RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer
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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.
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.
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.
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.
KeywordsRadioresistance DNA repair RhoC ROCK2 Cervical cancer
Dulbecco’s Modified Eagle Medium
Fetal Bovine Serum
ROCK2 high cells
ROCK2 low cells
Squamous cell carcinoma
SiHa Neo cells
SiHa RhoC cells
Cervical cancer is the most frequently diagnosed cancer in women in the underdeveloped and developing nations of the world today . This disease is on the rise with approximately 85% of new cases reported in the less developed regions of the world . As per the Cancer Facts & Figures 2019, American Cancer Society, the loco-regional response is very good (92%), however, the 5-year survival is 56% and 17% when diagnosed with regional and distant stage disease respectively. In India, women mostly present with a regional spread of the disease and have a poor 5 -year survival rate of 46% . Concurrent chemoradiation (CCRT) is the standard of care for patients in FIGO stages IB2 to IVA. However, it must be noted that CCRT is therapeutically limited by the stage of cancer as the size of the tumor has been shown to be an important prognostic factor [4, 5, 6]. Additionally, tumor heterogeneity due to intrinsic molecular mechanisms leading to radioresistance may contribute to further CCRT limitations. This leads to the question- what can be done besides CCRT to aid better prognosis of advanced tumors. Novel approaches in terms of radiosensitizers and molecular pathway targeted therapies, alone, or in combination with platinum compounds need to be identified to address this issue.
The radioresistance phenomenon has been extensively studied in several tumors and the role of various signaling pathways has been illustrated. In the lung cancer model, AKT1 has been implicated in double-strand break repair via phosphorylation of DNA-PKc . Also, the expression of a fragment of XRCC4 in breast cancer was seen to control radiation response by blocking the activity of Ligase IV of the NHEJ pathway . However, there is dearth of literature to explain the regulation of radioresistance in cervical cancer. One such signaling pathway that has contributed to tumor progression in several cancers is the Rho GTPase pathway.
Rho GTPases including RhoA and RhoC, are a class of highly conserved small molecule proteins that regulate various physiological processes and are also implicated in cancer progression and metastasis [9, 10]. RhoA has been associated with poor prognosis of prostate cancer  and its inhibition results in a decrease in tumor phenotype in gastric cancer . RhoB plays an opposing role in tumor progression mostly acting as a tumor suppressor . RhoC, another member of the Rho GTPase family of proteins, is known to govern both normal cell physiology and disease progression [14, 15, 16, 17, 18, 19]. Binding of GTP/GDP enables the protein to swiftly transition between “ON and OFF” states respectively, thus allowing it to efficiently control various downstream signal transduction pathways . RhoC has been demonstrated to regulate migration, cell cycle progression and various transcriptional networks in cancer cells [20, 21]. It is involved in the advancement of various tumor types like breast, gastric and ovarian cancer amongst others [14, 15, 22, 23, 24]. It has been ascertained that RhoC affects response to chemotherapy in the breast cancer model . Proteomic studies have revealed that RhoC is overexpressed in etoposide chemo-resistant non-small cell lung cancer . Interestingly, RhoC has also been reported to regulate stemness in ovarian, breast and head and neck cancers [23, 26, 27]. Our previous report shows that RhoC via Notch1 modulates angiogenesis, migration, invasion, metastasis, anoikis resistance and tumor growth in cervical cancer, leading to the progression of the disease . One of the most interesting observations was made by Hakem et al., who showed that RhoC is dispensable during the embryogenesis and initiation of a tumor but a mandate for metastasis . With such diverse functions it is intuitively easy to believe that RhoC may also regulate radioresistance, however there is no report on the role of RhoC in radioresistance.
Similarly, the role of Rho-associated kinases (ROCK) - effectors of Rho GTPases, has also been investigated extensively in cancers for more than a decade now. It has been reported that targeting ROCK signaling in melanomas led to decreased growth and metastasis . In bladder cancers, increased RhoA, RhoC, and ROCK signaling have been correlated with invasion and metastasis in clinical samples . ROCK1 and ROCK2 have been shown to play opposing roles in glioblastomas, where the knockdown of ROCK2 was seen to enhance proliferation whereas the inhibition of ROCK1 was seen to decrease the proliferation of glioblastoma cells . ROCK2 in association with CDK2, Cyclin E, NPM, and PLK2 has been shown to be important in the regulation of centrosomal duplication in varied cellular backgrounds [33, 34, 35, 36] suggesting its active role in cell cycle regulation.
In this study, our efforts are directed towards delineating the specific role of the RhoC-ROCK2 signaling in radiation response in cervical cancer. Radiation resistance remains to be the major challenge to successful treatment of cervical cancer. Ironically, there is no biomarker to predict the outcome of radiation therapy in cervical cancer. Considering that RhoC regulates several tumor phenotypes in cervical cancer and other tumors, we decided to explore the role of RhoC as a modulator of radiation response in cervical cancer using cell lines and patient-derived cells. Our results confirm that overexpression of RhoC induces radioresistance in cervical cancer cells and ROCK2 is the downstream target of RhoC in radiation response. We demonstrate that inhibition of ROCK2 sensitizes tumor cells to radiation therapy and that the RhoC-ROCK2 signaling pathway is of crucial importance in regulation of DNA repair in cervical cancer.
