lncRNA directs cooperative epigenetic regulation downstream of chemokine signals

Abstract

lncRNAs are known to regulate a number of different developmental and tumorigenic processes. Here, we report a role for lncRNA BCAR4 in breast cancer metastasis that is mediated by chemokine-induced binding of BCAR4 to two transcription factors with extended regulatory consequences. BCAR4 binding of SNIP1 and PNUTS in response to CCL21 releases the SNIP1's inhibition of p300-dependent histone acetylation, which in turn enables the BCAR4-recruited PNUTS to bind H3K18ac and relieve inhibition of RNA Pol II via activation of the PP1 phosphatase. This mechanism activates a noncanonical Hedgehog/GLI2 transcriptional program that promotes cell migration. BCAR4 expression correlates with advanced breast cancers, and therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 strongly suppresses breast cancer metastasis in mouse models. The findings reveal a disease-relevant lncRNA mechanism consisting of both direct coordinated protein recruitment and indirect regulation of transcription factors.

Figure 1. BCAR4 Correlates with Breast Cancer Metastasis

(A) Scatter plots of lncRNAs significantly up-regulated (red) or down-regulated (green) in two pairs of TNBC tissues compared to the matched adjacent normal tissues (NBT). The X- and Y-axes: averaged normalized signal values (Log₂ scaled); green lines: fold changes=4. (B) Commonly up-regulated lncRNAs in two pairs of TNBC compared to NBT. (C) RNAScope® detection of BCAR4 expression in human breast cancer and adjacent normal tissues. Left panel: representative images; Right panel: statistical analysis of training set (10 normal tissues vs. 222 cancer tissues) and validation set (10 normal tissues vs. 160 cancer tissues). (D) RNAScope® detection of BCAR4 expression in Non-metastasis (TnN0M0) vs. Metastasis (TnN>0M≥0) breast cancer tissue. Left panel: representative images; Right panel: statistical analysis of training set (167 Non-metastasis vs. 55 Metastasis) and validation set (66 Non-metastasis vs. 94 Metastasis). (E) Kaplan-Meier survival analysis of BCAR4 expression in breast cancer patients (n=160). (F) RNAscope® detection of BCAR4 expression in multiple human tissues. (G) RT-qPCR detection of BCAR4 expression in a panel of cell lines. (H) Nuclear localization of BCAR4 detected by RNA FISH in MDA-MB-231 cells. (I) Identification of signal pathways affected by BCAR4 knockdown in MDA-MB-231 cells. The X- and Y-axes: normalized ratio of firefly/Renilla luciferase activities. (J) RT-qPCR detection of GLI-target genes expression. Error bars, S.E.M. of three independent experiments (*p<0.05, **p<0.01 and ***p<0.001). See also Figure S1 and Tables S1-S3.

Figure 2. Identification and Biochemical Characterization of BCAR4-associated Proteins

(A) A list of top BCAR4-associated proteins identified by RNA pulldown and MS analysis in MDA-MB-231 cells: R1 and R2 (biological repeat 1 and 2). (B and C) Immunoblot (IB) detection of proteins retrieved by in vitro transcribed biotinylated BCAR4 from MDA-MB-231 cell lysates (B) and indicated recombinant proteins (C). (D and E) IB detection of Myc-tagged SNIP1 (D) and PNUTS (E) (wt vs. domain truncation mutants) retrieved by in vitro transcribed biotinylated BCAR4. Lower panels: graphic illustration of the domain structure of SNIP1 (D) or PNUTS (E). (F) In vitro RNA-protein binding followed by dot-blot assays. Bottom panel: schematic illustration of the BCAR4 sequence motifs that is recognized by SNIP1 and PNUTS, respectively. (G) IB detection of proteins retrieved by in vitro transcribed biotinylated BCAR4 (wt vs. Δ212-311 and Δ968-1087) from MDA-MB-231 cell lysates. (H) EMSA assay of recombinant SNIP1 and PNUTS binding to BCAR4 nt. 235-288 and nt. 991-1044 respectively. (I) In vitro kinase assay showing CIT-mediated phosphorylation of GLI2 (wt vs. S149A). *: unspecific band. (J) IHC staining of phospho-GLI2 (S149) in human breast cancer and adjacent normal tissues. Left panel: representative image. Right panel: statistics analysis based on (10 normal tissues vs. 222 cancer tissues). See also Figure S2, Tables S2 and S4.

Figure 3. Identification of A Noncanonical Hedgehog Signaling Pathway Mediated by CCL21/CIT/phospho-GLI2 Signaling Axis

(A and B) Immunoprecipitation (IP) and IB detection of CIT-RhoC interactions (A) and GLI2 phosphorylations (B) in cells treated with indicated growth factors, cytokines or chemokines. (C) IP and IB of GLI2 phosphorylations in cells transfected with indicated siRNAs followed by CCL21 treatment. (D and E) IP and IB detection of GLI2-SUFU (D) or GLI2-SNIP1 (E) interactions in MDA-MB-231 cells transfected with GLI2 (wt vs. S149A) followed by CCL21 treatment. (F and G) IP and IB detection of GLI2-SNIP1 interactions in cells transfected with SNIP1 (wt vs. Δ274-349) (F) or (wt vs. FHA domain point mutants) (G) followed by CCL21 treatment. (H and I)) IP and IB (H) and Immunofluorescence (I) detection of phospho-GLI2 nuclear translocation in cells treated with CCL21 treatment at different time points (H) or transfected with indicated siRNAs followed by CCL21 treatment (I). See also Figure S3.

