PPARγ and Agonists against Cancer: Rational Design of Complementation Treatments

PPARγ is a member of the ligand-activated nuclear receptor superfamily: its ligands act as insulin sensitizers and some are approved for the treatment of metabolic disorders in humans. PPARγ has pleiotropic effects on survival and proliferation of multiple cell types, including cancer cells, and is now subject of intensive preclinical cancer research. Studies of the recent decade highlighted PPARγ role as a potential modulator of angiogenesis in vitro and in vivo. These observations provide an additional facet to the PPARγ image as potential anticancer drug. Currently PPARγ is regarded as an important target for the therapies against angiogenesis-dependent pathological states including cancer and vascular complications of diabetes. Some of the studies, however, identify pro-angiogenic and tumor-promoting effects of PPARγ and its ligands pointing out the need for further studies. Below, we summarize current knowledge of PPARγ regulatory mechanisms and molecular targets, and discuss ways to maximize the beneficial activity of the PPARγ agonists

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc

Androgen Receptor Drives Cellular Senescence

The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence in vitro and in vivo. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor

miR-200b Inhibits Prostate Cancer EMT, Growth and Metastasis

<div><p>miRNA regulate gene expression at post-transcriptional level and fine-tune the key biological processes, including cancer progression. Here, we demonstrate the involvement of miR-200b in the metastatic spread of prostate cancer. We identified miR-200b as a downstream target of androgen receptor and linked its expression to decreased tumorigenicity and metastatic capacity of the prostate cancer cells. Overexpression of miR-200b in PC-3 cells significantly inhibited their proliferation and the formation of subcutaneous tumors. Moreover, in an orthotopic model, miR-200b blocked spontaneous metastasis and angiogenesis by PC-3 cells. This decreased metastatic potential was likely due to the reversal of the epithelial-to-mesenchymal transition, as was evidenced by increased pan-epithelial marker E-cadherin and specific markers of prostate epithelium, cytokeratins 8 and 18. In contrast, mesenchymal markers, fibronectin and vimentin, were significantly downregulated by miR-200b. Our results suggest an important role for miR-200b in prostate cancer progression and indicate its potential utility for prostate cancer therapy.</p></div

miRNA increased with AR expression and inhibitory effect in cancers with increased expression (p<0.05).

<p>miRNA increased with AR expression and inhibitory effect in cancers with increased expression (p<0.05).</p

Validation of select miRNA.

<p>(A). MiRNA expression was normalized to that of control cells (ctrl). PC3-AR and control cells were treated 5 days with doxycycline to induce AR expression and with R1881 to induce AR activation and nuclear translocation and total RNA collected for analysis. The comparison is to untreated control and RNU1A_1 non-coding RNA is used as an internal control. The statistical significance of observed differences compared to control is * p≤0.05, and **p≤0.01 as was determined by one-tailed Student’s T-test. The average values are calculated for two independent experiments performed in triplicate. (B) AR activation upregulates miR-200b. PC3-AR cells (grey bars) were treated 5 days with both doxycycline to induce AR expression and with R1881 to induce AR activation and nuclear translocation. The comparison is to untreated control. Flutamide was added where indicated to block AR activity. Control (ctrl) PC3-AR cells were left untreated. RNU1A_1 non-coding RNA was used as an internal control. *, p<0.05; **, p<0.01 as determined by Students T-test. The average values were calculated for two independent experiments performed in triplicate.</p

miRNA decreased with AR expression and growth-promoting effect in cancers with increased expression (p<0.05).

<p>miRNA decreased with AR expression and growth-promoting effect in cancers with increased expression (p<0.05).</p

miR-200b reverses EMT and decreases invasion and metastasis by PC3 cells.

<p>(A) Markers affected by miR-200b in PC-3 cells. E-cadherin, Fibronectin, and Vimentin were detected in whole cell lysates from PC3 ctrl and PC3 miR-200b cells by western blot. (B) Quantitative analysis of the experiment shown in (A) performed with Image J software. *, p<0.05 and, **, p≤0.01 as determined by Student’s T-test. Two independent experiments were pooled together. (C) Western blot for ZEB1 and quantification performed as above (the average of two experiments is shown). (D) End-point PCR for ZEB1. (E) <i>In vitro</i> transwell invasion assay: the comparison of PC3-ctrl and PC3 miR-200b cells. 10% FBS was used as chemoattractant and the experiment was performed in duplicate. *, p<0.05 by Student’s T-test. (F) The <i>in vivo</i> spontaneous metastases by PC3-ctrl and PC3 miR-200b cells. The cells were implanted orthotopically in the ventral prostates of male nude mice. The mice were subjected to whole body imaging for RFP-positive masses using Surface image. At the end of experiment, the animals were sacrificed, peritoneal cavity opened and metastasis visualized by fluorescence imaging after the removal of a primary tumor inside the peritoneal cavity and on the frontal wall of the abdomen (Peritoneal cavity). Bright field (BF) and fluorescence (RFP, red fluorescent protein) are shown. (G) Quantification of the experiment shown in (F). The metastases were counted and total fluorescence per metastasis calculated (left). Gross metastatic burden was estimated as total fluorescence per mouse. Ctrl indicates control and 200b indicates miR-200b. **, p≤0.01 by Student’s T-test.</p