Materials and methods
Cell lines and reagents
SiHa and CaSki (cervical squamous cell carcinoma (SCC)), cell lines used in this study were cultured using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum) at 37 °C in 5% CO2 conditions. The cultures were routinely tested for mycoplasma contamination. Linear accelerator (LINAC) was used for irradiation of cell lines and patient samples. Cell viability assays were performed using WST-1 reagent (Roche). Y27632, a general inhibitor of ROCK was obtained from Calbiochem (CAS 146986–50-7). The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724). Annexin-V FITC conjugate was used from BD (556420) and Propidium Iodide (PI) from Sigma (P4170). pCAG-ROCK2 construct was a kind gift from Professor Anne Ridley, King’s College London.
Cell survival assay
Equal number of control and experimental cells (1 × 103 cells) were seeded in a 96-well format, 10 μl of WST1 was used for every 200 μl of media and incubation was done at 37 °C, 5% CO2 for 30 min-1 h. The plate was read using a microplate reader at 450 nm with background subtraction of 655 nm.
Equal numbers (1 × 103 cells) of SiHa-Neo (SiHa-N) and SiHa-RhoC (SiHa-R) cells were seeded in 90 mm sterile dishes and cultured at 37 °C under 5% CO2 conditions for 2 weeks. In the case of irradiation preceding seeding, 35 mm dishes were used. The colonies thus formed were fixed in 4% paraformaldehyde (PFA), stained using 0.05% crystal violet, imaged and counted.
Real time quantitative PCR
Sequences of primers used
ROCK2 Forward primer
ROCK2 Reverse primer
BRCA2 Forward primer
BRCA2 Reverse primer
RAD50 Forward primer
RAD50 Reverse primer
NBS1 Forward primer
NBS1 Reverse primer
GAPDH Forward primer
GAPDH Reverse primer
RPLP Forward primer
RPLP Reverse primer
ROCK2 Forward primer (mouse)
ROCK2 Reverse primer (mouse)
Cells washed with cold 1xPBS were incubated with lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1%(v/v) NP-40, 1%(w/v) sodium deoxycholate, 0.1% (w/v) SDS, 50 mM NaF, 1mM Na3VO4, 50 mg/ml PMSF, 1 mg/ml leupeptin, 1 mg/ml pepstatin) for 30 min on ice, homogenized with a 23-G needle and centrifuged at 14,000 rpm for 10 min at 4 °C. The lysate was resolved using SDS–PAGE, blotted and probed using the appropriate antibody. Fractionation westerns were performed as described in Suzuki et al.,  Histone3 and alpha-Tubulin were used as loading controls for the nuclear fraction and cytoplasmic fractions respectively.
Pre-extraction of cells
Prextraction of cells was performed after washing cells twice with 1xPBS. The cells were treated with CSK buffer  twice for 5 min each and then washed with 1xPBS. The cells were then fixed in 2% PFA for further use.
Cells were fixed in 4% PFA and washed with 1xPBS prior to staining. The cells were permeabilized using 0.2% Triton-X in 1xPBS (PBST) for 5 min. The cells were blocked using 10% FBS for 1 h. Primary antibodies were added and incubated at RT for 1-2 h. The cells were washed with 1xPBST. Secondary fluorescent antibodies were used at 1:500 dilution and incubated at RT for 45 min. The cells were washed with 1xPBS and mounted using anti-fade gold (Thermofisher Scientific).
Immunofluorescence analysis was performed on cryo-sections as described previously  from patient samples using citrate buffer (pH 6.0) for antigen retrieval by boiling for 20 min. The slides were cooled at RT for 30 min prior to permeabilization using 0.2% Triton-X 100 in 1xPBS. Following this, blocking was done in 10% FBS and primary antibodies were used at the requisite dilutions. The slides were then incubated overnight at 4 °C. After washes in 1xPBS, the secondary staining was done using secondary Alexa fluorophores and vectashield was used for mounting. Images were taken using the Zeiss 710 confocal microscope.
Antibody inhibition assay
Antibody inhibition using saponin has been performed for the first time to the best of our knowledge. The cells were live permeabilized using 1 μg of the ROCK2 antibody, resuspended in 1xDMEM complete media (10%FBS) containing 0.0025% saponin. The media was replaced with 1xDMEM complete media devoid of saponin after 18 h. IgG was used as the isotype control. The reduction in protein levels of ROCK2 was confirmed by immunoblotting.
