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Cited by 108Prostate Cancer Treatment and ResearchProtease and Inhibitor MechanismsTGF-β signaling in diseases

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Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment

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Nguyen, Elizabeth V., Pereira, Brooke A., Lawrence, Mitchell G., Ma, Xiuquan, Rebello, Richard J., Chan, Howard, Niranjan, Birunthi, Wu, Yunjian, Ellem, Stuart, Guan, Xiaoqing, Wu, Jianmin, Skhinas, Joanna N., Cox, Thomas R., Risbridger, Gail P., Taylor, Renea A., Lister, Natalie L., Daly, Roger J.·2019·Molecular & Cellular Proteomics
DOI 10.1074/mcp.ra119.001496PMC PMC6601211
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Abstract

In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis.Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation.To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures.Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow.Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories "cell adhesion" and the "extracellular matrix."The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the "spliceosome" and "actin binding."STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling.It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domain-containing receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme that promotes collagen crosslinking.Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blotting analyses.Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay.Further, it significantly impaired the motility of cocultured RWPE-2 prostate tumor epithelial cells.These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.

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Graphical abstract Graphical Abstract Highlights Quantitative (phoshpo)proteome of primary cell cultures of patient-matched prostate CAF and NPF.Key CAF-associated proteins validated using orthogonal methodologies.LOXL2 inhibitors D-penicillamine and PXS-S2A impaired CAF migration and ECM alignment.Pre-treatment with LOXL2 inhibitors impaired migratory capacity of RWPE-2 cells in co-culture.
Graphical abstract Graphical Abstract Highlights Quantitative (phoshpo)proteome of primary cell cultures of patient-matched prostate CAF and NPF.Key CAF-associated proteins validated using orthogonal methodologies.LOXL2 inhibitors D-penicillamine and PXS-S2A impaired CAF migration and ECM alignment.Pre-treatment with LOXL2 inhibitors impaired migratory capacity of RWPE-2 cells in co-culture.CC BY · PubMed Central
Fig. 1. Comparison of the proteomes and phosphoproteomes of prostate CAF and NPF. Venn diagrams of overlapping proteins identified in the spectral library of the (A) CAF proteome and (B) NPF proteome. Bar graphs of common proteins (left axis) and the percentage of total identified proteins (right axis) in each number of patient cell lines in the spectral library of (C) CAF proteome and (D) NPF proteome. Scatter plots comparing the average log2 expression of (E) individual proteins and (F) phosphopeptides in matched CAF versus NPF patient-derived lines (n = 4).
Fig. 1. Comparison of the proteomes and phosphoproteomes of prostate CAF and NPF. Venn diagrams of overlapping proteins identified in the spectral library of the (A) CAF proteome and (B) NPF proteome. Bar graphs of common proteins (left axis) and the percentage of total identified proteins (right axis) in each number of patient cell lines in the spectral library of (C) CAF proteome and (D) NPF proteome. Scatter plots comparing the average log2 expression of (E) individual proteins and (F) phosphopeptides in matched CAF versus NPF patient-derived lines (n = 4).CC BY · PubMed Central
Fig. 2. Functional analysis of differentially abundant proteins in prostate CAF compared with NPF. The plot shows functional categories that are over-represented relative to all identified proteins using a permutation-based false discovery rate (FDR) analysis.
Fig. 2. Functional analysis of differentially abundant proteins in prostate CAF compared with NPF. The plot shows functional categories that are over-represented relative to all identified proteins using a permutation-based false discovery rate (FDR) analysis.CC BY · PubMed Central
Fig. 3. STRING network analysis of differentially abundant proteins. Networks formed by proteins with (A) increased abundance and (B) decreased abundance in CAF versus NPF.
