Gender related differences in polymorphism and miRNA targeting CLOCK genes in metastatic colorectal cancer patients

Background Experimental and clinical evidences support a role for the circadian timing system in metabolism and cancer. In patients treated with chronomodulated or conventional schedule, overall survival (OS) was influenced by both gender and delivery schedule (Levì 2006, Giacchetti 2014). Circadian rhythms are generated by a transcription-translation–based oscillatory loop and involves a set of clock genes including PERIOD (1 and 2), CLOCK, BMAL1, CRY (1 and 2). In this study we evaluated: the immunohistochemical (IHC) expression of PER-2 and proteins involved in proliferative activity (EGFR, ERβ1, ERβ2, Cyclin D1, β-catenin) the expression levels of miRNAs targeting CLOCK genes as well as polymorphism (SNPs) CLOCK genes related in a series of metastatic colorectal cancer (mCRC) patients, aimed to correlate phenotypic and molecular findings to clinico-pathological parameters and survival. Methods We retrospectively studied 83 mCRC patients treated as first line with chemotherapy (chronomodulated triplet combination of Irinotecan + Oxaliplatin + Folinic Acid + 5-Fluorouracil) plus cetuximab after RAS characterization. IHC phenotypic analysis, polymorphism (SNPs) (rs11133373C/G, rs11133391T/C, rs1801260T/C) and miRNAs expression level (miR-192-206-132-194-219) were performed on formalin fixed paraffin embedded tissues in 77 patients. In addition, using a custom panel, we studied the mutational status of 17 selected genes involved in CRC development and progression by next generation sequencing. OS was the primary study endpoint. Results Our series of patients had a median follow-up of 25 months (range 1-132), a median progression-free survival (PFS) of 14 months (CI 95% 10-19) and a median OS of 35 months (CI 95% 22-47). Fifty patients were male and 33 female with a median age of 59 years (30-85). PFS and OS were significantly better in females vs males (PFS M/F=12/19 months (CI 95% 8-16/11-27) p=0.03; OS M/F=31/50 months (CI 95% 22-39/35-64) p=0.03. Higher percentages of EGFR (73% p>0.0001), ERβ1 (77%, p=0.07), ERβ2 (88.5%, p>0.0001), Cyclin D1 (69.2%, p=0.06), β-catenin (84.6%, p=0.02) IHC expression have been observed in the subset of mCRC presenting PER-2 downregulation. Multiple Correspondence Analysis (MCA) analysis evidenced that better response to therapy is associated to PER-2 IHC positivity and low expression of EGFR, ERβ1, ERβ2, β-catenin . Increased OS in females is related to high expression level (H) of miR206 (p=0.003), miR219 (p=0.003) and miR194 (p=0.02), and low expression (L) of miR132 (p=0.06) considered as single factors. OS was also better in females vs males in the presence of genotype RS11133373C/C (p=0.01), RS1801260T/T (p=0.07) and RS11133391T/T (p=0.06). Subgroup analysis in females showed that a miRNA profile (≥2, 58 months vs 15 months, p=0.0002) together with the presence of RS11133391T/T polymorphism (median OS 87 months) additionally increased the reported beneficial effects. Of interest, OS significantly decreased in females when miR206 low expression level and miR132high expression level were simultaneously present (11 months vs 56 months, p=0.02). Conclusions For the first time a set of SNPs and miRNAs related to CLOCK genes, principal regulator of the molecular clock, identify a subgroup of mCRC patients who benefits more for a gender related difference in survival. Furthermore the biological profile made up by high PER-2 , low EGFR, ERβ1, ERβ2, β-catenin, miR-206 expression may identify a biological profile predictive of chemotherapy response. Correlation between these results and NGS mutational analysis will provide further information of clinical value.