Secretory phospholipase A2-IIa is a target gene of the HER/HER2-elicited pathway and a potential plasma biomarker for poor prognosis of prostate cancer

Abstract

Background: Our previous study showed that prostate cancer cells overexpress and secrete secretory phospholipases A2 group IIa (sPLA2-IIa) and plasma sPLA2-IIa was elevated in prostate cancer patients. The current study further explored the underlying mechanism of sPLA2-IIa overexpression and the potential role of sPLA2-IIa as a prostate cancer biomarker.

Methods: Plasma and tissue specimens from prostate cancer patients were analyzed for sPLA2-IIa levels. Regulation of sPLA2-IIa expression by Heregulin-α was determined by Western blot and reporter assay.

Results: We found that Heregulin-α enhanced expression of the sPLA2-IIa gene via the HER2/HER3-elicited pathway. The EGFR/HER2 dual inhibitor Lapatinib and the NF-kB inhibitor Bortezomib inhibited sPLA2-IIa expression induced by Heregulin-α. Heregulin-α upregulated expression of the sPLA2-IIa gene at the transcriptional level. We further confirmed that plasma sPLA2-IIa secreted by mouse bearing human prostate cancer xenografts reached detectable plasma concentrations. A receiver operating characteristic (ROC) analysis of patient plasma specimens revealed that high levels of plasma sPLA2-IIa, with the optimum cutoff value of 2.0 ng/ml, were significantly associated with high Gleason score (8-10) relative to intermediate Gleason score (6-7) prostate cancers and advanced relative to indolent cancers. The area under the ROC curve (area under curve, AUC) was 0.73 and 0.74, respectively.

Conclusion: We found that Heregulin-α, in addition to EGF, contributes to sPLA2-IIa overexpression in prostate cancer cells. Our findings support the notion that high levels of plasma sPLA2-IIa may serve as a poor prognostic biomarker capable of distinguishing aggressive from indolent prostate cancers, which may improve decision-making and optimize patient management.

Fig. 1. Determination of plasma sPLA2-IIa in prostate cancer patients

Plasma samples from prostate cancer patients were diluted ten times and then subjected to ELISA analysis in duplicate for each sample. The average was calculated as pg/ml based on the standard curve of each experiment. Two samples from stage T3 prostate cancer are above the scale. The dotted line indicates 2.0 ng/ml cutoff value.

Fig. 2. Association analysis of high levels of plasma sPLA2-IIa with high Gleason score and advanced cancer stage

18 specimens of stage T1 prostate cancer versus 101 specimens of advanced stage T2~T4 prostate cancer (A) and 85 specimens of Gleason score 6~7 prostate cancer versus 41 specimens of Gleason scores 8~10 prostate cancer (B), were subjected to ROC analysis. Area under the ROC curve (AUC) and 95% confidence interval (95% CI) were determined.

Fig. 3. Expression analysis of sPLA2-IIa in prostate cancer (A) and BPH (B) specimens

Open arrow (brown staining) indicates positivity for sPLA2-IIa in prostate cancer cells. Close arrow indicates normal epithelial cells. sPLA2-IIa is undetectable in the epithelial cells of normal acini and BPH.

Fig. 4. Heregulin-α activates HER3 and stimulates sPLA2-IIa expression in prostate cancer cells

LNCaP, LNCaP-AI, and LAPC4 cells were starved in 1% stripped medium for 24 hours. The cells were then treated with various concentrations of Heregulin-α for 24 hours (A). The cells were also treated with Lapatinib (10 uM) and Bortezomib (5 uM) without or with Heregulin-α (50 ng/ml) for 24 hours (B-D). The cell extracts were prepared and subjected to western blot analysis for sPLA2-IIa, HER2, P-HER2, HER3, and P-HER3.

Fig. 5. Heregulin-α regulates sPLA2-IIa promoter activity

LNCaP-AI (A) and LAPC4 (B) cells (10⁵ cells/well) were seeded in a 12 well plate. Next day, the cells were transiently transfected with sPLA2-IIa(−800)-Luc (0.25 ug/well) overnight. The cells were then treated with Lapatinib (10 uM) and Bortezomib (5 uM) without or with EGF (100 ng/ml) or Heregulin-α (50 ng/ml) for 24 hours. Luciferase assay was performed according to a standard protocol with Renilla luciferase as an internal control. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated for five times.