Biochemical activity induced by a germline variation in KLK3 (PSA) associates with cellular function and clinical outcome in prostate cancer

Abstract

Genetic variation at the 19q13.3 KLK locus is linked with prostate cancer susceptibility. The non-synonymous KLK3 SNP, rs17632542 (c.536T>C; Ile163Thr-substitution in PSA) is associated with reduced prostate cancer risk, however, the functional relevance is unknown. Here, we identify that the SNP variant-induced change in PSA biochemical activity as a previously undescribed function mediating prostate cancer pathogenesis. The 'Thr' PSA variant led to small subcutaneous tumours, supporting reduced prostate cancer risk. However, 'Thr' PSA also displayed higher metastatic potential with pronounced osteolytic activity in an experimental metastasis in-vivo model. Biochemical characterization of this PSA variant demonstrated markedly reduced proteolytic activity that correlated with differences in in-vivo tumour burden. The SNP is associated with increased risk for aggressive disease and prostate cancer-specific mortality in three independent cohorts, highlighting its critical function in mediating metastasis. Carriers of this SNP allele had reduced serum total PSA and a higher free/total PSA ratio that could contribute to late biopsy decisions and delay in diagnosis. Our results provide a molecular explanation for the prominent 19q13.3 KLK locus, rs17632542 SNP, association with a spectrum of prostate cancer clinical outcomes.

Keywords: Kallikrein-related peptidase 3/KLK3; Prostate cancer; diagnosis; disease aggressiveness; prostate-specific antigen/PSA; single nucleotide polymorphism.

Figure 1.

Thr¹⁶³ PSA abolished the effect of PSA on PC-3 cell proliferation and migration and is associated with reduced growth of primary tumours in-vivo PC-3 and MSK3 cells were transfected with furin activated Wt PSA, Thr¹⁶³ PSA or control plasmid (vector). A) Proliferation rate (confluence %) monitored in the IncuCyte live cell imaging system for PC3 cells expressing PSA variants and vector control over 48 h. Data were consistent across the three independent experiments. B) Proliferation of MSK3-PSA and vector control cells, measured by Prestoblue cell viability assays. C) Cell migration rate (relative wound density %) measured by the IncuCyte live cell imaging system for PC-3 cells expressing PSA variants compared to vector control over 48 h. D) Cell migration measured using the xCELLigence system for the PSA variant expressing MSK3 cells as compared to vector control. Three replicates were included unless otherwise indicated from three independent experiments. Data were consistent across the three independent experiments. Data are represented as mean ± SEM. Statistical significance for all these assays were analysed by Friedman test with Dunn’s multiple comparison test. E) Preclinical subcutaneous xenograft tumour model of PC-3-Luc cells transfected with furin activated Wt PSA, Thr¹⁶³ PSA or vector (n=7 mice/group). F) Mean volume ± SEM of subcutaneous tumours throughout the experiment, based on caliper measurements (mean ± SEM) (Dunnett’s multiple comparisons test). G) Representative photographs of resected subcutaneous tumours. H) Scatter plot of post-mortem weight of subcutaneous tumours at day 38; horizontal line indicates mean value (mean±SEM) (Mann-Whitney test). I) H&E staining of resected subcutaneous tumours. J) Serum concentration of total PSA at endpoint (mean±SEM) (Mann-Whitney test).

Figure 2.

Thr¹⁶³ PSA increased cancer cell invasive ability and increased metastasis in-vivo A) Schematic workflow of spheroid assay. B) Representative brightfield microscope images (4X magnification) of 3D spheroids formed by transfected PC-3 cells after 14 days of culture. C) Peripheral area (μm)² of invading cells outside the outer core. Also see Supplementary Figure 2A. D) Measure of invasiveness of the spheroid from 0 – 1. A circularity value of 1 (maximum) indicates the spheroid is perfectly circular and least invasive, while a decreasing value towards 0 indicates less circular spheroids. (N = 6 spheroids per condition) E) Representative fluorescent microscopy overlay images (10× magnification) of transfected MSK3 cells at 10 days with a magnified view. MSK3 cells were stained with calcein-AM (live cells, green) and ethidium heterodimer (dead cells, orange). Spheroid, Area (μm)² (F) and circularity (G) were measured. Also see Supplementary Figures 2B, 2C. At least two replicates (2 fields selected per well) from three independent experiments were analysed (Mann Whitney t-test). H) Schematic of a 3D osteoblast-derived bone matrix (OBM) co-culture with PC-3 cells, and the various analyses performed. I) Attachment of PC-3-mKO2 cells transfected with furin activated Wt PSA, Thr¹⁶³ PSA, Ala¹⁹⁵ PSA, or vector cells to OBM constructs after 12h co-culture. J) PC-3 proliferation on OBM constructs. Also see Supplementary Figures 3A, 3B. For J, 2 technical replicates were used, 4–5 fields of view/replicate, for a total of 120–230 cells per condition. P values on all groups were evaluated by one-way ANOVA followed by Games-Howell post hoc analysis. K) Intracardiac injection of PC-3-Luc-PSA cells in mice (n=7 mice/group). L) Reconstructed 3D microCT images of tumour-bearing hind legs from representative mice of each group; red arrows showing areas of significant bone degradation, indicating presence of tumour. M) Quantification of bone lesions per hind leg based on visual inspection of planar X-ray images (Dunnett’s multiple comparisons test with Two-way Anova). N) Representative bioluminescence images of tumour-bearing hind legs of mice (week 4). O) Scatter plots of tumour bioluminescence based on region of interest (ROI) drawn over individual hind legs (at week 4); horizontal line indicates median value (Dunnett’s multiple comparisons test). Also see Supplementary Figure 4.

