African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP: rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs. 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that Tp53 allele-specific gene expression may contribute to African American cancer disparities, TP53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild type for TP53 and heterozygous at the TP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of Tp53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for Tp53 allele-specific escape from Tp53-induced arrest was performed. Several novel RNAi suppressors of Tp53 were identified, one of which, PRDM1? (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1? silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing.
COPI, a coatomer protein complex of secretory vesicles, is involved in Golgi and endoplasmic reticulum traffic and in early endosome maturation. The loss of COPI results in the fragmentation of Golgi, accumulation of immature autophagosomes, inhibition of autophagy, and cell death. Since COPI is required by all cells, it would appear an unlikely target for cancer treatment. However, our recent function-based genomic screen unexpectedly identified a specific COPI subunit, ?1, as a cancer-specific target. The existing cancer drugs kill only proliferating but not growth-arrested tumor cells, but the depletion of ?1 induces cell death in both dividing and nondividing tumor cells, while sparing normal cells. The mechanism of this remarkable tumor selectivity turned out to be surprising and heretofore unprecedented.
Acquired intratumoral steroidogenesis is involved in progression of prostate cancer to castration resistant disease (CRPC) and a target for improved therapeutics. Recent work has shown that prostate cancer cells can acquire steroidogenic activity as they progress to a therapeutic-resistant state. However, benign prostate stromal cells (PrSCs) also have steroidogenic potential though they are often overlooked as a source of intratumoral androgens. Here, we present preliminary studies showing that the steroidogenic activity of primary human PrSCs is significantly increased by exposure to a Hedgehog agonist (SAG) or by transduction of PrSCs with lentiviruses that expresses active Gli2 (Gli2?N), a transcription factor that is triggered by Hh signaling. Comparative gene expression profiling on Chips, that was confirmed by quantitative real-time PCR, revealed that hedgehog agonist treatment induced in these cells expressions of hedgehog target genes (Gli1, Ptch1, and SCUBE1) plus a specific cadre of genes involved in cholesterol/steroid biosynthesis, metabolism, and transport. Genes involved downstream in steroid hormone generation, including CYP17A1 and CYP19A1 were also induced. Both the hedgehog agonist and the Gli2-expressing lentivirus significantly increased the output of testosterone (T) from PrSCs that were supplemented with dihydroepiandrosterone (DHEA), an adrenal precursor of T. Finally, knockdown of Gli2 by siRNA suppressed the ability of SAG to induce this response. Collectively, our data indicate that hedgehog/Gli signaling may be a factor in acquired intratumoral steroidogenesis of a prostate tumor through its actions on stromal cells in the tumor microenvironment and an influence for the development of CRPC.
Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ?1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ?2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.
The androgen receptor (AR) is expressed in a subset of prostate stromal cells and functional stromal cell AR is required for normal prostate developmental and influences the growth of prostate tumors. Although we are broadly aware of the specifics of the genomic actions of AR in prostate cancer cells, relatively little is known regarding the gene targets of functional AR in prostate stromal cells. Here, we describe a novel human prostate stromal cell model that enabled us to study the effects of AR on gene expression in these cells. The model involves a genetically manipulated variant of immortalized human WPMY-1 prostate stromal cells that overexpresses wildtype AR (WPMY-AR) at a level comparable to LNCaP cells and is responsive to dihydrotestosterone (DHT) stimulation. Use of WPMY-AR cells for gene expression profiling showed that the presence of AR, even in the absence of DHT, significantly altered the gene expression pattern of the cells compared to control (WPMY-Vec) cells. Treatment of WPMY-AR cells, but not WPMY-Vec control cells, with DHT resulted in further changes that affected the expression of 141 genes by 2-fold or greater compared to vehicle treated WPMY-AR cells. Remarkably, DHT significantly downregulated more genes than were upregulated but many of these changes reversed the initial effects of AR overexpression alone on individual genes. The genes most highly effected by DHT treatment were categorized based upon their role in cancer pathways or in cell signaling pathways (transforming growth factor-?, Wnt, Hedgehog and MAP Kinase) thought to be involved in stromal-epithelial crosstalk during prostate or prostate cancer development. DHT treatment of WPMY-AR cells was also sufficient to alter their paracrine potential for prostate cancer cells as conditioned medium from DHT-treated WPMY-AR significantly increased growth of LNCaP cells compared to DHT-treated WPMY-Vec cell conditioned medium.
