Vidal Fey

High-Throughput Cell-Based Screening of 4910 Known Drugs and Drug-like Small Molecules Identifies Disulfiram as an Inhibitor of Prostate Cancer Cell Growth

Purpose: To identify novel therapeutic opportunities for patients with prostate cancer, we applied high-throughput screening to systematically explore most currently marketed drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells. Experimental Design: We carried out a high-throughput cell-based screening with proliferation as a primary end-point using a library of 4,910 drug-like small molecule compounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant prostate epithelial cell lines (RWPE-1 and EP156T). The EC50 values were determined for each cell type to identify cancer selective compounds. The in vivo effect of disulfiram (DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial studies with copper or zinc were done in vitro for mechanistic exploration. Results: Most of the effective compounds, including antineoplastic agents, were nonselective and found to inhibit both cancer and control cells in equal amounts. In contrast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell proliferation at nanomolar concentrations. DSF reduced tumor growth in vivo, induced metallothionein expression, and reduced DNA replication by downregulating MCM mRNA expression. The effect of DSF was potentiated by copper in vitro. Conclusions: We identified three novel cancer-selective growth inhibitory compounds for human prostate cancer cells among marketed drugs. We then validated DSF as a potential prostate cancer therapeutic agent. These kinds of pharmacologically well-known molecules can be readily translated to in vivo preclinical studies and clinical trials. (Clin Cancer Res 2009;15(19):6070–8)

High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells.

The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional monolayers on plastic. However, many cellular features are impaired in these artificial conditions, and large changes in gene expression compared to tumors have been reported. Three-dimensional cell culture models have become increasingly popular and are suggested to be better models than two-dimensional monolayers due to improved cell-to-cell contact and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput three-dimensional drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in two dimensions, in poly(2-hydroxyethyl methacrylate) induced anchorage-independent three-dimensional models, and in Matrigel three-dimensional cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating, or they were allowed to grow in three-dimensional cultures for 4 days before the drug treatment. Large variations in drug responses were observed between the models indicating that comparisons of culture model-influenced drug sensitivities cannot be made based on the effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in two-dimensional cultures, while the responses of cells grown in poly(2-hydroxyethyl methacrylate) resembled those of the two-dimensional cultures. Furthermore, comparing the gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study, we also suggest that three-dimensional cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives to traditional two-dimensional screens for better comparability to the in vivo state.

Arachidonic Acid Pathway Members PLA2G7, HPGD, EPHX2, and CYP4F8 Identified as Putative Novel Therapeutic Targets in Prostate Cancer

The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.

Identification of MicroRNAs Inhibiting TGF-β-Induced IL-11 Production in Bone Metastatic Breast Cancer Cells

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3′ UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

Monensin Is a Potent Inducer of Oxidative Stress and Inhibitor of Androgen Signaling Leading to Apoptosis in Prostate Cancer Cells

Current treatment options for advanced and hormone refractory prostate cancer are limited and responses to commonly used androgen pathway inhibitors are often unsatisfactory. Our recent results indicated that sodium ionophore monensin is one of the most potent and cancer-specific inhibitors in a systematic sensitivity testing of most known drugs and drug-like molecules in a panel of prostate cancer cell models. Because monensin has been extensively used in veterinary applications to build muscle mass in cattle, the link to prostate cancer and androgen signaling was particularly interesting. Here, we showed that monensin effects at nanomolar concentrations are linked to induction of apoptosis and potent reduction of androgen receptor mRNA and protein in prostate cancer cells. Monensin also elevated intracellular oxidative stress in prostate cancer cells as evidenced by increased generation of intracellular reactive oxygen species and by induction of a transcriptional profile characteristic of an oxidative stress response. Importantly, the antiproliferative effects of monensin were potentiated by combinatorial treatment with the antiandrogens and antagonized by antioxidant vitamin C. Taken together, our results suggest monensin as a potential well-tolerated, in vivo compatible drug with strong proapoptotic effects in prostate cancer cells, and synergistic effects with antiandrogens. Moreover, our data suggest a general strategy by which the effects of antiandrogens could be enhanced by combinatorial administration with agents that increase oxidative stress in prostate cancer cells. Mol Cancer Ther; 9(12); 3175–85. ©2010 AACR.

