Genetic Variation Of Angiogenesis - Related Genes And The Efficacy And Toxicity Of Renal Cancer Incidence And Prognosis And Molecular Targeting Drugs

Renal cell carcinoma(RCC), which is also named renal carcinoma for short, is a common cancer of urinary system worldwide. It accounts for80%-90%of all renal space-occupying lesions and nearly3%of all new cancer diagnoses in the world. It is estimated that one hundred thousand people die of renal carcinoma each year. In china, there are about37.7men and16.6women in every one hundred thousand people per year are diagnosed with renal carcinoma. By contrast, the morbidity is higher in European and North American population and male incidence of renal carcinoma is significantly higher than the female’s. Currently, global incidence of renal carcinoma is increasing with the rate of25%per year on average and20%-30%patients have lost chance of radical operation at the time of diagnosis because of tumor metastasis. The rising morbidity and mortality of renal carcinoma bring not only serious health damage to patients, but also heavy economic burden the families and society. However, the etiology and pathogenesis of renal carcinoma remain unclear. In terms of the treatment, renal carcinoma is not sensitive to radiotherapy and chemotherapy and there are great individual differences with respect to molecular targeted therapy. Therefore, the research of proper biomarkers which can guide early diagnosis and individual therapy is vital significant.Recently, many studies have shown that renal carcinoma is associated with heredity, smoking, obesity, hypertension and antihypertensive therapy. However, only a few people will develop renal carcinoma among all people who possess similar predisposing factors, indicating that individual susceptibility to cancer is important in oncogenesis. The formation of renal carcinoma is a multifactor and multistage process. It is the outcome of combined impact of environmental risk factors and hereditary factors.Human Genome Project (HGP) has verified that genetic variation in human genome may have influence to the structure and function of genes which can further lead to changes of genetic biological function. Single nucleotide polymorphisms (SNPs) are one of the most common types in genetic variation. In the molecular genetics studies of disease, SNPs have been widely used as a molecular marker for gene location. Moreover, SNPs can help locate and identify specific disease related genes via comparing SNPs between patients and disease-free people. SNPs are able to affect the function of repairase by changing the genetic structure which results in increased risk of cancer. Present studies have discovered that angiogenesis is closely related to the occurrence and development of many tumors and a lot of new blood vessels are found in renal carcinoma tissues which demonstrates that angiogenesis plays an important role in the pathogenesis of renal carcinoma. In addition, our studies show that angiogenesis genes such as VEGFA and VEGFR are associated with the risk of renal carcinoma by regulating gene expression of themselves, and other genes such as VHL/HIF1A can affect the progress of renal carcinoma and the survival of patients through combined effects of several SNPs.This study aims to provide theoretical basis to reveal biological mechanisms of the development of renal carcinoma. Meanwhile, this study would supply important clues in screening of high-risk population susceptible to renal carcinoma and in individual prevention, intervention and treatment by looking for optimal biomarker.Part I Variants in angiogenesis-related genes and the risk of clear cell renal cell carcinomaClear cell renal cell carcinoma (ccRCC) accounts for about85%-95%of the malignant kidney tumor, to which angiogenesis plays a fundamentally important role in the pathogenesis. However, systemic studies on the association between angiogenesis-related genes and the risk of ccRCC are lacking. In the present study we expanded exploration to a greater number of genes putatively involved in angiogenesis. We selected7core genes (HIF1A, EPAS1, VEGFA, VEGFR1, VEGFR2, VEGFR3and PDGFRB) and analyzed24potentially functional SNPs in these genes for their association with the risk of RCC in a Chinese population.This case-control study comprised859ccRCC cases and1004cancer-free controls. We genotyped24potentially functional single nucleotide polymorphisms (SNPs) in7angiogenesis-related genes (HIF14, EPAS1, VEGFA, VEGFR1, VEGFR2, VEGFR3and PDGFRB) using the TaqMan or Snapshot method. Unconditional logistic regression was used to assess the associations. The functionality of selected SNPs was assessed by real-time quantitative RT-PCR and luciferase reporter gene assays.We found two SNPs (VEGFA rs2010963and VEGFR3rs448012) that were significantly associated with increased risk of ccRCC, after adjusting for multiple comparisons (rs2010963CC/GC vs. GG:P=0.048, OR=1.36,95%CI=1.12-1.66; rs448012CC/GC vs. GG:P=0.0448, OR=1.38,95%CI=1.13-1.69for). Real-time quantitative PCR revealed that the variant genotypes of rs2010963, but not rs448012, were associated with increased gene expression in ccRCC (CC/GC vs. GG:P=0.036). The luciferase reporter assay showed that the rs2010963C allele significantly increased luciferase activity than that of the rs2010963G allele.Our results