The specific antibody binding was confirmed by permeabilizing the cells either with the isotype or the ROCK2 antibodies using 0.0025% saponin in 1xPBS for 1 h, following which the cells were lysed and incubated with 1.5 mg of Dyna beads for overnight at 4 °C. The eluate from the Dyna beads was then immunoblotted and probed using ROCK2 antibody.
The primary tumor samples obtained from patients were subjected to collagenase treatment at a concentration of 0.2 mg/ml using a magnetic stirrer at 37 °C until single cells were obtained. The cells were passed through cell filtration unit and the single cells thus obtained were counted. The cells were lineage depleted using the manufacturer’s protocol (Miltenyi biotech-lineage depletion kit human). The cells were then inhibited using the ROCK2 antibody followed by 6Gy irradiation on the next day and the cell survival was assayed after 3 days.
For flow cytometric analysis cells were stained using the immunofluorescence protocol as described above. The cells were acquired using Gallios or FC500 flow cytometers. Cell cycle analysis was performed using DRAQ5/Hoechst33342 according to the manufacturer’s protocol. The cell viability tests were performed using either Annexin V, Propidium Iodide (PI-1 mg/ml) or both.
Live cell sorting
We developed a novel method to sort live cells based on the intracellular antigen ROCK2 followed by culturing for further assays. For live cell staining of the intracellular antigen prior to sorting, cells were detached using 5 mM EDTA, counted and 2 × 106 cells were used per tube. The cells were then spun at 1500 rpm for 5 min at 4 °C and 0.0025% saponin in 1xPBS was added to the cells for 10 min. The cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was completely drained. The saponin solution containing 1 μg of the antibody was added to the cells and incubated at RT for 1 h. Following incubation, the cells were washed with 1xPBS twice and the secondary antibody was added at 1:500 dilution for 30 min. After three washes with 1xPBS, the cells were taken for sorting in 2% serum containing 1xPBS. Live sorting was performed using BD Aria flow cytometer.
Migration assay was performed in a 6 well transwell chamber (8-μm pore size) using 1 x105cells that were seeded on the top chamber in 1% FBS containing 1xDMEM. The bottom chamber contained 10% FBS in 1xDMEM. The cells were incubated for 16 h in the incubator at 37 °C under 5% CO2 conditions. At the specified time point the cells were fixed using 4% PFA followed by DAPI staining of the cells that migrated across the membrane, towards 10% FBS containing media. DAPI stained cells that migrated were counted for the estimation of number of migratory cells.
Transcriptomic and bioinformatic analysis
The transcriptomic analysis was performed using Illumina paired end sequencing (150 × 2). The sequenced reads were aligned to the Homo sapiens DRCh38 build genome downloaded from Ensemble database. An average of 91.77% of the reads aligned to the reference genome. Tophat was used to align the transcript sequences and cufflinks were used to create a combined assembly. A Differential Gene Expression (DGE) analysis was performed using Cuffdiff package. Using DAVID, a gene ontology analysis was performed for the upregulated genes and the genes that were specifically expressed in the treated pool. Heatmap analysis was done for the DGE genes, using Clustvis, R based bioinformatic tool. The transcriptomic analysis was performed in replicates of n = 2. STRING database (version 11.0) was used to study the interaction networks.
2 × 106 cells of both irradiated (IR) and non-irradiated (NR) SiHa cells were embedded in Matrigel to grow tumors subcutaneously in SCID mice. After 4 weeks mice were sacrificed, tumors excised and weighed. The tumors were fixed using PFA, cryo-sectioned and stained using routine immunofluorescence procedures as described earlier for the patient sample sections. Imaging was done using Zeiss 710 confocal microscope.
The mean and standard deviations have been computed for the experiments performed in triplicates and the significance was calculated using the t-test. p < 0.05 was considered significant.
RhoC governs the transcriptional network in cervical cancer cell line
Heterogeneous response to concurrent chemoradiation therapy (CCRT) is governed by the tumor stage and molecular heterogeneity within the tumor, consequently leading to poor prognosis in cervical cancer. The challenge to successful treatment of this disease is dependent on identifying signaling pathway alterations which regulate the resistance phenotype. We have earlier published that RhoC regulates tumor progression in cervical cancer . In the present study, we explore the role of RhoC as a regulator of radioresistance.
An understanding of the cellular functions requires an in depth understanding of the functional interactions between the proteins. This can be achieved by the STRING network analysis. Thus, the enriched genes were further uploaded on STRING  for visualization of the interaction network that may be driven by RhoC. Selection of DNA repair pathway (Fig. 1b-iii) highlighted the corresponding protein nodes in the network, which indicate the functional connections detected in this gene set. The genes highlighted include the important DNA repair and cell cycle genes like CDK1, TP53 and RAD50. This indicated that the RhoC overexpression regulates the DNA repair machinery in the cervical cancer cells. The selected DNA repair genes as shown in Fig. 1(b-iv) are highly upregulated in the SiHa-R cells. A quantitative PCR based expression analysis of a select few genes including RAD50, BRCA2 and NBS1 confirmed that these genes were indeed significantly upregulated in SiHa-R cells as compared to SiHa-N (Fig. 1c-i). Additionally, immunoblot analysis of pH2Ax, MRE11 and RAD50 also illustrated an upregulation of these proteins in SiHa-R cells (Figs. 1c(ii-iv)). pH2Ax, MRE11-RAD50-NBS1 (MRN) complex are well known regulators of DNA repair and are involved in the sensing and repairing of double-strand breaks (DSBs) in DNA [43, 44, 45]. Combined, these findings begin to define the role of RhoC in DNA repair and radiation response in cervical cancer.