Figure 4. BCAR4 Is Required for CCL21-triggered, phospho-GLI2-mediated Gene Activation and Cell Migration

(A-C) ChIP-qPCR detection of GLI2 (A), phospho-GLI2 (B) or ChIRP-qPCR detection of BCAR4 (C) occupancy on the promoters of selected GLI2 target genes in MDA-MB-231 cells treated with CCL21. RPLP0 served as a non-GLI2 target gene control (A and B). (D) RT-qPCR detection of GLI2 target genes expression in MDA-MB-231 cells transfected with control or BCAR4 siRNA followed by CCL21 treatment. (E to G) Cell migration assays in MDA-MB-231 cells transfected with indicated siRNA (E and F) or treated with CCL21 neutralization antibody (G). (H) Matrigel cell invasion assay in cells transfected with indicated siRNAs. Left panel: representative images; right panel: statistical analysis. (I and J) Matrigel cell invasion assay (I) or RT-qPCR detection of GLI2 target genes (J) in MCF7 cells electroporated with indicated BCAR4 expression vectors followed by CCL21 treatment. Error bars, SEM of three independent experiments (*p<0.05, **p<0.01 and ***p<0.001). See also Figure S4.

Figure 5. Signal-induced BCAR4-SNIP1 Interaction Attenuates the Inhibitory Effect of SNIP1 on p300 HAT Activity

(A and B) RIP-qPCR detection of the indicated RNAs retrieved by Myc-specific antibody in MDA-MB-231 cells electroporated with indicated vectors followed by CCL21 treatment. (C) In vitro HAT activity assays of p300 in the presence of wt SNIP1, full-length (FL) BCAR4 and their corresponding mutants as indicated. (D) In vitro HAT activity assays of p300 in the presence of wt SNIP1 and its corresponding mutants as indicated. (E) IB detection of the interaction between SNIP1 (a.a. 97-274) and p300 (a.a. 1198-1806) wt or truncations. (F) In vitro HAT activity assays of p300 in the presence of SNIP1 a.a. 97-274 and BCAR4 sense or antisense RNAs. (G) ChIP-qPCR detection of acetylated histone marks occupancy on the promoters of selected GLI2 target genes in MDA-MB-231 cells treated with CCL21. (H) ChIP-qPCR detection of phospho-GLI2, p300 and H3K18Ac occupancy on PTCH1 promoter in MDA-MB-231 cells transfected with indicated siRNAs followed by CCL21 treatment. Error bars, SEM of three independent experiments (*p<0.05 and **p<0.01). See also Figure S5.

Figure 6. Recognition of BCAR4-dependent Histone Acetylation by PNUTS Attenuates Its Inhibitory Effect on PP1 Activity

(A) RIP-qPCR detection of the indicated RNAs retrieved by Myc-specific antibody in MDA-MB-231 cells transfected with indicated vectors followed by CCL21 treatment. (B) In vitro Phosphatase activity assays of PP1A in the presence of indicated proteins or nucleosome. (C) MODified Histone Peptide Array® detection of histone marks recognition by SNIP1 or PNUTS. Top panel: representative images; bottom panel: binding specificity. (D and E) IB detection of PNUTS retrieved by biotinylated histone peptides as indicated from lysate of MDA-MB-231 cell (D) or MDA-MB-231 cell electroporated with indicated vectors (E). (F) In vitro Phosphatase activity assays of PP1A in the presence of PNUTS and modified histones H3 as indicated. (G) ChIP-qPCR detection of H3K18Ac, PNUTS, Pol II Ser5 (normalized by Pol II occupancy) and PP1A occupancy on PTCH1 promoter in MDA-MB-231 cells pre-treated with C646 followed by CCL21 treatment. Error bars, SEM of three independent experiments (*p<0.05 and **p<0.01). See also Figure S6.

Figure 7. The Potential Therapeutic Role of BCAR4 in Breast Cancer Metastasis

(A-C) Representative bioluminescent (BLI) images (A), metastatic nodules numbers in the lungs (B) or isolated lung bright-field imaging (top panel) and H&E staining (middle and bottom panels) (C) of mice with fat pad injection of MDA-MB-231 LM2 cells harboring indicated shRNA. Data are means ± SEM (n=5). (D-F) Representative BLI images (D), lung colonization (E), and metastatic nodules numbers in the lungs (F) of mice with tail vein injection of MDA-MB-231 LM2 cells harboring indicated shRNA. Data are means ± SEM (n=5). (G and H) Representative BLI images (G) and lung colonization (H) of mice at day 30 after tail vein injection of MDA-MB-231 LM2 cells followed by intravenously LNA treatment. Data are means ± SEM (n=3). (I) RT-qPCR detection of BCAR4 expression in sorted GFP positive MDA-MB-231 LM2 cells dissociated from lung metastatic nodules of mice intravenously treated with LNA at day 30 (n=3). (J) A model for cooperative epigenetic regulation downstream of chemokine signals by lncRNA BCAR4.