Androgen-Regulated SPARCL1 in the Tumor Microenvironment Inhibits Metastatic Progression

Abstract

Prostate cancer is a leading cause of cancer death in men due to the subset of cancers that progress to metastasis. Prostate cancers are thought to be hardwired to androgen receptor (AR) signaling, but AR-regulated changes in the prostate that facilitate metastasis remain poorly understood. We previously noted a marked reduction in secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) expression during invasive phases of androgen-induced prostate growth, suggesting that this may be a novel invasive program governed by AR. Herein, we show that SPARCL1 loss occurs concurrently with AR amplification or overexpression in patient-based data. Mechanistically, we demonstrate that SPARCL1 expression is directly suppressed by androgen-induced AR activation and binding at the SPARCL1 locus via an epigenetic mechanism, and these events can be pharmacologically attenuated with either AR antagonists or HDAC inhibitors. We establish using the Hi-Myc model of prostate cancer that in Hi-Myc/Sparcl1(-/-) mice, SPARCL1 functions to suppress cancer formation. Moreover, metastatic progression of Myc-CaP orthotopic allografts is restricted by SPARCL1 in the tumor microenvironment. Specifically, we show that SPARCL1 both tethers to collagen in the extracellular matrix (ECM) and binds to the cell's cytoskeleton. SPARCL1 directly inhibits the assembly of focal adhesions, thereby constraining the transmission of cell traction forces. Our findings establish a new insight into AR-regulated prostate epithelial movement and provide a novel framework whereby SPARCL1 in the ECM microenvironment restricts tumor progression by regulating the initiation of the network of physical forces that may be required for metastatic invasion of prostate cancer.

Figure 1

Androgen represses SPARCL1 expression in prostate cancer. A, B, Sparcl1 expression in (A) UGE and (B) UGM (n≥3) of DHT treated UGS. C, WT male UGS were examined by IHC for SPARCL1 (n=3; 400× magnification). D, Co-occurrence of SPARCL1 loss and AR amplification or overexpression in MSKCC cohort analyzed from www.cbioportal.com and Mayo Clinic cohort. E, F, SPARCL1 and KLK3 expression as determined by qRT-PCR in (E) LNCaP and (F) VCaP cells in 10% FCS with vehicle, in 10% androgen depleted FCS (C/D serum), and 10% C/D serum with 100nM DHT (n≥3). G, SPARCL1 expression as determined by qRT-PCR in LNCaP cells treated with vehicle or 50μM MDV3100 (n=3). H, TMA containing localized tumor samples from controls (n=18) who did not receive therapy prior to radical prostatectomy (NT) and cases (n=46) who were treated with a luteinizing hormone-releasing hormone agonist (anti-androgen therapy) before sampling at radical prostatectomy was examined for SPARCL1 expression by IHC. I, Schematic of a potential AR binding site in a putative promoter/enhancer region of SPARCL1 located in the intron 3’ to the transcriptional start site (TSS). Black bars and numbers underneath represent genomic location in relation to the TSS amplified by qRT-PCR primers following ChIP of AR. J, ChIP of AR in LNCaP cells treated with vehicle or 100nM DHT (n≥3). Statistical analysis performed by One-way ANOVA with Tukey's Multiple Comparison Test (A, E, F), Student's t test (G), Student's t test with Welsh's Correction (H), and Two-way ANOVA (J) (mean ± SEM; *P≤0.05, **P≤0.01, ****P≤0.0001).

Figure 2

Androgen mediated chromatin remodeling at the SPARCL1 locus. A, SPARCL1 expression as determined by qRT-PCR in LNCaP, VCaP, CWR22RV1, and PC3 cells treated with vehicle or 1 μM 5-Aza-2’-deoxycytidine (DNA methyltransferase inhibitor) for 3 days (n=3). B, Methylation heatmap of SPARCL1 locus in prostate cancer cell lines (LNCaP, C42B, LAPC4, VCaP, PC3, CWR22RV1), primary prostate cells (PREC), and positive control (SSSI WBC). C, SPARCL1 expression as determined by qRT-PCR in LNCaP, VCaP, CWR22RV1 and PC3 cells treated with vehicle or Vorinostat (HDAC inhibitor) for 48 hours (n=3). D, Schematic of a potential acetylation site in the putative promoter/enhancer region of SPARCL1. Black bars and numbers underneath represent genomic location in relation to the TSS amplified by qRT-PCR primers following ChIP of AR or H3K27Ac. E, ChIP of H3K27Ac in LNCaP cells treated with vehicle or 100nM DHT (n≥3). F, Schematic of Androgen mediated suppression of SPARCL1 expression. Statistical analyses performed by Student's t test (A and C) (mean ± SEM; NS=not significant, **P≤0.01, ***P≤0.001).

Figure 3

Sparcl1 is not necessary for prostate development. A-C, Bud number and length determined from photomicrographs of WT and Sparcl1^−/− male UGS (n≥3) cultured in vitro for 5 days. D-E, Adult (8.5 months) prostates from WT and Sparcl1^−/− were weighed (n≥5). F-G, WT and Sparcl1^−/− prostates were examined by H&E (n=20 at ≥12 months; 40× magnification). Expression of p63, CK8 and Ki67 were examined in WT and Sparcl1^−/− prostates by IHC (n≥3; 400× magnification). Statistical analyses performed by Student's t test (mean ± SEM; NS=not significant).

