Xiaomei Liu

Dual action on promoter demethylation and chromatin by an isothiocyanate restored GSTP1 silenced in prostate cancer

Prostate carcinoma is characterized by the silencing of π‐class glutathione S‐transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1, due to aberrant methylation at the CpG island in the promoter/5′‐UTR, occurs in the vast majority of prostate tumors and precancerous lesions. It is a pathologic marker and probably an underlying cause of oxidative damage and inflammation at tumor initiation. Inhibition of the aberrant promoter methylation could therefore be an effective mean to prevent carcinogenesis. Several isothiocyanates, including phenethyl isothiocyanate (PEITC), found naturally in cruciferous vegetables, induced growth arrest and apoptosis in prostate cancer cells in culture and xenografts. The effects of PEITC to reactivate GSTP1 were investigated. Exposure of prostate cancer LNCaP cells to PEITC inhibited the activity and level of histone deacetylases (HDACs), and induced selective histone acetylation and methylation for chromatin unfolding. Concurrently PEITC demethylated the promoter and restored the unmethylated GSTP1 in both androgen‐dependent and ‐independent LNCaP cancer cells to the level found in normal prostatic cells, as quantified by methylation‐specific PCR and pyrosequencing. The dual action of PEITC on both the DNA and chromatin was more effective than 5′‐Aza‐2′‐deoxycytidine, sodium butyrate, or trichostatin A (TSA), and may de‐repress the methyl‐binding domain (MBD) on gene transcription. The PEITC‐mediated cross‐talk between the DNA and chromatin in demethylating and reactivating GSTP1 genes, which is critically inactivated in prostate carcinogenesis, underlines a primary mechanism of cancer chemoprevention. Consequently, new approaches could be developed, with isothiocyanates to prevent and inhibit malignancies.

LEF1 in Androgen-Independent Prostate Cancer: Regulation of Androgen Receptor Expression, Prostate Cancer Growth, and Invasion

A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease.

Natura-Alpha Targets Forkhead Box M1 and Inhibits Androgen-Dependent and -Independent Prostate Cancer Growth and Invasion

Purpose: The development of new effective therapeutic agents with minimal side effects for prostate cancer (PC) treatment is much needed. Indirubin, an active molecule identified in the traditional Chinese herbal medicine—Qing Dai (Indigo naturalis), has been used to treat leukemia for decades. However, the anticancer properties of Natura-alpha, an indirubin derivative, are not well studied in solid tumors, particularly in PC. Experimental Design: The growth kinetics and invasion ability of on human PC cell lines with or without Natura-alpha treatment were measured by cell proliferation and invasion assays. The antitumor effects of Natura-alpha were examined in nude mice tumor xenograft models, and in a patient with advanced hormone-refractory metastatic PC. Signal network proteins targeted by Natura-alpha were analyzed by using proteomic pathway array analysis (PPAA) on xenografts. Results: Natura-alpha inhibited the growth of both androgen-dependent (LNCaP) and androgen-independent (LNCaP-AI, PC-3, and DU145) PC cells with IC50 between 4 to 10 mmol/L, and also inhibited invasion of androgen-independent PC cells. Its antitumor effects were further evident in in vivo tumor reduction in androgen-dependent and androgen-independent nude mice tumor xenograft models and reduced tumor volume in the patient with hormone refractory metastatic PC. PPAA revealed that antiproliferative and antiinvasive activities of Natura-alpha on PC might primarily be through its downregulation of Forkhead box M1 (FOXM1) protein. Forced overexpression of FOXM1 largely reversed the inhibition of growth and invasion by Natura-alpha. Conclusion: Natura-alpha could serve as a novel and effective therapeutic agent for treatment of both hormone-sensitive and hormone-refractory PC with minimal side effects. Clin Cancer Res; 17(13); 4414–24. ©2011 AACR.

KLF6 Loss of Function in Human Prostate Cancer Progression Is Implicated in Resistance to Androgen Deprivation

Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy.

Induction of bicalutamide sensitivity in prostate cancer cells by an epigenetic Purα‐mediated decrease in androgen receptor levels

Increased androgen receptor (AR) levels support resistance to apoptosis and hormone therapy in advanced prostate cancer (PC). We recently linked the overexpression of AR in androgen‐independent LNCaP cells (AI‐cells) and tissues from castration‐resistant patients to decreased nuclear levels of Pur‐alpha (Purα) and loss from a protein complex bound to repressor sequences (ARS) in the 5′‐UTR of AR. Strategies to regain control of increased AR transcription may overcome resistance of AI‐cells and improve treatment outcomes.

Effect of inhibition of the PI3K/Akt/mTOR pathway on AR splicing and downstream targets.

101 Background: Increased androgen receptor (AR) and PI3K/Akt/mTOR signaling drive androgen-independent (AI) prostate cancer (PC) progression. AR splice variants (SV) lacking the ligand-binding domain activate ligand-independent transcriptional programs that support castration resistant proliferation and survival. Treatment of the androgen-dependent PTEN null LNCaP (AD-cells), its AI derivative (AI-cells) and wt PTEN RV1 cells with the dual PI3K/mTOR inhibitor BEZ235 (E) combined with the histone deacetylase inhibitor Panobinostat (PAN) induced synergistic growth inhibition and cell death in vitro and in vivo. The effect of these treatments on AR splicing is unknown. METHODS LNCaP AD, AI and RV1 cells were treated with PAN 20nM, E 350nM, rapamycin (Rapa) 20nM and PI3Ki BKM120 (K) 1.5µM for 24h. Total (t) AR, SVs and AR targets mRNA were measured by qtPCR and western. RV1 xenografts received 3 weeks E 35mg/kg/d or PAN 10mg/kg/QOD or PAN+E. RESULTS Baseline levels of tAR mRNA in AI were higher than AD and RV1 cells while SV1, SV7 and SV5-7 were higher in RV1 than AD and AI cells. Treatment with E, Rapa and K increased both tAR and SVs mRNAs and AR and PSA proteins. In contrast, treatment with PAN reduced tAR and SVs levels in AD and AI with no effect on RV1. PAN reduced the induction of tAR by Rapa or K in AD, AI and RV1 but did not abrogate induction of SVs. PAN inhibition was sufficient to abrogate the effect of E and decrease tAR and SVs levels as well as AR protein and AR canonical and non-canonical targets (PSA, TMPRSS2 and UBE2C) and PSA protein below baseline levels in all cells treated with PAN+E. Similarly, RV1 tumors treated with E had a 50% increase in tAR and 20% in SV5-7 but no induction of CDK1 or UBE2C and a 35% reduction in PSA while RV1 tumors treated with PAN+E showed 40% reduction in both SVs 1 and 7 and downstream targets CDK1 and UBE2C. CONCLUSIONS Inhibition of PI3K/mTOR with E increases overall levels of AR transcription that is more pronounced on SVs and uncouples ARs transcriptional activity on canonical downstream targets in vivo. PAN can abrogate the stimulation of E by decreasing both SVs expression and AR transcriptional activity on non-canonical targets. These effects may underlie their synergistic growth inhibition.