Identification Of Phf6 As A Temozolomide Resistance Factor In Glioblastoma Using Mirconnx

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Intrinsic and acquired resistance to temozolomide (TMZ) represents a major obstacle in the treatment of glioblastoma. MicroRNAs (miRNAs) have previously been implicated to play a role in chemoresistance including TMZ resistance. These short non-coding RNA sequences inhibit their mRNA targets, thereby contributing to treatment resistance. In this study, we examined the miRNA/mRNA interaction networks in TMZ resistant glioblastoma cell lines to identify drug targets for the reversal of TMZ resistance. Methods: TMZ resistance was induced in duplicate in the glioblastoma cell lines U87, Hs683, and LNZ308, creating two independent resistant subclones of each wildtype cell line. IC50 values were increased at least two-fold in the TMZ resistant subclones compared to their wildtype. mRNA and miRNA expression profiles of these cell lines were obtained by microarray data analysis. Next, we used these mRNA and miRNA expression profiles of the TMZ resistant cells to generate miRNA/mRNA interaction networks using the integrative network tool mirConnX. This tool combines sequence information with gene expression data analysis to create a condition-specific regulatory network. Results: In the obtained mRNA/miRNA network of our TMZ resistant cell lines, we identified the plant homeodomain (PHD)-like finger 6 (PHF6) mRNA to be targeted by the largest set of miRNAs, i.e. miR-143, miR-93, miR-183, miR-96, and miR-214. Analysis of PHF6 mRNA expression in the TMZ resistant subclones by qRT-PCR showed an increased expression of this mRNA in four of six resistant subclones. Protein expression analysis of PHF6 in these cell lines showed a modest increase in four of the six resistant subclones. Furthermore, analysis of mRNA and protein expression levels of PHF6 in glioblastoma patient datasets showed increased expression of PHF6 in glioblastoma tissue compared to normal brain tissue. In addition, knockdown of PHF6 using siRNA in combination with TMZ enhanced the TMZ response. These results suggest that PHF6 is a candidate drug target for the potentiation of TMZ efficacy. Conclusion: Altogether, these results demonstrate that mirConnX is a useful tool to investigate miRNA/mRNA interactions in TMZ resistant cells and can be used to identify new potential drug targets. Citation Format: Lotte Hiddingh, Rajiv S. Raktoe, Gertjan J.L. Kaspers, W. Peter Vandertop, David P. Noske, Pieter Wesseling, Thomas Wurdinger. Identification of PHF6 as a temozolomide resistance factor in glioblastoma using mirConnX. [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 3775. doi:10.1158/1538-7445.AM2014-3775