Cervical cancer is the second leading cause of cancer-associated mortality in women worldwide, with 530,000 cases and 275,000 deaths in 2008.1 More alarmingly, the highest incidence rate of cervical cancer, far surpassing any type of cancer, is observed in women younger than 45 years.1 It is also one of the biggest health threats to women in developing countries, especially in Asia, with more than 50% of cervical cancer cases worldwide.2 It is reported that tumor metastasis has led to as much as 90% of cancer-related deaths.3 For cervical cancer, invasion and migration play critical roles in the progression of cancer metastasis. Parametrial spread and lymph node metastases are important factors influencing therapeutic effect and prognosis of cervical carcinoma.4,5
Rho belongs to a subfamily of Ras-like small GTPases and is a major regulator of the actin cytoskeleton. As a member of the Rho family, RhoC is mainly involved in regulating the motility and migration of cells. Recent studies showed RhoC expressed in an abnormally high level in many malignant tumor tissues, such as breast cancer, gastric cancer, and hepatocellular carcinoma.6–8 Increasing evidences suggested that RhoC overexpression can promote invasion and metastasis of tumor cells.9
Rho-associated coiled coil–forming protein kinase (ROCK) is one of the classic downstream molecules of Rho. It is the first discovered regulatory protein of actin cytoskeletons. The activation of ROCK pathway may induce the polymerization of actin stress fibers, thereby enhancing cell migration.10 Moreover, this process does not rely on the extracellular proteolysis.11 Earlier studies also found that the messenger RNAs of RhoC and ROCK were significantly elevated in renal cancer cells, which were closely related to tumor histologic grades and clinical stages.12
Our previous study showed that the expressions of RhoC and ROCK1 were significantly up-regulated in cervical squamous cancer (SCC) tissues and precancerous lesions compared with normal cervical tissues. In addition, the expressions of the 2 proteins were positively correlated,13 implicating that it is valuable to investigate the role of this pathway in the pathogenesis of cervical carcinoma. In the present study, our results showed that RhoC and ROCK1 exactly influenced the process of cervical cancer cells CaSki invasion and migration, suggesting the potential involvement of RhoC/ROCK signaling pathway in the carcinogenesis and progression of SCC.
MATERIALS AND METHODS
Cervical Carcinoma Cell Lines and Plasmids
The CaSki SCC cell line was obtained from Beijing Cancer Hospital and Research Institute (Beijing, China). The pcDNA3.1+-HA vector and pcDNA3.1-HA-RhoC plasmid were gifts from Dr Sofia D Merajave of University of Michigan in the United States.
Cell Culture and Reagents
CaSki cells were cultured in 1× Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% CO2 atmosphere. All transfections were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following instructions provided by the manufacturer.
After washing with cold PBS, cells were incubated for 30 minutes on ice in a lysis buffer containing 20 mM/L Tris-HCl (pH 7.5), 150 mM/L NaCl, 1% NP-40, 1% (volume per volume) sodium deoxycholate, 0.1% (weight per volume) SDS, 50 mM/L NaF, 1 mM/L Na3VO4, 50 mg/mL PMSF, 1 mg/mL leupeptin, and 1 mg/mL pepstatin. Subsequently, cells were homogenized with a 23-G needle and centrifuged at 12,000 rpm for 15 minutes at 4°C. The lysate was resolved with a SDS-PAGE gel, which was then blotted and probed with appropriate antibodies. The primary antibodies used were EIF-5 (Clone AC-74; Santa Cruz Biotechnology Inc, Santa Cruz, CA), ROCK1 (N6786; Abcam Inc, Cambridge, MA), p-MYPT1 (PC-36; Cell Signaling Technology Inc, Cambridge, MA), MYPT (PC-36; Cell Signaling Technology Inc), and RhoC (Sc-26481; Santa Cruz Biotechnology Inc).
Cells were cultured to 95% confluence before scratched with a 10 μL pipette tip. Wounds were observed by a microscope and photographed at 0, 12, and 24 hours. The nude areas were measured using the Image-Pro-Plus program (Media Cybernetics Inc, Rockville, MD), and the percentage of wound closure was calculated.
