Genetic Variations in the HIF 1 A Gene Modulate Response to Adjuvant Chemotherapy after Surgery in Patients with Colorectal Cancer

Colorectal cancer (CRC) is the third most common malignancy with about 500, 000 new cases diagnosed each year and the fourth leading cause of cancer-related death worldwide (Jemal et al., 2009; Pearson et al., 2009). Estimated 5-year survival rates range from 85% to 90% for patients with stage I disease. However, the 5-year survival rate is less than 5% for patients with stage IV disease due to the high recurrence rate (Mancuso et al., 2005; Zhang et al., 2012). Due to different tumor characteristics and patients’ genetic backgrounds, the clinical outcomes of cancer patients show a great quantity of heterogeneity. Therefore, novel biomarkers associated with tumor progression is urgently needed to distinguish high-risk patients who would benefit from adjuvant therapy.


Introduction
Colorectal cancer (CRC) is the third most common malignancy with about 500, 000 new cases diagnosed each year and the fourth leading cause of cancer-related death worldwide (Jemal et al., 2009;Pearson et al., 2009). Estimated 5-year survival rates range from 85% to 90% for patients with stage I disease. However, the 5-year survival rate is less than 5% for patients with stage IV disease due to the high recurrence rate (Mancuso et al., 2005;Zhang et al., 2012). Due to different tumor characteristics and patients' genetic backgrounds, the clinical outcomes of cancer patients show a great quantity of heterogeneity. Therefore, novel biomarkers associated with tumor progression is urgently needed to distinguish high-risk patients who would benefit from adjuvant therapy.

Genetic Variations in the HIF1A Gene Modulate Response to Adjuvant Chemotherapy after Surgery in Patients with Colorectal Cancer
Yi Zhang 1& , Peng Wang 2& , Xing-Chun Zhou 2 , Guo-Qiang Bao 3 , Zhuo-Ming Lyu 4 , Xiao-Nan Liu 5 , Shao-Gui Wan 6 , Xian-Li He 3 *, Qi-Chao Huang 2 * Hypoxia Inducible Factor-1 (HIF-1) is a key transcription factor that responds to changes in environmental oxygen and cell energy, which allows the cell to adapt and survive in hostile environment of normal or tumor tissues. It plays an important role in regulating angiogenesis, cell adhesion, metastatic spread and apoptosis (Semenza, 2003). HIF-1 consists of two subunits, HIF-1α and HIF-1β, both of which belong to the basic loop-helix Per-Aryl hydrocarbon nuclear translocator-Sim (PAS) protein family (O'Donnell et al., 2006). Hypoxic conditions allow HIF-1α to bind to HIF-1β and then become an active transcriptional factor, while the presence of oxygen leads to the degradation of HIF-1α. In a previous study, the immunohistochemical analysis in 68 CRC patients has shown that high expression levels of HIF-1α is positively associated with TNM stage, lymph node involvement, and distant metastasis (Wu et al., 2010). Similarly, another study has shown that HIF-1α over-expression is associated with the poor prognosis in a cohort of 731 CRC patients (Baba et al., 2010). In addition, HIF-1α has also been found to be associated with poor prognosis in several types of cancer, including hepatocellular carcinoma (Wada et al., 2006), gastric tumors (Zhang et al., 2013), non-small cell lung cancer (Li et al., 2013), and neuroendocrine breast cancer (Marton et al., 2012).
Single-nucleotide polymorphisms (SNPs) are attractive molecular markers for translational studies. The HIF1A gene is located on chromosome 14 and a number of SNPs associated with tumor development and progression have been identified in this gene. Polymorphisms C1772T (rs11549465) comprising an amino acid change from proline to serine at position 582 and G1790A (rs11549467) resulting in the substitution of alanine with threonine at position 588 have been found to be correlated with the CRC risk (Kuwai et al., 2004;Kang et al., 2011). Another study has demonstrated that there is a significant association between SNP 191T>C (rs2057482) in HIF1A gene and the risk of rectal cancer (Frank et al., 2010). Considering the important role of HIF-1α in the progression of CRC, it is reasonable that polymorphisms of HIF1A may affect the biological behavior and prognosis of CRC. Although several studies have found that SNPs in HIF1A gene are associated with risk of CRC, however, few studys have investigated the association between SNPs in HIF1A gene and outcomes of CRC. Only one study suggested that HIF1A1 C1772T and G1790A polymorphisms were not involved in the progression or metastasis of CRC (Szkandera et al., 2010). However, many other functional SNPs in HIF1A gene may impact the outcomes of CRC patients in Chinese cohort. In this study, we selected 3 functional SNPs in the HIF1A gene and evaluated their associations with prognosis and efficacy of adjuvant chemotherapy after surgery in a Chinese cohort of 697 primary CRC patients receiving radical surgery.

