Background/purpose: Despite remaining one of the most active drugs in the treatment of advanced colorectal cancer (aCRC), oxaliplatin (OXA) resistance still is a multifactorial and complex process to decipher. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative OXA-resistance marker in CRC cell lines and patients. The purpose of this work was to identify molecular mechanisms underlying PKM2 role in resistance to OXA.
Material and methods: PKM2 transient gene silencing was achieved by transfecting specific siRNAs (Ambion) in HT29 and HCT116 CRC cell lines. Gene silencing was validated by qPCR and Western Blot with specific Taqman primers and probes (assay no. Hs00762869_s1) and antibody (Cell signaling), respectively. Cell proliferation was measured by the MTT assay (Roche Diagnostics). Inhibitory concentrations were determined in each cell line by the median-effect line method. Cell viability was studied with the use of trypan blue stain and apoptosis was detected with FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). PKM2 subcellular localization was studied by fluorescence microscopy in an Axiovision Z1 by using Apotome system at 40x immersion oil lens (Carl Zeiss, Heidelberg, Germany). Expression patterns of cell death genes were analyzed through Human Cell Death Pathway Finder PCR Array 384 HT (PAHS-212Z, SA Biosciences).
Results: In order to assess how PKM2 expression influences OXA response in CRC cells, we silenced PKM2 by using specific siRNAs in HT29 and HCT116 cells. Cells were then treated with OXA at different concentrations and we observed that PKM2 silencing induced resistance in HT29 (p53 mutated) cells and sensitivity in HCT116 (p53 wild type) cells. Moreover, PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to OXA in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that OXA and PKM2 silencing altered cell death gene expression patterns including BMF, which was significantly increased in HT29 cells in response to OXA in a dose and time-dependent manner but not in siPKM2-HT29 and HTOXAR3 cells.
Conclusion: Our data report new non-glycolytic roles of PKM2 in response to genotoxic damage, which depend on p53 mutational status and proposes BMF as a possible target gene of PKM2 to be involved in OXA response and resistance in CRC cells.
Citation Format: Alba Ginés, Anna Martínez-Cardús, Vicenç Ruiz de Porras, Eva Musulén, José Luis Manzano, Laura Layos, Cristina Bugés, Albert Abad, Eva Martinez-Balibrea. PKM2 subcellular localization is involved in oxaliplatin resistance acquisition in human colorectal cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3752. doi:10.1158/1538-7445.AM2014-3752