Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer

Abstract

Background: Polycomb repressive complex 2 (PRC2) mediates gene silencing through histone H3K27 methylation. PRC2 components are over-expressed in metastatic prostate cancer (PC), and are required for cancer stem cell (CSC) self-renewal. 3-Dezaneplanocin-A (DZNeP) is an inhibitor of PRC2 with broad anticancer activity.

Method: we investigated the effects of DZNeP on cell proliferation, tumorigenicity and invasive potential of PC cell lines (LNCaP and DU145).

Results: Exploring GEO and Oncomine databases, we found that specific PRC2 genes (EED, EZH2, SUZ12) predict poor prognosis in PC. Non-toxic DZNeP concentrations completely eradicated LNCaP and DU145 prostatosphere formation, and significantly reduced the expression of CSC markers. At comparable doses, other epigenetic drugs were not able to eradicate CSCs. DZNeP was also able to reduce PC cell invasion. Cells pre-treated with DZNeP were significantly less tumorigenic (LNCaP) and formed smaller tumors (DU145) in immunocompromised mice.

Conclusion: DZNeP is effective both in vitro and in vivo against PC cells. DZNeP antitumor activity is in part mediated by inhibition of CSC tumorigenic potential.

Figure 1

PRC2 gene expression in PC patients. A, comparison of PRC2 mRNA levels in normal prostate, primary and metastatic PC (GEO database). *p < 0.05, **P < 0.01. B, C: prognostic role of EZH2 and SUZ12 mRNA levels in PC sample (Oncomine database).

Figure 2

Effects of DZNeP on PC cells. A, Western blot analysis. (-), untreated cells. B, Annexin-PI staining on untreated and treated cells. Columns mean volume. C, D: cell cycle distribution after DZNeP treattment. Columns, mean volume. Cells were treated with 1 μM DZNeP for 3 and 5 days.

Figure 3

Effects of DZNeP on PS and CSC markers. A, representative picture of cells grown in SCM for 7 days. B, C PS number and volume in untreated and treated cells (1 μM, 7 days). Columns, mean volume; bars standard deviation. D, CSC marker expression. LNCaP cells were treated with DZNeP (1 μM, 3 days) and then sorted as described under "Flow cytometric analysis and separation".

Figure 4

Effects of DZNeP on invasion and in vivo tumor growth. A, Cells were treated with DZNeP (1 μM, 3 days) and assayed for cell viability at the end of the treatment (trypan blue staining). Alive cells were used for Matrigel invasion assay, as described in "Materials and Methods". Columns, mean volume; bars, standard deviation. **p < 0.01 with respect to untreated cells (T test). B, mRNA levels in invading and non-invading cells (QT-PCR). C, effects of DZNeP on LNCaP cell tumorigenicity. **p < 0.01 (log rank test) with respect to untreated cells. D, effects of DZNeP on DU145 xenograft tumor volume. *p < 0.05 with respect to untreated cells (U test). Star colors refer to dose treatment (blue, 1 μM; red, 10 μM). Number of animals: untreated: 6; 1 and 10 μM: 8. Point, mean value; bar, standard deviation.