RhoC regulates radiation response in cervical cancer cells
All irradiation experiments were carried out at 6Gy following a dose curve analysis which indicated that 6Gy was the LD50 for irradiation (Additional file 1: Figures S1b(i-ii)). The IR cells showed pH2Ax foci formation suggestive of radiation response in these cells (Additional file 1: Figure S1c). The resistance of the surviving fraction was confirmed by re-irradiating these cells followed by cell death analysis which suggested that there was no significant increase in cell death (Additional file 1: Figure S1d(i-ii)). The cells in the surviving fraction also showed enhanced clonogenic ability (Additional file 1: Figure S1e-i), and increased migration (Additional file 1: Figure S1e-ii). Considering that RhoC expression modulated radiation resistance of the tumor cells, we examined the changes in RhoC expression in the irradiated cells. We found an increased expression of this molecule as shown in Fig. 2a and Additional file 1: Figure S2a(i-ii).
To further demonstrate the contribution of RhoC to radiation response in cervical carcinoma, we used RhoC siRNA-based knockdown to evaluate its effect on cell survival (Fig. 2c-i). The SiHa cells treated with RhoC siRNA showed an increased apoptosis following irradiation thereby confirming that RhoC regulates radiation response in these cells. The specificity of the RhoC siRNA (previously published ;) was re-confirmed by immunodetection of both RhoC and RhoA (Fig. 2c-ii and Additional file 1: Figure S2b(i-ii)). Additionally, the knockdown of RhoC resulted in decreased pH2Ax foci formation in SiHa cells post-irradiation (Fig. 2c-iii). As shown in Fig. 1c-ii, the SiHa-R cells had increased pH2Ax as compared to SiHa-N cells.
We also analyzed the expression of DNA repair proteins using SiHa-R and SiHa-N xenografts, the tumor forming properties of which has been previously reported . Upon immunofluorescence staining, it was observed that RAD50, MRE11 and pH2Ax proteins were highly expressed in SiHa-R as compared to SiHa-N xenografts (Figs. 2d(i-ii) and Additional file 1: Figure S2c). Alternatively, RAD50 was seen to be down-regulated in CaSki-dnR cells as compared to the CaSki-N cells (Additional file 1: Figure S2d). Though the mechanism of regulation of gene expression by RhoC is not yet known, the above data reinforces our hypothesis that RhoC over-expression confers resistance to radiation by regulating the DNA repair protein expression. Combined, these findings begin to define the cellular consequences of RhoC driven transcriptional regulation of radioresistance.
ROCK2 regulates radioresistance in cervical cancer cells
Once the radioresistant phenotype of SiHa-R cells was confirmed by both molecular and functional approaches, we attempted to identify the downstream effector of RhoC in this context. The Rho-associated kinases, ROCK1 and ROCK2, are well established downstream targets of small GTPases including RhoC . These serine/threonine kinases are involved in several cellular mechanisms, with both ROCK1 and ROCK2 involved in regulation of cell migration , whereas ROCK2 plays a role in regulation of centromere duplication  and cell cycle progression via Cdc25A .
In order to study the effect of ROCK signaling on radiation response and radioresistance, we treated SiHa cells with Y27632 (ROCK inhibitor) . Treatment of SiHa cells with 10 μM Y27632 resulted in sensitization of these cells to radiation, resulting in increased cell death as seen by flow cytometric analysis (Fig. 3e). To specifically implicate ROCK2 in radioresistance, experiments were undertaken to alter the expression of ROCK2 alone and analyze the effect on cell survival post-irradiation. We noted that overexpression of ROCK2 in SiHa cells conferred resistance to these cells resulting in better survival post-irradiation, whereas inhibition of this protein sensitized cells to radiation, consequently leading to increased cell death. Transfection of pCAG-ROCK2  into SiHa cells, followed by irradiation resulted in decreased in cell death, indicating better resistance to irradiation in the background of ROCK2 over-expression (Fig. 3f). Q-PCR based analysis confirmed that transfection of pCAG-ROCK2 resulted in increased ROCK2 expression in SiHa cells (Additional file 1: Figure S3d). Alternatively, inhibition of ROCK2 via siRNA mediated silencing showed an increase in early apoptotic cells post-irradiation in comparison with the scrambled siRNA control (Additional file 1: Figure S3e(i-ii)).
