Browsing by Author "Hollenhorst, Peter"

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    4296 Targeting ERG Through Toll-Like Receptor 4 in Prostate Cancer
    (Cambridge University Press, 2020-07-29) Greulich, Ben; Plotnik, Josh; Hollenhorst, Peter; Medicine, School of Medicine
    OBJECTIVES/GOALS: The objective of this research was to learn how the oncogenic transcription factor, ERG, is regulated in prostate cancer. If we could learn how ERG is regulated and which genes are important for its oncogenic phenotype in prostate cells, we could design new therapeutic strategies against ERG, which has proven to be difficult to target. METHODS/STUDY POPULATION: We conducted an shRNA screen in prostate cells to determine candidate genes and pathways that are important for ERG function. To validate the findings of the screen, we performed a variety of cell-based functional assays, including trans-well migration, wound healing, and clonogenic survival assays. To further investigate the mechanism between ERG and the genes revealed by the screen, we performed biochemical and molecular biology experiments such as Western blotting and qRT-PCR for protein and mRNA expression, co-immunoprecipitation assays to determine protein-protein interactions, and chromatin immunoprecipitation (ChIP-qPCR) to determine transcription factor binding to DNA sites. RESULTS/ANTICIPATED RESULTS: The screen revealed that genes involved in the toll-like receptor 4 (TLR4) pathway are important for ERG-mediated migration. We tested the effect of a TLR4 inhibitor on ERG function and observed decreased migration and clonogenic survival exclusively in ERG-positive cells. Expression of pMEK and pERG was reduced when TLR4 was inhibited, which suggests a mechanism in which TLR4 upregulates pMEK, leading to the phosphorylation and activation of ERG. This is supported by functional assays in which cells expressing a phosphomimetic ERG are resistant to the TLR4 inhibitor. We demonstrated that ERG drives the transcription of TLR4 and its endogenous ligands HSPA8 and BGN. Therefore, ERG can sensitize the cell to TLR4 activation by increasing the number of receptors as well as providing the ligands needed for stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: This research provides a new therapeutic pathway for treating ERG-positive patients through TLR4 inhibition. This can be beneficial because many patients become resistant to the standard therapy, leaving very few treatment options. TLR4-based therapies could provide an alternative for patients who have developed resistance.
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    Activation of the oncogene ERG by the Ras/ERK and PI3K/AKT pathways
    (2019-08) Willhite, Sydney; Strittmatter, Brady; Hollenhorst, Peter
    Background and Hypothesis: The TMPRSS2-ERG re-arrangement occurs in ~50% of prostate cancers and results in aberrant expression of the transcription factor ERG in the prostate. ERG is known to be activated by the Ras/ERK and PI3K/AKT pathways, however, the exact mechanism of this activation is not fully understood. The aim of this project is to identify how activation of these signaling pathways differentially effect transcription of ERG target genes. Experimental Design or Project Methods: In order to test how the Ras/ERK and PI3K/AKT pathways effect ERG target gene transcription, normal prostate epithelial cells (RWPE1) were transfected with constitutively active AKT in combination with phospho-mutants of ERG. These cell lines were then used to conduct Quantitative Reverse Transcription PCR and Western blotting of known downstream ERG target genes to identify how the activation status of these signaling pathways affected transcription and protein production. Results: Overall, our results demonstrate that ERG mediated transcription of the VIM gene, a marker of EMT, was activated by the Ras/ERK pathway and was repressed by the PI3K/AKT pathway. In addition, we found that ERG expression decreased FOXO1 protein expression in our cell lines regardless of Ras/ERK and PI3K/AKT status. Transcription and protein quantification was also measured for ERG target gene VEGFA, a critical regulator of angiogenesis. Conclusion and Potential Impact: This project helps identify the molecular mechanisms by which a common oncogene in prostate cancer is activated. Our results demonstrate how upstream signaling pathways differentially regulate oncogenic transcription and cell transformation. Overall, this project will provide insight to the molecular mechanisms of possible therapeutic targets in prostate cancer, the most common cancer amongst men.
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    EZH2 modifies sunitinib resistance in renal cell carcinoma by kinome reprogramming
    (Cancer Research, 2017-12-01) Adelaiye-Ogala, Remi; Budka, Justin; Damayanti, Nur P.; Arrington, Justine; Ferris, Mary; Hsu, Chuan-Chih; Chintala, Sreenivasulu; Orillion, Ashley; Miles, Kiersten Marie; Shen, Li; Elbanna, May; Ciamporcero, Eric; Arisa, Sreevani; Pettazzoni, Piergiorgio; Draetta, Giulio F.; Seshadri, Mukund; Hancock, Bradley; Radovich, Milan; Kota, Janaiah; Buck, Michael; Keilhack, Heike; McCarthy, Brian P.; Persohn, Scott A.; Territo, Paul R.; Zang, Yong; Irudayaraj, Joseph; Tao, W. Andy; Hollenhorst, Peter; Pili, Roberto
    Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represent a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft (PDX) model that is intrinsically resistant to the RTKI sunitinib but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its anti-angiogenic and anti-metastatic activity but lost its direct anti-tumor effects due to kinome reprogramming, which resulted in suppression of pro- apoptotic and cell cycle regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTK, restoring the anti-tumor effects of sunitnib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease.