Basonuclin-2 regulates extracellular matrix production and degradation

Abstract

Epithelial-mesenchymal transition is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the ECM-a complex environment of proteoglycans and fibrous proteins essential for tissue homeostasis, signaling in response to chemical and biomechanical stimuli, and is often dysregulated under conditions such as cancer, fibrosis, and chronic wounds. Here, we demonstrate that basonuclin-2 (BNC2), a mesenchymal-expressed gene, that is, strongly associated with cancer and developmental defects across genome-wide association studies, is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases, and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the production and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers, which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.

Figure 1.. Basonuclin-2 (BNC2) is a mesenchymally expressed, epithelial–mesenchymal transition responsive gene, that is, targeted by miR-200.

(A) An epithelial or mesenchymal score is attributed to individual tumours (breast cancer, TCGA) based upon previously established gene signatures (52). (B, C) BNC2 expression is labelled for all tumours on an epithelial versus mesenchymal axis, or when epithelial (B) or mesenchymal (C) genes are considered separately. (D) BNC2 expression across breast cancer samples (TCGA) stratified by high versus low mesenchymal score. (E) BNC2 expression in TCGA samples across breast cancer subtypes, stratified by mesenchymal score. (F) BNC2 expression as measured by qRT-PCR across a panel of human breast cancer cell lines. CDH1 (E)-cadherin is an epithelial marker, FN1 (fibronectin) is a mesenchymal marker. (G, H, I) Relative BNC2 expression upon continuous TGF-β treatment of multiple epithelial-like cell lines (G) HMLE; (H) MCF10A; (I) MDCK). (J) Predicted miR-200–responsive target sites within the BNC2 3′UTR. Scores (in brackets) for miR-200a or miR-200b binding are taken from TargetScan 7.1, in which the most confidently predicted sites are indicated by scores closer to 100. (K) BNC2 expression as measured by qRT-PCR in response to miR-200c in mesHMLE cells. (L) Normalized activity of a BNC2-3′UTR Renilla/luciferase reporter gene in response to miR-200c. All data are representative of triplicate experiments. * indicates P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test) relative to control transfection.

Figure S1.. Commercial antibodies do not detect basonuclin-2 (BNC2).

(A) Three commercial anti-BNC2 antibodies were tested by Western blotting for the detection of BNC2–FLAG expression in Dox-inducible cell lines. BNC2–FLAG was only detected using the FLAG antibody. Further experiments (not shown) altering Western blotting conditions or attempting to detect endogenous BNC2 in BT549 or HS578T cells in the presence or absence of BNC2 siRNA were also unsuccessful. (B) Sigma anti-BNC2 antibody (middle panel) was used in a Western blot of cell extracts from BT549 and mesHMLE cells (both cell lines express high endogenous BNC2 mRNA). Total protein left panel, bands were quantitated and normalized to total protein on right graph. BNC2 siRNAs do not reduce band, despite reducing BNC2 mRNA. (C) Immunofluorescence using two of the above BNC2 antibodies. Again, signal is not reduced by BNC2 siRNA.

Figure S2.. Basonuclin-2 (BNC2) expression correlates with that of epithelial–mesenchymal transition markers.

(A, B) Correlation of expression between BNC2 and markers of (A) epithelial (CDH1, CRB3, EPCAM, ESRP1, ESRP2, GRHL2) or (B) mesenchymal (CDH2, FN1, PRRX1, SNAI2, VIM, ZEB1) cells are shown across the Cancer Cell Line Encyclopedia (CCLE) or The Human Genome Atlas (TCGA). Pearson correlation values are shown. (C) Heat map of relative expression of epithelial–mesenchymal transition–associated genes across breast cancer cell lines derived from CCLE. (D) BNC2 expression was correlated across the CCLE with the same 146 gene epithelial/mesenchymal signature used in Fig 1A.

Figure 2.. Basonuclin-2 (BNC2) is not a core regulator of epithelial–mesenchymal transition.

(A, B, C) Gene expression was measured by qRT-PCR after two rounds of transfection of BNC2 siRNAs across 6 d in (A) BT549, (B) HS578T, and (C) mesHMLE cells. (D) Gene expression was measured after Dox-mediated induction of a stably introduced BNC2 full-coding sequence in two MCF7 clonal cell lines. Gene expression of mesenchymal and epithelial markers are indicated. (E) BNC2 RNA induction upon Dox treatment. All data are representative of multiple experiments. (A, B, C, D) * indicates P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test) relative to either control transfection (A, B, C) or no Dox (D). (F) Fold change as assessed by RNA-Seq in the expression of epithelial or mesenchymal marker genes after BNC2 knockdown in BT549 and mesHMLE cells. From the 146 epithelial–mesenchymal transition signature genes, all genes that are expressed at >1 cpm in both cell lines are shown.

Figure S3.. Basonuclin-2 (BNC2) perturbation does not affect cell morphology.

