MUC1-C Induces the LIN28B→LET-7→HMGA2 Axis to Regulate Self-Renewal in NSCLC

Abstract

The LIN28B→let-7 pathway contributes to regulation of the epithelial-mesenchymal transition (EMT) and stem cell self-renewal. The oncogenic MUC1-C transmembrane protein is aberrantly overexpressed in lung and other carcinomas; however, there is no known association between MUC1-C and the LIN28B→let-7 pathway. Here in non-small cell lung cancer (NSCLC), silencing MUC1-C downregulates the RNA-binding protein LIN28B and coordinately increases the miRNA let-7. Targeting MUC1-C function with a dominant-negative mutant or a peptide inhibitor provided confirming evidence that MUC1-C induces LIN28B→let-7 signaling. Mechanistically, MUC1-C promotes NF-κB p65 chromatin occupancy of the LIN28B first intron and activates LIN28B transcription, which is associated with suppression of let-7. Consistent with let-7-mediated inhibition of HMGA2 transcripts, targeting of MUC1-C also decreases HMGA2 expression. HMGA2 has been linked to stemness, and functions as a competing endogenous RNA (ceRNA) of let-7-mediated regulation of the TGFβ coreceptor TGFBR3. Accordingly, targeting MUC1-C suppresses HMGA2 mRNA and protein, which is associated with decreases in TGFBR3, reversal of the EMT phenotype, and inhibition of self-renewal capacity. These findings support a model in which MUC1-C activates the ⇑LIN28B→⇓let-7→⇑HMGA2 axis in NSCLC and thereby promotes EMT traits and stemness.

Implications: A novel pathway is defined in which MUC1-C drives LIN28B→let-7→HMGA2 signaling, EMT, and self-renewal in NSCLC.

Figure 1. MUC1-C induces LIN28B expression

A. Lysates from H1975 cells were immunoblotted with the indicated antibodies. B. H1975 cells were infected with lentiviruses to stably express a control scrambled shRNA (CshRNA) or a MUC1 shRNA. The H1975/CshRNA and H1975/MUC1shRNA cells were analyzed for LIN28B mRNA levels by qRT-PCR (left). The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for H1975/CshRNA cells (assigned a value of 1). Lysates from the H1975/CshRNA and H1975/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). C. H1299 cells stably expressing a control scrambled shRNA (CshRNA) or a MUC1 shRNA were analyzed for LIN28B mRNA levels by qRT-PCR (left). The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for H1299/CshRNA cells (assigned a value of 1). Lysates from the H1299/CshRNA and H1299/MUC1shRNA cells were immunoblotted with the indicated antibodies (right). D. Lysates from MCF-7 and H1975 cells were immunoblotted with the indicated antibodies. E. MCF-7 cells stably expressing an empty vector or MUC1-C were analyzed for LIN28B mRNA levels by qRT-PCR (left). The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for MCF-7/vector cells (assigned a value of 1). Lysates from the MCF-7/vector and MCF-7/MUC1-C cells were immunoblotted with the indicated antibodies (right). F. LNCaP cells stably expressing an empty vector or MUC1-C were analyzed for LIN28B mRNA levels by qRT-PCR (left). The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for LNCaP/vector cells (assigned a value of 1). Lysates from the LNCaP/vector and LNCaP/MUC1-C cells were immunoblotted with the indicated antibodies (right).

Figure 2. Targeting MUC1-C with a MUC1-C(AQA) mutant or the GO-203 inhibitor suppresses LIN28B expression

A. Schematic representation of the MUC1-C subunit and the amino acid (aa) sequence of the cytoplasmic domain (CD). ED, extracellular domain. TM, transmembrane domain. The CQC motif is necessary and sufficient for MUC1-C homodimerization. Mutation of CQC to AQA thus blocks MUC1-C homodimer formation. D-aa sequences are shown for GO-203 and CP-2. B. Lysates from H1975 cells stably expressing an empty vector, MUC1-C or MUC1-C(AQA) were immunoblotted with the indicated antibodies (left). H1975/MUC1-C and H1975/MUC1-C(AQA) cells were analyzed for LIN28B mRNA levels by qRT-PCR (right). The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for H1975/MUC1-C cells (assigned a value of 1). C and D. H1975 (C) and H1299 (D) cells were treated with 5 µM GO-203 or CP-2 each day for 3 days and then analyzed for LIN28B mRNA levels by qRT-PCR. E. MCF-7/MUC1-C cells were treated with 5 µM GO-203 or CP-2 each day for 4 days and then analyzed for LIN28B mRNA levels by qRT-PCR. The results are expressed as relative LIN28B mRNA levels (mean±SD of three determinations) as compared to that obtained for CP-2-treated cells (assigned a value of 1) (left). Lysates from the GO-203- and CP-2-treated cells were immunoblotted with the indicated antibodies (right).

