Genetic Variants in Caveolin-1 and RhoA/ROCK1 Are Associated with Clear Cell Renal Cell Carcinoma Risk in a Chinese Population

Background The RhoA/ROCK pathway and Caveolin-1 (Cav-1) participate in the process of tumorigenesis in numerous types of cancer. Up-regulation of RhoA/ROCK and Cav-1 expression is considered to be associated with the development and progression of clear cell renal cell carcinoma (ccRCC). We investigated the association between genetic variations of RhoA/ROCK and Cav-1 and the risk of ccRCC in the Chinese population. Methods Between May 2004 and March 2014, a total of 1,248 clear cell renal cell carcinoma cases and 1,440 cancer-free controls were enrolled in this hospital-based case-control study. Nine SNPs in RhoA/ROCK and Cav-1 were genotyped using the TaqMan assay. Result We found two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) were significantly associated with the increasing risk of ccRCC (P = 0.002 and P < 0.001 respectively). The analysis of combined risk alleles revealed that patients with 2–4 risk alleles showed a more remarkable growth of ccRCC risk than the patients with 0–1 risk alleles(OR = 1.66, 95%CI = 1.31–2.11, P < 0.001). Younger subjects (P = 0.001, OR = 1.83, 95%CI = 1.30–2.57), higher weight subjects (P = 0.001, OR = 1.76, 95%CI = 1.25–2.47), female subjects (P = 0.007, OR = 1.75, 95% CI = 1.17–2.62), nonsmokers (P < 0.001, OR = 1.67, 95%CI = 1.26–2.23), drinkers (P = 0.025, OR = 1.75, 95% CI = 1.07–2.85), subjects with hypertension (P = 0.025, OR = 1.75, 95% CI = 1.07–2.85) and diabetes (P = 0.026, OR = 4.31, 95% CI = 1.19–15.62) showed a stronger association between the combined risk alleles and the risk of ccRCC by using the stratification analysis. Furthermore, we observed higher Cav-1 mRNA levels in the presence of the rs1049334 A allele in normal renal tissues. Conclusion Our results indicate that the two SNPs (Cav-1 rs1049334 and ROCK1 rs35996865) and genotypes with a combination of 2–4 risk alleles were associated with the risk of ccRCC. The functional SNP rs1049334 may affect the risk of ccRCC by altering the expression of Cav-1 and the relevance between the risk effects and the functional impact of this polymorphism needs further validation.


Introduction
Renal cell carcinoma accounts for the majority, which is more than 80%, of the malignancy of renal and the clear cell type is the most common subtype. It is reported that clear cell renal cell carcinoma(ccRCC) has become the seventh most common cancer type for its steady increase annually, accounting for 270,000 newly diagnosed cases and estimated 116,000 cancer deaths [1]. In China, ccRCC is also a big public health issue because of high incidence and limited medical infrastructure and awareness.
As with most human cancers, the genesis of ccRCC is very complex, and the molecular mechanisms underlying disease occurrence are still largely unknown. It is reported that western people show higher morbidity compared with Chinese population, which may be a result of geographic, lifestyle and genetic variations. Studies associated with the risk factors of ccRCC demonstrated that smoking, drinking, HBP and high BMI might contribute to the tumorgenesis. However, only a small number of people who share the same risk factors suffer from this disease, indicating that genetic variation may be an assignable reason of the origin of tumor.
RhoA is a small GTP-binding protein that acts as a molecular switch that plays important roles in a diversity of cellular processes including motility, mitosis, proliferation and apoptosis. RhoA has a distinct set of effector kinases, including the ROCK, CITRON, and PRK1, all of which regulate cellular processes that contribute to tumorigenesis, invasion, and metastasis [2]. he RhoA/ROCK pathway participates in the process of angiogenesis in numerous types of cancer, by controlling the permeability, migration, proliferation, proliferation and morphogenesis of tumor cells [3]. Cav-1 is secreted as a biologically active molecule of caveolae that promotes cell survival and angiogenesis within the tumor microenvironment, and is overexpressed in the metastatic and primary sites of several tumors. Previous studies demonstrated that Cav-1 functioned as a positive effector of RhoA activation through the phosphorylation of cav-1 by Src kinases under certain circumstances [4]. The association between RhoA/ROCK/Cav-1 and the genesis of tumor have raised increasing concerns.
To our knowledge, there is no report examining the association of single nucleotide polymorphisms (SNPs) in ROCK1/RhoA and Cav-1 and ccRCC risk. Considering that ROCK1/ RhoA and Cav-1 may play an important role in initiating the cancer, in the present study, we performed a hospital-based case-control study and selected and genotyped 9 tagging SNPs (tSNP) located in the functional regions of these genes to investigate the association of genetic polymorphisms of ROCK1/RhoA and Cav-1 with ccRCC risk in Chinese population.

