Zundel:Research
Research Interests & Ongoing Projects
Project 1) Mapping the Oxygen Sensing and Response Network. (aka the Hypoxeome/Ischemeome). In this project, we use various computational methods to examine thousands of hypoxic & ischemic gene expressions from normal tissue, cancers & various non-cancerous ischemic pathophysiologies. Using this approach, we seek to determine how normal tissue responds to hypoxia & ischemia (H/I) & how these normal responses are altered during tumorigenesis vs other non-cancerous pathophysiologies that also undergo hypoxic or ischemic situations. This approach thus suggests how tumors alter their normal responses to a critical pathophysiologic stress in ways that facilitate the tumorigenic processes. These studies also suggest various regulatory pathways & processes that are critical for cell function in response to H/I. This has allowed us to begin to map these pathways using high-throughput yeast-2 hybrid (HT-Y2H) & proteomic a0pproaches. Serendipitously, functional genomic approaches examining genes critical for either H/I survival or key tumorigenic H/I-regulated functions (invasiveness, genomic instability, metastasis, metabolism,… etc.) has frequently revealed genes regulated by H/I gene expression. Thus, a map of normal & cancerous H/I signaling/regulatory/functional networks is emerging with key nodes critical for specific functions identified. Examination of these nodes using proteomics reveals how the protein complexes are dynamically regulated (including novel post-translational modifications) & further identifies the key enzymes regulating these processes. Using this powerful mapping strategy, we are identifying critical proteins lying in non-redundant pathways that cannot be bypassed & have key “druggable” aspects. Once the targets are identified, we test the consequences of that protein’s loss or inhibition (i.e. knockdown, dominant negative) on the predicted aspect of tumorigenic processes. Then, following confirmation as a putative therapeutic target, we collaborate with notable structural biology, pharmacology & computational biology labs to use structure-based drug design (SBDD) to identify inhibitors of the protein in question. This begins the long process of drug validation moving eventually to clinical trials. Since the inception of my lab over the past 8 years, this huge project has generated the leads to all of my currently funded projects & is perhaps the most exciting project in which everyone in the lab contributes to, providing a common theme for all lab members.
Project 2) A novel functionality associated with pVHL. We have isolated an oxygen-labile ribonuclease inhibitor (pRI) and are in the process of characterizing it function. Interestingly, early studies of pVHL ascribed mRNA stabilization function to this tumor suppressor. We have found that the pVHL interaction controls the expression of greater than 400 genes, many of which are dependent on it’s interaction with pRI with 82% of gene expression that is regulated by pRI also being regulated by pVHL. Interestingly, a similar pattern is found under hypoxia, where pRI dissociates from pVHL. We have also found that pRI is a target for mono-ubiquitylation, leading to pRI subcellular localization and sequestration from the ribonuclease, angiogenin. This project will further investigate the regulation of pRI by pVHL, the role of pRI in pVHL-mediated vasculogenesis & ribosomal biogenesis. This is a significant finding as it mechanistically links a completely novel protein interaction & oncogenic functions to an important tumor suppressor.
Project 3) A mechanism controlling hypoxia- and reperfusion- induced G2/M arrest. We have recently identified a hypoxia/reperfusion responsive RING-H2 protein that forms a functional ubiquitin E3-ligase (UbE3) with the IAP, Survivin. This project will test the hypothesis that this UbE3 controls an oxygen-sensitive assembly of the Chromosomal Passenger Complex (CPC), acts as a Spindle checkpoint control in response to H/I, & if, when deregulated, can affect H/I mediated chromosomal/genomic instability &/or radiation sensitivity. We are also developing small molecule antagonists of the RING-H2 protein as radiation and chemo-sensitizers as antagonism of the RING-H2 protein leads to Spindle (G2/M) arrest which is the most damage-sensitive phase of the cell cycle.
Project 4) The role of secreted factors regulating renal cancer development. We have found that amongst the milieu of secreted factors controlled by pVHL, two factors are required for tumor growth and vasculogenesis, IL8 and angiogenin. IL8 is regulated by VHL in a RI independent fashion whereas, angiogenin is a direct target of RI. When examined in conjunction, high levels of IL8 and angiogenin predicted renal cancer in a stage-dependent manner in mouse models of renal cancer and simultaneous inhibition of both factors (but not each singly) results in significant inhibition of renal cancer xenograft growth. Current research will evaluate these factors further as biomarkers of renal cancer stage/grade and metastasis. As FDA-approved inhibitory antibodies to IL-8 and small molecule inhibitors of angiogenin are available, I will begin a clinical trial in metastatic renal cancer as soon as possible.
Project 5) H/I Phosphoproteome. In an effort to further understand early events in signaling during acute periods of H/I, we have conducted a phosphoproteomics screen in normal bronchial epithelial cells (NBEC) & cells from non-small cell lung cancer (NSCLC) having a diverse mutational spectrum. This screen, while in the early stages, has indicated that the H/I-induced phosphoproteome is altered in tumor cell lines vs NBEC potentially indicating altered kinase activity that impacts H/I mediated functions. This project will investigate what kinase are involved, their targets & whether acute H/I during tumorigenesis selects for altered kinase activity & how this impacts tumorigenesis in NSCLC.
Project 6) A mechanism controlling Cullin-dependent ubiquitin E3 ligases (Cul-UbE3) with particular focus on the COP9 Signalosome (CSN). We have found that the CSN plays a critical role in differentially regulating Cul-UbE3s. This is particularly important in tumor development as Cul-UbE3s regulate the degradation of multiple oncogenes and tumor suppressors including p27, p21, Cyclin B1, Cyclin D1, Cyclin E, Cdc25A, Wee1, c-myc, p53, HIF-1/2α, c-myb, E2F1, IkBa, NF-kB/p105, NF-kB/p100, SMAD4, b-catenin, IRS-1, IRS-2, Nrf2, CI, SMAD3, SMAD4, Notch1, Notch4,Vav, RhoBTB2, DDB2, CSA, Cdt1,...etc). Not surprisingly, the catalytic component of the CSN, CSN5, is one of the most highly amplified regions in several cancers, particularly breast cancer and is necessary and sufficient for the angiogenic phenotype in breast cancer. We are currently in the process of characterizing CSN regulation within various contexts and in a collaboration with the Trent lab have recently developed small molecule CSN antagonists that elicit specific tumor suppressive effects. These exciting studies have produced several lead compounds and are currently in phase 0 studies. This project has multiple potential focuses: A) Testing lead compounds for CSN5 inhibition & tumor suppression. B) Develop a novel screen using CSN5 &/or cullin mutants to globally identify the targets of Cul-UbE3s, many of which may be novel oncogenes or tumor suppressors. C) Further investigate the biology of the neddylation cycle.
Project 7) Tumor H/I Biomarker Validation. During the course of Project 1, many potential biomarkers specific to cancer, to normal tissue & to non-tumorigeneic H/I-related pathophysiologies have been identified. This project seeks to validate these biomarkers prior to their potential use as: A) Biomarkers of disease state B) Hypoxic-specific biomarkers C) Biomarkers for therapeutic targeting (i.e. nanotech) D) Biomarkers for unique imaging strategies
