Differential expression of apoptotic genes PDIA3 and MAP3K5 distinguishes between low- and high-risk prostate cancer

Abstract

Background: Despite recent progress in the identification of genetic and molecular alterations in prostate cancer, markers associated with tumor progression are scarce. Therefore precise diagnosis of patients and prognosis of the disease remain difficult. This study investigated novel molecular markers discriminating between low and highly aggressive types of prostate cancer.

Results: Using 52 microdissected cell populations of low- and high-risk prostate tumors, we identified via global cDNA microarrays analysis almost 1200 genes being differentially expressed among these groups. These genes were analyzed by statistical, pathway and gene enrichment methods. Twenty selected candidate genes were verified by quantitative real time PCR and immunohistochemistry. In concordance with the mRNA levels, two genes MAP3K5 and PDIA3 exposed differential protein expression. Functional characterization of PDIA3 revealed a pro-apoptotic role of this gene in PC3 prostate cancer cells.

Conclusions: Our analyses provide deeper insights into the molecular changes occurring during prostate cancer progression. The genes MAP3K5 and PDIA3 are associated with malignant stages of prostate cancer and therefore provide novel potential biomarkers.

Figure 1

Validation of PDIA3 and MAP3K5 mRNA expression via qRT PCR. (A) The housekeeping gene β-2 microglobulin (B2M) was chosen due to its even expression (mean Ct value) in each analyzed group (benign, GS 6 and GS 8-10 tissue). (B, C) Mean normalized expression levels (dCt) of GS 6 and GS 8-10 was determined for PDIA3 (B) and MAP3K5 (C). Results showed a significant increase of transcript abundance levels in GS 8-10 tumors in comparison to GS 6 tumors.

Figure 2

IHC analysis of Gleason grade-associated protein expression of PDIA3 and MAP3K5. Summary of PDIA3 and MAP3K5 protein expression quantification in tissue samples. Paraffin tissue sections were stained according to a standard IHC protocol using a staining automate and immunoreactivity of the different Gleason patterns identified in each specimen were scored by an uropathologist according to a 4 point scale (no - 0, weak - 1, intermediate - 2 and strong - 3 staining). For both antigens immunoreactivity was higher in tumors than in benign epithelium in accordance with the gene expression and real-time PCR data. Within the different tumor patterns staining intensity increased from CA3 to CA4 and decreased in the most dedifferentiated CA5 tumor regions. Interestingly, PDIA3 staining intensity in CA 3 regions within GS6 tumors (CA3 CS6) and within GS8 tumors (CA3 GS8) differed significantly, whereas this was not observed with MAP3K5. (B: Benign tissue; CA3: Gleason pattern 3, CA4: Gleason pattern 4, CA5: Gleason pattern 5).

Figure 3

IHC analysis for the proteins AMACR, MAP3K5 and PDIA3. Representative IHC pictures are shown for AMACR, MAP3K5 and PDIA3. AMARC was used as a marker for confirmation of the tumor. MAP3K5 and PDIA3 IHC revealed cytoplasmatic localization of both antigens and higher expression in tumors as compared to benign epithelium. Gleason pattern CA4 displayed highest staining levels, compared with CA3 (lower midst and right pictures; magnification upper panel: 400×; lower panel: 200×).

Figure 4

siRNA mediated knockdown of PDIA3 decreased apoptosis in prostate cancer cell line. (A) Knockdown efficiency measured via qRT PCR 48 h after transfection with 20 nM siRNA. (B) PC3 cells were treated with 20 nM scrambled siRNA control and PDIA3 siRNA. 48 h after transfection induction of apoptosis was performed with 1 μM Staurosporine (STS), 20 μM Fenretinide (FenR) or 1.5 μM Tapsigargin (TG) for 6 and 24 hours. Apoptosis was measured by determining caspase activation and compared to untreated control. Bar heights and error bars are means and upper range of triplicate samples relative to control treatment. * P < 0.05 (unpaired t-test).