Fig. 3. STRING network analysis of differentially abundant proteins. Networks formed by proteins with (A) increased abundance and (B) decreased abundance in CAF versus NPF.CC BY · PubMed Central
Fig. 4. Unsupervised hierarchical clustering of samples using proteins enriched in prostate CAF or NPF. Clustering was undertaken using differentially abundant proteins selected using a stringent cut-off of adjusted p value <0.05 and fold change >1.5. The positive or negative CAF weight indicates the degree of significance relative to NPF.
Fig. 4. Unsupervised hierarchical clustering of samples using proteins enriched in prostate CAF or NPF. Clustering was undertaken using differentially abundant proteins selected using a stringent cut-off of adjusted p value <0.05 and fold change >1.5. The positive or negative CAF weight indicates the degree of significance relative to NPF.CC BY · PubMed Central
Fig. 5. Characterization of proteins with differential phosphorylation between CAF and NPF. A, Functional analysis of differentially-phosphorylated proteins. Over-represented functional categories are relative to all identified proteins using a permutation-based false discovery rate (FDR) analysis. STRING network analysis for proteins with (B) increased and (C) decreased phosphorylation in CAF versus NPF. The differentially phosphorylated sites are shown.
Fig. 5. Characterization of proteins with differential phosphorylation between CAF and NPF. A, Functional analysis of differentially-phosphorylated proteins. Over-represented functional categories are relative to all identified proteins using a permutation-based false discovery rate (FDR) analysis. STRING network analysis for proteins with (B) increased and (C) decreased phosphorylation in CAF versus NPF. The differentially phosphorylated sites are shown.CC BY · PubMed Central
Fig. 6. Protein validation in patient-matched prostate CAF and NPF. A, Representative flow plots show surface expression of CD90 and CD166 antigen on NPF and CAF (gated on propidium iodide-negative live cells). B, Graph shows the mean percentage (± S.D.) of CD90 and CD166 double-positive cells in NPF and CAF from Patient 1–6 (P1–6; supplemental Fig. S2). *p value <0.05 C, Western blots show LOXL2 protein expression and α-tubulin loading control in patient-matched NPF and CAF (P1–6); 20 μg of protein was loaded per lane (10 μg was loaded for P2). LOXL2 blots on biological replicate samples are shown in supplemental Fig. S3A–S3B. D, Western blots show DDR2 and β-actin loading control in patient-matched NPF and CAF (P1–6); 20 μg of protein was loaded per lane. E, Western blots of phospho-FAK Y925 and total FAK levels; β-actin was used a loading control; 15 μg of protein was loaded per lane. Densitometry of biological replicate shown in supplemental Fig. S3C. Quantification of blots by densitometry shows the average fold-change and patient-matched expression levels for; F, LOXL2, (G) DDR2 and (H) pFAK925/total FAK levels in CAF and NPF from Patients 1–6. FAK and pFAK protein expression was first quantified relative to their respective β-actin loading controls. Bars represent the mean ± S.E. (n = 6 patients). *p value <0.05 compared with NPF.
Fig. 6. Protein validation in patient-matched prostate CAF and NPF. A, Representative flow plots show surface expression of CD90 and CD166 antigen on NPF and CAF (gated on propidium iodide-negative live cells). B, Graph shows the mean percentage (± S.D.) of CD90 and CD166 double-positive cells in NPF and CAF from Patient 1–6 (P1–6; supplemental Fig. S2). *p value <0.05 C, Western blots show LOXL2 protein expression and α-tubulin loading control in patient-matched NPF and CAF (P1–6); 20 μg of protein was loaded per lane (10 μg was loaded for P2). LOXL2 blots on biological replicate samples are shown in supplemental Fig. S3A–S3B. D, Western blots show DDR2 and β-actin loading control in patient-matched NPF and CAF (P1–6); 20 μg of protein was loaded per lane. E, Western blots of phospho-FAK Y925 and total FAK levels; β-actin was used a loading control; 15 μg of protein was loaded per lane. Densitometry of biological replicate shown in supplemental Fig. S3C. Quantification of blots by densitometry shows the average fold-change and patient-matched expression levels for; F, LOXL2, (G) DDR2 and (H) pFAK925/total FAK levels in CAF and NPF from Patients 1–6. FAK and pFAK protein expression was first quantified relative to their respective β-actin loading controls. Bars represent the mean ± S.E. (n = 6 patients). *p value <0.05 compared with NPF.CC BY · PubMed Central