Bats in the northeastern U.S. are affected by geomycosis caused by the fungus Geomyces destructans (Gd). This infection is commonly referred to as White Nose Syndrome (WNS). Over a million hibernating bats have died since the fungus was first discovered in 2006 in a cave near Albany, New York. A population viability analysis conducted on little brown bats (Myotis lucifugus), one of six bat species infected with Gd, suggests regional extinction of this species within 20 years. The fungus Gd is a psychrophile ("cold loving"), but nothing is known about how it thrives at low temperatures and what pathogenic attributes allow it to infect bats. This study aimed to determine if currently available antifungal drugs and biocides are effective against Gd. We tested five Gd strains for their susceptibility to antifungal drugs and high-throughput screened (HTS) one representative strain with SpectrumPlus compound library containing 1,920 compounds. The results indicated that Gd is susceptible to a number of antifungal drugs at concentrations similar to the susceptibility range of human pathogenic fungi. Strains of Gd were susceptible to amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. In contrast, very high MICs (minimum inhibitory concentrations) of flucytosine and echinocandins were needed for growth inhibition, which were suggestive of fungal resistance to these drugs. Of the 1,920 compounds in the library, a few caused 50%--to greater than 90% inhibition of Gd growth. A number of azole antifungals, a fungicide, and some biocides caused prominent growth inhibition. Our results could provide a theoretical basis for future strategies aimed at the rehabilitation of most affected bat species and for decontamination of Gd in the cave environment.
As a general strategy for function-based gene identification, an shRNA library containing approximately 150 shRNAs per gene was enzymatically generated from normalized (reduced-redundance) human cDNA. The library was constructed in an inducible lentiviral vector, enabling propagation of growth-inhibiting shRNAs and controlled activity measurements. RNAi activities were measured for 101 shRNA clones representing 100 human genes and for 201 shRNAs derived from a firefly luciferase gene. Structure-activity analysis of these two datasets yielded a set of structural criteria for shRNA efficacy, increasing the frequencies of active shRNAs up to 5-fold relative to random sampling. The same library was used to select shRNAs that inhibit breast carcinoma cell growth by targeting potential oncogenes. Genes targeted by the selected shRNAs were enriched for 10 pathways, 9 of which have been previously associated with various cancers, cell cycle progression, or apoptosis. One hundred nineteen genes, enriched through this selection and represented by two to six shRNAs each, were identified as potential cancer drug targets. Short interfering RNAs against 19 of 22 tested genes in this group inhibited cell growth, validating the efficiency of this strategy for high-throughput target gene identification.
Castration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.
Inhibition of mTOR by rapamycin prevents cellular senescence. Here we investigated the effects of MEK and PI-3K on cellular senescence. Unlike LY294002 (PI-3K inhibitor), both U0126 and PD98059 (MEK inhibitors) did not significantly decrease beta-Gal staining in aging human fibroblasts and fibrosarcoma cells. However, using a sensitive, functional method, we identified that not only LY294002 but also U0126 prevented irreversible loss of proliferative potential associated with cellular senescence. At concentrations that blocked S6 phosphorylation, rapamycin, U0126 and LY294002 equally prevented senescence. Furthermore, there was no additive effect by combining of rapamycin with either U0126 or LY294002. Taken together this suggests that (a) simultaneous activation of PI-3K and MEK is required to ensure cellular senescence and (b) U0126 and LY294002 suppress senescence via the rapamycin-sensitive pathway.
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