Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgen-independent prostate cancer cells

Abstract

Previously, we have developed a unique in vitro LNCaP cell model, which includes androgen-dependent (LNCaP-C33), androgen-independent (LNCaP-C81) and an intermediate phenotype (LNCaP-C51) cell lines resembling the stages of prostate cancer progression to hormone independence. This model is advantageous in overcoming the heterogeneity associated with the prostate cancer up to a certain extent. We characterized and compared the gene expression profiles in LNCaP-C33 (androgen-dependent) and LNCaP-C81 (androgen-independent) cells using Affymetrix GeneChip array analyses. Multiple genes were identified exhibiting differential expression during androgen-independent progression. Among the important genes upregulated in androgen-independent cells were PCDH7, TPTE, TSPY, EPHA3, HGF, MET, EGF, TEM8, etc., whereas many candidate tumor suppressor genes (HTATIP2, CDKN2A, CDKN2B, CDKN1C, TP53, TP73, ICAM1, SOCS1/2, SPRY2, PPP2CA, PPP3CA, etc.) were decreased. Pathway prediction analysis identified important gene networks associated with growth-promoting and apoptotic signaling that were perturbed during androgen-independent progression. Further investigation of one of the genes, PPP2CA, which encodes the catalytic subunit of a serine phosphatase PP2A, a potent tumor suppressor, revealed that its expression was decreased in prostate cancer compared to adjacent normal/benign tissue. Furthermore, the downregulated expression of PPP2CA was significantly correlated with tumor stage and Gleason grade. Future studies on the identified differentially expressed genes and signaling pathways may be helpful in understanding the biology of prostate cancer progression and prove useful in developing novel prognostic biomarkers and therapy for androgen-refractory prostate cancer.

Figure 1

Expression analysis of differential gene expression by real-time RT-PCR. Total RNA was reverse-transcribed and subsequently amplified by a real-time PCR using gene-specific primers. A fold-change in expression was calculated relative to LNCaP-C33 cells using GAPDH as an internal control by ΔΔC_T method. Each experiment was repeated at least three times and data presented as mean ± standard deviation.

Figure 2

Immunoblot analysis of PPP2CA (alpha isoform of catalytic subunit of protein phosphatase 2A). Consistent with the microarray analysis, a reduced expression of PPP2CA is observed in androgen-independent (AI) LNCaP-C81 cells compared to androgen-dependent (AD) LNCaP-C33 cells. Except DU145, a reduced expression of PPP2CA is also evident in other AI cell lines (C4-2 and PC3). Phospho-glycerol kinase (PGK) was used as an internal control.

Figure 3

Immunohistochemical analysis of PPP2CA expression on a prostate cancer tissue array. A and B, Normal/benign tissue at 10X and 40 × magnifications, respectively. C, Prostate cancer section with various grade lesions (10× magnification), D and E, magnified (40X) view of section depicting lesions with Gleason grade 6 (3+3) and 8 (4+4), respectively. A decreased expression of PPP2CA is observed in prostate cancer, which further correlates with Gleason grade.