To further confirm that ROCK2 is indeed important and regulates radioresistance in cervical cancer, we decided to use another approach to inhibit ROCK2 and observe the effect on cell survival. Given that Y27632 is an inhibitor of both ROCK2 and ROCK1 and siRNA has limitation with transfection of primary cells, we chose to use antibody mediated inhibition to this effect.
The use of antibodies to inhibit surface protein functions has been well explored and is currently in clinical use as targeted therapeutics . As early as 1994, the technique of inhibition of intracellular cyclin D was performed in cells by microinjection of the specific antibody . Also, the use of other detergents like TRITON-X have been employed for the delivery of macromolecules into the live cells . We worked on similar principles and used Saponin, which is a glycoside with mild detergent properties to perform reversible permeabilization of cells. Saponin at a concentration of 0.0025% (w/v) was used for the subsequent antibody mediated inhibition to study the effect of ROCK2 inhibition on cell survival. Flow cytometric analysis of cell death proved that saponin treatment alone did not affect cell viability under both non-irradiated and irradiated conditions (Figs. 3g-i and Additional file 1: Figure S3f). Cells were then treated with the ROCK2 and isotype IgG antibody in the presence of saponin. Western blot analysis shows the specific inhibition of ROCK2 by the antibody with no effect on the ROCK1 levels (Fig. 3g-ii). Additionally, in order to establish that the antibody did indeed permeate the cells treated cells were lysed after 1 h of treatment and dynabeads were used to immunoprecipitate ROCK2. Immunoblot analysis of the immunoprecipitate confirmed that the ROCK2 protein was pulled down in ROCK2 treated cells only, with no corresponding band in the IgG-treated lane, thus confirming that the antibodies entered and bound to the ROCK2 protein within live cells (Additional file 1: Figure S3 g). To test the effect of ROCK2 inhibition on radiation response, the treated SiHa cells were irradiated and cell death analysis was performed. Significant cell death was observed in the ROCK2 inhibited (ROCK2In) cells as compared to the IgG control (IgGIn), indicating sensitization of these cells to irradiation following ROCK2 antibody treatment (Fig. 3g (iii-iv)).
Additionally, we further tested the expression of ROCK2 in xenografts formed using non-irradiated (NR) versus irradiated (IR) SiHa cells. We found that the xenografts obtained from the surviving fraction of irradiated SiHa cells were significantly heavier (Figs. 3h-i and Additional file 1: Figure S3 h-i). An expression analysis using immunofluorescence and immunoblotting respectively, in sections and xenograft lysates, showed a marked increase in ROCK2 expression in the IR xenografts (Fig. 3h-ii and Additional file 1: Figure S3 h-ii). These data collectively suggest a role of ROCK2 in radiation response in cervical cancer.
Transcriptional gene networks support DNA repair in ROCK2 high cells
DNA repair and radioresistance in tumors are closely associated  and therefore, we tested the association of ROCK2 with DNA repair. To understand this relationship, we used transcriptomics-based approach to understand the status of DNA repair machinery in the ROCK2 expressing cells. A transcriptomic analysis was performed on the cells sorted based on ROCK2 expression, as high ROCK2 (ROCK2hi) and low ROCK2 populations (ROCK2lo).
The transcriptomic analysis of these two populations, ROCK2lo and ROCK2hi reflected global changes in the gene expression profiles. As shown in Additional file 1: Figure S4d, there was a distinct transcriptional network operational in ROCK2hi cells as compared to ROCK2lo cells. A GO annotation revealed that the differentially expressed genes clustered in several biological processes and significant amongst them were the genes involved in cellular response to DNA damage, mitotic cell cycle checkpoint, DNA damage checkpoint, DNA repair, DNA damage response-p53 and several others including WNT signaling and MAPK signaling pathways (Fig. 4c). The GO analysis provided an insight into the transcriptional status of the ROCK2hi cells and indicated that these cells have an enriched DNA repair transcriptional network. STRING network analysis was used to identify the transcriptional networks in these cells. String database allows building of networks and understanding the cellular functions using information about the interaction between expressed proteins. As shown in Fig. 4d-i, the genes enriched in the GO biological processes formed a well-defined network, with some of the DNA repair pathway genes forming a tightly clustered network. This analysis confirms the transcriptional status of the ROCK2hi cells and suggests a better DNA repair activity by these cells. The network also mapped the genes that were involved in cell cycle regulation and the TP53 signaling which is important in mediating a cell cycle response to DNA damage stimulus. A heatmap representation of the genes that formed a tight cluster, as shown by STRING network analysis depicts the increased expression levels of those genes in the ROCK2hi population (Fig. 4d-ii). These data support the existence of better DNA repair machinery for the regulation of resistance in the cervical cancer tumor cells with higher ROCK2 expression.
