Abstract PR04: LIN28B promotes growth and tumorigenesis of the intestinal epithelium primarily via Let-7 repression

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 miRNAs, although Let-7 independent effects have been surmised. Our past studies have shown that LIN28B expression in colorectal cancers is associated with poor prognosis and cancer recurrence, and that LIN28B promotes migration, invasion, and metastasis of CRC cell lines in mouse xenograft models. Considering these studies, and the observation that LIN28B is more frequently up-regulated in a variety of other cancers, we sought to elucidate the molecular mechanisms of LIN28B function in intestinal homeostasis and colorectal cancer. We report that intestine-targeted expression of Lin28b in mice causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Lgr5-positive crypt base columnar cells were not expanded, nor compromised by the depletion of Paneth cells. However, Lin28b fostered intestinal polyp and adenocarcinoma formation. We observed that higher Lin28b levels also correlated with increased incidence of adenocarcinoma. This is consistent with human epidemiological data illustrating that increased LIN28B levels are observed at early stages of CRC, with high expression at both stages I and II disease correlating inversely with patient survival and higher probability of recurrence. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-Seq) to identify direct RNA targets. This revealed that LIN28B protein binds a substantial number of mRNAs, many of which are specific to epithelial cells. LIN28B modestly augmented protein levels of these target mRNAs, in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. Previous reports have linked Let-7 to the regulation of the PI3K-Akt-mTOR signaling pathway via regulation of Insr, Igf1r, and Irs2 mRNAs, yet we found that Lin28b did not have a significant impact on these mRNAs or insulin-PI3K-mTOR signaling. Kras and Myc mRNAs, which are also reported to be Let-7 targets, were likewise unaffected. Instead, we found profound up-regulation of Hmga2, Igf2bp1, Igf2bp2, and E2f5 in the context of Let-7 depletion or Lin28b over-expression. Our study reveals that Let-7 miRNAs are critical for repressing tissue growth and promoting Paneth cell differentiation. These results highlight the critical nature of LIN28B and Let-7 dosage, where a stepwise reduction of Let-7 levels has a direct effect on target mRNAs and adenocarcinoma risk. These Let-7 dependent effects of LIN28B appear to supersede any Let-7 independent effects. In sum, LIN28B can definitively act as an oncogene in the absence of canonical genetic alteration in intestinal carcinogenesis. This abstract is also presented as poster A42. Citation Format: Blair Madison, Qi Liu, Xue Zhong, Christopher M. Hahn, Nan Lin, Matthew Emmet, Ben Z. Stanger, Anil K. Rustgi. LIN28B promotes growth and tumorigenesis of the intestinal epithelium primarily via Let-7 repression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR04.