Ethics statement
The present study was approved by the Institutional Review Board of the Nanjing Medical University, Nanjing, China and each participant involved in this study gave a written informed consent prior to inclusion in the study.

Study Population
In this study, 1248 patients with clear cell renal cell carcinoma and 1440 cancer-free agematched controls were consecutively recruited from May 2004 to March 2014 at the First Affiliated Hospital of Nanjing Medical University. Patients with blood relationship or from the same region or the same families were excluded from this study in advance. All the diagnosis of clear cell renal cell carcinoma was established by pathological examination of samples resected by surgery and none of the patients had history of other cancers or relative therapy before. Each patient's ccRCC classification and staging were according to the TNM staging system by American Joint Committee on Cancer (AJCC). The Furman scale was used to assess nuclear grade of ccRCC. The 1440 controls were people who visited for regular health examination in the outpatient departments and declared free of any cancers. They were recruited frequency matched to the cases on age (±5 years) and gender. A written informed consent was given to each patient and all of them approved to donate 5ml venous blood, restoring in a condition of -20°C anticoagulated by EDTA.

DNA extraction and Genotyping
Genomic DNA of each individual was isolated and purified from 500 ml EDTA-anticoagulated peripheral blood samples using a DNA extraction kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions. Genotyping of the selected SNPs was conducted using the TaqMan SNP Genotyping Assay, which were performed on the 384-well LightCycler 480 Real Time PCR System (Roche, Penzberg, Germany). PCR was performed in a mixture containing 1.5μL SNP Genotyping Assay Mix, 1.5μL TaqMan Universal Master Mix, 1μL Dnase-free water and 1μL genomic DNA. The PCR conditions were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. The LightCycler480 Software (Version 1.5.0) was used to automatically collect and analyze the data and to generate the genotype calls. The quality control was performed in each plate by four negative controls to ensure the genotyping accuracy. Regenotyping was conducted in approximately 10% of the samples and all the results were 100% concordant.

