High SPDEF may identify patients who will have a prolonged response to androgen deprivation therapy

Abstract

Background: Due to the indolent nature of prostate cancer, new prognostic measures are needed to identify patients with life threatening disease. SAM pointed domain-containing Ets transcription factor (SPDEF) has been associated with good prognosis and demonstrates an intimate relationship with the androgen receptor (AR), however its role in prostate cancer progression remains unclear.

Methods: A tissue microarray constructed from cores of 713 consecutive radical prostatectomy specimens were immunohistochemically stained for SPDEF and correlated with progression free and metastatic free survival. In vitro studies assessed growth rate, migration, and sensitivity to bicalutamide to explore mechanisms behind the tissue microarray observations.

Results: Patients with high SPDEF demonstrate longer metastases free survival after receiving the standard of care (HR = 9.80, P = 0.006). SPDEF expression corresponded with bicalutamide growth inhibition and apoptosis induction in all cell lines studied. In addition, a feedforward loop of AR-SPEF expression regulation is observed.

Conclusions: SPDEF may be clinically useful to identify patients who will have extended benefits from androgen deprivation therapy. In vitro observations suggest SPDEF mediates initial sensitivity to androgen deprivation therapy through both AR regulation and downstream events.

Keywords: SPDEF; androgen receptor; metastasis; prostate.

Fig. 1

Example SPDEF IHC staining of TMA tumor core. TMA was constructed and IHC stained by RCPI PRN. A: Analysis consisted of 0.6 mm tumor and normal tissue cores from 713 patients arrayed in paraffin blocks. A: Example of SPDEF intensity = 0, (B) example of SPDEF intensity = 5. C: Cox proportional hazard modeling of SPDEF staning intensity and outcome rates. SPDEF does not associate with biochemical failure, however significantly associates with metastatic disease incidence in the univaritate analysis; the multivariate shows a trend, although it is not significant.

Fig. 2

KaplanˆMeier survival analysis demonstrate significantly longer metastasis free survival in patients with high SPDEF. TMA was assembled from consecutive RP specimens of CaP patients at RPCI treated with the standard of care (n = 713). Cores IHC stained in triplicate with a-SPDEF and given a score on a1ˆ5 scale based on intensity. Metastasis disease defined as distant metastasis to lymph or organ sites. A median of 105 months follow-up was available for analysis. Analysis performed with SAS. A: Kaplan survival analysis. B: Numerical representation of patients and events analyzed at each time point.

Fig. 3

ARˆSPDEF Feed-forward Loop. Cells were grown in RPMI 1640 + 10% with either 10 nM DHT or 20 mM bicalutamide or 10% CSS FBS and SPDEF and AR expression is indicated by (A) protein by western blot, or (B) mRNA by RT-qPCR. Cells infected with pGIPz lentivirus particles encoding shRNA towards AR results in a similar down-regulation of SPDEF expression as indicated by (C) protein by western blot and (D) mRNA by qPCR. AS = Androgen Starvation, *P < 0.05, OE= overexpression, EV=empty vectors, KD = knockdown. E: Reciprocal regulation of AR by SPDEF Cells were either transfected with pcDNA3.1-SPDEF (SPDEF OE) or infected with pGIPz-shRNA SPDEF (SPDEF KD). Western blot was used to detect protein expression for AR, SPDEF, and HSP70. SPDEF OE can be seen to increase AR expression in all three-cell lines tested, however SPDEF KD only downregulated the short form of AR expressed in LNCaPC4-2 cells. Error bars indicate standard deviation and *P < 0.05 by students t-test. F: Hypothetical mechanism for SPDEFˆAR regulation. AR activation induces SPDEF expression. SPDEF potentially binds elements in AR promoter to induce full-length receptor in normal conditions In castrate conditions SPDEF may bind at elements within the AR gene to activate the truncated form of the receptor.

Fig. 4

SPDEF effects on disease progression. After selection, cells were plated at 25,000 cells well and allowed to grow for 96 hr in RPMI1640 + 10% FBS and antibiotic. At indicated timepoints wells were harvested in triplicate. Cell number was assessed by trypan blue exclusion in (A) LNCaP, (B) LNCaP C4-2, and (C) PC-3-M. D: SPDEF inhibits migration in androgen independent CaP lines After infection/transfection and subsequent selection, 20,000 cells were plated in RPMI1640 without FBS in the top well of a 96-well modified Boyden-chamber assay plate and let migrate into FBS overnight. Cell number was estimated by Calcein-AD fluorescence. Error bars indicate standard deviation. *P < 0.05 OE= Overexpression, EV=empty vectors, KD = knockdown, AS= androgen sensitive.

Fig. 5

A: SPDEF expression corresponded with sensitivity to bicalutamide growth inhibition. After SPDEF transfection/infection and selection, cells were treated with 50 mM bicalutamide for 72 hr. Cell number was estimated by MTT. No significant difference was observed between EV controls, one EV bar was omitted. All numbers are reported compared to untreated control. B: SPDEF expression correlates with sensitivity to bicalutamide. Cells treated with 50 mM Casodex for 72 hr in triplicate, stained with HD and PI, and then analyzed by flow cytometry. Error bars indicate standard deviation.*P < 0.05, OE=overexpression, EV=empty vectors, KD = knockdown.

Fig. 6

Bicalutamide induces caspase 7 cleavage in cells expression SPDEF. LNCaP and LNCaP C4-2 cells were transfected/infected with vectors containing SPDEF cDNA or shRNA or control vectors and subsequently treated with 50 mM bicalutamide. SPDEF, AR, and cleaved caspase 7 protein expression were detected by western blot. HSP 70 was used as a loading control. OE= overexpression, EV= empty vectors, KD = knockdown.