Cell-Type-Specific Signalling Networks Impacted by Prostate Epithelial-Stromal Intercellular Communication

Abstract

Prostate cancer is the second most common cause of cancer death in males. A greater understanding of cell signalling events that occur within the prostate cancer tumour microenvironment (TME), for example, between cancer-associated fibroblasts (CAFs) and prostate epithelial or cancer cells, may identify novel biomarkers and more effective therapeutic strategies for this disease. To address this, we used cell-type-specific labelling with amino acid precursors (CTAP) to define cell-type-specific (phospho)proteomic changes that occur when prostate epithelial cells are co-cultured with normal patient-derived prostate fibroblasts (NPFs) versus matched CAFs. We report significant differences in the response of BPH-1 benign prostate epithelial cells to CAF versus NPF co-culture. Pathway analysis of proteomic changes identified significant upregulation of focal adhesion and cytoskeleton networks, and downregulation of metabolism pathways, in BPH-1 cells cultured with CAFs. In addition, co-cultured CAFs exhibited alterations in stress, DNA damage, and cytoskeletal networks. Functional validation of one of the top differentially-regulated proteins in BPH-1 cells upon CAF co-culture, transglutaminase-2 (TGM2), demonstrated that knockdown of this protein significantly reduced the proliferation and migration of prostate epithelial cells. Overall, this study provides novel insights into intercellular communication in the prostate cancer TME that may be exploited to improve patient management.

Keywords: fibroblast; mass spectrometry; prostate cancer; signalling; tumour microenvironment.

Figure 1

Application of CTAP to benign prostate epithelial cell–prostate fibroblast intercellular communication. (A) CTAP provides spatial resolution, identifying which cell type the MS-detected peptides are derived from. Patient-derived fibroblasts (NPFs and CAFs) and BPH-1 cells were modified to express DDCM.tub-KDEL (DDC) and LyrM37-KDEL (Lyr), respectively. Cells were cultured with differentially isotopically-labelled amino acid precursors to allow identification of the cell-type of origin during LC-MS/MS proteomic analysis. Discrimination between additional variables was achieved by differential isobaric tandem mass tag (TMT) labelling of peptides. Adapted from Tape et al., 2014 [10]. (B) Western blot validation that the BPH-1 and NPF/CAFs express HA-tagged Lyr and DDC, respectively.

Figure 2

X-plot of differentially abundant proteins in BPH-1/fibroblast co-culture compared to monoculture. Scatter plots representing mean log2-transformed protein abundance; x-axis represents abundance in monoculture, and y-axis represents abundance in co-culture. Proteins significantly upregulated (red dots; protein level in co-culture/corresponding monoculture) and downregulated proteins (blue dots; protein level in monoculture/corresponding co-culture) in co-culture were selected using a cut-off value of FC > 2. Proteins closer to the y-axis represent an increase of expression, while proteins closer to the x-axis represent a decrease of expression upon co-culture. Grey background: BPH-1 protein expression, yellow background: fibroblast protein expression. FC: fold change. Quadrant I: BPH-1 proteins in monoculture compared to BPH-1 proteins in co-culture with NPFs, Quadrant II: NPF proteins in monoculture compared to NPF proteins in co-culture with BPH-1 cells, Quadrant III: CAF proteins in monoculture compared to CAF proteins in co-culture with BPH-1 cells, Quadrant IV: BPH-1 proteins in monoculture compared to BPH-1 proteins in co-culture with CAFs. Data represent mean protein abundance across 3 biological replicates with NPFs and CAFs from a single patient.

Figure 3

BPH-1 and fibroblast proteins differentially expressed in co-culture. A summary of BPH-1 proteins with (A) increased and (B) decreased expression following co-culture with CAFs, and comparison with effect of NPF co-culture, and a summary of CAF proteins with (C) increased and (D) decreased expression following co-culture with BPH-1 cells, and a comparison with effects in NPFs when they are co-cultured with BPH-1 cells. Only proteins with a >twofold change in either direction in CAF co-culture conditions presented. Data represent mean protein abundance across 3 biological replicates with NPFs and CAFs from a single patient. Mean ± SD, n = 3. Student’s t-test. * p < 0.05, ** p <0.01, ***p < 0.001.