Figure 3. MUC1-C activates LIN28B by an NF-κB-mediated mechanism

A. Schema of the Lin28B-luciferase reporter (Lin28B-P1-Luc) showing location of the NF-κB binding site in the first intron relative to the transcription start site (upper panel). H1975/CshRNA and H1975/MUC1shRNA cells were transfected with the empty Luc vector or Lin28B-P1-Luc. Cells were also transfected with the SV-40-Renilla-Luc plasmid as an internal control. Luciferase activity was measured at 48 h following transfection (lower panel). The results are expressed as relative luciferase activity (mean±SD of three determinations) compared with that obtained from cells transfected with the empty Luc vector (assigned a value of 1). B. H1975/MUC1-C and H1975/MUC1-C(AQA) cells were transfected with the empty Luc vector or Lin28B-P1-Luc. Cells were also transfected with SV-40-Renilla-Luc as an internal control. Luciferase activity was measured at 48 h following transfection. Results are expressed as relative luciferase activity (mean±SD of three determinations) compared with that obtained from cells transfected with the empty Luc vector (assigned a value of 1). C. H1975 cells were co-transfected with the Lin28B-P1-Luc and SV-40-Renilla-Luc plasmids. At 48 h after transfection, cells were left untreated (Control) or treated with 5 µM GO-203 or CP-2 for an additional 36 h. Results are expressed as relative luciferase activity (mean±SD of three determinations) as compared to that obtained for the control untreated cells (assigned a value of 1). D. H1975 cells were transfected with empty Luc, wild type (WT) Lin28B-P1-Luc or mutant Lin28B-P1-Luc plasmids. Cells were also transfected with SV-40-Renilla-Luc plasmid. Luciferase activity was measured at 48 h following transfection. Results are expressed as relative luciferase activity (mean±SD of three determinations) compared with that obtained from cells transfected with the empty Luc vector (assigned a value of 1). E. H1975 cells were transfected to express a p65 siRNA or a control siRNA (CsiRNA). After 48 h, lysates were immunoblotted with the indicated antibodies. F. H1975 cells were co-transfected with Lin28B-P1-Luc and SV-40-Renilla-Luc plasmids. At 48 h following transfection, cells were left untreated (Control), treated with DMSO as vehicle control, or treated with 5 µM BAY11-7085 for an additional 12 h. Results are expressed as relative luciferase activity (mean±SD of three determinations) as compared to that obtained for the control untreated cells (assigned a value of 1).

Figure 4. MUC1-C occupies the LIN28B gene with NF-κB

A. Lysates from H1975 cells were precipitated with a control IgG or either anti-p65 (left) or anti-MUC1-C (right) antibody. Precipitates were immunoblotted with the indicated antibodies. B–C. Soluble chromatin from the indicated H1975 cells was precipitated with anti-NF-(B or a control IgG. The final DNA precipitates were amplified by qPCR with pairs of primers for the NF-(B binding region (NBR; +313 to +415) in intron 1 or a control region (CR; −4964 to −4823). The results (mean±SD of three independent experiments each assayed in triplicate) are expressed as the relative fold enrichment compared to that obtained with the IgG controls. D. Soluble chromatin from H1975/MUC1-C and H1975/MUC1-C(AQA) cells was precipitated with anti-NF-(B or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). The results (mean±SD of three independent experiments each assayed in triplicate) are expressed as the relative fold enrichment compared with the IgG controls. E. H1975 cells were left untreated or treated with 5 µM GO-203 or CP-2 each day for 3 days. Soluble chromatin was precipitated with anti-NF-(B or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). The results (mean±SD of three independent experiments each assayed in triplicate) are expressed as the relative fold enrichment compared with the IgG controls. F. Soluble chromatin from MCF-7/vector and MCF-7/MUC1-C cells was precipitated with anti-NF-(B or a control IgG (left). For re-ChIP analysis, complexes were released and re-immunoprecipitated with anti-MUC1-C (right). The results (mean±SD of three independent experiments each assayed in triplicate) are expressed as the relative fold enrichment compared with the IgG controls.