Matrigel-coated transwell filters with 8 mm pores (BD Biosciences, San Jose, CA) were used for the invasion assay. Cells (1 × 105/L) suspended in serum-free DMEM were layered in the upper compartment of transwell inserts. The bottom chambers contained 1× DMEM with 10% fetal bovine serum and served as the chemoattractants. After incubation for 12 or 24 hours at 37°C, invaded cells at the bottom of the upper chamber were stained with hematoxylin and counted under a phase contrast microscope.
Cells (3 × 103/L) were seeded in each well of a 96-well plate and incubated for different periods. At a given time point, cells were washed with PBS before 200 μL of MTT (Sigma Aldrich Inc, St Louis, MO) working solution (0.5 mg/mL) was added. The mixture was incubated at 37°C for 4 hours. Subsequently, the medium was removed, and pellets were dissolved in 200 μL of isopropanol. The absorbance was then measured at the wavelength of 490 nm.
All data are presented as mean ± SD (x ± s). One-way analysis of variance was performed with the SPSS 17.0 software package (Chicago, IL). Results were considered statistically significant when P value is less than 0.05.
Effect of RhoC on the Invasion and Migration of CaSki Cells
RhoC Overexpression in CaSki Cells
To understand the potential effects of RhoC overexpression on cervical carcinoma cells, pcDNA3.1-HA-RhoC plasmid was used to transfect the CaSki cells for 48 hours, whereas empty pcDNA3.1-HA vector was used as negative control. Western blot analysis was performed to confirm the overexpression of RhoC. RhoC protein expression was significantly elevated in CaSki cells transfected with the wild-type RhoC plasmid compared with the parental CaSki cells (control) or cells transfected with empty pcDNA3.1-HA vector (Fig. 1).
Effects of RhoC on the Migration of CaSki Cells
We further determined the impact of RhoC overexpression on the migration of CaSki cells by scratch wound healing assay. CaSki cells overexpressing wild-type RhoC showed significantly higher migration rate than those transfected with the control vector after scratching for 24 hours, suggesting that RhoC overexpression could remarkably promote the migration of CaSki cells (Fig. 2).
Effects of RhoC on the Invasion of CaSki Cells
Transwell invasion assay was conducted to examine the effect of RhoC expression on the invasiveness of cervical carcinoma cells. The ability of CaSki cells to invade through the transwell Matrigel was observed and recorded. As shown in Figure 3, CaSki cells with different levels of RhoC expression showed different invasiveness in 12 and 24 hours. Compared with the control, more RhoC-overexpressing CaSki cells invaded through the Matrigel, indicating their increased invasiveness.
RhoC Enhanced the Expression of ROCK1
Rho-associated serine threonine protein kinase 1 is a typical downstream protein of RhoC. We measured the protein levels of RhoC, ROCK1, and p-MYPT1, a protein directly downstream of ROCK1, after transient transfection of RhoC for 48 hours. RhoC overexpression significantly up-regulated the protein expressions of ROCK1 and p-MYPT compared with the control (Fig. 4), indicating that RhoC may mediate the expressions of the 2 protein kinases.
Rho-Associated Serine-Threonine Protein Kinase Signaling Pathway Enhanced the Invasion and Migration of CaSki Cells
Inhibitory Effect of Y-27632 on CaSki Cell Proliferation
A specific inhibitor of ROCK, Y-27632, can inhibit its effector Rho kinase. MTT assay was performed to determine the appropriate concentration of Y-27632 to inhibit ROCK signaling pathway (Fig. 5), 10 μM were selected according to the assay. We further confirmed the validity of this concentration by examining the phosphorylation level of MYPT1 (Fig. 6A and B). MYPT1 phosphorylation was significantly reduced in CaSki cells, suggesting that the selected concentration were effective in blocking the ROCK pathway.
Rho-Associated Serine-Threonine Protein Kinase Signaling Pathway Was Involved in the RhoC-Mediated Invasion and Migration
We blocked the ROCK pathway with Y-27632 and then performed scratch and transwell assay on CaSki cells to investigate whether the ROCK signaling pathway was involved in the process of RhoC-enhanced metastasis. The increased invasion and migration induced by RhoC overexpression could be reversed by the blockage of the ROCK signaling pathway with Y-27632 (Fig. 6C and D), suggesting that ROCK participated in the migration process. Furthermore, transwell invasion assay also showed that RhoC overexpression could remarkably increase the invasiveness of CaSki cells, whereas the addition of Y-27632 could reverse this process (Fig. 6E and F).