indicate that VEGFA rs2010963and VEGFR3rs448012are associated with risk of ccRCC. Furthermore, rs2010963is a functional SNP that may affect ccRCC susceptibility by modulating endogenous VEGFA expression.Part Ⅱ The polymorphisms in the VHL and HIF1A genes are associated with the prognosis, but not development of renal cell carcinoma Hypoxia-inducible factor (HIF) could act as a transcription factor by modulating a lot many genes which were involved in angiogenesis and proliferation, survival, spread and apoptosis of the tumor cells. As a result, overexpression of HIF, activating such genes including the VEGF gene, the PDGF gene, the CXCR gene, the TGF-a gene and the EGFR gene, may be a key factor affecting on genesis and development of RCC. Protein VHL (pVHL) is fundamental to the process of the degradation of VHL. pVHL is coded by the von Hippel-Lindau tumor suppressor gene. Abnormality in expression and activity of pVHL caused by variability like mutation and deletion in VHL gene could give negative effect on the degradation of HIF-la, which play a pivotal role in renal carcinogenesis and prognosis. This study aimed to clarify the influence of VHL and HIF1A polymorphisms on RCC susceptibility and survival.We genotyped four potentially functional SNPs (rs779805located in the promoter region in VHL, rs11549465located in the exon region, rs11549467located in the exon region and rs2057482located in the3’-untranslated region in HIF1A) by Taqman-MGB probes.No significant differences in VHL or HIF1A genotypes were observed between RCC cases and controls. However, individuals with≥2variant alleles of the four polymorphisms were associated with less frequent lymph node metastasis and lower clinical stage (P=0.032and P=0.041, respectively). And the number of variant alleles were associated with improved survival in a dose-response manner (P trend=0.013). Furthermore, multivariate Cox regression analysis showed that the number of variant alleles (≥1versus0) was an independent prognostic factor for RCC survival (P=0.025) together with clinical stage and tumor grade.The VHL and HIF1A polymorphisms may not influence RCC susceptibility but may jointly influence RCC progression and survival. Given the vital role of VHL/HIF1A/VEGF pathway in angiogenesis and RCC progression and survival, combined with the influence of genetic variation in this pathway on the targeted agents efficacy, prediction of the RCC patients will be more accurate. It will also provide guidance to the molecular targeting treatment of advanced renal cell carcinoma.Part III Single nucleotide polymorphism associations with response and toxic effects in patients with advanced renal-cell carcinoma treated with sorafinibSorafenib is a new oral targeted anti-tumor agent, which has been approved by FDA in December2005applying to metastatic renal cell carcinoma treatment, and is the first molecular targeted drug used for patients with advanced renal cancer clinically in China. Sorafenib suppressed the growth of tumor through two synergistic pathways:(1) By suppressing the tyrosine kinase activity of Kit and Flt-3receptor, and suppressing the Ras/Raf/Erk/MEK signal pathway mediated by EGF as the inhibitor of the serine/threonine kinase of Raf in the downstream to inhibit the proliferation of tumor cell.(2) By suppress the function of VEGFR and PDGFR to inhibit the neo-angiogenesis. In this study, we investigate the association of the polymorphism of angiogenesis related and drug-metabolizing enzyme genes and the response and toxic effect of patients with advanced renal cancer who received Sorafenib treatment.This study comprised100patients with advanced renal cancer validated by pathology, among which63cases were male and the others were female. Clinical statistics was fully and accurately collected. A total of42potential function SNPs located in VEGF、VEGFR1, VEGFR2, VEGFR3、PDGFRA、PDGFRB、IL8、CYP3A4、 CYP3A5、CYP1A1、CYP1A2、ABCB1、ABCB2、HIF1A、EPAS1genes were selected: According to the characteristics of SNPs, three methods, PCR-RFLP, SNaPshot and PCR-sequencing, were applied in genotyping. The median progression free survival (PFS) and overall survival (OS) of the100cases were31.8and46.7months, respectively.The results showed that rs1570360located in5’flanking region of VEGF gene and rs2239702located in5’flanking region of VEGFR2gene were significantly associated with PFS of renal carcinoma patients. PFS of individuals with A allele was significantly shorter than the patients with G allele. Herein, rs2239702was significantly related to OS of renal carcinoma patients. OS of patients carry A allele is significantly shorter than patients with G allele (Log-Rank P=0.041). Besides, rs2010963located in promoter region of VEGF A gene is found to be closely relevant to risk of hand-foot-skin reaction after using sorafinib in advanced renal carcinoma patients(P=0.005), and rs1045642located in exon region of ABCB1gene is associated with hypertension, another side effect(P=0.028).Genetic polymorphisms may have an influence in tumor-targeted therapy. Specific genotypes of VEGF、VEGFR2and so on are likely to improve the patients’ reponse to drugs in some degree and lengthen PFS and OS of patients which can benefit to prognosis. In the meanwhile, specific genotypes of these genes are capable to reduce toxic effects of molecular targeted drug sorafinib.