Lin28b promotes head and neck cancer progression via modulation of the insulin-like growth factor survival pathway

Abstract

Lin28 is a developmentally regulated RNA binding protein which has recently emerged as key regulator in the biogenesis of the let-7 micro-RNA family. While the expression of Lin28b has been linked to advanced tumor stage, the precise molecular mechanism(s) by which Lin28b drives disease progression is still being unraveled. Herein, we generated a let-7-resistant Lin28b ORF, stably expressed in the FaDu head and neck cancer (HNC) cell line. FaDu-Lin28b cells exhibited enhanced tumor growth in vitro and in vivo. Global gene and micro-RNA expression analyses revealed significant enrichment in several pathways involved in cell migration, chromatin remodeling, and cellular stress response. Direct regulation of selected genes (HMGA2, CCND2, IGF1R, and IGF2BP2) via a let-7-Lin28b mechanism was validated. Notably, up-regulation of several genes in the IGF pathway in Lin28b-expressing cells was observed. Functional studies revealed significant increase in the survival of Lin28b-expressing cells when cultured under stress conditions, which was dependent on the presence of IGF1. Therefore, our data identified several novel gene targets for Lin28b-let7, and revealed a novel mechanism by which Lin28b promote tumorigenesis. Concordantly, clinical examinations of Lin28b, IGF2BP2 and IGF2 revealed a significant association between the expression of these genes with disease relapse, thereby corroborating the potential relevance for the Lin28b/IGF axis in HNC progression.

Figure 1. Stable expression of Lin28b enhanced HNSCC tumorigenicity in vitro and in vivo

Expression of Lin28b in FaDu cells stably transfected with Lin28b-expression vector was detected by (A) qRT-PCR; and (B) Western blotting (27 kd), using GAPDH (37 kd) for loading control. (C) Representative images depicting enhanced migration ability of FaDu~Lin28b cells compared FaDu~GFP cells. Data were presented as mean ± S.E, n=10. (D) FaDu~Lin28b cells exhibited enhanced tumor formation and radiation resistance in vivo. Data were presented as mean ± S.E, n=4-5 mice/group. **p<0.005; ***p<0.0005.

Figure 2. Characterizing the miRNA and mRNA changes in FaDu~Lin28b cells

(A) FaDu~Lin28b or FaDu~GFP cells were subjected to global miRNA expression profiling using the nCounter NanoString platform, and the normalized expression value for each miRNA were plotted for the FaDu~GFP (x-axis) vs. the FaDu~Lin28b (y-axis) cells. (B) Expression of two representative let-7 miRNAs (miR-98 and Let7-g) was assessed in two stable FaDu~Lin28b clones (A1 and D1) using single-well Taqman miRNA assay. Data were presented as mean ± S.E, n=3. (C) Venn diagram depicting the intersection between the up regulated gene list in FaDu~Lin28b cells, in silico predicted Let-7 gene targets, and up regulated gene list from a publicly available microarray dataset (GSE6631) on primary HNSCC. (D) Validation of a selected group of genes identified from the microarray analyses. Data were presented as mean ± S.E, n=6. *p<0.05, **p<0.005, ***p<0.0005.

Figure 3. Activation of the IGF pathway in FaDu~Lin28b cells through a Let-7 dependent mechanism

(A) Schema depicting the construction of luciferase reporter vectors carrying the indicated predicted let-7 miR binding sites downstream of the firefly luciferase gene in the pMIR-REPORT vector. The indicated number of Let-7 binding sites is shown in black bars. (B) The indicated wild type or mutant reporter vector was co-transfected with a pre-miR control (100 nM) or pre-miR Let-7b (100 nM) in HEK-293 cells, and luciferase activity was measured 24 hours later. Renilla luciferase activity was used for normalization. Data were presented as mean ± S.E, n=6. (C) FaDu~GFP, FaDu~Lin28b A1, and FaDu~Lin28b D1 cells were cultured in serum-free medium supplemented with 0, 10, or 50 ng/ml IGFI, and cell viability was measured on day 6 by MTS assay. Data were presented as mean ± S.E, n=6. *p<0.05, **p<0.005, ***p<0.0005.

Figure 4. Expression of Lin28b, IGF2BP2, and IGF2 was associated with higher risk of recurrence in primary HNSCC samples

(A) Expression of Lin28b (nuclear, or cytoplasmic), IGF2BP2 (cytoplasmic), and IGF2 (cytoplasmic) was assessed in 39 HNSCC samples by immunohistochemistry. The relative expression level of each protein was plotted as a function of patient outcome: recurrent (R); non-recurrent (NR). (B) Positive correlations were observed between Lin28b with IGF2BP2 and IGF2, as well as between IGF2BP2 with IGF2 expression in the same cohort of HNSCC specimens as presented in A.

Figure 5. Schema describing a model for Lin28b-mediated tumor progression

Over-expression of Lin28b in HNSCC cells inhibits let-7 miRNA family biogenesis. In turn, down-regulation of mature Let-7 miRNAs in Lin28b-expressing cells allows the over-expression of several Let-7 gene targets with known roles in tumor progression, such as HMGA2, and CCND2. Furthermore, down-regulation of Let-7 miRNAs in Lin28b-expressing cells also leads to up regulation of several genes in the IGF pathway such as IGF1R, and IGF2BP2, which promotes tumor survival under stress conditions.