Cervical cancer is still one of the greatest threats to women health, although the incidence in Europe14 and the United States15 has been declining because of the effectiveness of screening by cytology. Invasiveness in peritonsillar tissue and metastasis to other organs are important factors for the high recurrence rate of cervical cancer.4,5 The molecular mechanism of cervical cancer cell metastasis has become the key topic in current research. Hölters et al16 and Shi et al17 demonstrated that tetraspanin 1 was related to cell surface proteins integrin and aquaporin 8 participated in the process of cervical cancer cell motility and invasion. The loss of ZBRK1, a zinc finger protein that interacts with breast cancer 1 (BRCA1) and KRAB-ZFP–associated protein 1 (KAP1), contributed to the increased expression of KAP1, potentiating its role in enhancing the metastasis and invasion of cervical cancer cells.18
Recent findings indicated that RhoC was associated with the SCC.19,20 RhoC was found highly expressed in squamous carcinoma of cervical intraepithelial neoplasia II/III than the normal cervical epithelium and cervical intraepithelial neoplasia I. The expression of RhoC in SCC was significantly correlated with pelvic lymph node metastasis and deep stromal infiltration. Results in our study showed that the expression level of RhoC could affect the invasion and migration of CaSki cells, suggesting RhoC’s regulatory role in SCC progression. This is consistent with previous findings in ductal adenocarcinoma,21 liver cancer,22 and breast cancer,23 which demonstrated that RhoC expression is linked to tumor progression and prognosis. The higher level of RhoC expression appeared in the later tumor stage and accompanied with worse prognosis.
RhoC could regulate the actin cytoskeleton through activating the profilin to form actin stress fibers and inhibiting actin depolymerization.24–26 In addition, RhoC-mediated myofilament contraction plays an important role in the retraction and migration of cells.27 Rho-associated serine-threonine protein kinase is involved in cellular processes of cell morphogenesis, adhesion, and migration. It has been shown to activate actin myosin contractility and focal adhesion involved in cell movement. Rho-associated serine-threonine protein kinase also promotes migration by restricting the integrin activity and membrane protrusions to the leading edge.28,29
The Rho/ROCK signaling pathway plays an important role in tumor growth and metastasis by regulating actin cytoskeleton reorganization.30 This effect has been proved in non–small cell lung cancer, human malignant glioblastoma, and bladder cancer.31–33 However, the specific mechanism of RhoC/ROCK signaling pathway in cervical cancer is still unclear. Our study revealed that RhoC could regulate the invasion and migration of CaSki cells through the ROCK signaling pathway, and ROCK1 played an important role in this process. The results suggest that the RhoC/ROCK1 pathway may play a key role in mediating SCC progression. This finding is consistent with the results from Croft et al10 and Sahai et al,11 who concluded that ROCK was involved in various cellular biologic behaviors through regulating cytoskeleton. The activation and inhibition of ROCK could respectively promote and inhibit the migration of tumor cells.34–36
Clinical data showed that SCC, one of the most pathologic types in cervical cancers, accounts for approximately 80% to 90% of cervical cancers.37 Human papillomaviruses (HPV) infection is the most important risk factor for cervical carcinoma. Some epidemiological studies of cervical cancer have showed that more than 99% of patients with SCC were infected by high-risk HPV (HPV 16 [46%–63%], HPV 18 [10%–14%], and HPV 31 [2%–7%]).38–40 Considering the high incidence of SCC caused by HPV 16 infections, we selected the HPV 16+ CaSki cell line, an SCC cell line, as the cell model in this study. However, a limitation to this study is that adenocarcinoma of the uterine cervix, a fraction of cervical carcinoma caused by other HPV subtypes (HPV 18, HPV 31, and HPV 35), was not included, which need further investigation for the role of RhoC/ROCK pathway.
Invasion and migration are signs of advanced tumor progression. They are also the major causes of clinical treatment failures and patient deaths. This study shows that the RhoC/ROCK pathway plays an important regulatory role in the progression of cervical cancer metastasis, and suggests that blocking the RhoC downstream signaling pathway by small molecule inhibitors could be a feasible treatment strategy to inhibit tumor invasion and migration.
This research was supported by grants from the National Natural Science Foundation of China (nos. 11072006, 10772007, 81070078, and 81270157) and National Basic Research Program of China (973 Program, 2013CB933702).
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Keywords:© 2014 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.
Cervical squamous cell carcinoma; RhoC; ROCK1; Invasion; Migration