ROCK2 crosstalk with DNA repair assembly proteins to regulate radiation response in cervical cancer cells
ROCK2 expression results in an increased cell survival and cell cycle transition
To test if the ROCK2hi cells have an advantage in cell cycle progression and transition from G2 to M post-irradiation induced arrest, we investigated the expression of CDK1 in both populations. As shown in Fig. 4c, the transcriptomic data indicates that the genes facilitating G2/M transition are overexpressed in ROCK2hi cells. CDK1 is an indicator of the progression of cells from the G2 to the M phase of the cell cycle [69, 70, 71, 72]. Christopher Marshall’s group earlier reported that ROCK and CDK1 co-operate in tumor progression in melanoma and non-small cell lung cancer, in mice models . Consistently, we found that the cell population, co-expressing ROCK2 and CDK1, increased post irradiation as observed by both immunofluorescence and flow cytometry (Fig. 6c(i-ii)). Significantly the ROCK2hi cells had higher expression of phospho-Thr161 CDK1 (pCDK1)  as compared to ROCK2lo cells, under both irradiated and non-irradiated conditions as revealed by flow cytometry (Fig. 6c-iii and Additional file 1: Figure S6b-iii). The existence of a pool of cells with high ROCK2 and pCDK1 reveals the enhanced cell cycling capability of these cells.
Reports from Wang et al., suggest a functional interaction between ROCK2 and BRCA2 in late events of the cell cycle, particularly during centromere duplication . Such association was also observed in our study wherein BRCA2 was shown to co-express with ROCK2 in irradiated cells. The ROCK2 and BRCA2 proteins were found to have an increased interaction when subjected to irradiation (Additional file 1: Figure S6c and S6d). Our data thus affirms a role for ROCK2 in radiation response supported by better DNA repair, cell survival and cell cycle machinery.
ROCK2 is the downstream effector of RhoC in the context of radiation response
To further explore if ROCK2 could be a potential radiosensitization target, clinical samples were inhibited for ROCK2 and their response to radiation was analyzed, in vitro. The clinical specimens were disintegrated into a single cell suspension which was followed by a ROCK2 inhibition, using the antibody-based technique described earlier. The results suggest a significant increase in cell death over the IgG treated control cells (Fig. 8c-i). Concomitantly; cells were also treated with Cisplatin (20 μM) as concurrent chemo-radiation is the current treatment modality for cervical cancer. It was observed that additive treatment of cells with Cisplatin and ROCK2 inhibition resulted in a significant increase in cell death upon irradiation as compared to cells with Cisplatin treatment alone (Fig. 8c-ii).
Thus, our study establishes that RhoC regulates radio-resistance in cervical cancer via ROCK2’s crosstalk with DNA repair assembly proteins, however the exact mechanism of regulation of ROCK2 by RhoC is still under investigation.
Existing literature supports the hypothesis that heterogeneous response of tumor cells to radiation is due to existence of a subpopulation of cancer cells which are resistant to radiation . The combination of intrinsic properties including DNA repair, cell cycle status, survival pathways and extrinsic properties like stimulus from the microenvironment enable these cells to withstand radiation assault. There is surmounting data suggesting that a cell’s increased genomic stability is attained by utilizing DNA repair machinery. Understanding these processes and their mechanisms contribute to development of effective therapeutic strategies targeting advanced cancers. Although it is well accepted that RhoA plays an important role in radiation response , the role of RhoC, a RhoGTPase family protein, has not been explored in this context. RhoC and its downstream effector ROCK2 have been independently shown to regulate tumor progression in several tumor types [22, 24, 35, 49, 78, 79, 80]. Although ROCK2 plays an important role in cell division, the precise role of ROCK2 in DNA repair remains elusive. Our data proposes a novel role of RhoC in cervical cancer radioresistance and presents evidence to support ROCK2 as the downstream effector of RhoC in radioresistance.
Our key finding is that enhanced RhoC levels contribute to radioprotection in cervical cancer cells. It modifies the transcriptional network associated with DNA repair and cell cycle progression. ROCK2, the downstream effector of RhoC, crosstalks with DNA repair machinery to regulate radiation response, and its inhibition in primary tumors results in radiosensitization, in vitro. We also report that both RhoC and ROCK2 are localized in the nuclear compartment in a subset of tumor cells.
A recent report implicates RhoC in chemoresistance in non-small cell lung cancer , however its role in radioresistance is not elucidated in any tumor model. We have earlier reported that RhoC regulates several tumor phenotypes, and in this study, we extend the role of RhoC to radioresistance mediated through transcriptional regulation of DNA repair machinery.