Fig. 7. LOX/LOXL2 enzymatic function in prostate CAF. (A) LOX/LOXL and (B) LOXL2 enzymatic activity was measured in the conditioned media from patient-matched NPF and CAF (P4–6). Specific LOX/LOXL and LOXL2 enzymatic function was determined via the addition of BAPN (100 μm) and PXS-S2A (1 μm) inhibitors respectively. Bars represent mean ± S.E. (n = 3). *p value <0.05 compared with NPF.
Fig. 7. LOX/LOXL2 enzymatic function in prostate CAF. (A) LOX/LOXL and (B) LOXL2 enzymatic activity was measured in the conditioned media from patient-matched NPF and CAF (P4–6). Specific LOX/LOXL and LOXL2 enzymatic function was determined via the addition of BAPN (100 μm) and PXS-S2A (1 μm) inhibitors respectively. Bars represent mean ± S.E. (n = 3). *p value <0.05 compared with NPF.CC BY · PubMed Central
Fig. 8. LOX/LOXL2 inhibition abrogates extracellular matrix orientation in prostate CAF. A, Representative images of fibronectin staining show ECM fiber alignment for cell-derived matrices produced by NPF and CAF or CAF treated with the LOX/LOXL2 inhibitor DPEN or selective LOXL2 inhibitor PXS-S2A. Images were processed and color-coded to represent the degree of fiber orientation distribution within each sample; scale bar = 50 μm. Quantification of fiber alignment for NPF and CAF treated with B, DPEN or (C) PXS-S2A from Patient 4 and 6. Lines represent mean values (n = 3). *p value <0.05 compared with CAF. Raw images are shown in supplemental Fig. S7.
Fig. 8. LOX/LOXL2 inhibition abrogates extracellular matrix orientation in prostate CAF. A, Representative images of fibronectin staining show ECM fiber alignment for cell-derived matrices produced by NPF and CAF or CAF treated with the LOX/LOXL2 inhibitor DPEN or selective LOXL2 inhibitor PXS-S2A. Images were processed and color-coded to represent the degree of fiber orientation distribution within each sample; scale bar = 50 μm. Quantification of fiber alignment for NPF and CAF treated with B, DPEN or (C) PXS-S2A from Patient 4 and 6. Lines represent mean values (n = 3). *p value <0.05 compared with CAF. Raw images are shown in supplemental Fig. S7.CC BY · PubMed Central
Fig. 9. LOX/LOXL2 inhibition impairs CAF migration. A, Representative images of CAF cultures in the wound healing/scratch assay exposed to vehicle control (control; ctr) or PXS-S2A treatment for 36 h. Scale bar = 100 μm. The percentage gap closure was normalized and presented relative to control CAF (Ctr) at 36 h in the presence of (B) the LOXL/LOXL2 inhibitor d-Penicillamine (Patient 4–6) or (C) the selective LOXL2 inhibitor PXS-S2A (Patient 4 and 6). Graph bars represent mean ± S.E. (n = 6). *p value <0.05 relative to control CAF.
Fig. 9. LOX/LOXL2 inhibition impairs CAF migration. A, Representative images of CAF cultures in the wound healing/scratch assay exposed to vehicle control (control; ctr) or PXS-S2A treatment for 36 h. Scale bar = 100 μm. The percentage gap closure was normalized and presented relative to control CAF (Ctr) at 36 h in the presence of (B) the LOXL/LOXL2 inhibitor d-Penicillamine (Patient 4–6) or (C) the selective LOXL2 inhibitor PXS-S2A (Patient 4 and 6). Graph bars represent mean ± S.E. (n = 6). *p value <0.05 relative to control CAF.CC BY · PubMed Central
Fig. 10. LOX/LOXL2 inhibition in CAF impairs prostate epithelial cell motility. A, Representative images of fluorescently-labeled transformed prostate epithelial cells (RWPE2; green) in co-culture with patient CAF stained with fibronectin (red). Scale bar = 50 μm. CAF cultures were pre-treated with (B) 10 or 50 μm of d-Penicillamine (Patient 4–6), C, 10 and 100 nm of PXS-S2A (Patient 4 and 6) or vehicle control (C) for 24 h before drug wash-out. RWPE2 migration was assessed over 12 h in CAF co-cultures and presented as mean speed and track length (normalized to control CAF; C). Bars represent the mean ± S.E. (n = 3). *p value <0.05 compared with control CAF.
Fig. 10. LOX/LOXL2 inhibition in CAF impairs prostate epithelial cell motility. A, Representative images of fluorescently-labeled transformed prostate epithelial cells (RWPE2; green) in co-culture with patient CAF stained with fibronectin (red). Scale bar = 50 μm. CAF cultures were pre-treated with (B) 10 or 50 μm of d-Penicillamine (Patient 4–6), C, 10 and 100 nm of PXS-S2A (Patient 4 and 6) or vehicle control (C) for 24 h before drug wash-out. RWPE2 migration was assessed over 12 h in CAF co-cultures and presented as mean speed and track length (normalized to control CAF; C). Bars represent the mean ± S.E. (n = 3). *p value <0.05 compared with control CAF.CC BY · PubMed Central

