SPINK1 protein expression was evaluated in two cohorts (University of Michigan (UM) and Swedish Watchful Waiting (SWW)) using immunohistochemsitry (IHC) on tissue microarrays that have previously been evaluated for TMRPSS2-ERG status by fluorescence in situ hybridization (FISH). A. Representative SPINK1 positive and negative cores are shown, along with cells from the same cores negative and positive for TMRPSS2-ERG rearrangement by FISH. A TMRPSS2-ERG rearrangement through intrachromosomal deletion is indicated by loss of one 5’ (green) ERG signal. B. Contingency tables for SPINK1 expression and TMRPSS2-ERG status and p-values for Fisher’s exact tests for both cohorts are indicated.

Non-invasive detection of SPINK1 outlier-expression in men with TMRPSS2-ERG negative prostate cancer. Total RNA was isolated from the urine of 148 men with prostate cancer and assessed for TMRPSS2-ERG and SPINK1 expression by quantitative PCR. Samples above the dashed red line show SPINK1 outlier expression (See Methods).

Meta-COPA analysis of 7 prostate cancer gene expression profiling datasets in Oncomine. The expression of SPINK1 and scatter plots of ERG vs. SPINK1 and ETV1 vs. SPINK1 expression are shown from two studies (Glinsky et al., 2004; Yu et al., 2004) where SPINK1 ranked as a top 100 COPA outlier. A. The expression of SPINK1, in normalized expression units (non-median centered), for all profiled samples including benign prostate tissue (blue) and clinically localized prostate cancer (PCa, red), as well as Gleason pattern 6, 7, 8 or 9 prostate cancer (magenta, orange, light blue and purple, respectively) are shown. B–C. Scatter plots are shown for B) ERG vs. SPINK1 and C) ETV1 vs. SPINK1 for all samples in both studies. Outlier-expression is delineated by the dashed gray lines (See Methods). See Figure S2 for SPINK1 outlier expression in additional prostate cancer profiling studies.

Relationship between SPINK1 outlier expression and biochemical recurrence after surgical resection. Kaplan-Meier analyses of outlier SPINK1 expression from the (A) Glinsky et al. DNA microarray dataset (Glinsky et al., 2004) and SPINK1 IHC from the (B) UM and (C) Memorial Sloan Kettering Cancer Center (MSKCC) cohorts and biochemical recurrence after surgical resection are shown.

A–B. To recapitulate the outlier-expression of SPINK1, we generated adenoviruses expressing SPINK1 or LACZ (control). The benign immortalized prostate cell line RWPE was infected with SPINK1 or LACZ adenovirus as indicated and assayed for (A) proliferation or (B) invasion through a modified basement membrane. C. As these results suggest that SPINK1 may require co-existing genetic lesions to function in prostate cancer, we assayed prostate cancer cell lines by qPCR for SPINK1 (yellow), ERG (blue) and ETV1 (green) outlier expression. D–F. SPINK1 mediates invasiveness in 22RV1 cells. To investigate the role of SPINK1 in the outlier-expressing cell line 22RV1, cells were treated with transfection reagent alone (untreated), or transfected with non-targeting or siRNA against SPINK1, ETV1 or ERG as indicated. Cells were assayed for (D) proliferation and (E) invasion. Photomicrographs of invaded cells treated with the indicated siRNA are shown in F. G–H. VCaP (TMPRSS2-ERG positive) and LNCaP (ETV1 rearrangement positive) prostate cancer cell lines as indicated were treated with transfection reagent alone (untreated), or transfected with non-targeting or siRNA against SPINK1, ETV1 or ERG as indicated and assayed for invasion. For all proliferation and invasion experiments, mean (n = 3) + S.E. are shown, and P-values < 0.05 are given.