Following irradiation, the DNA damage checkpoint responses and DNA repair machinery are important in cellular response to radiation [59, 81]. We show that RhoC contributes to radiation response in tumor cells by regulating the expression of important genes involved in DNA repair machinery. The comparison of DNA repair genes in SiHa cells with RhoC overexpression showed a marked upregulation of important DNA repair proteins including RAD50 and MRE11, essential components of the MRN complex . The overexpression of RhoC also led to increased levels of pH2Ax in SiHa cells while its inhibition resulted in reduced pH2Ax foci formation. The pH2Ax foci formation is the hallmark for radiation-induced DSBs in DNA and is also used to understand DNA repair kinetics . Our in vitro observations were supported by a transcriptomic analysis of the SiHa cells with stable overexpression of RhoC, which also revealed enrichment of genes regulating the DNA repair pathway. It is important to note that elevated RhoC expression resulted in a gamut of changes in various biological processes including cell proliferation, chromatin remodeling, planar cell polarity and cell cycle arrest, but importantly several biological processes related to DNA repair were also highlighted. G1/S transition, DSB repair via NHEJ pathway and cellular response to DNA damage were biological processes that were enriched in SiHa-R cells. Irrespective of detailed mechanistic, the findings herein demonstrate that RhoC selectively upregulates the transcriptional network of DNA repair.
Amongst the various downstream effectors of Rho GTPases, the Rho associated coiled coil kinases (ROCKs) play an important role in cancer progression and ROCK2 has been shown to be present in the nuclear compartment and is involved in the cell division process [34, 83, 84]. Further experiments were thus directed to discern the role of ROCK2 as the downstream effector of RhoC in cervical cancer radioresistance. A major consequence of inhibition of ROCK2 was increased radio-sensitization and consequently increased cell death of cancer cells, in vitro. Additionally, increased expression of ROCK2 enhanced cell survival post-irradiation. Importantly, the inhibition of ROCK2 resulted in abolition of radioresistant phenotype gained by SiHa-R cells. The finding that ROCK2 and RhoC have phenotypically similar consequences on radioresistance suggested that this pathway regulates DNA damage response induced by ionizing radiation. Intriguingly, a transcriptomic profiling of the ROCK2hi cells showed similar biological process enrichment as that observed in SiHa-R cells.
There are several reports on the role of ROCK2 in tumor progression and in normal differentiation [85, 86, 87, 88]. However, its role in radioresistance in the context of cervical cancer reported herein is a novel finding. It has been reported in hepatocellular carcinoma that ROCK2 regulates the cell division process by regulation of the ubiquitination of Cdc25A, a G1/S transition protein . The most interesting observation was reported by Wang et al., wherein they showed that BRCA2 and nucleophosmin (NPM) regulate centrosome duplication by forming a complex with ROCK2 . Also, ROCK2 has been shown to regulate centrosome duplication by modulation of CDK2/CyclinE complex . Though its interaction with the cell cycle regulatory machinery is well explored, the cross talk with DNA repair proteins has not been reported till date.
Radiation induced repair of damaged DNA by activation of DNA damage checkpoints, results in cell cycle arrest to enable the DNA repair process. The initial events comprise of sensing the damage followed by activation of p53 which results in cell cycle arrest at G1/S and G2/M allowing the cells to repair the DNA. A number of proteins like ATM, BRCA2/1, CHEK1/2 and RAD family proteins are involved in these processes . Additionally, pH2Ax foci formation is the hallmark DSB marker and also an early sensor of DNA damage . Our study extends the role of ROCK2 to DNA repair by presenting evidence of its crosstalk with the early DNA repair assembly proteins including pH2AX and the MRN complex. Inhibition of ROCK2 resulted in reduced pH2AX foci formation and MRE11-RAD50 proteins of the MRN complex which was also supported by transcriptomic analysis of the ROCK2hi and ROCK2lo cells, which displayed an upregulation of DNA repair proteins and enrichment of the DNA repair related biological processes. Our data clearly indicates that ROCK2 intercalates with the early DNA damage response machinery. However, irrespective of the mechanism of interaction with the early repair machinery, these findings support the role of ROCK2 in the early DNA repair process. It is intriguing to note that ROCK2 cooperates with several nuclear proteins to participate in DNA homeostasis related processes and is thus a promising target for biomarker development.
The consequences of ROCK2 inhibition was also validated using clinical samples wherein we found an increased radiosensitization of tumor cells. But the observation that ROCK2 inhibition and Cisplatin together radiosensitized the tumor cells better than Cisplatin alone, was the most exciting revelation. It enables us to take this study further and check the potential of the use of ROCK2 inhibition and Cisplatin, in vivo in mice models, and on a larger cohort to establish it as a therapeutic target.
