MUC1-C regulates NEAT1 lncRNA expression and paraspeckle formation in cancer progression

Abstract

The MUC1 gene evolved in mammals for adaptation of barrier tissues in response to infections and damage. Paraspeckles are nuclear bodies formed on the NEAT1 lncRNA in response to loss of homeostasis. There is no known intersection of MUC1 with NEAT1 or paraspeckles. Here, we demonstrate that the MUC1-C subunit plays an essential role in regulating NEAT1 expression. MUC1-C activates the NEAT1 gene with induction of the NEAT1_1 and NEAT1_2 isoforms by NF-κB- and MYC-mediated mechanisms. MUC1-C/MYC signaling also induces expression of the SFPQ, NONO and FUS RNA binding proteins (RBPs) that associate with NEAT1_2 and are necessary for paraspeckle formation. MUC1-C integrates activation of NEAT1 and RBP-encoding genes by recruiting the PBAF chromatin remodeling complex and increasing chromatin accessibility of their respective regulatory regions. We further demonstrate that MUC1-C and NEAT1 form an auto-inductive pathway that drives common sets of genes conferring responses to inflammation and loss of homeostasis. Of functional significance, we find that the MUC1-C/NEAT1 pathway is of importance for the cancer stem cell (CSC) state and anti-cancer drug resistance. These findings identify a previously unrecognized role for MUC1-C in the regulation of NEAT1, RBPs, and paraspeckles that has been co-opted in promoting cancer progression.

Fig. 1. MUC1-C activates NEAT1 expression by MYC- and NF-κB-mediated mechanisms.

BT-549/tet-MUC1shRNA (A) and MDA-MB-468/tet-MUC1shRNA (B) cells treated with vehicle or DOX for 7 days were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR using primers listed in Supplementary Table S1. The results (mean±SD of 5 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). C BT-549/CshRNA and BT-549/MUC1shRNA#2 cells were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of at least 3 independent biological replicates) are expressed as relative levels compared to that obtained for CshRNA-expressing cells (assigned a value of 1). D BT-549/tet-MUC1shRNA cells expressing a tet-MUC1-CD vector were treated with vehicle or DOX for 7 days and analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of at least 3 independent biological replicates) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). E BT-549 cells treated with vehicle or 5 μM GO-203 for 48 h were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of 5 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). F Schema of the NEAT1 gene with highlighting of the dELS and PLS regions. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-MUC1-C and anti-MYC (left) or with anti-MUC1-C and anti-NF-κB p65 (right). The DNA samples were amplified by qPCR with primers for the NEAT1 dELS region (left) and the PLS region (right). The results (mean ± SD of 3 determinations) are expressed as percent input. G BT-549/tet-MYCshRNA cells treated with vehicle or DOX for 7 days were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). H BT-549/CshRNA and BT-549/NF-κBshRNA cells were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean ± SD of 6 determinations) are expressed as relative levels compared to that obtained for CshRNA-expressing cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right).

Fig. 2. NEAT1 regulates MUC1-C expression in an auto-inductive pathway.

A BT-549/CsiRNA and BT-549/NEAT1siRNA cells were analyzed for MUC1-C transcripts by qRT-PCR. The results (mean±SD of 3 biological replicates) are expressed as relative levels compared to that obtained for CsiRNA cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). B BT-549/CshRNA and BT-549/NEAT1shRNA cells were analyzed for MUC1-C transcripts by qRT-PCR. The results (mean±SD of 3 independent biological replicates) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). C, D GSEA of RNA-seq data from NEAT1siRNA vs CsiRNA cells using the indicated HALLMARK gene signatures. E Venn diagrams of common downregulated and upregulated genes in NEAT1- and MUC1-C-silenced cells. F, G GSEA of RNA-seq data from NEAT1siRNA vs CsiRNA cells using the indicated HALLMARK gene signatures. H, I BT-549/CshRNA, BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days, and BT-549/NEAT1shRNA cells were analyzed for DUSP2 (H) and IL-6 (I) transcripts by qRT-PCR. The results (mean ± SD of 3 independent biological replicates) are expressed as relative levels compared to that obtained for DOX- and CshRNA cells (assigned a value of 1).

Fig. 3. MUC1-C regulates SFPQ, NONO, and FUS expression by MYC-dependent mechanisms.

A BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for nascent SFPQ and NONO transcription. The results (mean±SD of at least 3 independent biological replicates) are expressed as relative gene transcription compared to that obtained in vehicle-treated cells (assigned a value of 1). B BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 3 biological replicates) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). C BT-549/tet-MYCshRNA cells treated with vehicle or DOX for 7 days were analyzed for SFPQ and NONO transcripts by qRT-PCR (left). The results (mean±SD of at least 3 replicates) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). D Schema of the SFPQ gene with highlighting of the dELS1 and dELS2 regions. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-MYC. The DNA samples were amplified by qPCR with primers for the SFPQ dELS1 and dELS2 regions. The results (mean ± SD of 3 replicates) are expressed as percent input. E Schema of the NONO gene with highlighting of the PLS region. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle of DOX for 7 days was precipitated with anti-MYC. The DNA samples were amplified by qPCR with primers for the NONO PLS region. The results (mean ± SD of 3 replicates) are expressed as percent input. F BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 3 biological replicates) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). G BT-549/tet-MYCshRNA cells treated with vehicle or DOX for 7 days were analyzed for FUS transcripts by qRT-PCR. The results (mean±SD of at least 3 biological replicates) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right).

Fig. 4. MUC1-C activates NEAT1 and RBP genes by PBAF-mediated increases in chromatin accessibility.

A BT-549 cells expressing a CshRNA, BRG1shRNA or PBRM1shRNA were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of three independent biological replicates) are expressed as relative RNA levels compared to that obtained for CshRNA-expressing cells (assigned a value of 1). B Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-BRG1 and anti-PBRM1. The DNA samples were amplified by qPCR with primers for the NEAT1 PLS region. The results (mean ± SD of 3 biological replicates) are expressed as percent input. C Genome browser snapshot of ATAC-seq data from the NEAT1 PLS in BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean ± SD of at least 3 biological replicates) are expressed as % undigested chromatin. D Genome browser snapshot of ATAC-seq data from the NEAT1 PLS in BT-549/CshRNA and BT-549/PBRM1shRNA cells (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean ± SD of 3 replicates) are expressed as % undigested chromatin. E BT-549 cells expressing a CshRNA, BRG1shRNA or PBRM1shRNA were analyzed for SFPQ and NONO transcripts by qRT-PCR. The results (mean±SD of 3 independent replicates) are expressed as relative RNA levels compared to that obtained for CshRNA-expressing cells (assigned a value of 1). F Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-MUC1-C, anti-BRG1, and anti-PBRM1. The DNA samples were amplified by qPCR with primers for the SFPQ dELS2 region. The results (mean ± SD of three independent biological replicates) are expressed as percent input. G. Genome browser snapshot of ATAC-seq data from the SFPQ dELS2 in BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean ± SD of 3 replicates) are expressed as % undigested chromatin. H Genome browser snapshot of ATAC-seq data from the SFPQ dELS2 in BT-549/CshRNA and BT-549/PBRM1shRNA cells (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean ± SD of 3 replicates) are expressed as % undigested chromatin.

Fig. 5. MUC1-C-induced regulation of RBP proteins.

A Lysates of BT-549 cells were immunoprecipitated with a control IgG or anti-MUC1-C. The input lysate and precipitates were immunoblotted with antibodies against the indicated proteins. B Immunofluorescence staining of MUC1-C, SFPQ, and FUS in BT-549 cells. Nuclei were stained with DAPI. The enlarged inset images and colocalization analyses are on the right. Pearson’s coefficients of colocalization: MUC1-C + SFPQ = 0.625, MUC1-C + FUS = 0.594. C Cytoplasmic and nuclear lysates from BT-549/CsiRNA and BT-549/NEAT1siRNA cells were immunoblotted with antibodies against the indicated proteins. D Total cell lysates of BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. E Cytoplasmic and nuclear lysates from BT-549/CsiRNA and BT-549/NEAT1siRNA were immunoblotted with antibodies against the indicated proteins. F Total cell lysates from BT-549/CsiRNA and BT-549/NEAT1siRNA cells were immunoblotted with antibodies against the indicated proteins. G Representative NEAT1 RNA-FISH images of BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days with highlighting of red NEAT1 foci. H Representative NEAT1 RNA-FISH images of BT-549/CshRNA and BT-549/MUC1shRNA#2 cells with highlighting of red NEAT1 foci.

Fig. 6. MUC1-C/NEAT1 pathway contributes to chemoresistance.

