We found the major let-7 isoforms expressed in normal colonic epithelium (let-7a and let-7b) are down-regulated in approximately two-thirds of colon tumors. In a subset of these tumors, we found concomitant upregulation of the post-transcriptional inhibitor of let-7 processing, namely LIN28B. In order to determine the role of LIN28B in colon cancer, we constitutively expressed LIN28B in immortalized rat colonic epithelial cells and transformed human colon cancer cell lines. We found that constitutive LIN28B expression fosters cell migration, invasion, and cellular transformation, demonstrating a previously unrecognized role for LIN28B in colon tumorigenesis.
Furthermore, we found that constitutive LIN28B expression in immortalized colonic epithelial and colon cancer cells results in decreased levels of mature let-7 isoforms, thereby relieving suppression of let-7 targets. Interestingly, expression of LGR5 and PROM1 – intestinal/colonic stem cell markers that are not predicted let-7 targets – is also increased in the presence of Lin28b. LGR5 and PROM1 expression remains upregulated following restoration of let-7 in cells constitutively expressing LIN28B, and Lin28b protein is capable of binding LGR5 and PROM1 transcripts. Taken together, these results suggest that a subset of Lin28b functions may occur via let-7 independent mechanisms.
We have demonstrated increased LIN28B
expression in colon tumors, which likely occurs as a result of increased LIN28B
transcriptional activity mediated by c-myc. Since c-myc is a transcriptional target of canonical Wnt signaling, it is possible that LIN28B
is up-regulated in colon tumors as a consequence of APC
mutation (or other changes that deregulate Wnt signaling), which occurs in the vast majority of colon tumors (Pino and Chung, 2010
). Alternatively, up-regulation of LIN28B
in colon tumors may occur as a result of increased mRNA stabilization. Interestingly, this may occur as a result of decreased let-7
in colon tumors, since LIN28B
is also a predicted let-7
target. The ability of let-7
to regulate one another other likely represents a feedback loop that allows the cell to tightly regulate levels of each, further highlighting their importance in cellular processes.
We have shown that Lin28b does not function exclusively via repression of let-7
biogenesis. Interestingly, recent evidence demonstrates that Lin28 also does not function exclusively through inhibition of let-7
processing, but blocks gliogenesis in favor of neurogenesis in undifferentiated cells by stabilizing IGF2
mRNA (Balzer et al., 2010
). Considering the high degree of homology between Lin28 and Lin28b, and the RNA-binding activity inherent to both, it is possible that both Lin28 and Lin28b modulate expression of a number of genes in addition to IGF2
, and PROM1
, independent of their ability to inhibit let-7
biogenesis. As both Lin28 and Lin28b are implicated in multiple processes, including tumorigenesis, pluripotency, and cell fate decision, it becomes increasingly important to fully elucidate the mechanisms by which these homologs function as pursued in this study.
Mutations that specifically ameliorate the ability of Lin28b to inhibit let-7
would be useful in further elucidating the mechanisms of Lin28b's functions. One might consider introducing point mutations into the cold-shock domain of Lin28b; this approach has been utilized previously for evaluating let-7
independent functions of Lin28 (Balzer et al., 2010
). However, cold-shock domain mutations may disrupt all RNA-binding activities of LIN28B
, for example, LGR5
binding, thereby precluding assessment of specific let-7
independent functions. Thus, determining the specific domains and/or amino acid residues of LIN28B
essential for repression of let-7
biogenesis is critical.
Our finding that LIN28B
points to a potential specific function of LIN28B
in intestinal and colonic epithelial stem cells. Within the intestine, expression of the cell surface protein PROM1
is restricted to the crypts and adjacent epithelial cells (Snippert et al., 2009
), while expression of the orphan receptor LGR5
occurs exclusively in cycling columnar cells within the crypt base (Barker et al., 2007
). Since co-expression of LGR5
marks intestinal and colonic epithelial stem cells, up-regulation of these factors by Lin28b suggests a possible role for Lin28b in establishment and/or maintenance of intestinal stem cells.
Interestingly, adenomas may arise in the colon from PROM1+
crypt cells (Zhu et al., 2009
), and overexpression of LGR5
in colorectal adenocarcinomas correlates with late-stage tumorigenesis, invasion, and metastasis (Uchida et al., 2010
). We have demonstrated that constitutive LIN28B
expression promotes both tumorigenesis as well as induction of LGR5
in colonic epithelial cells. LGR5
up-regulation occurring in the context of tumorigenesis fostered by LIN28B
overexpression may support the emerging premise of stem cells in sustaining tumorigenesis. Targeting stem cell-like tumor cells within colon cancers is potentially an effective therapeutic strategy, and overexpression of LIN28B
may serve as an indicator of stem cell-like tumor cells that could be targeted. This underscores the importance of fully elucidating the role of LIN28B
in both tumorigenesis and pluripotency within the colon, as well as in other tissues where LGR5
mark stem cell populations.