Igf2 Is Essential For Tumor Initiating Cell Activity In Human Colorectal Cancer

A small fraction of all cells within individual tumours from colorectal cancer (CRC) patients drives long term tumor growth and metastases in immune-compromised mice. Targeting these tumor-initiating cells (TIC) may improve the long-term outcome in advanced CRC. To identify candidate genes which drive proliferation and survival of TIC, we have performed a large scale high throughput loss of function shRNA screen in three-dimensional TIC enriched patient spheroids. Spheroids were transduced with the barcoded Cellecta decipher library comprising 27,500 shRNAs targeting 5043 genes associated with cell signaling pathways (Module 1). Two weeks later, cells were harvested for DNA isolation, barcode amplification and high-throughput barcode sequencing. Amongst others, we found 5/6 shRNAs targeting Insulin growth factor 2 (IGF2) scoring within a 20% depletion threshold, presenting depletion levels very similar to positive control shRNAs. mRNA expression profiling and qPCR analyses demonstrated low to moderate expression of the IGF2 gene product in the majority of patient spheroid cultures analyzed (n=17). In contrast, two out of 15 patient derived spheroid cultures analyzed demonstrated very pronounced IGF2 overexpression (u003e250 fold). Integrative SCNA profiling, expression and TAD profiling using the CESAM algorithm, followed by 4C Seq, demonstrated a tandem duplication of the IGF2 locus in these two patients which interrupts a IGF2 adjacent TAD boundary and results in de novo contact domain formation between the IGF2 promoter and a normally hidden distant super-enhancer. A dual luciferase reporter assay revealed that the hijacked enhancer is functionally active in human CRC cells and thereby may drive unphysiological IGF2 expression following enhancer hijacking. To assess the functional relevance of IGF2 tandem duplications, spheroids were transduced with RFP expressing lentiviral vectors encoding for 3 shRNAs targeting IGF2 as well as control shRNAs targeting EIF3A or scrambled shRNA. Strikingly, IGF2 knockdown led to a marked reduction of RFP + cells in competitive proliferation assays and markedly reduced viability assessed by ATPlight assay. Moreover, IGF2 knockdown strongly reduced tumor formation following xenotransplantation into immune deficient NSG mice. These data demonstrate that IGF2 is required for survival, proliferation and tumor-initiation of primary human TIC enriched spheroids. Notably, oncogenic miRNA-483 is encoded within intron 8 of IGF2, however, its function in IGF2 locus tandem duplicated cells remains elusive. Understanding the mechanisms of IGF2 dependency and the role of miRNA-483 in this context will be essential for the future development of therapeutic approaches targeting IGF2 expression in this patient subset harbouring tandem-duplications of the IGF2 locus. Citation Format: Taronish D. Dubash, Christine Siegl, Sebastian M. Dieter, Joachim Weischenfeldt, Alexandros P. Drainas, Laura Schwarzmueller, Malgorzata Oles, Balca Mardin, Mikolaj Slabicki, Huber Wolfgang, Martin Schneider, Jan Korbel, Hanno Glimm, Claudia R. Ball. IGF2 is essential for tumor initiating cell activity in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2017-2893