Study population
Between January 2007 and June 2012, a total of 697 patients who were diagnosed as primary colorectal cancer and had complete follow-ups and clinical information were recruited. This cohort consisted of 454 patients from Department of General Surgery at Tangdu Hospital and 243 patients from Department of General Surgery at Xijing Hospital and both hospitals are affiliated with Fourth Military Medical University, Xi'an, China. All the patients who had no prior history of other cancers were newly diagnosed and confirmed by pathologists. None of the patients had been treated by surgery, chemotherapy and/or radiotherapy before enrollment into the study. All patients' demographic and clinical data were collected from medical record. There were no age, gender, and cancer stage restrictions on recruitment. 5ml of blood were extracted from all patients for genomic DNA extraction using the E.Z.N.A. Blood DNA MidiKit (Omega Bio-Tek, Norcross, GA) in the laboratory. In this study, all analyses were restricted to Han Chinese because more than 99% of the ethnicity was Han. The Institutional Review Board at Fourth Military Medical University approved this study and all patients signed an informed consent form before enrollment.

SNP selection and genotyping
SNPs in HIF1A gene were selected using a set of web-based SNP selection tools (http://snpinfo.niehs. nih.gov/snpinfo/snpfunc.htm) according to the previous description (Zhou et al., 2012). Briefly, only validated SNPs were selected, and SNPs with minor allele frequency (MAF) <5% in Han Chinese population (CHB) were excluded. Potential functional SNPs were identified to meet the following criteria: SNPs in miRNA binding sites of 3' untranslated region (UTR), SNPs in the transcription factor binding site of the 5' flanking region (2000 bp upstream from the transcript start site), SNPs in splice sites and non-synonymous SNPs in exons. If there were multiple potential functional SNPs within the same haplotype block (defined by the linkage coefficient r 2 > 0.8), only 1 SNP was included. We also included the SNPs which are associated with other diseases in previous studies. Finally, we identified 2 SNPs in HIF1A gene were selected, including 1 SNP in the 3'-UTR (rs2057482) and 1 SNP in intron region (rs2301113) which have been previously reported to be associated with disease susceptibility (Doring et al., 2010). All selected SNPs were genotyped by iPLEX genotyping system (Sequenom, San Diego, CA, USA). Genotyping was performed by laboratory personnel blinded to patients' information. Internal quality controls and negative controls were used to ensure genotyping accuracy, and 5 samples were randomly selected and genotyped in duplicate with 100% concordance.

Statistical analysis
For each SNP, additive, dominant (WV+VV vs WW) and recessive (VV vs WW+WV) genetic models were selected for analysis. Two major endpoints were evaluated in this study: overall survival (OS) and recurrence-free survival (RFS). OS was defined as the time from surgery to death from any cause. RFS was defined as the time from the date of surgery to the first date of recurrence. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated by the Cox proportional hazard model with adjusting for age, gender, smoking status, differentiation, clinical stage, histology and treatment after surgery. Kaplan-Meier curves and a log-rank test were used to assess the differences of patient groups in OS and RFS. Statistical significance was set at a level of 0.05 and all analyses were done using the SPSS software package (version 19.0, SPSS, Inc.).

