and invasion by RhoC has been shown in several tumors including breast, cervix, inflammatory breast cancer, lung and ovarian to name a few (Bravo-Cordero, Hodgson, & Condeelis, 2014; Chen, Cheng, Zhang, Li, & Geng,; Faried et al, 2006; Gou et al,; Han et al, 2014; Li et al, 2013; Srivastava et al, 2010; Tumur et al,; Vega, Fruhwirth, Ng, & Ridley, 2011; Yang et al).…”

ROCK2 promotes osteosarcoma growth and glycolysis by up-regulating HKII via phospho-PI3K/AKT signalling

“…The stimulation of contraction and adhesion is achieved by inducing the formation of actin stress bres and focal adhesions of broblasts, as well as by modulating surface integrins to aggregate platelets and lymphocytes. Recent research has con rmed that ROCK2 is highly expressed in liver, colon and kidney cancer, and that its expression is closely associated with poor patient prognosis [36,37]. And ROCK2 silencing signi cantly inhibits tumour cell growth and metastasis, and promotes cell apoptosis.…”