A KRAS variant is a biomarker of poor outcome, platinum chemotherapy resistance and a potential target for therapy in ovarian cancer

Abstract

Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.

Figure 1. The KRAS-variant predicts significantly worse overall survival for post-menopausal ovarian cancer patients over 52 years of age

Overall survival for ovarian cancer patients with (n=59) and without (n=220) the KRAS-variant are compared using Kaplan Meier analysis. Outcome is significantly worse for KRAS-variant positive EOC patients over 52 years of age by log-rank test (p = 0.0399).

Figure 2. The KRAS-variant is associated with suboptimal debulking after neoadjuvant chemotherapy

Surgical debulking after neoadjuvant chemotherapy is compared in ovarian cancer patients (n=116) with the KRAS-variant (n=26) or without (n=90). By chi-squared analysis the KRAS-variant patients are significantly more likely to be suboptimally debulked with greater residual disease (RD) than non-variant patients (p=0.044).

Figure 3. Differential Gene Expression in KRAS-variant (KV) EOC Tumors

A. A signature of 50 differentially expression gene candidates in KV triple negative breast tumors shows higher scores in KV EOC samples than in non-variant samples. B. Genes associated with KRAS-addicted tumors were used to create a corresponding signature, which is up-regulated in KV EOC tumors. C. Re-analysis of differential gene expression in carboplatin-sensitive and resistant EOC cells shows differential expression of the top 20 genes in KV EOC tumors. D. Top differentially expressed genes between KV (green) and non-variant (blue) tumor samples.

Figure 4. The KRAS-variant is associated with resistance to Carboplatin and Carboplatin/Taxol chemotherapy in cell lines

Cell lines with the KRAS-variant (BG1) and without the KRAS-variant (CAOV3) were treated with chemotherapy and half maximal inhibitory concentration (IC50) is shown on the Y-axis, and chemotherapeutic agent on the X-axis. Higher IC50 represents resistance to the tested chemotherapeutic agent. BG1 = KRAS-variant/BRCA wild-type cell line; CAOV3 = non-variant/BRCA wild-type cell line; IGR-OV1 = KRAS-variant/BRCA1 mutant cell line. Error bars are RSE.

Figure 5. Targeting the KRAS-variant impacts cell survival

Cell lines with (BG1) and without (CAOV3) the KRAS-variant were treated with siRNA/miRNA combinations that bind selectively to the variant allele. A. Decreased cell survival in the KRAS-variant line, BG1 (p<0.001), with no effect on the non-variant line, CAOV3. B. Decreased KRAS protein expression in BG1 (right) concordant with the decrease in cell survival, with no effect on CAOV3 (left). Different siRNAs are denoted by numbers.