A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. (Journal of Biomolecular Screening 2009:936-943)

High-Throughput Transcriptomic and RNAi Analysis Identifies AIM1, ERGIC1, TMED3 and TPX2 as Potential Drug Targets in Prostate Cancer

Prostate cancer is a heterogeneous group of diseases and there is a need for more efficient and targeted methods of treatment. In this study, the potential of gene expression data and RNA interference technique were combined to advance future personalized prostate cancer therapeutics. To distinguish the most promising in vivo prevalidated prostate cancer drug targets, a bioinformatic analysis was carried out using genome-wide gene expression data from 9873 human tissue samples. In total, 295 genes were selected for further functional studies in cultured prostate cancer cells due to their high mRNA expression in prostate, prostate cancer or in metastatic prostate cancer samples. Second, RNAi based cell viability assay was performed in VCaP and LNCaP prostate cancer cells. Based on the siRNA results, gene expression patterns in human tissues and novelty, endoplasmic reticulum function associated targets AIM1, ERGIC1 and TMED3, as well as mitosis regulating TPX2 were selected for further validation. AIM1, ERGIC1, and TPX2 were shown to be highly expressed especially in prostate cancer tissues, and high mRNA expression of ERGIC1 and TMED3 associated with AR and ERG oncogene expression. ERGIC1 silencing specifically regulated the proliferation of ERG oncogene positive prostate cancer cells and inhibited ERG mRNA expression in these cells, indicating that it is a potent drug target in ERG positive subgroup of prostate cancers. TPX2 expression associated with PSA failure and TPX2 silencing reduced PSA expression, indicating that TPX2 regulates androgen receptor mediated signaling. In conclusion, the combinatorial usage of microarray and RNAi techniques yielded in a large number of potential novel biomarkers and therapeutic targets, for future development of targeted and personalized approaches for prostate cancer management.

Integrative omics reveals MYCN as a global suppressor of cellular signalling and enables network-based therapeutic target discovery in neuroblastoma

Despite intensive study, many mysteries remain about the MYCN oncogenes functions. Here we focus on MYCNs role in neuroblastoma, the most common extracranial childhood cancer. MYCN gene amplification occurs in 20% of cases, but other recurrent somatic mutations are rare. This scarcity of tractable targets has hampered efforts to develop new therapeutic options. We employed a multi-level omics approach to examine MYCN functioning and identify novel therapeutic targets for this largely un-druggable oncogene. We used systems medicine based computational network reconstruction and analysis to integrate a range of omic techniques: sequencing-based transcriptomics, genome-wide chromatin immunoprecipitation, siRNA screening and interaction proteomics, revealing that MYCN controls highly connected networks, with MYCN primarily supressing the activity of network components. MYCNs oncogenic functions are likely independent of its classical heterodimerisation partner, MAX. In particular, MYCN controls its own protein interaction network by transcriptionally regulating its binding partners. Our network-based approach identified vulnerable therapeutically targetable nodes that function as critical regulators or effectors of MYCN in neuroblastoma. These were validated by siRNA knockdown screens, functional studies and patient data. We identified β-estradiol and MAPK/ERK as having functional cross-talk with MYCN and being novel targetable vulnerabilities of MYCN-amplified neuroblastoma. These results reveal surprising differences between the functioning of endogenous, overexpressed and amplified MYCN, and rationalise how different MYCN dosages can orchestrate cell fate decisions and cancerous outcomes. Importantly, this work describes a systems-level approach to systematically uncovering network based vulnerabilities and therapeutic targets for multifactorial diseases by integrating disparate omic data types.

Wnt signalling is a bi-directional vulnerability of cancer cells

Wnt signalling is involved in the formation, metastasis and relapse of a wide array of cancers. However, there is ongoing debate as to whether activation or inhibition of the pathway holds the most promise as a therapeutic treatment for cancer, with conflicting evidence from a variety of tumour types. We show that Wnt/β-catenin signalling is a bi-directional vulnerability of neuroblastoma, malignant melanoma and colorectal cancer, with hyper-activation or repression of the pathway both representing a promising therapeutic strategy, even within the same cancer type. Hyper-activation directs cancer cells to undergo apoptosis, even in cells oncogenically driven by β-catenin. Wnt inhibition blocks proliferation of cancer cells and promotes neuroblastoma differentiation. Wnt and retinoic acid co-treatments synergise, representing a promising combination treatment for MYCN-amplified neuroblastoma. Additionally, we report novel cross-talks between MYCN and β-catenin signalling, which repress normal β-catenin mediated transcriptional regulation. A β-catenin target gene signature could predict patient outcome, as could the expression level of its DNA binding partners, the TCF/LEFs. This β-catenin signature provides a tool to identify neuroblastoma patients likely to benefit from Wnt-directed therapy. Taken together, we show that Wnt/β-catenin signalling is a bi-directional vulnerability of a number of cancer entities, and potentially a more broadly conserved feature of malignant cells.

Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.