Epithelial splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) upregulation predicts poor prognosis in prostate cancer

Radical prostatectomies of 17,747 patients treated at the Department of Urology and the Martini Clinic at the University Medical Center Hamburg-Eppendorf between 1992 and 2014 were available. All prostatectomies were processed according to a standardized procedure, including a complete embedding of the entire prostate for histological analysis [17]. Histopathological parameters were available from the patients’ records, including Gleason grade, pathological tumor stage (pT), presents of lymph node metastasis (pN), and presents of tumor cells in the resection margin (R). In addition to the classical Gleason score categories, “quantitative” Gleason grading was done as described before [18]. In short, for every individual prostatectomy specimen, the percentage of Gleason 4 patterns in neoplastic tissues were estimated and the group of Gleason score 3 + 4 and 4 + 3 cancers were subdivided for practical use in 8 subgroups as follows: 3 + 4 with ≤5% Gleason 4, 3 + 4 with 6–10% Gleason 4, 3 + 4 with 11–20% Gleason 4, 3 + 4 with 21–30% Gleason 4, 3 + 4 with 31–49% Gleason 4, 4 + 3 with 50–60% Gleason 4, 4 + 3 with 61–80% Gleason 4 and 4 + 3 with > 80% Gleason 4. In addition, two subgroups were defined by the presence of a tertiary Gleason 5 pattern (3 + 4 Tert. 5 and 4 + 3 Tert. 5). For 14,464 patients, follow-up data were available (median: 48.0 months; range: 1 to 276 months; Table 1). PSA recurrence was defined as the time point at which the postoperative PSA level rose to at least 0.2 ng/ml. The production of TMAs has already been described in detail [19]. In brief, from each individual patient a 0.6 mm core was removed from a cancer containing tissue block. The molecular database associated with the TMA include IHC results on ERG expression in 13,089 [20] and ESRP1 in 12,140 tumors [16], and fluorescence in situ hybridization (FISH) results on ERG breakage in 7225 (expanded from [20]) as well as on deletion status of 3p13 (FOXP1) in 7201 (expanded from [21]), 5q21 (CHD1) in 8074 (expanded from [22]), 6q15 (MAP 3 K7) in 6171 (expanded from [23]), 8p21 (NKX3.1) in 7001 [24], PTEN (10q23) in 6803 (expanded from [25]), 12p13 (CDKN1B) in 6187 [26], 12q24 (NCOR2) in 7435 [20], 13q14 (ENOX1) in 7499 [27], 16q23 (WWOX) in 3928 [28], 17p13 (TP53) in 8307 [29], and 18q24 in 7032 [30] cancers.

Freshly cut TMA sections were processed in a single run in 1 day. TMAs were deparaffinized and exposed to heat-induced antigen retrieval for 5 min in an autoclave in pH 7.8 Tris-EDTA buffer at 121 °C. Primary antibodies specific against ESRP2 protein (rabbit polyclonal antibody, Sigma-Aldrich, St. Louis, Missouri, USA, HPA0485597; dilution 1:450) and ESRP1 protein (rabbit polyclonal antibody, Sigma Aldrich Germany, cat#HPA023720; dilution 1:450) were incubated for 60 min at 37 °C. The EnVision Kit (Agilent, CA, USA) was used to visualize bound antibody according to the manufacturer’s instructions. ESRP2 staining was seen in the nuclei of prostate epithelial cells and was sometimes accompanied by cytoplasmic staining. Since splicing occurs in the nucleus, we assumed nuclear expression to be biologically relevant and only scored nuclear staining. ESRP2 positive staining was usually seen in all tumor cells (100%). Therefore, the staining intensity was estimated in three categories, i.e. negative (not detectable), low (1–2+) and high (3+) staining. Immunohistochemical ESRP1 data were available from a previous study [16]. To test the combined impact of ESRP1 and ESRP2 expression on prostate cancer prognosis an ESRP1/ESRP2 score was generated as follows: score 0: ESRP1 and ESRP2 negative, score 1: ESRP1 low and ESRP2 negative or ESRP1 negative and ESRP2 low, score 2: ESRP1 and ESRP2 low, score 3: ESRP1 high and ESRP2 negative or ESRP1 negative and ESRP2 high, score 4: ESRP1 high and ESRP2 low or ESRP1 low and ESRP2 high, and score 5: ESRP1 high and ESRP2 high.

ESRP1 and ESRP2 antibody specificity was validated in control cell lines with ectopic ESRP1 and ESRP2 protein overexpression (Supplementary Fig. 2). To produce these cells, cDNAs encoding ESRP1/RBM35A (# HG13708-UT, Sino Biological Inc., Wayne PA, USA) and ESRP2 (#HG23639-UT, Sino Biological Inc., Wayne PA, USA) were transformed in competent Escherichia coli cells (One ShotTM Top10, TermoFisher Scientifc, Germany), the plasmid DNA was isolated (NucleoBond BAC 100 Kit, #740579, Macherey-Nagel, Düren, Germany) and transfected to cultivated HeLa cells (15 μg/70% confluence/1500 mm dish) using JetPEI DNA Transfection Reagent (Polyplus-transfection, #101-10 N S.A., Illkirch, France). Transfected cells were harvested after 24 h, centrifuged at 1000×g for 5 min, stabilized in agarose, fixed in 4% buffered formalin overnight and embedded in paraffin (FFPE fixation). Non-transfected HeLa cells were cultivated (37 °C and 5% CO₂) in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S), harvested and FFPE fixed and served as negative control. For immunohistochemistry, freshly cut section of negative and positive control cell lines were stained as described above.