Figure 3.

Biochemical characterization of the effect of the Thr¹⁶³ variant on PSA activity. A) Schematic for PSA proteolytic activity analysis. B) Rate of hydrolysis by mature PSA proteins (Wt PSA, Thr¹⁶³ PSA, and catalytically inactive mutant control Ala¹⁹⁵ PSA, all at 0.1 μM) were compared using the peptide substrates MeO-Suc-RPY-MCA (10 μM) and Mu-HSSKLQ-AMC (1 μM) over 4 h at 37°C. Proteolytic activity derived from assaying a constant amount of PSA with increasing concentration (0–250 mM) for these two substrates were used to estimate K_cat values using nonlinear regression analysis in Graphpad Prism. Results are shown as the mean ± SEM from two experiments, each with three replicates (Welch’s t-test ***P=0.0001). Also see Supplementary Figure 5B. C) Time (mins) versus relative absorbance (OD) corrected to the substrate alone controls was plotted indicating the activity of pro-MMP2 (0.14 μM) when pre-incubated with PSA protein variants (Wt, Thr¹⁶³ and Ala¹⁹⁵ at 0.07 μM) at 37°C and then the activity analysed with the chromogenic substrate (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC₂H_5, 40 μM) for active MMP2 over 2 h. Results are shown as the mean ± SEM of three experiments analysed using Kruskal-Wallis test. ****P<0.0001. D) Intact/total IGFBP-3 (2.2 μM) after 24h incubation at 37°C with PSA variants (0.25 μM) as shown relative to IGFBP-3 control without added PSA. Also see Supplementary Figure 5C. Results are shown as mean ± SEM (n=3, Welch’s t-test, **P=0.003 and ***P=0.0006). E) Fluorescent activity observed for the furin generated active PSA captured from serum free conditioned media of furin-PSA overexpressing PC-3 cells (Wt, Thr¹⁶³, inactive mutant Ala¹⁹⁵ and vector) using the peptide substrate MeO-Suc-RPY-MCA. (n=2, Mean ± SEM *P=0.03). F) Inhibition of HUVEC tube formation on Matrigel by treatment of HUVECs with serum free conditioned media from the PC-3 cells overexpressing (Wt, Thr¹⁶³ and Ala¹⁹⁵ PSA) and PBS control (negative). Scale bar is 500 μm. The graph to the right represents the effect of these PSA protein variants on HUVEC tube formation expressed as an angiogenesis index. The angiogenesis index, reflecting the extent of tube formation or angiogenic potential of the cells 39,53 is shown in relation to the control (mean + SEM, *P=0.02 as compared to control (t-test), n=2). Also see Supplementary Figure 5D.

Figure 4.

rs17632442 SNP association with PSA levels and prostate cancer survival. A) PSA-inhibitor (ACT) complex, free and total PSA. B) A representative silver stain analysis of recombinant wild type (Wt) and Thr¹⁶³ and Ala¹⁹⁵ PSA (0.1 μM) incubated with ACT (0.5 μM) at room temperature for 3 h before resolving on gel showed lower complexing potential of Thr¹⁶³ PSA with ACT compared to the Wt PSA. Inactive mutant Ala¹⁹⁵ does not complex with ACT. C) Representative immunohistochemical results for Gleason Grade 4 adenocarcinoma tissues, showing strong staining for PSA for the TT compared to the CC genotype. Graph on the right shows difference in PSA expression scores between [T] and [C] allele (CC=2, CT=10, TT=10) for the immunohistochemical samples. D-E) Genotype correlation of total PSA (tPSA) levels and f/t PSA ratio in prostate cancer cases (PRACTICAL consortium) and disease-free controls (MDC and VIP cohorts). D) PRACTICAL consortium. N= 31,770; genotype status TT=28,399, CT=3,272 and CC=99 for tPSA levels comparison. N=976; genotype status TT=878, CT=96 and CC=2 for f/t PSA ratio comparison. E) MDC cohort with genotype status TT=2,092, CT=348 and CC=18; and VIP cohort with genotype status TT=4,305, CT=489 and CC=16. (****P<0.0001, Kruskal-Wallis Test). F) Survival analysis for the rs17632542 SNP (c.536T>C) in 37,316 cases of PRACTICAL consortium with follow-up on cause specific death. Of these, 4,629 died of prostate cancer, 3,456 died of other causes. Cases by carrier status, TT=33,281, CT=3,909 and CC=126. The cumulative incidence of death from prostate cancer, Hazards ratio (HR)=1.33, 95% CI=1.24–1.45, P<0.001 (left panel) and all causes other than prostate cancer, HR=1.08, 95% CI=0.98–1.19, P=0.431 (right panel) are indicated. Number at risk are also indicated.