LIN28B fosters colon cancer migration, invasion and transformation through let-7-dependent and -independent mechanisms

Abstract

Lin28b is an RNA-binding protein that inhibits biogenesis of let-7 microRNAs. LIN28B is overexpressed in diverse cancers, yet a specific role in the molecular pathogenesis of colon cancer has to be elucidated. We have determined that human colon tumors exhibit decreased levels of mature let-7 isoforms and increased expression of LIN28B. To determine LIN28B's mechanistic role in colon cancer, we expressed LIN28B in immortalized colonic epithelial cells and human colon cancer cell lines. We found that LIN28B promotes cell migration, invasion and transforms immortalized colonic epithelial cells. In addition, constitutive LIN28B expression increases expression of intestinal stem cell markers LGR5 and PROM1 in the presence of let-7 restoration. This may occur as a result of Lin28b protein binding LGR5 and PROM1 mRNA, suggesting that a subset of LIN28B functions is independent of its ability to repress let-7. Our findings establish a new role for LIN28B in human colon cancer pathogenesis, and suggest LIN28B post-transcriptionally regulates LGR5 and PROM1 through a let-7-independent mechanism.

Figure 1. Expression of let-7 isoforms in isolated human colonic epithelia

Levels of mature let-7 microRNA family members were measured in four isolated normal human colonic epithelia via qPCR. Levels of mature microRNAs are depicted relative to let-7a. let-7a and let-7b account for the vast majority of let-7 microRNAs present in human colonic epithelia.

Figure 2. Mature let-7a and let-7b levels and LIN28B expression in human colon tumors

LIN28B mRNA and mature let-7a and let-7b microRNAs measured in human colon tumors via qPCR. LIN28B expression inversely correlates with levels of mature let-7a (r=−0.47, p=0.0297) and let-7b (r=−0.41, p=0.0637) mature levels in colon tumors. Data summarized in Table 1.

Figure 3. LIN28B constitutive expression promotes invasion, migration, and soft-agar colony formation in IEC-6 cells

(A) Western blot for Lin28b in IEC-6 cells transduced with MSCV-PIG-LIN28B retrovirus. (B) Decreased levels of mature let-7a and let-7b in IEC-6 cells following LIN28B constitutive expression. (C) Lin28b increases migration and invasion in IEC-6 cells. (D) LIN28B expression confers the ability of IEC-6 cells to form colonies in soft-agar.

Figure 4. LIN28B constitutive expression promotes invasion, migration, and soft-agar colony formation in colon cancer cells

(A) Western blot for Lin28b in LoVo colon cancer cells transduced with MSCV-PIG-LIN28B retrovirus. (B) Decreased levels of mature let-7a and let-7b in LoVo cells following LIN28B constitutive expression. (C) LIN28B overexpression in LoVo cells increases migration and invasion. (D) LIN28B expression in colon cancer cells increases soft-agar colony formation.

Figure 5. let-7 restoration abrogates increased migration and invasion phenotype of colon cancer cells constitutively expressing LIN28B

(A) qPCR for mature let-7a in LIN28B-LoVo cells transiently transfected with Lin28b-resistant let-7a hairpin precursors. Mature let-7a levels are restored with decoy hairpin expression in cells that constitutively express LIN28B. (B) Restoration of let-7a and let-7b reduces in vitro migration of LIN28B-LoVo cells. (C) Restoration of let-7a and let-7b reduces in vitro invasion of LIN28B-LoVo cells.

Figure 6. let-7 dependent and independent gene regulation in cells constitutively expressing LIN28B

(A) Western blot for let-7 targets CDC34, IGF2BP1 and HMGA2 in IEC-6, DLD-1, and LoVo cells that constitutively express LIN28B. (B) Increased mature let-7 levels in LIN28B-LoVo cells restores repression of let-7 targets. (C) Up-regulation of LGR5 and PROM1 in LIN28B-expressing cells is maintained following let-7 restoration.

Figure 7. LGR5 and PROM1 mRNAs interact with Lin28b protein

(A) RNAs co-immunoprecipitating with Lin28b in colon cancer cells were reverse transcribed with an oligo-dT primer and qPCR performed for GAPDH, IGF2, LGR5, and PROM1 mRNAs. Fold change is relative to mRNA levels immunoprecipitated in the absence of Lin28b antibody, following normalization to ACTB (β-actin) mRNA levels. Error bars depict standard deviation from the mean. (B) Luciferase assays were performed to assess association of Lin28b protein with LGR5 and PROM1 3′ UTR sequences in colon cancer cells. Luciferase activity is enhanced by LGR5 and PROM1 3′ UTRs in the presence of increased LIN28B expression. Error bars depict standard deviation from mean.