Differential gene expression upon shRNA-mediated silencing of APC in HT-29 colorectal cancer cells

Wnt signaling plays a pivotal role in colorectal cancer. Intrinsic activation of Wnt by mutational events, such as mutations in the tumor suppressor gene APC, represents the most frequent initiating event in this disease background. Long truncated versions of APC retain partial functionality, which leads to a sub-maximal, “just right” activation state of Wnt signaling supposed to be beneficial for disease initiation. In order to study the transcriptomic alterations of an over-stimulated Wnt signaling pathway, conditional shRNA-mediated silencing of APC was performed in chromosomal instable HT-29 CRC cells which express a 1555 amino acid variant of APC protein able to bind and partially inactivate β-catenin. To achieve this, cells were stably transduced with lentiviral particles encoding for a doxycyline-inducible shRNA directed against APC, or, as a control, a non-silencing shRNA (pTRIPZ inducible shRNA vectors, RHS4740-EG324, Horizon Dharmacon, CO, USA). 72 hours after APC silencing, total RNA was isolated and quality controlled for subsequent RNA-Seq Analysis (DKFZ Heidelberg, Genomic and Proteomic Core Facility) on a HiSeq 2000 instrument (Illumina). Overall, we observed bona-fide Wnt target genes, such as NKD1, AXIN2, PTK7, ASCL2, and SMOC2, and additional putative direct or indirect targets of Wnt signaling up-regulated upon shRNA-mediated APC silencing. HT-29 cells stably transduced to conditionally express a shRNA directed against APC or, as a control, a non-silencing (NonS) shRNA were treated with 500 ng/ml doxycycline for 72 hrs prior to RNA isolation. The experiment was repeated with the same setting, and biological duplicates were analyzed by RNA-Seq. HT-29 cells induced to express the non-silencing RNA were used as the reference sample for analysis and gene expression in these cells was set to “1” in order to examine relative gene expression upon APC silencing.