RASSF1A encodes a tumor suppressor that inhibits the RAS→RAF→MEK→ERK pathway and is one of the most frequently inactivated genes in human cancers. MUC1-C is an oncogenic effector of the cancer cell epigenome that is overexpressed in diverse carcinomas. We show here that MUC1-C represses RASSF1A expression in KRAS wild-type and mutant cancer cells. Mechanistically, MUC1-C occupies the RASSF1A promoter in a complex with the ZEB1 transcriptional repressor. In turn, MUC1-C/ZEB1 complexes recruit DNA methyltransferase 3b (DNMT3b) to the CpG island in the RASSF1A promoter. Targeting MUC1-C, ZEB1, and DNMT3b thereby decreases methylation of the CpG island and derepresses RASSF1A transcription. We also show that targeting MUC1-C regulates KRAS signaling, as evidenced by RNA-seq analysis, and decreases MEK/ERK activation, which is of importance for RAS-mediated tumorigenicity. These findings define a previously unrecognized role for MUC1-C in suppression of RASSF1A and support targeting MUC1-C as an approach for inhibiting MEK→ERK signaling.
The RAS Association Domain Family 1A (RASSF1A) tumor suppressor gene (TSG) is localized to a region in chromosome 3 (3p21.3) that is deleted in human lung and certain other cancers [1, 2]. RASSF1A expression is also repressed in diverse cancers by promoter hypermethylation [3, 4]. Importantly, RASSF1A is one of the most frequently downregulated TSGs in human cancers [5,6,7,8]. RASSF1A forms a complex with KRAS and regulates multiple downstream effectors, including suppression of the canonical RAF→MEK→ERK pathway [8,9,10]. RASSF1A thereby relieves RAS→RAF-mediated suppression of the MST2 kinase [11, 12], linking RASSF1A to the HIPPO tumor suppressor pathway [13, 14]. RASSF1A also promotes the formation of a complex between YAP and p73, resulting in the transcriptional activation of cell differentiation [15, 16]. In addition, RASSF1A links KRAS to MOAP-1 and thereby activation of the proapoptotic BAX pathway [17, 18]. Other studies have shown that RASSF1A depletion induces the epithelial–mesenchymal transition (EMT) and the metastatic potential of lung cancer cells . RASSF1A deficiency thus enhances the development of KRAS-driven lung tumors in association with induction of a proinflammatory response . These findings have supported the importance of RASSF1A in integrating (i) regulation of the KRAS pathway, (ii) activation of proapoptotic signaling, and (iii) suppression of inflammation, EMT, and tumorigenesis.
MUC1-C is an oncoprotein that associates with receptor tyrosine kinases (RTKs) at the cell membrane and promotes activation of their downstream signaling pathways [21,22,23,24]. MUC1-C also localizes to the nucleus , where it interacts with transcription factors, such as β-catenin/TCF4 [26,27,28] and p53 , and regulates expression of their target genes . The role of nuclear MUC1-C extends to the epigenetic repression of TSGs by activating (i) DNA methyltransferase 1 (DNMT1) and DNMT3b, and thereby DNA methylation  and (ii) function of polycomb repressive complex 1 (PRC1)  and PRC2  with downregulation of TSG transcription . MUC1-C thereby represses expression of the Crumbs CRB3 polarity factor , which functions as a tumor suppressor by activating the HIPPO cascade of MST1/2 and LATS1/2 signaling [35, 36]. In this way, MUC1-C activates YAP and YAP/β-catenin-mediated induction of WNT target genes, such as MYC . In contrast to RASSF1A, MUC1-C binds directly to the BAX BH3 domain with inhibition of BAX function  and is of importance to induction of EMT and the cancer stem cell (CSC) state [33, 38]. These findings have collectively supported the notion that MUC1-C plays an opposing role to that of RASSF1A in the regulation of pathways linked to cancer progression.
The RASSF1A promoter contains a CpG island that is frequently hypermethylated in lung , breast  and diverse other carcinomas . MUC1-C has been linked to TSG repression ; however, there is no known association between MUC1-C and hypermethylation of the RASSF1A promoter. In addressing this issue, the present studies demonstrate that MUC1-C forms a complex with ZEB1 on the RASSF1A promoter, recruits DNMT3b and suppresses RASSF1A transcription. The results support a model in which MUC1-C is necessary for RASSF1A promoter methylation, downregulation of RASSF1A expression and activation of MEK→ERK signaling.
