Germline Variants in Asporin Vary by Race, Modulate the Tumor Microenvironment, and Are Differentially Associated with Metastatic Prostate Cancer

Abstract

Purpose: Prostate cancers incite tremendous morbidity upon metastatic growth. We previously identified Asporin (ASPN) as a potential mediator of metastatic progression found within the tumor microenvironment. ASPN contains an aspartic acid (D)-repeat domain and germline polymorphisms in D-repeat-length have been associated with degenerative diseases. Associations of germline ASPN D polymorphisms with risk of prostate cancer progression to metastatic disease have not been assessed.

Experimental design: Germline ASPN D-repeat-length was retrospectively analyzed in 1,600 men who underwent radical prostatectomy for clinically localized prostate cancer and in 548 noncancer controls. Multivariable Cox proportional hazards models were used to test the associations of ASPN variations with risk of subsequent oncologic outcomes, including metastasis. Orthotopic xenografts were used to establish allele- and stroma-specific roles for ASPN D variants in metastatic prostate cancer.

Results: Variation at the ASPN D locus was differentially associated with poorer oncologic outcomes. ASPN D14 [HR, 1.72; 95% confidence interval (CI), 1.05-2.81, P = 0.032] and heterozygosity for ASPN D13/14 (HR, 1.86; 95% CI, 1.03-3.35, P = 0.040) were significantly associated with metastatic recurrence, while homozygosity for the ASPN D13 variant was significantly associated with a reduced risk of metastatic recurrence (HR, 0.44; 95% CI, 0.21-0.94, P = 0.035) in multivariable analyses. Orthotopic xenografts established biologic roles for ASPN D14 and ASPN D13 variants in metastatic prostate cancer progression that were consistent with patient-based data.

Conclusions: We observed associations between ASPN D variants and oncologic outcomes, including metastasis. Our data suggest that ASPN expressed in the tumor microenvironment is a heritable modulator of metastatic progression.

Figure 1

Kaplan-Meier metastasis-free survival curves for germline ASPN D 13/14 compared to ASPN D13/13 (P=0.012) or to all other germline ASPN D genotypes (P=0.003).

Figure 2

ASPN expression in prostate cancer associated fibroblasts is associated with Gleason grade and oncologic outcomes. A, ASPN expression as determined by IHC in prostate tissue following radical prostatectomy. B, ASPN expression in prostate cancer associated fibroblasts in prostate tissue following radical prostatectomy as determined by IF of ASPN and pancytokeratin at 400× magnification. C, ASPN expression in human primary prostate cancer associated fibroblasts (PCAF), human primary benign prostate associated fibroblasts (BAF), an immortalized human benign prostate fibroblast cell line (WPMY1), primary human benign prostate epithelial cells (PreC), and human prostate cancer cell lines (LNCaP, 22RV1, PC3) as determined by qRT-PCR (top) and immunoblotting (bottom). D, ASPN expression as determined by IHC in JHU TMAs containing stroma adjacent to benign (n=48), Gleason sum ≤6 (n=31), Gleason sum 7 (n=6), and Gleason sum 8–9 (n=27) prostate tissue. Statistical analysis performed by one-way ANOVA with Tukey’s multiple comparison test (mean ± SEM; **P≤ 0.001, ***P ≤ 0.0001; upper). ASPN staining in human prostate cancer sections at 200× magnification (lower). E, ASPN expression is associated with biochemical recurrence (BCR). Kaplan–Meier survival curves of ASPN in MSKCC and in Mayo High Risk Cohort for BCR-free survival. F, ASPN expression is associated with metastatic recurrence. Kaplan–Meier survival curves of ASPN in Mayo High Risk Cohort for metastatic recurrence.

Figure 3

ASPN D13 is protective of metastatic progression while ASPN D14 promotes metastatic progression. A, ASPN expression in WPMY1 (A and B), WPMY1 ASPN D13 (A and B), and ASPN D14 (A and B) as measured by immunoblotting. B and C, Photomicrographs and GU weight of WPMY1 (A) + PC3 (n=7), WPMY1 (B) + PC3 (n=5), WPMY1 ASPN D13 (A) + PC3 (n=6), WPMY1 ASPN D13 (B) + PC3 (n=7), WPMY1 ASPN D14 (A) + PC3 (n=7), and WPMY1 ASPN D14 (B) + PC3 (n=5) prostate orthotopic xenografts (mean ± SEM). D, Average number of visible metastases to other organs and distant lymph nodes in WPMY1 (A) + PC3 (n=7), WPMY1 (B) + PC3 (n=5), WPMY1 ASPN D13 (A) + PC3 (n=6), WPMY1 ASPN D13 (B) + PC3 (n=7), WPMY1 ASPN D14 (A) + PC3 (n=7), and WPMY1 ASPN D14 (B) + PC3 (n=5) prostate orthotopic xenografts (mean ± SEM). E, Representative images of metastases taken at 40× magnification from WPMY1 ASPN D13 + PC3 and WPMY1 ASPN D14 + PC3 prostate orthotopic xenografts. Inset is a 400× magnification of the tumor region. Statistical analyses performed by One-way ANOVA with Tukey’s multiple comparison test to compare WPMY1 (A+B), WPMY1 ASPN D13 (A+B), and WPMY1 ASPN D14 (A+B) (mean ± SEM; *P≤0.004 and **P≤0.0003).