For statistical calculations the JMP® 14 software (SAS Institute Inc., NC, USA) was used. Chi²-tests and contingency tables were used to find associations between ESRP1/ESRP2 and molecular or histopathological parameters. Survival curves were calculated according to Kaplan-Meier. Significant differences between groups were detected by the Log-Rank test. The statistical independence and significance between pathological, molecular and clinical variables was tested by different Cox proportional hazards regression analyses.

In our TMA analyses, 12,140 (68.4%) were interpretable for ESRP1 and 12,962 (73.0%) for ESRP2 (out of a total of 17,747 tumor samples). Reasons for non-informative cases (n = 5607; 31.6% for ESRP1 and n = 4785; 27.0% for ESRP2) included lack of tissue samples or absence of unequivocal cancer tissue in the TMA spot.

In normal prostate glands, both ESRP1 and ESRP2 staining in the nucleus was rare and, if present, faint. In prostate cancers, nuclear ESRP1 and ESRP2 staining was more common and also more intense (Fig. 1). For ESRP1, positive nuclear staining was recorded in 4689 (38.6%) of 12,140 interpretable tumors, including 36.0% with low and 2.6% with high staining intensity. For ESRP2, positive nuclear staining was seen in 5393 (41.6%) of 12,962 interpretable cancers. Of these cancers, 36.4% showed low and 5.2% showed high staining intensity. A combined staining for ESRP1 and ESRP2, was detectable in 2503 (21.6%) of 11,597 for ESRP1 and ESRP2 interpretable cancers.

The TMPRSS2:ERG fusion status of the prostate cancers on the TMA has been determined previously by FISH and IHC [20]. The combined data revealed 5100 (ERG status by FISH)/9786 (ERG status by IHC) tumors with evaluable ESRP1 immunostaining and 5416 (ERG status by FISH)/10,380 (ERG status by IHC) tumors with evaluable ESRP2 immunostaining. Data on ERG status determined by FISH as well as IHC were available for 4236 tumors with ESRP1 and 4484 tumors with ESRP2 immunostaining. Identical results for ERG status determined by FISH or IHC were shown in 3850 (90.9%; ESRP1) and in 4073 (90.8%; ESRP2) of these cases. Nuclear staining of both ESRP1 and ESRP2 was linked to TMPRSS2:ERG fusion and ERG expression. For example, the fraction of tumors with detectable ESRP1 expression increased from 35.1% in ERG-negative cancers to 42.8% in ERG-positive cancers and with detectable ESRP2 expression from 36.7% in ERG-negative cancers to 51.6% in ERG-positive cancers (Fig. 2).

For most of 11 analyzed chromosomal regions, ESRP1 and ESRP2 expression was significantly more common in deleted than in non-deleted cancers in all analyzed cancers (11/11 for ESRP1 and 9/11 for ESRP2, p < 0.03 each, data not shown). In ERG-negative cancers these statistically significant associations were retained for ESRP1 in 8 and for ESRP2 in 9 chromosomal regions. In ERG-positive cancers, a statistically significant difference was found for ESRP1 in 5 and for ESRP2 in 1 of the analyzed loci (Fig. 3 and Supplementary Fig. 1).

Both, high ESRP1 and high ESRP2 staining were significantly associated with adverse tumor features, including advanced tumor stage, high Gleason grade, presence of lymph node metastasis (p < 0.0001 each, Table 2), and high early PSA recurrence (p < 0.0001 each; Fig. 4a-b). Most of these associations were also seen in the subsets of ERG-negative and ERG-positive cancers (Fig. 4c-f, Supplementary Tables 1 and 2). The ESRP1/ESRP2 score analysis showed a striking combined impact of combined ESRP1 and ESRP2 expression on prostate cancer prognosis. The higher the score (score 5 = both markers high), the more likely was an early PSA recurrence (Fig. 4g).

Four different multivariate analyses were applied to evaluate whether ESRP1 or ESRP2 expression as well as our ESRP1/ESRP2 score is a statistically independent prognostic marker in all prostate cancers and the subset of ERG-negative and ERG-positive cancers (Table 3). Scenario 1 evaluated all parameters available after surgery, including pathological tumor stage (pT), pathological nodal stage (pN), surgical margin status, preoperative PSA value and Gleason grade obtained after evaluation of the entire prostate. In scenario 2, all postoperatively available parameters with exception of nodal status were included. This was because the indication and extent of lymph node dissection is not standardized in the surgical therapy of prostate cancer and more often executed if aggressive cancer is expected based on biopsy results. This may introduce a bias towards high grade cancers in the cohort with available lymph nodes. Two additional scenarios had the purpose to model the preoperative situation to the best possible extent. Scenario 3 included preoperative PSA, clinical tumor stage, and Gleason grade obtained on the prostatectomy specimen. Since a postoperative determination of the Gleason grade is “better” than the preoperatively determined Gleason grade (subjected to sampling errors and consequently under-grading in more than one third of cases), this parameter was replaced by the original preoperative biopsy Gleason grade in Scenario 4. These analyses identified ESRP1 expression, ESRP2 expression as well as our ESRP1/ESRP2 score as independent prognostic parameters in all cancers as well as the subset of ERG-negative and ERG-positive cancers in all four scenarios (p ≤ 0.05, Table 3).