MUC1-C suppresses RASSF1A expression
RASSF1A is repressed in diverse cancers [5,6,7,8]. To investigate if MUC1-C is involved in RASSF1A regulation, we first studied the effects of silencing MUC1-C in BT-549 and MDA-MB-231 TNBC cells and, notably, found upregulation of RASSF1A mRNA and protein (Fig. 1a, b; Supplemental Fig. S1A). Similar results were obtained in KRAS mutant A549 and H460 NSCLC cells (Fig. 1c, d; Supplemental Fig. S1B), indicating that the effects of MUC1-C on RASSF1A are independent of KRAS status. These findings were not limited to TNBC and NSCLC cells in that downregulation of MUC1-C also induced RASSF1A expression in PC-3 prostate cancer cells (Fig. 1e, f). In concert with these results, enforced expression of MUC1-C in MUC1-null HEK293 cells was associated with suppression of RASSF1A mRNA and protein (Fig. 1g, h). These findings supported a role for MUC1-C in the repression of RASSF1A expression.
MUC1-C forms a complex with ZEB1 on the RASSF1A promoter
MUC1-C induces the ZEB1 transcriptional repressor in human cancer cells . In turn, MUC1-C binds to ZEB1 and promotes repression of ZEB1 target genes, such as miR-200c . The RASSF1A gene includes potential ZEB1 binding motifs upstream to the CpG island in the promoter region and in intron 1 (Fig. 2a). ChIP-qPCR studies of chromatin from BT-549 (Fig. 2b) and A549 (Fig. 2c) cells demonstrated that (i) MUC1-C and ZEB1 occupy the RASSF1A promoter region, and (ii) silencing MUC1-C decreases ZEB1 occupancy (Fig. 2d, e). We also found that MUC1-C and ZEB1 are detectable on RASSF1A intron 1 (Fig. 2f) and that MUC1-C silencing decreases the occupancy of ZEB1 in this region (Fig. 2g). Similar results were obtained in PC-3 cells (Supplemental Fig. S2A, B), consistent with a role for MUC1-C in enhancing ZEB1 binding to its target genes.
MUC1-C suppresses RASSF1A activation by a ZEB1-mediated mechanism
As shown for MUC1-C, stable silencing of ZEB1 in BT-549 cells was associated with upregulation of RASSF1A mRNA and protein (Fig. 3a, b). We also found that silencing MUC1-C or ZEB1 was associated with comparable increases in RASSF1A expression (Fig. 3c). ZEB1 silencing in A549 cells similarly resulted in RASSF1A induction (Fig. 3d, e). In the HEK293 cell model, MUC1-C-induced repression of RASSF1A was attenuated by silencing ZEB1, confirming involvement of the MUC1-C→ZEB1 pathway in suppressing RASSF1A expression (Fig. 3f, g). In concert with these findings, overexpression of MUC1-C in MCF-10A breast epithelial cells was associated with induction of ZEB1 and repression of RASSF1A (Supplemental Fig. S3).
To further assess these effects of MUC1-C and ZEB1, BT-549 cells were transfected to express a RASSF1A promoter-luciferase reporter (pRASSF1A-Luc) containing the ZEB1 binding site (Fig. 4a). pRASSF1A-Luc activity was induced by silencing MUC1-C (Fig. 4b) or ZEB1 (Fig. 4c). Similar studies in A549 cells confirmed these effects of MUC1-C (Fig. 4d) and ZEB1 (Fig. 4e) on pRASSF1A-Luc activation, supporting the premise that MUC1-C represses the RASSF1A promoter by a ZEB1-mediated mechanism.
MUC1-C/ZEB1 recruit DNMT3b to the RASSF1A promoter
Methylation of RASSF1A promoter has been identified as one mechanism responsible for suppression of RASSF1A expression [3, 4]. Carcinoma cells under study here were therefore treated with decitabine (DEC; 5-aza-2′-deoxycytidine) to assess whether DNA methylation contributes to RASSF1A repression. As anticipated, we found upregulation of RASSF1A in response to DEC treatment (Supplemental Fig. S4A-C). These and the above findings that the MUC1-C→ZEB1 pathway represses RASSF1A activation thus invoked the possibility that MUC1-C/ZEB1 complexes contribute to RASSF1A promoter methylation. MUC1-C drives DNMT3b expression and changes in DNA methylation patterns in cancer cells . In addition, ZEB1 has been associated with recruitment of DNMT3b [42, 43], supporting a potential model in which MUC1-C/ZEB1 complexes associate with DNMT3b on the RASSF1A promoter. Indeed, ChIP studies demonstrated that, like MUC1-C and ZEB1, DNMT3b occupies the RASSF1A promoter (Fig. 5a). In re-ChIP experiments, we also found that MUC1-C and ZEB1 form complexes with DNMT3b on the RASSF1A promoter (Fig. 5b, c). Moreover, silencing MUC1-C (Fig. 5d) or ZEB1 (Fig. 5e) was associated with decreases in DNMT3b occupancy, indicating that MUC1-C/ZEB1 complexes recruit DNMT3b to the RASSF1A promoter. In support of these findings, DNMT3b occupancy was significantly increased in HEK293/MUC1-C, as compared with HEK293/vector, cells (Fig. 5f).
