Limits...
DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

No MeSH data available.


Related in: MedlinePlus

MUC1-C induces DNMT1 and DNMT3b, but not DNMT3a, in human carcinoma cells(A–B) BT-549 (A) and MDA-MB-231 (B) breast cancer cells stably expressing a Control shRNA (CshRNA) or a MUC1 shRNA (MUC1shRNA) were analyzed for MUC1 and DNMT1 mRNA levels by qRT-PCR with pairs of primers listed in Table S1. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1) (left and middle panels). Lysates were immunoblotted with the indicated antibodies (right panels). (C) MCF-7 breast cancer cells stably expressing an empty vector (MCF-7/vector) or MUC1-C (MCF-7/MUC1-C) were analyzed for DNMT1 mRNA levels by qRT-PCR. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vector expressing cells (assigned a value of 1) (left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D–E) The indicated BT-549 (D) and MDA-MB-231 (E) cells were analyzed for (i) DNMT3a (left panels) and DNMT3b mRNA (middle panels). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1). Lysates were analyzed by immunoblotting with the indicated antibodies (right panels). (F) MCF-7/vector and MCF-7/MUC1-C cells were analyzed for DNMT3a (left panel) and DNMT3b (middle panel) mRNA levels by qRT-PCR (mean±SD of 3 determinations) as compared to that obtained for vector expressing cells (assigned a value of 1). Lysates from the indicated cells were analyzed by immunoblotting (right panel).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5121097&req=5

Figure 1: MUC1-C induces DNMT1 and DNMT3b, but not DNMT3a, in human carcinoma cells(A–B) BT-549 (A) and MDA-MB-231 (B) breast cancer cells stably expressing a Control shRNA (CshRNA) or a MUC1 shRNA (MUC1shRNA) were analyzed for MUC1 and DNMT1 mRNA levels by qRT-PCR with pairs of primers listed in Table S1. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1) (left and middle panels). Lysates were immunoblotted with the indicated antibodies (right panels). (C) MCF-7 breast cancer cells stably expressing an empty vector (MCF-7/vector) or MUC1-C (MCF-7/MUC1-C) were analyzed for DNMT1 mRNA levels by qRT-PCR. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vector expressing cells (assigned a value of 1) (left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D–E) The indicated BT-549 (D) and MDA-MB-231 (E) cells were analyzed for (i) DNMT3a (left panels) and DNMT3b mRNA (middle panels). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1). Lysates were analyzed by immunoblotting with the indicated antibodies (right panels). (F) MCF-7/vector and MCF-7/MUC1-C cells were analyzed for DNMT3a (left panel) and DNMT3b (middle panel) mRNA levels by qRT-PCR (mean±SD of 3 determinations) as compared to that obtained for vector expressing cells (assigned a value of 1). Lysates from the indicated cells were analyzed by immunoblotting (right panel).

Mentions: Stable silencing of MUC1-C in BT-549 breast cancer cells was associated with suppression of DNMT1 mRNA and protein (Fig. 1A). A similar response to MUC1-C silencing was observed in MDA-MB-231 breast cancer cells (Fig. 1B), indicating that MUC1-C promotes DNMT1 expression. To confirm this notion, we overexpressed MUC1-C in MCF-7 breast cancer cells (24) and observed an increase in DNMT1 mRNA and protein (Fig. 1C), indicating that MUC1-C, and not the MUC1 N-terminal subunit (MUC1-N), is sufficient for this response. In extending these observations to other types of carcinomas, we found that silencing MUC1-C in KRAS mutant A549 (Fig. S1A) and H460 (Fig. S1B) non-small cell lung cancer (NSCLC) cells similarly results in decreased DNMT1 expression. In addition, MUC1-C conferred DNMT1 expression in SK-CO-1 colon cancer cells (Fig. S1C), indicating that this association is found in diverse types of carcinomas. In further studies, we found that silencing MUC1-C in BT-549 cells suppressed DNMT3b, but not DNMT3a, expression (Fig. 1D). The same pattern of response was observed in MDA-MB-231 (Fig. 1E), A549 NSCLC (Fig. S2A), H460 NSCLC cells (Fig. S2B), and SK-CO-1 colon cancer cells (Fig. S2C). We also found that overexpression of MUC1-C in MCF-7 cells increases DNMT3b and not DNMT3a (Fig. 1F). To provide additional support for these findings, we used other approaches for targeting MUC1-C expression. In this way, silencing MUC1 in H460 cells with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing resulted in the downregulation of DNMT1 and DNMT3b (Fig. S3A). Moreover, targeting MUC1-C function in BT-549 and A549 cells with the GO-203 inhibitor (26) decreased DNMT1 and DNMT3b levels (Figs. S3B and S3C). These results demonstrate that MUC1-C selectively drives DNMT1 and DNMT3b, but not DNMT3a, expression in breast and other carcinoma cells.


DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS
MUC1-C induces DNMT1 and DNMT3b, but not DNMT3a, in human carcinoma cells(A–B) BT-549 (A) and MDA-MB-231 (B) breast cancer cells stably expressing a Control shRNA (CshRNA) or a MUC1 shRNA (MUC1shRNA) were analyzed for MUC1 and DNMT1 mRNA levels by qRT-PCR with pairs of primers listed in Table S1. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1) (left and middle panels). Lysates were immunoblotted with the indicated antibodies (right panels). (C) MCF-7 breast cancer cells stably expressing an empty vector (MCF-7/vector) or MUC1-C (MCF-7/MUC1-C) were analyzed for DNMT1 mRNA levels by qRT-PCR. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vector expressing cells (assigned a value of 1) (left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D–E) The indicated BT-549 (D) and MDA-MB-231 (E) cells were analyzed for (i) DNMT3a (left panels) and DNMT3b mRNA (middle panels). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1). Lysates were analyzed by immunoblotting with the indicated antibodies (right panels). (F) MCF-7/vector and MCF-7/MUC1-C cells were analyzed for DNMT3a (left panel) and DNMT3b (middle panel) mRNA levels by qRT-PCR (mean±SD of 3 determinations) as compared to that obtained for vector expressing cells (assigned a value of 1). Lysates from the indicated cells were analyzed by immunoblotting (right panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5121097&req=5

Figure 1: MUC1-C induces DNMT1 and DNMT3b, but not DNMT3a, in human carcinoma cells(A–B) BT-549 (A) and MDA-MB-231 (B) breast cancer cells stably expressing a Control shRNA (CshRNA) or a MUC1 shRNA (MUC1shRNA) were analyzed for MUC1 and DNMT1 mRNA levels by qRT-PCR with pairs of primers listed in Table S1. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1) (left and middle panels). Lysates were immunoblotted with the indicated antibodies (right panels). (C) MCF-7 breast cancer cells stably expressing an empty vector (MCF-7/vector) or MUC1-C (MCF-7/MUC1-C) were analyzed for DNMT1 mRNA levels by qRT-PCR. The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained for vector expressing cells (assigned a value of 1) (left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D–E) The indicated BT-549 (D) and MDA-MB-231 (E) cells were analyzed for (i) DNMT3a (left panels) and DNMT3b mRNA (middle panels). The results (mean±SD of 3 determinations) are expressed as relative mRNA levels compared to that obtained with cells expressing MUC1shRNA (assigned a value of 1). Lysates were analyzed by immunoblotting with the indicated antibodies (right panels). (F) MCF-7/vector and MCF-7/MUC1-C cells were analyzed for DNMT3a (left panel) and DNMT3b (middle panel) mRNA levels by qRT-PCR (mean±SD of 3 determinations) as compared to that obtained for vector expressing cells (assigned a value of 1). Lysates from the indicated cells were analyzed by immunoblotting (right panel).
Mentions: Stable silencing of MUC1-C in BT-549 breast cancer cells was associated with suppression of DNMT1 mRNA and protein (Fig. 1A). A similar response to MUC1-C silencing was observed in MDA-MB-231 breast cancer cells (Fig. 1B), indicating that MUC1-C promotes DNMT1 expression. To confirm this notion, we overexpressed MUC1-C in MCF-7 breast cancer cells (24) and observed an increase in DNMT1 mRNA and protein (Fig. 1C), indicating that MUC1-C, and not the MUC1 N-terminal subunit (MUC1-N), is sufficient for this response. In extending these observations to other types of carcinomas, we found that silencing MUC1-C in KRAS mutant A549 (Fig. S1A) and H460 (Fig. S1B) non-small cell lung cancer (NSCLC) cells similarly results in decreased DNMT1 expression. In addition, MUC1-C conferred DNMT1 expression in SK-CO-1 colon cancer cells (Fig. S1C), indicating that this association is found in diverse types of carcinomas. In further studies, we found that silencing MUC1-C in BT-549 cells suppressed DNMT3b, but not DNMT3a, expression (Fig. 1D). The same pattern of response was observed in MDA-MB-231 (Fig. 1E), A549 NSCLC (Fig. S2A), H460 NSCLC cells (Fig. S2B), and SK-CO-1 colon cancer cells (Fig. S2C). We also found that overexpression of MUC1-C in MCF-7 cells increases DNMT3b and not DNMT3a (Fig. 1F). To provide additional support for these findings, we used other approaches for targeting MUC1-C expression. In this way, silencing MUC1 in H460 cells with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing resulted in the downregulation of DNMT1 and DNMT3b (Fig. S3A). Moreover, targeting MUC1-C function in BT-549 and A549 cells with the GO-203 inhibitor (26) decreased DNMT1 and DNMT3b levels (Figs. S3B and S3C). These results demonstrate that MUC1-C selectively drives DNMT1 and DNMT3b, but not DNMT3a, expression in breast and other carcinoma cells.

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

No MeSH data available.


Related in: MedlinePlus