Figure 5. Targeting MUC1-C increases let-7 expression

A. H1975/CshRNA and H1975/MUC1shRNA cells were analyzed for let-7a-c miRNA levels by qRT-PCR. Results are expressed as relative let-7a-c levels (mean±SD of three determinations) as compared to that obtained for CshRNA cells (assigned a value of 1). B. H1975/MUC1shRNA cells were transfected to express a control vector or MUC1-C. Lysates were immunoblotted with the indicated antibodies (left). Total RNA was analyzed for let-7c expression (right). Results are expressed as relative let-7c levels (mean±SD of three determinations; H1975/MUC1shRNA/vector cells assigned a value of 1). C. Total RNA from H1975/MUC1-C and H1975/MUC1-C(AQA) cells (left) and from H1975 cells treated with 5 µM GO-203 or CP-2 each day for 3 days (right) was analyzed for let-7c by qRT-PCR. Results are expressed as relative let-7c levels (mean±SD of three determinations; H1975/MUC1-C cells assigned a value of 1). D. Lysates from H1975/CshRNA and H1975/MUC1shRNA cells were immunoblotted with the indicated antibodies. E. Lysates from H1975/MUC1-C and H1975/MUC1-C(AQA) cells (left) and from H1975 cells treated with 5 µM GO-203 or CP-2 each day for 3 days (right) were immunoblotted with the indicated antibodies.

Figure 6. MUC1-C induces EMT and tumorsphere formation

A. Lysates from H1975/CshRNA and H1975/MUC1shRNA cells were immunoblotted with the indicated antibodies. B. H1975 cells were infected with lentiviruses expressing a control scrambled shRNA or a Lin28B shRNA. Lysates were immunoblotted with the indicated antibodies. C. Representative images of tumorspheres derived from H1975/CshRNA and H1975/MUC1shRNA cells (left). Bar represents 50 microns. The percentage sphere forming efficiency (%SFE) is expressed as the mean±SD of three determinations (right). D. Lysates from H1975/MUC1-C and H1975/MUC1-C(AQA) cells were immunoblotted with the indicated antibodies. E. Representative images of tumorspheres derived from H1975/MUC1-C and H1975/MUC1-C(AQA) cells (left). Bar represents 100 microns. The percentage SFE is expressed as the mean±SD of three determinations (right). F. Tumorspheres established from H1975 cells (left) were left untreated or treated with 5 µM GO-203 or 5 µM CP-2 for 48 h. Bar represents 100 microns. The percentage SFE is expressed as the mean±SD of three determinations (right). G. Representative images of tumorspheres derived from H1975/CshRNA and H1975/LIN28BshRNA cells (left). Bar represents 100 microns. The percentage SFE is expressed as the mean±SD of three determinations (right).

Figure 7. Targeting MUC1-C decreases TGFBR3 expression

A and B. The indicated H1975 (A) and H1299 (B) cells were analyzed for HMGA2 and TGFBR3 mRNA levels by qRT-PCR. The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared to that obtained for cells expressing the CshRNA (assigned a value of 1). C. H1975/MUC1-C and H1975/MUC1-C(AQA) cells were analyzed for HMGA2 and TGFBR3 mRNA levels by qRT-PCR. The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared to that obtained for H1975/MUC1-C cells (assigned a value of 1). D. H1975 cells treated with 5 µM GO-203 or CP-2 each day for 3 days were analyzed for HMGA2 and TGFBR3 mRNA levels by qRT-PCR. The results are expressed as relative mRNA levels (mean±SD of three determinations) as compared to that obtained for CP-2-treated cells (assigned a value of 1). E. Schema depicting the proposed MUC1-C→⇑LIN28B→⇓let-7→⇑HMGA2→⇑TGFBR3 pathway that drives EMT and self-renewal.