MUC1-C drives DNMT3b-mediated methylation of the RASSF1A promoter
To assess function of the MUC1-C/ZEB1/DNMT3b complexes, we studied the effects of silencing MUC1-C on methylation of the CpG island in the RASSF1A promoter (Fig. 6a). Immunoprecipitation of methylated DNA (MeDIP) followed by qPCR demonstrated that silencing MUC1-C (Fig. 6b), ZEB1 (Fig. 6c), or DNMT3B (Fig. 6d) decreases CpG island methylation. In addition, RASSF1A promoter methylation was increased in HEK293 cells expressing MUC1-C (Fig. 6e), confirming involvement of the MUC1-C/ZEB1/DNMT3b pathway. In concert with these findings, silencing DNMT3b was associated with increases in RASSF1A expression in BT-549 (Fig. 6f), A549 (Supplemental Fig. S5) and HEK293/MUC1-C (Fig. 6g) cells. Other work has demonstrated that RASSF1 CpG island methylation is linked to activation of RASSF1C expression . In concert with those findings, silencing MUC1-C with decreases in RASSF1 promoter methylation was associated with suppression of RASSF1C mRNA levels (Supplemental Fig. S6).
MUC1-C regulates the RAS→MEK→ERK pathway
RNA-seq analysis further demonstrated that targeting MUC1-C in BT-549 cells is highly associated with regulation of KRAS signaling as determined by gene set enrichment analysis from the Hallmarks Molecular Signature Database (Fig. 7a; Supplemental Fig. S7) . Targeting MUC1-C expression in A549 cells was also significantly associated with the Hallmark RAS Signaling gene set (Fig. 7b; Supplemental Fig. S7). In concert with this involvement of MUC1-C in KRAS signaling and MUC1-C-mediated repression of RASSF1A, we found that downregulation of MUC1-C in BT-549 cells has no apparent effect on KRAS activity (Supplemental Fig. S8), but is associated with decreases in MEK and ERK phosphorylation (Fig. 7c), consistent with the role of RASSF1A in suppression of the MEK→ERK pathway [8,9,10]. Moreover, silencing RASSF1A in BT-549/MUC1shRNA cells attenuated the suppression of pMEK and pERK levels (Fig. 7d), confirming dependence on RASSF1A for this response. Similar effects of targeting MUC1-C signaling on downregulation of pMEK and pERK were observed in A549 (Fig. 7e; Supplemental Fig. S9) and PC-3 (Fig. 7f) cells. As further support for MUC1-C→ZEB1→RASSF1A signaling in driving the MEK→ERK pathway, expression of MUC1-C in HEK293 cells increased pMEK and pERK levels (Supplemental Fig. S10A) and this response was attenuated by ZEB1 silencing (Supplemental Fig. S10B).
Epigenetic silencing of TSGs is considered an early event in oncogenesis and is universally found in human cancers . MUC1-C, a widely overexpressed oncogenic protein in human carcinomas [23, 24], has been linked to the epigenetic downregulation of TSGs, such as CDH1, CDKN2A, PTEN, and BRCA1, by mechanisms involving in part PRC1/2-mediated suppression [30,31,32,33, 47]. The present findings have identified a role for MUC1-C in downregulation of the RASSF1A TSG, which is reportedly one of the most frequently inactivated genes in over 30 types of cancers [5, 7]. Studies in normal human mammary epithelial cells identified a role for the Sp1 transcription factor in activation of the RASSF1A promoter, such that decreases in Sp1 occupancy were associated with downregulation of RASSF1A expression . Our results demonstrate that silencing MUC1-C in breast, NSCLC and prostate cancer cells is associated with induction of RASSF1A mRNA and protein. In support of these observations, enforced expression of MUC1-C in MUC1-low MCF-10A mammary epithelial cells or in MUC1-null HEK293 cells resulted in suppression of RASSF1A expression. We also found that MUC1-C occupies the RASSF1A promoter and intron 1, suggesting that MUC1-C plays a direct role in repressing RASSF1A transcription. In concert with this notion, we found that MUC1-C suppresses activation of the RASSF1A promoter. These findings provided support for the premise that overexpression of MUC1-C, as found in human carcinomas, contributes to repression of the RASSF1A gene.