Distribution of patients' characteristics and prognosis analysis
In this study, a total of 697 patients with resected CRC were included (Table 1). The median age at the time of diagnosis was 60 years (range, 15-88) for all patients, and 383 (54.9%) were males. 319 (45.8%) patients were colon cancer and 378 (54.2%) patients were rectum cancer. The pathological stages were as follows: 453 at stage I/ II (65.0%) and 244 at stage III/IV (35.0%). The majority of patients (79.6%) had poor differentiated tumors, 543 (77.9%) received adjuvant chemotherapy after surgery. During the median follow-up of 29.2 months (ranging from 1.7 to 72.5), 185 (26.5%) patients died of CRC, and 208 (29.8%) patients developed recurrence. Six patients died from other causes and thus were treated as censored data in survival analysis.
Multivariate Cox regression analyses were performed to assess the prognostic effects of clinical characteristics and we found that patients with advanced TNM stage (stage III and IV) had significant higher death risk (HR=3.00, 95%CI: 2.15-4.19) and recurrence risk (HR=2.96, 95%CI: 2.15-4.07) than patients with early TNM stage.
In comparison, patients receiving chemotherapy after surgery showed a significantly decreased risk of death and recurrence (both HR were 0.54) when compared with those treated by surgery alone (Table 1). There was no association between CRC outcome and other clinical characteristics.

Association of SNPs with clinical outcome in CRC patients
Cox regression analyses were used to assess the associations of HIF1A SNP genotypes with CRC survival in 3 genetic models (Table 2). We found that there was no significant association between the two polymorphisms (rs2057482 and rs2301113) and OS or RFS in the multivariate analysis. Notably, our data showed that SNP rs2301113 was associated with OS or RFS of CRC patients with borderline significances in recessive model. Comparing to patients with the AC and CC genotype, those with variant alleles (CC genotype) had lower  The Kaplan-Meier curve of rs2057482 and rs2301113 were showed in Figure 1. And the log-rank tests showed no significant results for these two SNPs. We then performed a stratified analysis to evaluate OS and RFS in different strata according to CRC patients' treatment after surgery (Table 3), and found that CT/TT genotypes of rs2057482 exhibited a borderline significant association with better overall survival when compared with CC genotype in those without chemotherapy.

Association analysis of treatment protocol with outcomes in CRC patients stratified by SNPs
Although SNPs in HIF1A gene were not significantly associated with CRC patients' outcomes, SNPs may have modifying effect on the prognostic effects of adjuvant chemotherapy in our study. To further evaluate this hypothesis, we analyzed the association between treatment protocol and outcomes in CRC patients stratified by genotypes of SNP rs2057482 and rs2301113, respectively. Surprisingly, as shown in Table 4