Cell morphology of multiple breast cell lines in response to BNC2 perturbation are shown as labelled. Cells (BT549, HS578T, mesHMLE) were either transfected with two rounds of BNC2 siRNA across 6 d or subjected to Dox-induction of full-length BNC2–FLAG (MCF7, T47D) for 3 d.

Figure S4.. Basonuclin-2 (BNC2) perturbation does not affect actin or epithelial–mesenchymal transition marker localisation.

Localisation of the mesenchymal marker FN1 (in mesenchymal BT549, HS578T, and mesHMLE cells), or the epithelial marker CDH1 (in epithelial MCF7 and T47D cells), were examined along with F-actin (phalloidin) after the transfection of either control or BNC2 siRNAs (upper six panels) or Dox-induction of stably transfected BNC2 (lower four panels). Nuclei are visualized by DAPI.

Figure 3.. Basonuclin-2 is a central component of an epithelial–mesenchymal transition and ECM gene expression module.

(A) Graph visualization of a network derived from weighted gene co-expression network analysis performed using the 5,000 most differentially expressed genes among TCGA breast cancer patients. The co-expression modules identified (shown as colours) are labelled with general functions based upon GO analysis. Basonuclin-2 was the fifth most highly connected gene within the “red” cluster. (B) GO analysis results for the 170 most centrally associated genes (correlation with module eigengene of >0.7) within the “red” co-expression module.

Figure S5.. Ontologies enriched in the basonuclin-2 (BNC2) containing “red” weighted gene co-expression network analysis gene expression module.

Weighted gene co-expression network analysis was performed among the 5,000 most differentially expressed genes across TCGA breast cancer cohort. Genes were then clustered into 10 co-expression groups. The full-gene ontology enrichment analysis results of genes in the BNC2-containing “red” module is shown, including log₂ fold enrichment (enrichment) and statistical significance thereof (P Val, FDR). The total number of genes in the listed ontology (reference) and the number of genes in the list of BNC2 co-expressed genes present in that ontology (red mod) are also shown.

Figure 4.. Endogenous basonuclin-2 (BNC2) regulates the ECM and ECM degradation.

(A) Highly enriched ontologies of genes that were responsive to BNC2 siRNA transfection in BT549 cells (up- or down-regulated >3x) as measured by RNA-seq. (B) Epithelial–mesenchymal transition was identified as particularly strongly enriched among BNC2-responsive genes by Gene Set Enrichment Analysis. These genes largely represent the matrisomal component of epithelial–mesenchymal transition. (C) Heat map of gene expression from RNA-seq showing core matrisomal genes. (D) Volcano plot generated from triplicate RNA-seq experiments after BNC2 knockdown. A selection of key matrisomal genes and ECM regulators are indicated. (E) Genes that contribute to gene ontologies that are enriched in both co-expression with BNC2 in patient weighted gene co-expression network analysis (Fig 3A) and that are regulated by BNC2 knockdown in BT549 cells (Fig 4A). (F, G) Select collagens and matrix metalloproteases that are responsive to BNC2 knockdown in RNA-seq (F) were measured by qRT-PCR in BT549, HS578T and mesHMLE cells (G). * indicates P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test) relative to control transfection.

Figure S6.. Ontologies enriched after basonuclin-2 (BNC2) knockdown in BT549 cells.

Full-gene ontology (Gene Panther) enrichment for all genes up- and down-regulated at least fourfold after BNC2 siRNA transfection of BT549 cells (as measured by RNA-seq) including log₂ fold enrichment (enrichment) and statistical significance thereof (P Val, FDR). Columns with colour name labels are shown for each WGNCA gene co-expression module (Fig 3A), with coloured horizontal bands signifying that the identical gene ontology term was also enriched among these co-expressed genes in breast cancer patients (Fig S5). The red co-expression module containing BNC2 (Fig 3A) has the greatest overlap in Gene Ontology terms with genes which are differentially regulated in cells after BNC2 knockdown. The total number of genes in the listed ontology (reference), and the number of genes in the list of BNC2-regulated genes present in that ontology (BNC2 kd) are also shown.

Figure 5.. Endogenous basonuclin-2 controls collagen expression.

(A, B) Localisation of endogenous collagen type-I or type-V and F-actin (phalloidin) after basonuclin-2 siRNA transfection in (A) BT549 and (B) HS578T cells. Bar indicates 10 μm length. (C) Quantitation of collagen expression (488 nm fluorescence per cell). Individual measurements indicate average quantitation per cell from separate fields of view (∼8–15 cells in each case).

Figure 6.. Endogenous basonuclin-2 (BNC2) controls collagen deposition and matrix degradation by fibroblasts.

