E2F Family Transcription Factors are Directly Regulated by TRIM28 to Promote Castration-Resistance in Prostate Cancer

E2F family transcription factors are directly regulated by TRIM28 to promote castration-resistance in prostate cancer. Specific Aim Prostate cancer (PCa) is the second-leading cause of cancer-related death in American men. Androgen deprivation therapies that target the androgen receptor (AR) are the mainstay treatment for metastatic PCa. New generation anti-androgen enzalutamide (Enz) significantly increases PCa patient survival. However resistance to Enz develops rapidly. Importantly, castration-resistant prostate cancer (CRPC) is driven primarily by aberrant activation of the AR in the milieu of low androgen. The AR exerts its tumorigenic roles mainly through genomic regulation of target gene expression. This genomic action is tightly regulated by a number of cofactors, and one of which is TRIM24. We recently reported that TRIM28 is an upstream regulator of TRIM24 protein stability in CRPC, where both TRIM24 and TRIM28 expression is highly elevated. Mechanistically, TRIM28 stabilizes TRIM24 by preventing TRIM24 from SPOP-driven ubiquitination and degradation, and thereby TRIM24 is able to reactivate AR signaling in CRPC. To explore the TRIM28-mediated downstream signaling, genome-wide transcriptome analysis revealed that TRIM28 engaged in various oncogenic pathway including cell cycle and DNA replication. By interrogating cell cycle signature genes, we showed that TRIM28 induces E2F1, E2F2 and E2F3 gene expression in PCa. In addition, TRIM28 ChIP-seq revealed that TRIM28 directly binds to promoter region of transcription factor E2F1-E2F3, which play a pivotal role in activating transcription of the genes required for cell cycle progression. Further, deregulated expression or activity of the E2F family has often been detected in many human cancers, which leads to uncontrolled cell proliferation. In this proposal, we hypothesize that E2F transcription factor is a critical downstream target of TRIM28 and clinically-available E2F inhibitor LY101-4B may suppress CRPC progression. To test this hypothesis, three specific aims are proposed. In Aim1 we will first validate the direct regulation of E2F1-E2F3 gene expression by TRIM28 with the use of genomic approaches. Aim2 will examine clinical expression of E2F and evaluate the efficacy of E2F inhibitor in CRPC cell lines and xenografts. Aim 1. To demonstrate the direct regulation of E2F1-E2F3 gene expression by TRIM28 Hypothesis: TRIM28 directly regulates E2F1-3 expression in PCa. To validate TRIM28-ChIP Seq, we will perform TRIM28 ChIP in LNCaP (androgen-dependent PCa) and 22Rv1 (CRPC) cell lines, we will run qPCR to confirm the enrichment of TRIM28 at E2F1-E2F3 promoter region. We will also perform CRISPR-mediated deletion of the promoter region of E2F1-E2F3 where TRIM28 binds, followed by TRIM28 ChIP and qPCR to confirm if the CRISPR technology can abolish TRIM28 binding at the promoters of E2F1-E2F3. Lastly, we will collect RNA and protein lysate from parental and CRISPR-edited cells and qPCR analysis and western blot will be conducted respectively to test if E2F1-E2F3 expression was altered in CRISPR-edited cells. In another approach, we will first construct a luciferase reporter containing the promoter of E2Fs, and we will perform luciferase reporter assay using cell lysate from control or TRIM28 knockdown cells to reveal if TRIM28 is required for promoter activation for E2Fs. To investigate which functional domain of TRIM28 is required for transcription activation function, different mutants of TRIM28 including RING domain, HP1, PHD or BROMO domain deletion mutants will be constructed. We will employ TRIM28 knockdown approach with rescuing experiments using TRIM28 mutants in prostate cancer cells. RNA will be extracted from the cells and subjected to qPCR analysis to quantitate E2F1- E2F3 expression. To examine if TRIM28 binding partners TRIM24 and TRIM33 is involved in regulating E2F1- E2F3 expression, we will knockdown the expression of TRIM24 and TRIM33 in prostate cancer cells, respectively. Cells will be harvested, and thus E2Fs expression will be analyzed by qPCR. Aim 2. To establish E2F as a therapeutic target in CRPC Aim 2a. To examine E2F1 expression using patient biospecimen Hypothesis: E2F1 expression is aberrantly upregulated in CRPC. We will examine the expression of E2F1 and the correlation of TRIM28 and E2F1 expression in a number of PCa patient cohorts. We will then obtain tissue microarray distributed by Prostate Cancer Biorepository Network (PCBN) consisting a large patient cohort of localized and CRPC, and we will perform immunohistochemistry using those specimen with the IHC-validated E2F1 antibodies. Aim 2b. To target E2F activity in CRPC Hypothesis: E2F transcription activity is required for CRPC growth. We will knockdown E2F1-E2F3 with shRNA followed by rescuing the knockdown cells by E2F1 wildtype, E2F1 mutant (E132) which is DNA binding deficient or E2F-?TA lacking transactivation domain in 22Rv1 and C4-2B cell lines. We will examine the cell growth, cell proliferation and xenograft tumor growth of E2F1 wildtype and mutants-rescued cells. In another approach, we will treat CRPC cell lines with vehicle or E2F inhibitor LY101-4B and evaluate the PCa growth by cell proliferation assay, colony formation assay and xenograft assay.