The observation which was equally important to note was the expression of RhoC in nuclear compartments of the tumor cells. RhoC is classically a cytosolic protein which regulates cytoskeletal organization  and cancer cell motility . There is only one report which indicates the presence of RhoC in nuclear compartment in breast cancer cell lines . The observation that a fraction of RhoA is found in the nucleus and is activated by Net1 upon irradiation  lends support to our finding that RhoC, which is homologous to RhoA, may exist in the nucleus. Irrespective of the lack of exact mechanism of activation of nuclear RhoC, it is important to note that a subset of tumor cells, in the clinical samples, exhibit strong co-expression of ROCK2 and RhoC. The presence of RhoC is important in this context, wherein it might be required for activation of ROCK2 in the nucleus. The co-expression of the two proteins intuitively points towards the existence of a molecularly distinct tumor cell population. Similarly, the cells that are dual positive for ROCK2 and DNA repair may be identified as cells with better DNA repair abilities which may have intrinsic resistance ability, consequently leading to therapy evasion and relapse. Such cells with enhanced radioresistance are believed to be cancer stem cells which exhibit heterogeneity in their radiation response [92, 93, 94, 95, 96]. The co-immunoprecipitation of ROCK2, from nuclear fraction of the cells, along with RhoC supports our finding. However, the co-precipitated ROCK2 is ~ 120 kDa as opposed to 160 kDa of ROCK2. Studies on Rho kinases (ROCK1 and ROCK2) [97, 98] have shown the presence of a smaller fragment of ROCK, ~ 130 kDa, in the cells under various cytological conditions. However, in the context of radioresistance the form of ROCK2 that binds to RhoC needs thorough investigation.
Finally, findings herein strongly provide robust evidence, both clinical and cell line based, that RhoC and ROCK2 regulate radiation response and contribute to radioresistance in cervical cancer. Presently, there is no specific biomarker to stratify cervical cancer tumor for therapy response. As compared to breast tumors where expression of various markers (ER, PR, and Her2) can be a determinant of the therapy outcome , cervical cancer does not have a robust marker. RhoC and ROCK2 have the potential to be developed as candidate biomarkers for radioresistant tumors to predict therapy outcome. Combined, our findings suggest a non-canonical signaling pathway for RhoC which regulates radiation response in cervical cancer via ROCK2 mediated DNA repair regulation. Future work would aim at evaluating ROCK2 as a biomarker and therapeutic target in the context of radioresistance in cervical cancer and understanding the mechanistic of RhoC and ROCK2 in DNA repair regulation.
In this report, we investigated the role of RhoC and ROCK2 in radioresistance in cervical cancer. We have shown that the inhibition of RhoC and ROCK2 sensitizes tumor cells to irradiation. Based on transcriptomic analysis, we find that the gene network supporting DNA repair is enriched in RhoC overexpressing cells and this is also true for ROCK2hi cells, which have an enrichment of genes supporting DNA repair, cell survival and cell division. ROCK2 operates by crosstalk with early DSB repair proteins. We also propose that inhibition of ROCK2 abrogates the gain of phenotype, radioresistance, which is conferred by overexpression of RhoC, thus placing it as the downstream effector of RhoC in this context. Most interestingly, the inhibition of ROCK2 along with Cisplatin induced a better sensitization to irradiation as compared to cisplatin alone, in a fraction of clinical samples. Thus, the two molecules have the potential to be developed as biomarkers or therapeutic targets for the enhancement of response to radiation therapy in this cancer.
We thank Prof. Anne Ridley and Prof. Lawrence Banks for their valuable comments and suggestions. We would like to thank the NCBS Central Flow Facility for support with usage of the Flow Cytometer, NCBS animal house facility, and the Radiation Oncology Department, St. John’s Medical College Hospital for support with the LINAC radiation facility.
AP has contributed to experiment planning and execution, data analysis and manuscript preparation, PT has contributed to experiment planning and execution, data analysis and manuscript preparation, AHU has contributed to clinical sample collection, data collection and analysis, and manuscript preparation, CB has performed the bioinformatic analysis of the transcriptomic data, SBS has contributed to live sorting assays using Flow Cytometer, Prof. SK has contributed towards manuscript preparation, JT has contributed to experiment execution and data analysis, NS has contributed to clinical data analysis, CR has contributed to clinical data analysis, SS has conceptualized the project, contributed to experiment planning, data analysis and manuscript preparation. All authors read and approved the final manuscript.
This work was partly funded by the RGUHS advanced research wing, DST-SERB and DBT-BioCARE award granted to Dr. Sweta Srivastava and Dr. Avinash HU. This work was initially supported by a grant from DBT (Glue grant) awarded to Prof. Sudhir Krishna (National Centre for Biological Sciences) and Dr. Cecil Ross (St. John’s National Academy of Health Sciences). DST-ITS and ICMR travel grants were awarded to Dr. Sweta Srivastava. Ms. Annapurna Pranatharthi and Pavana Thomas have been supported by fellowships from CSIR-UGC and CSIR respectively.
All human patient sample studies have been approved by the Institutional Ethics Committee of St. John’s Medical College Hospital, Bangalore, India. Experiments were done in compliance with ethical guidelines and without any clinical interventions. Patient consent was obtained for all the cases in the study.
Mice experiments were performed with ethical clearance from the Institutional Animal ethics Committee (IAEC), National Centre for Biological Sciences, India.
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The authors declare that they have no competing interests.
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