A BT-549 and BT-549/PTX-R cells were analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of three replicates) are expressed as relative levels compared to that obtained for BT-549 cells (assigned a value of 1). B BT-549/PTX-R cells expressing tet-MUC1shRNA were treated with vehicle or DOX for 7 days and analyzed for NEAT1_1 and NEAT1_2 transcripts by qRT-PCR. The results (mean±SD of three replicates) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). C BT-549/PTX-R cells expressing CshRNA or NEAT1shRNA were analyzed for ABCC4 and ABCC5 transcripts by qRT-PCR. The results (mean±SD of three independent replicates) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1). D BT-549/PTX-R cells expressing CshRNA or NEAT1shRNA were treated with 1 nM PTX for 24, 48, and 72 h. Cell viability was assessed by Alamar blue assay. The results (mean ± SD of 3 biologic replicates each with 5 determinations) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1). E BT-549/PTX-R cells expressing CshRNA or NEAT1shRNA treated with PTX were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three biologic replicates) are expressed as relative absorbance compared to that for untreated cells (assigned a value of 1) (right). F Analysis of the TCGA BRCA cohort assessing the correlation of MUC1 with NEAT1 in patient samples. Disease-free survival of patients with grade 3 TNBC tumors treated with cytotoxic chemotherapy expressing high vs low levels of MUC1 (G) and NEAT1 (H).

Fig. 7. MUC1-C/NEAT1 pathway drives the CSC state.

A RNA-seq was performed in triplicate on BT-549/NEAT1siRNA and BT-549/CsiRNA cells. GSEA was performed using the BENPORATH ES 2 gene signature. B Candidate box plots showing common NEAT1 and MUC1-C driven stemness genes from RNA-seq data. C Lysates of BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. D Lysates from BT-549/CsiRNA and BT-549/NEAT1siRNA cells were immunoblotted with antibodies against the indicated proteins. E BT-549 cells grown as monolayers in 2D culture and as tumorspheres in 3D culture were analyzed for the indicated RNA levels by qRT-PCR. The results (mean±SD of three replicates) are expressed as relative levels compared to that obtained for BT-549 2D cells (assigned a value of 1). F BT-549/tet-MUC1shRNA 3D cells treated with vehicle or DOX for 7 days were analyzed for the indicated RNA levels. The results (mean±SD of three replicates) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). G BT-549/CshRNA 3D and BT-549/NEAT1shRNA 3D cells were analyzed for tumorsphere formation. Photomicrographs are shown for the tumorspheres (bar represents 100 μm; left). The results (mean ± SD of three determinations) are expressed as tumorsphere number (right). H BT-549/NEAT1shRNA 3D cells transfected with an empty or MUC1-C-expressing vector were analyzed for tumorsphere formation. Photomicrographs are shown for the tumor spheres (bar represents 100 μm; left). The results (mean ± SD of three determinations) are expressed as tumorsphere number (right).

Fig. 8. Schema depicting MUC1-C-mediated regulation of NEAT1 and NEAT1-binding proteins.

A MUC1-C binds directly to NF-κB p65 and MYC in regulating their target genes. Based on the present results, MUC1-C/NF-κB complexes occupy the NEAT1 gene PLS region and recruit BRG1 and PBRM1 with increases in chromatin accessibility and expression. As confirmation of MUC1-C dependence, silencing MUC1-C decreased occupancy of NF-κB, BRG1, and PBRM1, and chromatin accessibility of NEAT1 gene PLS region. Silencing MUC1-C similarly decreased occupancy of MYC, BRG1, and PBRM1 on the NEAT1 dELS region. In concert with these results, MUC1-C, NF-κB, MYC, BRG1, and PBRM1 were necessary for NEAT1_1 and NEAT1_2 expression. B MUC1-C/MYC complexes integrate activation of NEAT1 with regulation of the SFPQ, NONO and FUS genes. MUC1-C was shown to be necessary for occupancy of MYC, BRG1, and PBRM1 on the (i) SFPQ dELS region, and (ii) NONO and FUS PLS regions. MUC1-C, MYC, BRG1, and PBRM1 were also necessary for chromatin accessibility of the SFPQ, NONO, and FUS genes and their expression. C MUC1-C forms complexes with SFPQ and FUS, but not NONO, and MUC1-C localizes with SFPQ and FUS in paraspeckles. As found for NEAT1_2, SFPQ, NONO, and FUS expression, silencing MUC1-C decreased paraspeckle formation. In terms of functional significance, MUC1-C and NEAT1 drive gene signatures associated with intrinsic chronic inflammation and cancer progression. MUC1-C and NEAT1 were necessary for self-renewal capacity and drug resistance in concert with driving inflammatory memory and the CSC state.