(A) Cell-specific BNC2 expression (transcripts per million) derived from single-cell sequencing of human tissues (obtained from the Human Protein Atlas). (B) Gene expression of selected collagens and matrix metalloproteases (as in Fig 4D) in response to knockdown of BNC2 in human fibroblasts (as in Fig 4D). (C) Immunofluorescence analysis of endogenous collagen I and F-actin in fibroblasts in response to BNC2 siRNA. Bar indicates 10 μm length. (D) Immunofluorescence analysis of collagen I in cell-derived matrix prepared from fibroblasts in which control or BNC2 siRNAs were transfected. (D, E) Motility of GFP-labelled MDA-MB-231 breast cancer cells on a 3D matrix derived from fibroblasts (shown in (D)) monitored. Speed and total distance traveled is shown (P-Val = 0.074 and 0.093, respectively). (F) BNC2 was knocked down in fibroblasts and digestion of fluorescent gelatin matrix was visualized. (G) BNC2 expression after knockdown was measured by qRT-PCR. (H) Quantitation of gelatin degradation from >250 cells. * indicates P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test) relative to control transfection.

Figure 7.. Basonuclin-2 (BNC2) promotes cell motility and invasion.

(A) Representative siRNA-mediated knockdown of BNC2 expression across immortalized breast and breast cancer cell lines. (B, C, D, E, F) Phenotypic effect of BNC2 knockdown on (B, C) migration, (D, E) invasion, and (F) wound healing comparing control to BNC2 siRNA–transfected cells. (B, C, D, E, F) siRNAs were obtained from either GenePharma (B, D, F) or QIAGEN (C, E). (G, H) Wound closure in BT549 cells in which BNC2 was knocked down using an inducible shRNA vector. (I, J, K, L) Wound closure was measured 1–3 d after the induction of full-length BNC2 by Dox in (I, J) MCF7 and (K, L) T47D cells. (H, J, L) Similar trends to those shown in (H, J, L) were seen with two independent clonal cell lines. * indicates P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test) relative to control transfection, in each case making pair-wise comparisons between control and BNC2-perturbed experiments.

Figure S7.. Basonuclin-2 promotes cell motility.

Images from migration, invasion, and wound healing assays presented in Fig 7.

Figure S8.. Basonuclin-2 promotes motility in independent clonal cell lines.

Independent clonal cell lines yield reproducible results. (A) BT549 cells matching Fig 7H. (B) MCF7 cells matching Fig 7J.

Figure S9.. Basonuclin-2 (BNC2) knockdown has minimal effect on cell number.

(A, B, C) Crystal violet assays were performed across multiple cell lines in response to (A) BNC2 siRNA, (B) BNC2 shRNA, or (C) inducible BNC2 induction by Dox.

Figure 8.. Basonuclin-2 (BNC2) expression correlates with more aggressive cancer.

(A) BNC2 expression in the bulk sequencing of matched breast invasive carcinoma (BRCA) and normal tissue was determined using Gene Expression Profiling Interactive Analysis. (B) Violin plot of BNC2 expression across different breast cancer grades. (C) Kaplan–Meier plots indicating survival of patients with four different cancers, split by whether BNC2 high/low expression is classified as the upper versus lower half of expression (50/50) or whether only the top/bottom quartiles (75/25) or top/bottom 10th percentiles (90/10) of expression are considered. (D) Relative expression of BNC2 mRNA across all breast or fibroblast cell lines within the cancer cell line encyclopedia. Breast cancer lines have been sub-divided into epithelial and mesenchymal-like based upon largely mutually exclusive E-cadherin and ZEB1 expression. (E) Gene expression derived from single-cell sequencing of 26 breast cancer patients (58). (C, F) Kaplan–Meier plots correlating patient survival with FAP (fibroblast marker) expression as described in (C).

Figure S10.. Expression and prognostic significance of basonuclin-2 (BNC2) across cancer types.

(A) BNC2 expression across tumour and normal cells in multiple cancer types using Gene Expression Profiling Interactive Analysis. Numbers indicate number of patient samples in each cohort. (B) Violin plot of BNC2 expression across different breast cancer grades. (C) Kaplan–Meier plots (from KMplot) indicating survival of patients across all cancers, split by whether BNC2 high/low expression is classified as the upper versus lower half of expression (50/50) or whether only the top/bottom quartiles (75/25) or top/bottom 10th percentiles (90/10) are considered. (D) As with (C), except patients were grouped by FAP expression.

Figure S11.. High basonuclin-2 (BNC2) expression corresponds to poorer breast cancer prognosis.

(A, B, C) Kaplan–Meier survival curves derived from (A) KMplot of 2,976 breast cancer patients, subdivided by Nottingham histological tumour grade (comparing highest and lowest quartile of BNC2 expression), (B) TCGA (1,108 patients), and (C) the Metabric dataset (2,508 patients). Plots in (B, C) were derived from Cbioportal, comparing selected highest, lowest, and remaining BNC2 expression cohorts.

Figure S12.. Basonuclin-2 knockdown regulates genes associated with fibrinolysis, semaphorin signaling, and cytokines.

RNA-Seq from BT549 cells transiently transfected with control or basonuclin-2 siRNAs. (A, B, C) The expression of genes associated with (A) the plasminogen activation system (fibrinolysis), (B) semaphorin ligands and their receptors, and (C) cytokines are shown as heat maps and volcano plots.