Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription

Abstract

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.

Figure 1. Fibroblast-derived CDM induces sustained growth inhibition of cancer cells.

(a) Collagen I and fibronectin staining of CDM generated by NFs (TIFFs). Scale bar, 20 μm. (b) Proliferation of MDA-MB-231 and HeLa cells plated on TIFF CDM or on plastic in full medium for the indicated times. n(HeLa)=10, n(MDA-MB-231)=8. (c) Proliferation of MDA-MB-231 and HeLa cells after detachment from TIFF matrices (6 days on matrix before detachment) and replating on plastic in full medium for the indicated times n(HeLa)=7, n(MDA-MB-231)=10. (d) Representative images of MDA-MB-231 cell morphology on CDM and plastic. Shown are maximum intensity projections of confocal images. Scale bar, 10 μm. (e) Schematic representation of the experimental set-up. Red arrows indicate the time points of sample collection for Illumina gene expression analysis. (f) Common gene expression changes in MDA-MB-231 and HeLa cells on CDM and 5 days after CDM detachment (CDM to plastic (pl.)). The numbers of commonly regulated genes (up- or downregulated) in both cell lines are indicated in the table. Upregulated genes are marked with red and downregulated genes with blue. All data are mean±s.e.m. Unpaired t-test was used for statistical analyses.

Figure 2. Normal ECM restrains JMJD1a expression, cell proliferation and tumour growth.

(a,b) Western blot (a) and quantification (b) of JMJD1a protein levels normalized to loading control. (c) JMJD1a mRNA expression (qRT–PCR) relative to GAPDH mRNA in MDA-MB-231 and HeLa cells after matrix detachment (6 days on TIFF CDM and 5 days on plastic; CDM to plastic) and on plastic. (d,e) Western blot (d) and quantification of JMJD1a protein levels (e) normalized to loading control. (f) JMJD1a mRNA expression (qRT–PCR) relative to GAPDH mRNA (f) in the indicated cells plated on either TIFF CDM or on plastic for 4 days. (g) Western blot quantification showing JMJD1a stability on CDM and plastic 24 h after plating. Time of the cycloheximide (CHX) treatment is indicated and P values are calculated between 0 and 5 h. Paired t-test was used for statistical analysis, n=3. (h) Proliferation of MDA-MB-231 cells upon JMJD1a silencing on plastic, n=3. (i) Proliferation of JMJD1a-GFP or GFP-overexpressing MDA-MB-231 cells on plastic. Cells were sorted by FACS (JMJD1a: high and low; GFP: high), n=3. (j) Proliferation of GFP control and JMJD1a-GFP-overexpressing MDA-MB-231 cells on TIFF-derived CDM. n (GFP)=11 CDMs and n(JMJD1a-GFP)=12 CDMs. Two-way analysis of variance (ANOVA) was used to calculate the P value. Data are mean±s.e.m. (k) Control or JMJD1a siRNA-transfected MDA-MB-231 cells (1 × 10⁶) were implanted on CAM membranes inside a plastic ring to analyse tumour growth in vivo for 3 days. Shown are quantified tumour areas from three individual experiments n(siControl)=25, n(siJMJD1a)=23 eggs. (l) Orthotopic tumour growth assay. Control or JMJD1a siRNA-transfected MDA-MB-231 cells (2 × 10⁶) were injected into the fat pad of nude mice (n=19) and tumour growth was measured 8 days after injection. Western blot in showing the silencing efficacy of JMJD1a siRNA on the day of the injection (Day 0) and at the end of the experiment (Day 8). Shown are mean±s.d. and (g–i) mean±s.e.m. Paired t-test was used for statistical analyses in b–h and non-paired t-test in j,k.

Figure 3. Patient-derived CAF and NF CDMs are architecturally and functionally distinct.

(a,b) Representative western blots showing SMA-α (a) and YAP/TAZ expression (b) in NFs and CAFs isolated from three SCC patients. (c,d) Quantification of YAP (c) and TAZ (d) expression in NFs and CAFs normalized to loading control. Data are mean±s.d. (e) Collagen I (red) and fibronectin (blue) staining of patient #1 and #3 NF and CAF CDM. Scale bar, 20 μm. (f) Representative SEM images of TIFF, Patient #1 NF and CAF CDM. Scale bar, 5 μm. (g) Proliferation of MDA-MB-231 and HeLa cells on Patient #1 NF and CAF CDM. n(MDA-MB-231)=10–11 and n(HeLa)=19 from three independent experiments. Data are mean±s.e.m. and P values are calculated from the doubling times. (h) JMJD1a expression in MDA-MB-231 cells cultured on TIFF, NF and CAF CDMs and on plastic. Quantification shows relative JMJD1a amount normalized to loading control. (c,d) Non-parametric Mann–Whitney test was used for statistical analyses in c,d and non-paired t-test in g.

Figure 4. JMJD1a levels correlate with α-SMA-positive stroma in human tumours.

(a,b) JMJD1a and α-SMA staining from sections of the same normal breast tissue and primary breast carcinomas (a) or HNSCC (b) tissue. For breast carcinomas, examples of the immunostaining of different expression levels and two different magnifications of the same tissue are shown. Scale bar, 200 μm. S, stroma; T, tumour. Inset in a highlights the α-SMA localization in basal cells in the normal mammary gland (B, basal; L, luminal). Quantification of the incidence of JMJD1a or α-SMA positivity in the analysed samples is shown. (c,d) Stiffness of the patient-derived NF or CAF (c) and TIFF (d) CDMs is expressed by the Young's modulus, which was measured using AFM indentation. Each grey dot represents individual measurement and black line indicates the mean. Non-paired t-test was used for statistical analysis. Non-paired t-test was used for statistical analysis.