RASSF1A is epigenetically silenced by promoter hypermethylation . In this respect, studies in human carcinoma cells have shown that MYC/EZH2/DNMT3b complexes occupy the RASSF1A promoter and are necessary for its methylation and inactivation . Of potential relevance to those findings, MUC1-C drives MYC [34, 50, 51], EZH2 , and DNMT3b  expression in cancer cells and could thereby contribute to the formation of MYC/EZH2/DNMT3b complexes. MUC1-C also activates the inflammatory NF-κB p65 pathway, binds to NF-κB p65 and induces transcription of ZEB1 [38, 41, 52] (Fig. 7g). In turn, MUC1-C forms a complex with ZEB1 and promotes ZEB1-mediated transcriptional repression . MUC1-C and ZEB1 thus cooperate in suppression of the miR-200c gene and thereby the induction of EMT in human cancer cells [30, 38]. The present studies extend the importance of the MUC1-C→NF-κB p65→ZEB1 pathway by demonstrating that MUC1-C and ZEB1 also occupy the RASSF1A promoter and suppress its activation. In addition, we found that MUC1-C/ZEB1 complexes recruit DNMT3b to the RASSF1A promoter and that MUC1-C, ZEB1 and DNMT3b are necessary for its methylation (Fig. 7g). In this way, MUC1-C/ZEB1-mediated recruitment of DNMT3b could integrate with that conferred by MYC/EZH2  and thereby further enhance methylation of the RASSF1A promoter. Moreover, MUC1-C binds to EZH2 and increases H3K27 trimethylation . Therefore, MUC1-C could directly contribute to the function of MYC/EZH2/DNMT3b complexes by interacting with EZH2 . Our findings thus (i) support a model in which MUC1-C→ZEB1→DNMT3b signaling contributes to repression of RASSF1A, (ii) invoke the possibility for functional integration of MUC1-C/ZEB1/DNMT3b and MYC/EZH2/DNMT3b complexes on the RASSF1A promoter, and (iii) provide evidence for a potential link between ZEB1-mediated induction of EMT and downregulation of RASSF1A expression (Fig. 7g). Our findings may also provide the basis for studies of other TSGs, such as HIC1 , that are hypermethylated in cancer cells.
RASSF1A plays an important role in the regulation of RAS signaling and downstream effectors, such as the MEK→ERK pathway [8, 12, 15, 54,55,56]. In this capacity and as determined using the Hallmarks Molecular Signature Database, we found that targeting MUC1-C in TNBC and NSCLC cells is highly associated with the RAS signaling gene set. To our knowledge, MUC1-C has not been previously linked to regulation of the RAS pathway. Notably, these findings do not preclude a role for MUC1-C in other pathways, such as GRB2/SOS , that like RASSF1A contribute to the control of RAS signaling. Further studies will thus be needed to more precisely address other potential relationships between MUC1-C and RAS. Along these lines, we found that (i) targeting MUC1-C in carcinoma cells is associated with suppression of the MEK→ERK pathway, and (ii) overexpression of MUC1-C in HEK293 cells with suppression of RASSF1A results in activation of MEK and ERK. Importantly, RAF→MEK→ERK signaling is necessary for RAS-induced oncogenesis  and inhibiting this pathway has represented a major focus of drug development [58, 59]. However, additional weapons are needed for the treatment of RAS-driven carcinomas. Therefore, targeting the MUC1-C→ZEB1→DNMT3b pathway with derepression of RASSF1A could represent an alternative strategy for inhibiting MEK→ERK signaling in cancer cells (Fig. 7h). In this context, previous work demonstrated that targeting MUC1-C is associated with marked synergy in combination with MEK inhibitors . These findings were attributed to the effects of targeting MUC1-C on downregulation of BCL-XL . The present results demonstrating that targeting MUC1-C induces RASSF1A and suppresses pMEK and pERK therefore provide new insights regarding the potential basis for synergy with MEK inhibitors. Of additional importance, RAS signaling in cancer is MYC dependent [57, 61]. In this respect, MUC1-C drives MYC expression in carcinoma cells [34, 50, 51] and, accordingly, targeting MUC1-C could suppress integration of the RAS and MYC pathways in promoting cancer progression.
