Use of a proteomic approach to link pyruvate kinase M2 expression to oxaliplatin resistance in colorectal cancer patients and human cell lines

4121 Background: Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. The aim of this work was to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer (CRC) cells by using a proteomic approach and translate it to the clinical setting. Methods: An oxaliplatin resistant cell line (HTOXAR3) was established from human HT29 CRC cells. Proteins were extracted and analyzed through comparative proteomics. Results were confirmed by western blotting (WB) and Real Time Quantitative PCR. A tissue microarray constructed with 41 metastatic paraffin-embedded colorectal adenocarcinomas from patients treated with oxaliplatin + 5FU was used to determine p53, Ki-67 and bcl-2 expression. RNA from these tumors was subjected to RTQPCR to assess PKM2 mRNA expression. Differences in response were analyzed by contingency tables and chi-square test. Results: HTOXAR3 were 6 fold-resistant as compared to HT29 cells. Of the 47 spots >4 fold over-expressed or down-regulated in HTOXAR3 vs. HT29 cells, Mass Spectrometry, WB and RTQPCR confirmed down-regulation of PKM2 in HTOXAR3. In a panel of 8 CRC cell lines, we found a negative correlation between OXA resistance and PKM2 mRNA levels (spearman r = -0.846, p = 0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PKM2 mRNA up-regulation. Patients with the lowest PKM2 levels had lower response rates to oxaliplatin-based treatment (20% vs. 64.5% p =0.026) and overexpressed p53 (90% vs. 50%, p = 0.032). High PKM2 levels were associated with high ki-67 levels (p = 0.048). Conclusions: By using proteomics we have identified PKM2 as a putative predictive marker of response in CRC patients treated with oxaliplatin plus 5FU as first line chemotherapy. No significant financial relationships to disclose.