Figure 4

X-plot of differentially abundant phosphosites in BPH-1/fibroblast co-culture compared to monoculture. Scatter plots representing mean log2-transformed phosphosite abundance; x-axis represents abundance in monoculture, and y-axis represents abundance in co-culture. Significantly upregulated phosphosites (red dots; phosphosite level in co-culture/corresponding monoculture) and downregulated phosphosites (blue dots; phosphosite level in monoculture/corresponding co-culture) in co-culture were selected using a cut-off value of FC > 2. Phosphosites closer to the y-axis (red) represent a significant increase of expression, while phosphosites closer to the x-axis (blue) represent a decrease of expression upon co-culture. Grey background: BPH-1 phosphosite levels, yellow background: fibroblast phosphosite levels. Quadrant I: BPH-1 phosphoproteins in monoculture compared to BPH-1 phosphoproteins in co-culture with NPFs, Quadrant II: NPF phosphoproteins in monoculture compared to NPF phosphoproteins in co-culture with BPH-1 cells, Quadrant III: CAF phosphoproteins in monoculture compared to CAF proteins in co-culture with BPH-1 cells, Quadrant IV: BPH-1 phosphoproteins in monoculture compared to BPH-1 phosphoproteins in co-culture with CAFs. FC, fold change. Data represent phosphosite abundance across 3 biological replicates from NPFs and CAFs from a single patient.

Figure 5

STRING/Cytoscape network analysis of differentially regulated BPH-1 proteins and phosphosites. Enriched signalling networks of (A) increased and (B) decreased BPH-1 (phospho)proteins in CAF co-culture. Node size is representative of protein expression fold change (co-culture/monoculture); node border width is representative of the p-value for co-culture/monoculture comparison for each protein; node connection line thickness is representative of STRING score between connected proteins.

Figure 6

STRING/Cytoscape network analysis of differentially regulated CAF proteins and phosphosites. Enriched signalling networks of (A) increased and (B) decreased CAF (phospho)proteins in BPH-1 co-culture. Node size is representative of protein expression fold change (co-culture/monoculture); node border width is representative of the p-value for co-culture/monoculture comparison for each protein; node connection line thickness is representative of STRING score between connected proteins.

Figure 7

Functional interrogation of selected candidates via proliferation assays. (A) Representative Western blot images demonstrating siRNA-mediated knockdown. (B) MTS assays for BPH-1 and RWPE-1 benign prostate epithelial cells after 72 h of monoculture. Data are expressed relative to 0 h and normalized to the control (siOTP). (C) Epithelial cell proliferation in co-culture. GFP-positive BPH-1 and RWPE-1 cells were cultured with GFP-negative CAF cells for 72 h. GFP fluorescence intensity was used as a measure of epithelial cell number, relative to 0 h and normalized to the control. Mean ± SEM, n = 3. One-way ANOVA. * p < 0.05, ** p < 0.01.

Figure 8

Functional interrogation of selected candidates via migration assays. (A) Scratch closure assay of BPH-1 and RWPE-1 benign prostate epithelial cells. Data represent scratch area 16 h post-scratch relative to 0 h timepoint. Mean ± SEM, n = 3. (B) Random cell migration assay of prostate epithelial cells in co-culture with CAFs. GFP-positive BPH-1 and RWPE-1 cells were cultured with GFP-negative CAF cells over 16 h. GFP-positive cells were tracked for total distance travelled. Mean ± SEM, n = 4. One-way ANOVA. * p < 0.05. Representative plots of x- and y-axis movements for BPH-1 and RWPE-1 cells in co-culture with CAFs are shown underneath.