Materials and methods
Human BT-549 TNBC, A549 (mutant KRAS) NSCLC, H460 (mutant KRAS) NSCLC, and embryonic kidney HEK293 cells were cultured in RPMI1640 medium (ATCC, Manassas, VA, USA). MDA-MB-231 (mutant KRAS) TNBC cells were grown in Dulbecco’s modified Eagle’s medium (Corning, Manassas, VA, USA). PC-3 prostate cancer cells were grown in F-12K medium (ATCC). MCF-10A cells were cultured in MEGM medium (Lonza, Walkersville, MD, USA). Media were supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cell authentication was performed by short tandem repeat analysis. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells stably expressing a control scrambled shRNA (CshRNA), MUC1shRNA, ZEB1shRNA, DNMT3bshRNA, empty vector or MUC1-C were generated as described [30,31,32]. Cells were transfected to express a control siRNA (AM4611; ThermoFisher Scientific, Waltham, MA, USA) or RASSF1A siRNA (AM16708; ThermoFisher Scientific) in the presence of Lipofectamine RNAimax reagent (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative reverse-transcription PCR (qRT-PCR)
Total RNA was isolated using Trizol reagent (Invitrogen). cDNAs were synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY, USA) . Samples were amplified using the Power SYBR Green PCR Master Mix (Applied Biosystems) and the 7300 Realtime PCR System (Applied Biosystems). Primers used for qRT-PCR analysis are listed in the Supplemental Table S1.
Whole cell lysates were prepared in NP-40 buffer containing protease inhibitor cocktail (ThermoFisher Scientific). Immunoblotting was performed with anti-MUC1-C , anti-RASSF1A (Abcam, Cambridge, MA, USA), anti-β-actin (Sigma), anti-ZEB1, anti-DNMT3b, anti-pMEK(S217/S221), anti-MEK, anti-pERK(T202/Y204), and anti-ERK (Cell Signaling Technologies, Danvers, MA, USA).
RASSF1A promoter-luciferase reporter assays
Cells were transfected with (i) an empty pGL3 vector, (ii) a pRASSF1A-Luc vector containing RASSF1A promoter sequences –600 to +19 relative to the TSS, and (iii) SV-40-Renilla-Luc in the presence of Lipofectamine 3000 Reagent (Invitrogen). At 48 h after transfection, cell extracts were prepared using the Luciferase Assay System (Promega, Madison, WI, USA). Luminescence was detected with the Dual-Luciferase Reporter Assay System (Promega).
Chromatin immunoprecipitation (ChIP) assay
Soluble chromatin was precipitated with anti-MUC1-C (NeoMarkers, Fremont, CA, USA), anti-ZEB1, anti-DNMT3b, or a control non-immune IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In re-ChIP studies, complexes from the primary anti-MUC1-C or anti-ZEB1 ChIPs were eluted and re-immunoprecipitated with anti-DNMT3b. The precipitates were analyzed by ChIP-PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems) and the 7300 Realtime PCR System (Applied Biosystems). Primers used for ChIP-PCR are listed in the Supplemental Table S2. Data are reported as the fold enrichment relative to IgG .
Promoter methylation analysis was performed using the Methylation DNA IP (MeDIP) kit (Active Motif) as described . Primers used for MeDIP are listed in Supplemental Table S3. Data are reported as the fold enrichment relative to IgG .
Total RNA from cells cultured in triplicates was isolated using Trizol reagent (Invitrogen). TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) was used for library preparation.
RNA-seq data analysis
Raw sequencing reads were aligned to the human genome (GRCh38.74) using STAR (20.1 × 106 uniquely mapped reads per sample). Raw feature counts were normalized and differential expression analysis using DESeq2. Differential expression rank order was utilized for subsequent GSEA, performed using the fgsea (v1.8.0) package in R. Gene sets queried included the Hallmark Gene Sets available through the Molecular Signatures Database (MSigDB) .
KRAS activation assays
Lysates were assayed for KRAS activation according to the manufacturer’s instructions (Cat. #STA-400-K; Cell Biolabs, San Diego, CA).
Each experiment was repeated at least three times. Data are expressed as the mean ± SD. The unpaired Student’s t-test was used to examine differences between means of two groups. A p-value of < 0.05 was considered a statistically significant difference.
Data and software availability
The accession number for the RNA-seq data reported in this paper is GEO ACCESSION GSE123860.
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This work was supported by Grants from the National Cancer Institute of the National Institutes of Health under award numbers CA97098, CA166480, CA216553, CA233084, and U24CA232979.
Conflict of interest
Regarding potential conflicts of interest, D. Kufe has equity interests in, serves as a member of the board of directors of and is a paid consultant to Genus Oncology. The other authors declare that they have no conflict of interest.
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Rajabi, H., Hata, T., Li, W. et al. MUC1-C represses the RASSF1A tumor suppressor in human carcinoma cells. Oncogene 38, 7266–7277 (2019). https://doi.org/10.1038/s41388-019-0940-1
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