Figure 5. Mechanosensitive regulation of JMJD1a on soft and stiff ECM and CDM.

(a,b) Representative western blot (a) and quantification (b) showing JMJD1a expression in cells plated on 0.5 and 50 kPa hydrogels and on plastic (PL). Tubulin was used as loading control, n=4 (mean±s.d.). (c) JMJD1a (red) and YAP/TAZ (green) and DAPI (blue) staining in MDA-MB-231 cells on large (800 μm²) and small (400 μm²) spreading area micropatterns (adhesive area is the same). Cell morphology is shown as DIC. (d) Quantification of cytoplasmic and nuclear JMJD1a and YAP/TAZ localization on small and large micropatterns. n(cells)=20 per pattern size. (e,f) Immunofluorescence staining showing (e) and quantifying (f) YAP/TAZ and JMJD1a localization on collagen I and fibronectin-coated hydrogels of varying stiffness (0.5, 4 and 50 kPa) and on plastic. Scale bar, 10 μm. (g) Representative western blot showing JMJD1a and YAP/TAZ nuclear (N) and cytoplasmic (CP) localization in cells plated on 0.5 and 50 kPa hydrogels. Lamin A/C and GAPDH were used as fractionation controls. (h,i) Immunofluorescence staining quantification (i) and representative images (h) of JMJD1a localization in cells plated on TIFF CDM, plastic or collagen and fibronectin ligands (2.5 μg ml⁻¹ collagen and 2.5 μg ml⁻¹ fibronectin). Nuclear localization of JMJD1a was quantified with the CellProfiler software. n(cells): CDM=74 cells; plastic=33 cells and collagen+FN=61. Scale bar, 10 μm. (j) Representative immunofluorescence images showing JMJD1a localization in MDA-MB-231 cells growing on NF and CAF CDMs for 3 days. Scale bar, 10 μm. (k) YAP/TAZ and JMJD1a localization in CA-SRC-expressing MDA-MB-231 cells growing on soft 0.5 kPa hydrogels or on plastic. Representative images from three independent experiments. Scale bar, 10 μm. (l) Model of distinct mechanotransductional regulation of YAP/TAZ and JMJD1a on soft and stiff. Red arrows indicate the pathway, which we and others have shown to regulate YAP/TAZ nuclear localization. Blue arrow indicates the SRC kinase-mediated and stiffness-dependent regulation of JMJD1a and YAP/TAZ.

Figure 6. JMJD1a regulates YAP/TAZ transcription

(a) Representative western blot showing YAP/TAZ expression in MDA-MB-231 on CDM and on plastic. (b) Taqman qRT–PCR of CTGF (n=4) and THBS1 (n=5) mRNA levels in MDA-MB-231 cells grown on CDM or on plastic. (c) Quantification of JMJD1a and YAP/TAZ staining intensity from immunofluorescence images of MDA-MB-231 cells on plastic. Intensity (int den) was quantified using the CellProfiler software. n(cells)=146. R-value indicates correlation. (d) ChIP showing the binding of JMJD1a to the TAZ promoter. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. n=3 (mean±s.d.). Paired t-test was used to calculate P value. (e) ChIP showing H3K9me2 levels on TAZ promoter of siControl and siJMJD1a_3-transfected MDA-MB-231 cells. ChIP was performed with two independent H3K9me2 antibodies. Analysis was performed by SYBR green-based detection and fold increase in signal relative to the background signal (IgG control antibody) is shown. Representative results from two independent experiments. (f,g) A representative western blot (e) and quantification (f) showing YAP/TAZ expression in JMJD1a-silenced MDA-MB-231 cells after 3 days of silencing, n=7. (h) Taqman qRT–PCR of JMJD1a (KDM3A), CTGF and THBS1 mRNA levels in JMJD1a-silenced MDA-MB-231 cells, n=4. (i) Taqman qRT–PCR of YAP and TAZ mRNA levels in JMJD1a siRNA-transfected MDA-MB-231 cells. n(JMJD1a and TAZ)=4, n(YAP)=3. (j,k) Representative western blot (i) and quantification (j) showing YAP/TAZ expression in JMJD1a-overexpressing MDA-MB-231 cells normalized to loading control, n=4. (l) Taqman qRT–PCR of JMJD1a (KDM3A), CTGF and THBS1 mRNA levels in JMJD1a-overexpressing MDA-MB-231 cells, n=4. (m) A representative western blot and quantification of YAP/TAZ expression on TIFF-derived CDM in GFP control and JMJD1a-GFP-overexpressing cells after 4 days on CDM. (n) Representative western blot of JMJD1a and YAP/TAZ expression in GFP control and JMJD1a-overexpressing MDA-MB-231 cells plated on 4 or 50 kPa hydrogels or on PL.

Figure 7. JMJD1a and YAP/TAZ expression correlates in human carcinomas.

(a) Example images for the scoring of immunohistochemical stainings for JMJD1a and YAP/TAZ expression from primary breast cancer tissue samples. Scale bar, 100 μm. The tables show the association between cytoplasmic and nuclear expression of JMJD1a and YAP/TAZ. The χ²-test was used for the statistical analyses. (b) A proposed model for the normal and cancer-associated matrix-induced control of cancer cell proliferation via mechanosensitive regulation of JMJD1a and transcriptional regulation of YAP/TAZ.