Analysis of Cav-1 and ROCK1 expressions
To further assess the correlations between the Cav-1 and ROCK1 mRNA expressions and the polymorphisms of rs1049334 and rs35996865 in vivo, a total of 64 paratumor renal tissues adjacent to tumour containing 100% normal cells were obtained from patients. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The 1.5-μg total RNA was reverse transcribed in a final volume of 20 μl using random primers under standard conditions using the PrimeScript RT Master Mix (Invitrogen).
Statistical Analysis SPSS 16.0 (IL, CA, USA) was used for the statistical analysis. To assess the Hardy-Weinberg equilibrium of the genotype distribution, we performed a goodness-of-fit χ 2 test. The differences in frequency distributions of epidemiological factors like lifestyles and personal health condition between ccRCC cases and controls were tested by using the t test for continuous variables and the χ 2 -test for categorical variables, respectively. Odds ratios (ORs) and the 95% confidence intervals (CIs) for the association between the polymorphisms and risk of ccRCC was analyzed by unconditional logistic regression, adjusting for age, smoking and drinking status, family history and other factors. Additionally, the false discovery rate (FDR) was used to adjust the P value for multiple comparisons, based on the Benjamini-Hochberg method. The associations were considered statistically significant when Bonferroni FDR-adjusted P values were <0.05. We used the Student's t test or one-way analysis of variance (ANOVA) to explore associations between the two polymorphisms (rs35996865 and rs1049334) and the mRNA levels of ROCK1 and Cav-1. All statistical analyses were two-sided and P <0.05 was considered statistically significant.
Association between renal cell carcinoma risk and genetic polymorphisms of Cav-1 and ROCK1/RhoA A few genetic characteristics of the 9 selected tSNPs were presented in Table 2. The tSNP of rs1049337 in Cav-1 was excluded from further analysis because of the allele frequencies in control group not conforming to HWE (P <0.001). Genotype and allele distributions of the remaining 8 tSNPs in the patients and controls were detailed in Table 3. No significant differences in genotype and allele distributions of tSNPS in RhoA were observed between the cases and controls (rs2269736 P = 0.155, rs2410 P = 0.388, rs2625955 P = 0.595). We found two polymorphisms were significantly associated with ccRCC: rs1049334 in Cav-1 and rs35996865 in ROCK1 (P = 0.002 and P <0.001 respectively).  We found rs1049334 (G>A), which is located in Cav-1, was significantly associated with risk of ccRCC. Subjects with GA and AA genotypes had a significant increased ccRCC risk compared with those with GG (AA vs GG: adjusted OR = 1.68, 95% CI = 1.17-2.41; GA vs GG: adjusted OR = 1.30, 95% CI = 1.09-1.54). When combined the subjects with GA and AA genotypes, an increased risk of ccRCC was also observed in the combined group (adjusted OR = 1.33, 95% CI = 1.14-1.59). Alleles comparison showed similarly ccRCC risk rising (P < 0.001, OR = 1.32, 95%CI = 1.15-1.51). Another SNP (rs35996865) located in ROCK1 was also found to be associated with ccRCC risk. For this SNP, significant association was observed in the subjects with GG genotype and dominant model (GG vs.TT, OR = 5.41, 95%CI = 2.47-11.83); GG+ GT vs.TT, OR = 1.30, 95%CI = 1.09-1.54). A similar result was observed when compared G alleles with T alleles (P < 0.001, OR = 1.39, 95%CI = 1.17-1.65).

The analysis of combined polymorphisms and ccRCC risk
The two polymorphisms (rs1049334 and rs35996865) were combined based on the number of the risk alleles to explore the genetic power on the risk of ccRCC. As is shown in the Table 4, with the increasing of the number of the risk alleles, elevation of the ccRCC risk was observed and the difference was statistically significant. What's more, when we divided the patients with two groups, patients with 2-4 risk alleles showed a more remarkable growth of ccRCC risk than the patients with 0-1 risk alleles(OR = 1.66, 95%CI = 1.31-2.11, P < 0.001) comparing with other group combinations. After that, stratification analysis indicated that the increased  (Table 5). However, in patients with localized (stage I and II) or advanced stage (stage III and IV) and moderately (grade I and II) or poorly differentiated (grade III and IV) nuclear grade, no significant difference was observed (S1 Table).
Associations between Cav-1 rs1049334 and ROCK1 rs35996865 and the expression levels of corresponding mRNA In our study, 64 normal tumor-adjacent tissue samples were obtained to assess the associations between these two polymorphisms and the expression of Cav-1 and ROCK1 (Fig 1). According to the results, subjects with rs1049334 AA or AG genotypes had higher expression levels of  Cav-1 compared with those with GG genotypes (P = 0.003). However, the ROCK1 expression level in patients carrying three types of rs35996865 genotypes was similar (P = 0.713), suggesting that the influence of rs3599686 polymorphism on gene expression is weak.

