Protocadherin-PC promotes androgen-independent prostate cancer cell growth

Abstract

Background: Protocadherin-PC (PCDH-PC) expression is upregulated in apoptosis-resistant sublines of the LNCaP human prostate cancer (CaP) cell line. Here, we assess the role of PCDH-PC in CaP cells and its mRNA expression in human prostate tissues.

Methods: LNCaP cells transfected with PCDH-PC were tested for their ability to grow in vitro and in vivo in androgen-deprived conditions. PCDH-PC mRNA expression was evaluated by semi-quantitative RT-PCR and by in situ hybridization.

Results: PCDH-PC expression induced Wnt signaling in CaP cells and permitted androgen-independent growth of hormone-sensitive CaP cells. Expression of PCDH-PC-homologous transcripts was low and restricted to some epithelial cells in normal tissue and to CaP cells in tumors. However, hormone-resistant CaP cells expressed significantly higher levels of PCDH-PC-related mRNA.

Conclusions: Our findings suggest a novel mechanism for the progression of CaP involving expression of PCDH-PC. This novel protocadherin induces Wnt signaling, promotes malignant behavior and hormone-resistance of CaP cells.

Fig. 1

PCDH-PC mRNA expression in human prostatic tissue, hormone sensitive, and apoptosis resistant cell lines, a: Expression of PCDH- PC mRNA was investigated by semi-quantitative RT-PCR and by comparison with an internal control, the TBP mRNA. PCDH-PC mRNA expression was higher in apoptosis resistant lines LNCaP-TR and LNCaP-SSR compared to LNCaP parental cell line, b: Relative expression of PCDH-PC mRNA in prostatic tissues was also determined by semi-quantitative RT-PCR. Values corresponded to the mean of PCDH-PC expression levels in RNAs extract from different groups: the peripheral (n = 7), central (n = 9), and transitional (n = 6) zones of the normal prostate, the benign hyperplastic prostate (n = 15), untreated (n = 10), and treated (n = 8) prostate tumors and hormonal refractory patients (n = 9). Of note, PCDH-PC expression is significantly upregulated in hormone failure group as compared to either non-CaP, treated, or untreated CaP groups.

Fig. 2

In situ localization of PCDH-PC m RNA in prostatic tissues. In situ hybridization technique was performed on formalin fixed paraffin-embedded tissue using digoxigenin-labeled PCDH-PC antisense probes, a: Tissues from normal prostate. Note the staining corresponding to PCDH-PC mRNA was mainly localized in the basal epithelial cells. Differentiated glandular cells were faint or negative-stained. Representative results of ISH performed on primary (untreated) cancers were presented in (b). Tumor cells (indicated by arrows) were strongly positive for PCDH-PC staining compared to adjacent normal epithelial cells. In tissues obtained from patients treated by hormonal therapy (c) and from hormone-refractory human CaPs (d), strong staining corresponding to PCDH-PC mRNA was localized in all tumor cells (indicated by arrows) and in normal (atrophic) epithelial cells. Magnification: a–d 200×.

Fig. 3

PCDH-PC mRNA is expressed in some human normal tissues. Relative expression of PCDH-PC mRNA in different human normal tissues (brain, duodenum, kidney, liver, lung, placenta, prostate, skeletal muscle, spleen, and urothelium) was determined by semi-quantitative RT-PCR.

Fig. 4

Inducible expression of PCDH-PC causes dramatic changes in β-catenin localization, a: LNCaP-pVgRXR/PCDH-PC cell line generated from Ecdysone system was treated with either ponA (IO µM) or ethanol 0.05% for 48 hr. Relative expression of PCDH-PC transcripts in both conditions was evaluated with a standard RT-PCR procedure. Note expression of PCDH-PC is very low in absence of ponasterone and extends in presence of PonA demonstrating inducibility of the system, b: LNCaP-pVgRXR/PCDH-PC cells were treated with vehicule (ethanol) or with ponA (10 µM) for 48 hr, and stained for β-catenin. Interestingly, immunostaining shows that hormone exposure is concomitant with a change in β-catenin expression pool from the cell-cell border to the cytoplasm and occasionally in the nucleus. Magnification 400×.

Fig. 5

PCDH-PC augments β-catenin /TCF-mediated transcription in various cell lines. β-catenin/TCF-mediated transcription activation was measured by luciferase assay subsequent to 48 hr transfection with a TOPFLASH or FOPFLASH reporter construct (1 µg) together with a β-gal reporter plasmid (100 ng). Various cells lines were used: (a) LNCaP-pVgRXR-PCDH-PC, stable treated with ethanol at 0.05% or with ponA (10 µM) for 48 hr; (b) LNCaP that stably expresses PCDH-PC (LNCaP-PCDH-PC) and Mock-transfected control cells; (c) DUI45 and (d) HEK 293. HEK 293 and DUI45 were co-transfected with various doses of either pcDNA3 or pcDNA3-PCDH-PC expression plasmids (0,400, or 750 ng) as indicated. TCF-dependent transcription was defined as the ratio TOPFLASH/FOPFLASH luciferase activities each corrected internally for β-gal activities.

Fig. 6

PCDH-PC promotes anchorage and androgen-independent CaP cell growth, a: Soft agar assays of PC-3 stably expressing PCDH-PC (PC-3/PCDH-PC) or mock transfected PC-3 (control PC-3) cells. Cells (20,000) were seeded in triplicate in 6-wells plates. The colonies were counted after 2 weeks of culture in completed medium (RMPI/5% FCS). The number of soft agar colonies presented is the mean of colony counts in 10 40× microscopic fields from three wells, b: Growth curve assays. LNCaP/PCDH-PC cells or empty-vector-transfected LNCaP cells (3,500) were seeded in triplicate in 12-well plates. The cells were cultivated in hormone-deprived medium (RMPI/5% CS-FCS). At 2–3 day intervals, the cell number was analyzed. In contrast to control cells, LNCaP/PCDH-PC continued to proliferate in androgen-depleted medium, c: Anchorage-independent growth in hormone-deprived medium. LNCaP/PCDH-PC cells or empty-vector-transformed LNCaP cells (20,000) were seeded in triplicate in 6-well plates. The colonies were counted after 2 weeks of culture in RMPI/5% CS-FCS. Error bars indicate standard errors. *P < 0.001 compared with control-cells. d: Tumorigenicity of PCDH-PC overexpressed LNCaP cells in castrated nude mice. 2 × 10⁶ of either control LNCaP cells or LNCaP/PCDH-PC cells were injected into eight nude mice castrated 1 week before injection. After 7 weeks, mice injected with control cells had no visible or palpable tumor (0/8) whereas 100% of castrated mice xenografted with LNCaP/PCDH-PC cells formed tumors (8/8). Hematoxylin and eosin staining showed these tumors were highly vascularized. Magnification: 200×.