Jane E. Ishmael - US grants

DESCRIPTION (Adapted from the Candidate's Abstract) Human exposure to peroxisome proliferators is widespread and may occur in a variety of environmental, occupational and clinical settings. Although peroxisome proliferators are rodent carcinogens the hazard that they pose to humans is uncertain as the molecular mechanism underlying their effects is unknown. The biological effects of peroxisome proliferators appear to be mediated by a direct interaction with peroxisome proliferator activated receptor alpha (PPAR-alpha), which is a member of the steroid/thyroid hormone receptor superfamily of ligand-dependent transcription factors. Mice that are devoid of this receptor fail to show typical hepatocytic alterations in response to treatment with peroxisome proliferators, such as peroxisome proliferation, hepatomegaly, increased DNA synthesis, increased expression of PPAR-alpha regulated genes and hepatocarcinogenesis. The overall objectives of this proposal are to characterize a novel PPAR-alpha interacting protein (clone D7) and to determine the role of nuclear receptor coactivators in PPAR- alpha-mediated disruption of cell cycle. It is currently believed that regulated transcription factors (such as PPAR-alpha and the tumor suppressor protein p53) utilize a common group of coactivator proteins which may be expressed in limited amounts in the cell. Thus, it is possible that chronic activation of PPAR-alpha by peroxisome proliferators could sequester coactivators at a multiplicity of PPAR-alpha target gene promoters and thereby interfere with p53 signaling through p300. The specific hypothesis to be tested is that nuclear receptor-associated coactivator proteins (such as p300) play an integral and essential role in the basic mechanism underlying peroxisome proliferator-induced carcinogenesis by exerting effects on specific stages of the cell cycle. Toward this goal the specific aims of the proposal are to: (1) characterize the interaction of PPAR-alpha with a novel transcriptional coactivator protein (clone D7) in the presence and absence of peroxisome proliferators and to define the mechanism by which the protein encoded by D7 may function in the PPAR-alpha signaling pathway; (2) establish that peroxisome proliferators influence the rate of cell replication by exerting specific effects on stages of the cell cycle using Hepa cells as a model, and (3) test the hypothesis that PPAR-alpha-interacting proteins (such as p300 and clone D7) play a critical role in mediating cell cycle changes that occur in response to peroxisome proliferators. In general it is hoped that these studies will increase our understanding of the relationship between cell proliferation and hepatocarcinogenesis induced by nongenotoxic carcinogens.

Loss of homeostasis in the cellular secretory pathway is implicated in major human diseases such as cancer, diabetes and inflammation. Secreted and cell-wall proteins are also critical for host infection by human pathogenic bacteria and fungi, and secreted proteases play a role in biofilm formation. Macrocyclic natural products (NPs) HUN-7293 (from fungi) and the apratoxins (from cyanobacteria), as well as the cotransin synthetic analogs of HUN-7293, are reported to block cotranslational protein translocation at an early stage of the secretory pathway with varying degrees of selectivity, but exactly how they influence protein biogenesis is unknown. Our discovery of a mechanistically distinct inhibitor of protein translocation, coibamide A (CbA, from cyanobacteria), has led to the observation that HUN-7293, apratoxins and CbA share a common cellular target, the Sec61 protein channel (translocon), yet inhibit the biogenesis and secretion of different proteins. This presents the opportunity to discover and use new NPs to elucidate the cellular secretory pathway as a therapeutic target, and to provide a reservoir of potential drug leads to reinforce the dwindling pharmaceutical pipelines of new chemical entities. We plan to utilize a multidisciplinary approach involving natural products and synthetic chemistry, chemical biology, pharmacology and evolutionary genomics to pursue the following three aims: 1) Expand and define the class of NPs that target proteostasis; 2) Elucidate the specific Sec61 binding site and inhibitory mechanism of CbA and two active synthetic analogs; 3) Utilize a genomics workflow for evolution- based prediction of new fungal NPs? function. In Aim 1, existing NP libraries likely to be rich in non-polar depsipeptides will be screened for new proteostasis modulators using a primary functional screen in U87MG cells engineered to express Gaussia luciferase (Gluc) and a secondary target-based assay for Sec61-dependent inhibition of ER translocation. Preliminary data give a hit rate of 0.3% for the Gluc secretion assay. In Aim 2, chemogenetic screening approaches will be used to determine how Sec61 function is perturbed by CbA, followed by biochemistry and structural biology to resolve the mechanistic basis for ER translocation inhibition. The comparative selectivity profile of two new CbA analogs, and prioritized new NPs from aim 1, relative to CbA- ApxA and cotransin, will be determined in cell-free and cell-based assays. In Aim 3, the genomic diversification of NPs will be investigated in an evolutionary context using phylogeny and ecology of fungi. For example, NRPS (Adenylation) A-domain phylogenies will reveal evolutionary relationships of biosynthetic gene clusters (BGCs), and will be used to predict structure, function in human cells, and correlation between ecology and NP diversity. This multidimensional approach will reveal the feasibility of targeting cellular proteostasis for therapeutic needs, while avoiding toxicities due to non-specific inhibition of secretory protein biosynthesis. It is also to expected to provide evolutionary and ecological rationale for targeting fungal producers of protein secretion inhibitors.

Abstract Glioblastoma multiforme (GBM) remains refractory to current standard-of-care treatment and is associated with a bleak prognosis and poor quality of life in the final months. Endoplasmic reticulum (ER) stress, and consequently a constitutive activation of the unfolded protein response (UPR), is a common feature of GBM and has been linked to increased aggressiveness and therapeutic resistance. The Sec61 translocon is a protein-conducing channel which spans the ER membrane and is essential for cotranslocational translocation of client proteins. Sec61 subunits are also upregulated by ER stress in GBM and the gene encoding the gamma subunit (SEC61G) is considered a GBM proto-oncogene. We now present evidence that Sec61 is directly implicated in ER stress/UPR signaling in GBM cancer stem cells (GSCs). Overall, our results suggest that pharmacological inactivation of Sec61 results in depletion of BiP mRNA levels and prevents pro- survival UPR signaling particularly through the UPR sensor inositol-requiring enzyme 1 (IRE1). Our central hypothesis is that Sec61 translocon is an essential regulator of UPR signaling and proteostasis in GBM. We will test this hypothesis through the following specific aims: 1) Define the role of Sec61 in UPR signaling in GBM. 2) Evaluate the therapeutic potential of pharmacological targeting Sec61 in orthotopic GSCs mouse models. This multidimensional approach will reveal the tumor-supportive properties of Sec61 translocon in GBM and GSCs and explore the feasibility of safely targeting Sec61 for therapeutic advantage while avoiding toxicities due to non-specific inhibition of secretory protein biosynthesis. This early stage pre-clinical research is expected to inform the future advancement of newly designed synthetic Sec61 inhibitors for the treatment of GBM and other aggressive human cancers characterized by high levels of adaptive ER stress.