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. 2012 Sep 11;109(37):14977-82.
doi: 10.1073/pnas.1203525109. Epub 2012 Aug 27.

Secreted Protein, Acidic and Rich in Cysteine-Like 1 (SPARCL1) Is Down Regulated in Aggressive Prostate Cancers and Is Prognostic for Poor Clinical Outcome

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Secreted Protein, Acidic and Rich in Cysteine-Like 1 (SPARCL1) Is Down Regulated in Aggressive Prostate Cancers and Is Prognostic for Poor Clinical Outcome

Paula J Hurley et al. Proc Natl Acad Sci U S A. .
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Abstract

Prostate cancer is the second leading cause of cancer death among United States men. However, disease aggressiveness is varied, with low-grade disease often being indolent and high-grade cancer accounting for the greatest density of deaths. Outcomes are also disparate among men with high-grade prostate cancer, with upwards of 65% having disease recurrence even after primary treatment. Identification of men at risk for recurrence and elucidation of the molecular processes that drive their disease is paramount, as these men are the most likely to benefit from multimodal therapy. We previously showed that androgen-induced expression profiles in prostate development are reactivated in aggressive prostate cancers. Herein, we report the down-regulation of one such gene, Sparcl1, a secreted protein, acidic and rich in cysteine (SPARC) family matricellular protein, during invasive phases of prostate development and regeneration. We further demonstrate a parallel process in prostate cancer, with decreased expression of SPARCL1 in high-grade/metastatic prostate cancer. Mechanistically, we demonstrate that SPARCL1 loss increases the migratory and invasive properties of prostate cancer cells through Ras homolog gene family, member C (RHOC), a known mediator of metastatic progression. By using models incorporating clinicopathologic parameters to predict prostate cancer recurrence after treatment, we show that SPARCL1 loss is a significant, independent prognostic marker of disease progression. Thus, SPARCL1 is a potent regulator of cell migration/invasion and its loss is independently associated with prostate cancer recurrence.

Conflict of interest statement

Conflict of interest statement: N.E., I.A.V., M.G., and E.D. are employees of and have stock in GenomeDx Biosciences, Inc.