Discussion
In the present study, we evaluated the associations between genetic polymorphisms in the HIF1A gene and clinical outcomes in a cohort with 697 CRC patients who received curative surgery. We do not find any statistically significant association between HIF1A SNPs and OS or RFS of CRC patients. However, we obtained an important finding that SNPs (rs2057482 and rs2301113) in HIF1A gene may modulate the protective effects of adjuvant chemotherapy on CRC patients. To the best of our knowledge, this is the first study to report that HIF1A gene polymorphisms may serve as an independent biomarker to predict the protective treatment response of adjuvant   Several studies have performed case-control study to investigate the association between SNP rs2057482 in HIF1A gene and cancers. For example, a case-control study with 518 cervical cancer patients has found that a significantly increased risk of cervical cancer is associated with the CC genotype of rs2057482, and carriers of CT/ TT genotypes have significantly decreased HIF1A mRNA expression levels compared to those with CC genotype (Fu et al., 2014). In another case-control study, they have observed the significant associations of SNP rs2057482 with risk of rectal cancer (Frank et al., 2010). In addition, another study has demonstrated that the combined variant genotypes of rs2057482 and rs11549467 are associated with increased prostate cancer risk (Li et al., 2012). However, hardly any studies have been performed to find the association between SNP rs2301113 and cancers.
A few previous studies have focused on the association between the polymorphisms including two SNPs, C1772T (rs11549465) and G1790A (rs11549467), in HIF1A gene and risk of CRC. For example, one study has found that there is a significant association between increased risk of developing colorectal cancer and T allele genotype of rs11549465 (Kang et al., 2011). Another study has found that rs11549465 exhibits similar results, indicating a significant association with CRC risk, but has suggested that this SNP may not be involved in progression or metastasis of CRC (Kuwai et al., 2004). In a study with a cohort of Korean patients, these two SNPs are further found not to be an independent prognostic marker for CRC patients with surgical treatment (Lee et al., 2011). Because minor allele frequencies of them are less than 5% in CHB population, we exclude these two SNPs in our study. Consistent with these reports, our study does not find any statistically significant association between two HIF1A SNPs and OS or RFS of CRC patients, suggesting that there may be different roles of HIF1A SNPs in cancer development and progression.To date, few biomarkers for prediction of adjuvant chemotherapy response in CRC have been established and proven to be valid for clinical application. In our study, we for the first time found that the patients with CC genotype of rs2057482 and CT/TT genotypes of rs2301113 can obviously benefit from adjuvant chemotherapy. Similarly, a previous study (Havelund et al., 2012) have analyzed the correlation between the rs2057482 and response to chemoradiotherapy (CRT) in rectal cancer and found positive results. However, this finding has not been replicated in validation cohort.
The mechanisms underlying the modulating role of HIF1A SNPs in adjuvant chemotherapy response remain to be determined. Previous studies have found that HIF-1α overexpression indicates unfavorable prognosis and may serve as an potential prognostic factor in several cancers (Zhong et al., 1999;Marton et al., 2012;Wan et al., 2012;Li et al., 2013;Luan et al., 2013;Zhang et al., 2013;), including colon, breast , gastric, lung , skin, ovarian, pancreatic, prostate, and renal carcinomas. Furthermore, down-regulated HIF-1α expression in transplanted esophageal squamous cell carcinoma in vivo could enhance the cytotoxicity of cisplatin (Liao et al., 2012). One of the major mechanisms underlying the association of HIF-1α with patients' outcomes in these cancers may be that HIF-1α or its target genes can induces chemoresistance in many types of cancer. For example, one study has found that HIF-1α contribute to metastasis and chemoresistance of prostate cancer and the targeted reduction of HIF-1α expression has significantly increased the responsiveness of prostate cancer to chemotherapy (Ranasinghe et al., 2013). Also, upregulation of HIF-1α expression appears to be a significant mechanism underlying the resistance to antiangiogenic therapies in neuroblastoma (Hartwich et al., 2013). In addition, another study has demonstrated that HIF-1α regulates the expression of XPA and thus contributes to cisplatin resistance in lung cancer . The SNP rs2057482 is located in the 3' UTR region of HIF1A gene, which could potentially affect the microRNA binding affinity and thus influence the expression of HIF1A. In addition, although SNP rs2301113 is located in intron, the CHB data (http://snpinfo.niehs.nih.gov/snpinfo/ snptag.htm) showed that rs2301113 was a tagging SNP. Many SNPs which possibly can affect the expression of HIF-1α may have high linkage disequilibrium (LD) with rs2301113. This hypothesis needs to be clarified. These two significant SNPs in our study may change the expression of HIF-1α and thus contribute to chemotherapy resistance in CRC patients with specific genotypes. Further functional experiments are warranted to confirm our hypothesis.
The present study had several limitations. Firstly, only 697 patients with complete clinical information and acceptable quality of genotyping assay were included for statistical analysis, so our findings need to be confirmed by additional studies with large sample size. Secondly, the exact biological significance of these SNPs needs further clarification by functional study. Moreover, our results also should be validated among different ethnic populations in the future.
In conclusion, this is the first study to suggest that polymorphisms in HIF1A gene may have significant impact on treatment response of adjuvant chemotherapy in Chinese CRC patients with radical surgery. Once validated, our finding would have important clinical significance for the treatment decision-making of surgical CRC patients.