Faculty Bibliography

Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear beta-catenin activity (called C3) can inhibit both the AR and beta-catenin-signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both beta-catenin/T-cell factor and beta-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on beta-catenin. Given that AR interacts with, and is transcriptionally regulated by beta-catenin, C3 treatment also resulted in decreased occupancy of beta-catenin on the AR promoter and diminished AR and AR/beta-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and beta-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of beta-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach.

MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila beta-catenin) expression and activity by directly targeting the 3'-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/beta-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of beta-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that beta-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of beta-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/beta-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that beta-catenin, but not its transcriptional activity, is central to pluripotency acting through a beta-catenin/Oct4 complex.

Signaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and beta-catenin (beta-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.

The Wnt signaling pathway is implicated in major physiologic cellular functions, such as proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Proliferation and migration are important responses of T-cells, which are major cellular targets of HIV infection. Using an informatics screen, we identified a previously unsuspected interaction between HIV's Nef protein and beta-catenin, a key component of the Wnt pathway. A segment in Nef contains identical amino acids at key positions and structurally mimics the beta-catenin binding sites on endogenous beta-catenin ligands. The interaction between Nef and beta-catenin was confirmed in vitro and in a co-immunoprecipitation from HEK293 cells. Moreover, the introduction of Nef into HEK293 cells specifically inhibited a Wnt pathway reporter.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

BACKGROUND: Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is mediated through the transcriptional effector beta-catenin (beta-cat) is commonly de-regulated in many cancers. Cells with active beta-cat-regulated transcription (CRT) are protected against apoptosis; conversely, inhibition of CRT may prevent cell proliferation. MATERIALS AND METHODS: In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-beta-cat response in MM cells MM.1, U266, BMSC and primary BMMC obtained from patient samples (n=16). RESULTS: We demonstrated that iCRTs we used, block Wnt/beta-cat reporter activity, down regulate beta-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 muM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target beta-cat in MM cells. Additionally, iCRT-treatment of MM cells, co-cultured with BMSC, showed an inhibitory effect on VEGF and cell migration. CONCLUSION: This study provides the first in vitro data evaluation of newly-described iCRTs as potential Wnt-beta-cat/VEGF pathway antagonists in multiple myeloma.

The recent development of high-throughput sequencing technologies and the availability of whole genome sequences of a variety of living organisms, including that of humans, have led to an enormous push in the quest for a comprehensive inquiry for the function of each and every gene discovered in different model organisms. A major conclusion from the sequencing projects was that while forward genetics had been extremely successful in identifying key genes/components of many biological processes, such as signal transduction cascades, the function(s) of the majority of genes in the genome remains a mystery. In this article, we discuss the use of a variety of high-throughput postgenomic tools, including functional genomics, proteomics, and chemical genetics that are being implemented in an exhaustive molecular dissection of a key evolutionarily conserved signal transduction pathway, namely the Wnt/wingless (wg) pathway and its associated signaling network. WIREs Syst Biol Med 2011 3 649-665 DOI: 10.1002/wsbm.140 For further resources related to this article, please visit the WIREs website