Figure 4

SPARCL1 suppresses adenocarcinoma formation in the prostate. A, Representative image of SPARCL1 expression examined by IHC in human benign adjacent (1), PIN (2), and adenocarcinoma (3) in the prostate. B, Representative images of SPARCL1 expression examined by IHC in benign adjacent, PIN, and prostate cancer from Hi-Myc mice at 6 months of age (400×). C, Sparcl1 expression examined by qRT-PCR in WT murine prostates at 5 months (n=3), Hi-Myc murine prostates at 4.5 (n=3) and 6 months (n=3), and Myc-CaP cells. D, Prostates from 4.5 month old WT (n=6), Sparcl1^−/− (n=8), Hi-Myc (n=5), Hi-Myc/Sparcl1^+/− (n=8) and Hi-Myc/Sparcl1^−/− (n=11) mice were weighed. E, F, Prostates from 4.5 month old WT (n=7), Sparcl1^−/− (n=10), Hi-Myc (n=60), Hi-Myc/Sparcl1^+/− (n=10) and Hi-Myc/Sparcl1^−/− (n=11) mice were examined by H & E by the study pathologist (100× magnification). G, SPARCL1 expression in Myc-CaP-EV stable clones (A and B) and Myc-CaP-mSPARCL1 clones (A and B) as determined by immunoblotting. H, In vitro proliferation of Myc-CaP-EV and Myc-CaP-mSPARCL1 stable clones as measured by MTT assay. I, J, Orthotopic allografts of Myc-CaP-EV (B) and Myc-CaP-mSPARCL1 (A) at 21 days (n≥4). Statistical analyses performed by Oneway ANOVA (C), Chi-squared test (F), and Student's t test (J) (mean ± SEM; *P≤0.05; **P≤0.01).

Figure 5

SPARCL1 in the tumor microenvironment restricts metastatic progression. A, Photomicrographs of Myc-CaP orthotopic allografts in WT and Sparcl1^−/− prostates. B, Average GU weight of Myc-CaP orthotopic allografts in WT (n=7) and Sparcl1^−/− prostates (n=7). C, H&E (100× magnification) of metastases from Myc-CaP orthotopic allografts in WT and Sparcl1^−/− prostates. D, Average number of metastases from Myc-CaP orthotopic allografts in WT (n=19) and Sparcl1^−/− (n=20) prostates. Statistical analyses performed by Student's t test (mean ± SEM; NS=not significant; *P≤0.05).

Figure 6

SPARCL1 modulates cytoskeletal stiffness and remodeling dynamics. A, Representative bright field images of RGD-coated ferrimagnetic microbeads incubated with PC3 cells adhered on a collagen or collagen+rSPARCL1 matrix. B, Forced motions using Magnetic Twisting Cytometry were applied to determine mechanical properties including cytoskeletal stiffness (g’) and friction (g”) of cells adhered on a matrix of collagen (geometric mean ± SE; n=219) or collagen+rSPARCL1 (geometric mean ± SE; n=257; *P≤0.05, Student's t-test). The solid lines are the fit of experimental data to the structural damping equation with addition of a Newtonian viscous term as described previously (16). Accordingly, the exponent in the power law, or the slope of the solid lines, describes the material behavior of living cells and is an index along spectrum of solid-like (a Hookean elastic solid) to fluid-like (a Newtonian viscous fluid) states. Fitting was performed by nonlinear regression analysis. C-F, Spontaneous motions of RGD-coated ferrimagnetic beads incubated with PC3 cells adhered on a collagen (n=635) or collagen+rSPARCL1 matrix (n=542). C, The exponent α of cells adhered on Collagen vs. Collagen+rSPARCL1. D-F, SPARCL1 in the collagen matrix decreased the speed of cytoskeletal remodeling as measured by (D) the Diffusion coefficient and computed mean square displacement (MSD) (E) at 10s and (F) at 300s (mean ± SEM; NS=not significant; *P≤0.05).

Figure 7

SPARCL1 restricts traction force and focal adhesion assembly. The contractile stress arising at the interface between each adherent PC3 cell and its substrate (collagen or collagen+rSPARCL1) was measured by Fourier transform traction microscopy. A, Representative phase contrast and traction images of PC3 adhered on collagen or collagen+rSPARCL1. B, Projected cell area of PC3 cells on collagen (n=16) or collagen+rSPARCL1 (n=21). C-D, SPARCL1 in the collagen matrix decreased RMS traction and net contractile moment, a scalar measure of cell's contractile strength (mean ± SEM). E, RMS tractions near RGD-coated beads were significantly increased compared to those near SPARCL1-coated and uncoated. RMS tractions were normalized to number of beads within the defined area. Bars represent median (n=8 randomly chosen localized areas per group). F, Focal adhesion assembly measured by immunofluorescence of Paxillin (focal adhesion), Phallodin (actin fibers), and DAPI (nuclei) of PC3 cells adhered on a matrix of collagen (n=35) or collagen+rSPARCL1 (n=59) (600x magnification). G, Quantification of focal adhesion number per cell. Statistical analyses performed by Student's t test (B, C, D and G) and One-way ANOVA (E) (mean ± SEM; NS= not significant; *P≤0.05, **P≤0.005, ****P≤0.0001).