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  1. Bruderer, 2015, Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues, Mol. Cell. Proteomics, 14, 1400, 10.1074/mcp.M114.044305 · DOI · Paper page

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Authors
Nguyen, E. V., Pereira, B. A., Lawrence, M. G., Ma, X., Rebello, R. J., Chan, H., Niranjan, B., Wu, Y., Ellem, S., Guan, X., Wu, J., Skhinas, J. N., Cox, T. R., Risbridger, G. P., Taylor, R. A., Lister, N. L., & Daly, R. J.
Affiliations
  • Peter MacCallum Cancer Centre (AU)
  • University of Southern Queensland (AU)
  • Peking University Cancer Hospital (CN)
  • Garvan Institute of Medical Research (AU)
  • The University of Melbourne (AU)
Corresponding author
Daly, Roger J.
ORCID
Venue
Molecular & Cellular Proteomics
Publisher
Elsevier BV
Published
2019-07
Volume
18
Issue
7
Pages
1410-1427
ISSN
1535-9476 (ISSN-L)
DOI
10.1074/mcp.ra119.001496
PMC
PMC6601211
OpenAlex
W2944168058
Cited by
107
References
98
Citations by year
2026: 12 · 2025: 16 · 2024: 15 · 2023: 17 · 2022: 10 · 2021: 17 · 2020: 19 · 2019: 1
FWCI
8.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular & Cellular Proteomics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Southern Queensland ePrints (University of Southern Queensland) (acceptedVersion)
  • Monash University Research Portal (Monash University) (submittedVersion, other-oa)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by)
Funders
  • Cancer Australia (ID NCG2616)
  • Faculty of Medicine, Nursing and Health Sciences, Monash University (SGS16–0295)
  • Department of Health | National Health and Medical Research Council (NHMRC) (1102752, 1058540)
  • Victorian Cancer Agency (VCA) (15023)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Prostate Cancer Treatment and Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
Keywords
prostate cancer, cancer-associated fibroblasts, LOXL2, DDR2, tumor microenvironment, extracellular matrix, collagen cross-linking, phosphoproteome, proteomic profiling, intercellular communication, therapeutic target
MeSH terms
Cancer-Associated Fibroblasts, Amino Acid Oxidoreductases, Cell Movement, Epithelial Cells, Extracellular Matrix, Humans, Male, Neoplasm Proteins, Phosphoproteins, Phosphorylation, Prostate, Prostatic Neoplasms, Reproducibility of Results, Signal Transduction, Autocrine Communication, Paracrine Communication, Proteome, Proteomics, Cell Line, Tumor, Tumor Microenvironment

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