Discussion
Caveolin-1 (Cav1) is a principal functional constituent of caveolae, which are invaginated plasma membrane microdomains and function as a regulator of signal transduction events and cytoskeletal dynamics [5,6]. Glenney first reported that Caveolin-1 was a novel substrate for the src kinase oncogene in virally transformed fibroblasts by binding to several key proteins [7]. Cav-1 has been proved to modulate multiple cancer-associated processes including cellular transformation, tumor growth, cell migration and metastasis, cell death and survival, multidrug resistance and angiogenesis in a number of signaling pathways [8,9]. Collective evidence from researches indicated that the elevated level of Cav-1 was involved in some unfavorable clinical characteristics like larger size, higher grade and stage, resistance to conventional therapies and poor prognosis of various types of malignancy in several organs including colon, liver, stomach, prostate, breast, lung, brain and kidney [10][11][12][13][14][15][16][17][18][19][20]. Although the role of Cav-1 plays in cancer is controversial [12], it is widely confirmed that overexpression of Cav-1 in renal cell carcinoma is associated with poor disease-free survival and metastasis [21][22][23]. Study in genetic variants revealed a significant association between Cav-1 polymorphisms and ccRCC susceptibility [24]. RhoA, which is a predominant member of the well-known Ras superfamily of small guanosine triphosphatases (GTPases), is a small G protein that can exhibit intrinsic GTPases activities that can function as a molecular switch in cellular processes including cytoskeletal dynamics, migration, vesicle trafficking, cell proliferation, apoptosis and transcription [25][26][27]. A number of studies have shown that RhoA had abilities to control cancer metastasis and progression [28][29][30] and the expression of RhoA was up-regulated in several common malignancies including gastric, pancreatic and breast cancer [31][32][33]. Rho-associated coiled-coil forming kinase, which is often referred to as ROCK, is a major effector in the Rho signaling way. ROCK1 is one of the ROCK family and plays a critical role in mediating the effects of RhoA, activated by binding to the Active (GTP-loaded) Rho [34,35]. The Rho/Rho-kinase pathway plays an important role in various cellular functions and is involved in several proinvasive pathways including src [36]. Accumulating evidences have shown that the interaction between Caveolin-1 and Rho-GTPases could regulate metastasis by controlling the activation of src in malignancies [37,38]. However, study on the association between genetic variants in Cav-1 with RhoA/ ROCK and susceptibility of ccRCC is insufficient. In our study, we genotyped nine polymorphisms in RhoA/ROCK1 and Cav-1 to explored the association between RhoA/ROCK1 and Cav-1 genetic variants and ccRCC susceptibility in Chinese population. The Cav-1 rs1049334 and the ROCK1 rs35996865 significantly differed between ccRCC patients and control participants, indicating that the risk of ccRCC is increased in participants with the A allele of rs1049334 and G allele of rs35996865. The further analysis of the combining risk alleles showed that the group of patients with 2-4 risk alleles was more susceptible to ccRCC compared with those with 0-1 risk alleles. Environmental and epidemiological factor also had certain effects on the risk of ccRCC according to our stratification analyses in combination alleles and Cav-1 rs1049334. Age, BMI, gender, smoking and drinking status, the history of HBP and diabetes are all related with the ccRCC susceptibility, implying that the interaction of the environment, hereditary background and genetic variants may be a complex system contributed to the occurrence of ccRCC.
Additionally, increasing Cav-1 mRNA level was found in individuals who carried the rs1049334 A allele in the preliminary functional analysis of the variant. Interestingly, stratification analyses of the association between the rs1049334 and the risk of ccRCC revealed a little different in history of hypertension and history of diabetes compared with that of risk alleles. We supposed that the role of RhoA/ROCK1 in influencing the level of plasma glucose and vascular contractility may contribute to this procedure [39][40][41]. No significant association between the polymorphisms and clinicopathological characteristics of ccRCC was observed, we surmise that the effect of rs1049334 on the expression of Cav-1 is not power enough to influence the disease progression.
Limitations should not be ignored in the present study. First, the sample is not large enough that bias there may exist in the analyzing of very low-penetrance SNPs and reduce the statistical power of combined analysis and stratification. Second, selection bias of subjects associated with a particular genotype could not be eliminated because our case-control study is hospitalbased. However in present study, except for rs1049337, the rest 8 tSNPs all conformed to HWE. The selection bias might not be substantial because the distribution of genotypes was all similar to the Hapmap database of Chinese population.
In conclusion, we investigated an association between Cav-1 and RhoA/ROCK1 polymorphisms and susceptibility, clinical characteristics in a large sample population of ccRCC patients. Cav-1 rs1049334 (G>A) and ROCK1 rs35996865 (T>G) were significantly associated with the elevated risk of ccRCC in Chinese population, and the combination of risk alleles indicated a positive influence on the ccRCC risk. Furthermore, functional polymorphism in Cav-1 rs1049334 (G>A) altered Cav-1 expression, which may contribute to the genesis of ccRCC. Further functional investigations are expected to confirm our results.
Supporting Information S1