LIN28B promotes colorectal cancer differentiation and metastasis

Differentiation is a fundamental process underlying cellular identity and plasticity. Traditionally, differentiation is a transition between less committed/specialized cell states into more committed/specialized state, and this is believed to be unidirectional. Differentiation is evident in development, division of stem cells, homeostasis or normal turnover, regeneration following injury, and malignant transformation. Clusters of differentiated cells are necessary for tumor cell invasion (collective invasion), intravasation, circulation, extravasation and metastasis (colonization in a distant organ). We have unraveled the critical function of LIN28B (human), an RNA binding protein, as a master regulator of reprogramming of differentiation in colorectal cancer (CRC) and metastasis. Indeed, LIN28B plays essential roles in embryonic stem cell differentiation and inducible pluripotent stem cell differentiation but its role in tumor metastasis is not elucidated. Having published that Lin28b (mouse) is oncogenic in a genetic mouse model with the induction of colonic adenomas and CRC, and about one-third of human CRC cancers overexpress LIN28B (in turn associated with worse outcomes), we have determined that LIN28B induces the CDX2 transcriptional factor in promoting primary CRC differentiation. In addition, we have new evidence that LIN28B directly targets Claudin-1 (CLDN1) mRNA to foster the formation of tight junctions and cell-cell adhesion, which in turn induces NOTCH3 to modulate metastasis to the liver. We also have new evidence that Lin28b endows in vivo mouse orthotopically implanted tumoroids that harbor Apc/Kras/p53 mutations to metastasize to the liver as they do not metastasize without Lin28b. Thus, we are defining new mechanisms that reveal how LIN28B promotes differentiation at the primary CRC site and then leverages previously unappreciated mechanisms to promote metastasis. We hypothesize that LIN28B promotes primary CRC differentiation and mediates metastasis through induction and maintenance of a tumor differentiation program that may involve distinct subclones destined for metastasis. This hypothesis will be pursued through the following in vivo physiologically relevant interrelated Specific Aims: (1) To identify the molecular mechanisms by which CLDN1 facilitates CRC metastatic progression in the context of LIN28B overexpression, with NOTCH3 being an attractive target and determination of other targets; (2) To determine the contribution of Lin28b to invasion, circulation and distant organ (liver) colonization in a syngeneic in vivo trackable model of metastatic CRC, allowing us to span the entire metastatic cascade; and (3) To identify subpopulations in primary CRC, potentially fated for metastasis (e.g. those cells that may display hybrid/partial EMT), and determine the dependence of their phenotype on Lin28b by integrating single-cell RNA-seq with lineage tracing (bar-coding). Our novel, mechanistic findings and innovative approaches allow us to provide insights into the fundamental aspects of cellular differentiation in the context of primary CRC, and elucidate why such differentiated tumor cells have a proclivity for metastasis, and thus address gaps in the field.