Figures

Fig. 1.
Fig. 1.
Sparcl1 inhibits androgen-induced fetal prostate bud elongation. (A) Sparcl1 expression in male mouse E17.5 UGS as detected by IHC. (B) Sparcl1 expression examined by quantitative PCR during prostate development. Statistical analysis performed by one-way ANOVA with Newman–Keuls post hoc test (mean ± SEM; n ≥ 3; **P < 0.0001). (C) Male E15.5 UGS cultured in vitro with vehicle or Sparcl1 (10 μg/mL) for 7 d (n ≥ 13) and examined by IHC. The black box indicates bud. (D) Sparcl1 inhibits bud number, but not significantly as measured by IHC (n = 4). (E) Sparcl1 significantly inhibits bud length in UGS cultured in vitro. Bud length determined from photomicrographs for vehicle (n = 43) and Sparcl1 (n = 30) -treated UGS (n = 3 UGS); *P = 0.01. (F and G) Sparcl1 does not inhibit epithelial proliferation as examined by IHC for Ki67 in UGS in vitro cultures. Ki67-positive and -negative cells within the epithelial bud were counted from IHC sections of E15.5 male UGS cultured in vitro with vehicle or Sparcl1 (n = 3 UGS). Statistical analysis for C and G performed by Student t test (mean ± SEM). n.s., not significant.
Fig. 2.
Fig. 2.
Sparcl1 expression is decreased during prostate regeneration in adult mouse. Decreased Sparcl1 protein (A) and gene (B) expression during androgen-induced regrowth determined by IF (A) and quantitative PCR (B) as compared in adult mouse prostate, adult mouse prostate 3 wk following castration, and adult mouse prostrate treated with dihydrotestosterone (DHT) for 3 d following castration (regenerating prostate) (mean ± SD; n = 3).
Fig. 3.
Fig. 3.
Sparcl1 restricts benign prostate epithelial cell invasion. Sparcl1 inhibits prostasphere number (A) and size (B). Adult mouse prostate epithelial cells disassociated into single cells, cultured in Matrigel and treated with Sparcl1 (10 μg/mL) or vehicle for 14 d to form prostaspheres. Statistical analysis performed by Student t test (mean ± SEM; n = 4; *P ≤ 0.005).
Fig. 4.
Fig. 4.
SPARCL1 inhibits adhesion, migration, and invasion of prostate cancer cells. (A and B) Adhesion of PC3 cells following incubation on a type I collagen matrix containing BSA (10 μg/mL) or SPARCL1 (10 μg/mL) (n = 3). Arrows indicate adhered cells. Magnification in A, 400×. (C) Migration of PC3 cells incubated with BSA (10 μg/mL) or SPARCL1 (10 μg/mL) across a filter for 20 h (n = 3). (D) Cell adhesion and migration recorded by time-lapse microscopy for 22 h of PC3 cells on a type I collagen matrix containing SPARCL1 (10 μg/mL) or BSA (10 μg/mL). (E) Invasion of PC3 cells incubated with BSA (10 μg/mL) or SPARCL1 (10 μg/mL) of type I collagen- or Matrigel-coated filters for 20 h (n = 3). Statistical analysis performed by Student t test (mean ± SEM; *P ≤ 0.005).
Fig. 5.
Fig. 5.
SPARCL1 inhibits type I collagen-induced RHOC-mediated migration. (A) PC3 cells grown on a type I collagen matrix containing SPARCL1 (10 μg/mL) or BSA (10 μg/mL). Specific IP of activated (GTP-bound) RHOA/B/C and immunoblot (IB) for RHOA and RHOC. Pre-IP lysates were examined for total RHOC, RHOA, GAPDH, and SPARCL1 expression. ImageJ quantification of activated RHOC (normalized to total pre-IP RHOC). Statistical analysis performed by Student t test (mean ± SEM; n = 4; *P = 0.02). (B) PC3 cells transiently transfected with RHOC or constitutively active RHOC (G14V), treated with SPARCL1 (10 μg/mL) or vehicle and allowed to migrate across a filter for 20 h. Statistical analysis performed by Student t test (mean ± SEM; n = 3; *P = 0.013). (C) PC3 cells transiently transfected with pcDNA3.1- or hSPARCL1/pcDNA3.1, treated with isotype control or a α2β1-integrin blocking antibody, and then grown on a type I collagen matrix. Specific IP of activated (GTP-bound) RHOA/B/C and IB for RHOC. Pre-IP lysates IB for total RHOC and GAPDH expression.
Fig. 6.
Fig. 6.
Loss of SPARCL1 expression correlates with Gleason grade and is an independent marker for prostate cancer recurrence. (A and B) SPARCL1 expression is inversely proportional to prostate cancer Gleason grade as determined by IHC in prostate adenocarcinoma Gleason sum 5 (n = 4), 6 (n = 16), 8 (n = 10), and 9 (n = 8), and benign adjacent glands (n = 20) from radical prostatectomies as JHU Gleason grade TMAs. Statistical analysis performed by one-way ANOVA with Bonferroni post hoc test (mean ± SEM; *P ≤ 0.002). (C) SPARCL1 expression is inversely proportional to prostate cancer Gleason grade. Analysis performed on data sets from Taylor et al. for SPARCL1 gene expression (27). Statistical analysis performed by one-way ANOVA. *Prostrate cancer (PCA) vs. benign adjacent and **Met vs. PCA P ≤ 0.01. (D and E) Loss of SPARCL1 expression is prognostic of prostate cancer recurrence. (D) Kaplan–Meier curves for SPARCL1 in a high-risk prostate cancer cohort from the Mayo Clinic for BCR (P = 0.007), MET (P = 0.0009), and PCSM (P = 0.07) endpoints (n = 235). (E) Kaplan–Meier curves for SPARCL1 in a Gleason sum 7 cohort (n = 119, P = 0.046) and a Gleason sum ≥8 cohort (n = 98, P = 0.011) from the Mayo Clinic for MET free survival. Statistical analysis found in SI Materials and Methods.

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