Secretory phospholipase A2-IIa is involved in prostate cancer progression and may potentially serve as a biomarker for prostate cancer

Abstract

The majority of prostate cancers are indolent, whereas a significant portion of patients will require systemic treatment during the course of their disease. To date, only high Gleason scores are best associated with a poor prognosis in prostate cancer. No validated serum biomarker has been identified with prognostic power. Previous studies showed that secretory phospholipase A2-IIa (sPLA2-IIa) is overexpressed in almost all human prostate cancer specimens and its elevated levels are correlated with high tumor grade. Here, we found that sPLA2-IIa is overexpressed in androgen-independent prostate cancer LNCaP-AI cells relative to their androgen-dependent LNCaP cell counterparts. LNCaP-AI cells also secrete significantly higher levels of sPLA2-IIa. Blocking sPLA2-IIa function compromises androgen-independent cell growth. Inhibition of the ligand-induced signaling output of the HER network, by blocking PI3K-Akt signaling and the nuclear factor-kappaB (NF-κB)-mediated pathway, compromises both sPLA2-IIa protein expression and secretion, as a result of downregulation of sPLA2-IIa promoter activity. More importantly, we demonstrated elevated serum sPLA2-IIa levels in prostate cancer patients. High serum sPLA2-IIa levels are associated significantly with high Gleason score and advanced disease stage. Increased sPLA2-IIa expression was confirmed in prostate cancer cells, but not in normal epithelium and stroma by immunohistochemistry analysis. We showed that elevated signaling of the HER/HER2-PI3K-Akt-NF-κB pathway contributes to sPLA2-IIa overexpression and secretion by prostate cancer cells. Given that sPLA2-IIa overexpression is associated with prostate development and progression, serum sPLA2-IIa may serve as a prognostic biomarker for prostate cancer and a potential surrogate prostate biomarker indicative of tumor burden.

Fig. 1.

sPLA2-IIa was overexpressed in androgen-independent prostate cancer cells. (A) Real-time reverse transcription–PCR analysis showed the relative sPLA2-IIa messenger RNA level compared with β-actin in LNCaP-AI and LNCaP cells. The data represent one of five repeated experiments. (B) sPLA2-IIa protein expression by western blot analysis. (C) Quantitative analysis of sPLA2-IIa in condition medium from LNCaP-AI and LNCaP cells by ELISA assay. The cells (500 000 cells per well in six-well plate) were cultured in stripped medium for 2 days. Then, the condition medium samples were collected and subjected to ELISA analysis in duplicate for each sample. The condition medium samples from LNCaP-AI cells, but not LNCaP cells, were diluted 10 times. Average of duplicate samples was converted to nanogram per milliliter against standard curve. The data represents one of five repeated experiments. (D) The role of sPLA2-IIa on growth of prostate cancer cells. LNCaP-AI cells were cultured in 10% stripped medium in the presence of cFLSYR or c(2NapA)LS(2NapA)R for 4 days, followed by MTT assay.

Fig. 2.

Expression analysis of sPLA2-IIa in prostate cancer cells. (A–C) LNCaP-AI cells were starved in 1% stripped medium for 24 h. The cells were then treated with Erlotinib (20 μM), Gefitinib (20 μM), Lapatinib (20 μM), CI-1033 (8 μM), LY294002 (20 μM) and Bortezomib (20 μM) without or with EGF (100 ng/ml) for 24 h. The cell extracts were prepared and subjected to western blot analysis for sPLA2-IIa, P-Akt, Akt and β-actin. (D–E) LAPC-4 cells (D) and LNCaP cells (E) were starved in 1% stripped medium for 24 h. The cells were then treated with Lapatinib (20 μM) (D) or Heregulin-α (50 ng/ml) (E) for 24 h. The cell extracts were prepared and subjected to western blot analysis for sPLA2-IIa, P-Akt, Akt, and β-actin. (F) LNCaP-AI cells were starved in 1% stripped medium for 24 h. The cells were then treated with Erlotinib (20 μM), Gefitinib (20 μM), Lapatinib (20 μM), CI-1033 (8 μM), LY294002 (20 μM) and Bortezomib (20 μM) for 24 h. Cell culture medium was collected from each sample and subjected to ELISA for sPLA2-IIa. The condition medium samples were diluted 10 times for ELISA. Average of duplicate samples was converted to nanogram per milliliter against standard curve. The data represent one of five repeated experiments.

Fig. 3.

HER/HER2-PI3K-Akt-NF-κB signaling regulates sPLA2-IIa promoter activity. (A) Human sPLA2-IIa promoter sequence (accession number NC_000001). The C/EBP-binding site in the core promoter region is indicated by bold letter. The consensus sequence of the NF-κB site located at −782 bp was indicated by underline. Most NF-κB sites appear to be 10 bp in length with the consensus sequence 5′-GGGRNWYYCC-3′, where R denotes a purine base, N denotes any base, W denotes an adenine or thymine and Y denotes a pyrimidine base. (B–C) LNCaP (B) and LNCaP-AI (C) cells were transiently transfected with sPLA2-IIa(-800)-Luc (0.5 μg). The cells were then treated with Erlotinib (20 μM), Gefitinib (20 μM), Lapatinib (20 μM), CI-1033 (8 μM), LY294002 (20 μM) and Bortezomib (20 μM) without or with EGF (100 ng/ml) for 24 h. 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.

Fig. 4.

Determination of serum sPLA2-IIa in prostate cancer patients. Plasma samples from healthy donors and prostate cancer patients were diluted 10 times and then subjected to ELISA analysis with duplicate for each sample. Average of the duplicate sample was calculated to present as picogram per milliliter based on the standard curve of each experiment.

Fig. 5.

Expression analysis of sPLA2-IIa in prostate cancer specimens. Solid arrow indicates benign prostatic glands, which are negative and serve as the built in controls, whereas open arrow indicates prostate cancer cells. A moderate cytoplasmic granular staining is shown in Gleason score 6 prostatic adenocarcinoma (A), a strong and diffuse cytoplasmic granular staining in Gleason score 7 prostatic adenocarcinoma glands (B) and a strongest staining in Gleason score 8 prostatic adenocarcinoma glands (C).

Fig. 6.

The HER/HER2-PI3K-Akt-NF-κB-elicited pathway in prostate cancer cells. PI3K-Akt signaling cross talks with the NF-κB-mediated pathway by Akt phosphorylation and activation of IKK, an upstream kinase of NF-κB. Small molecule inhibitors Erlotinib, Gefitinib, Lapatinib, CI-1033, LY294002, Bortezomib, cFLSYR and c(2NapA)LS(2NapA)R against EGFR, HER2, PI3K, NF-κB and sPLA2 are indicated.