The goal of the present proposal is to investigate the mechanism by which TCF4 regulates neuroendocrine differentiation (NED) and induces enzalutamide resistance in castration-resistant prostate cancer (CRPC). In treating patients with CRPC, enzalutamide, the second-generation AR antagonist, has been considered a cornerstone of care. However, clinical benefits are limited to a median time of 4.8 months because resistance to enzalutamide inevitably emerges. In our preliminary RNA sequence study, it was found that the expression levels of NE markers such as chromogranin A and parathyroid hormone-related peptide (PTHrP) were highly elevated in an enzalutamide-resistant human prostate cancer (CaP) cell line when compared to the parental cell line. After analyzing the promoters of the NED-related genes, we found that TCF4, a transcription factor that has not been linked to CaP previously, mediated NED in response to enzalutamide treatment and was elevated in the enzalutamide-resistant CaP cell line. Importantly, the NED marker PTHrP mediated enzalutamide resistance in tissue culture. When tissues obtained from men who died of metastatic CaP were examined, a positive correlation was found between the expression levels of TCF4 and PTHrP. Inducible TCF4 overexpression also caused enzalutamide resistance in mouse xenograft model. Importantly, enzalutamide stimulated the expression of the AR splice variant, AR-V7, which then removed the suppressive effect of AR on TCF4 and increased its expression levels. Based on these observations, we hypothesize that TCF4 is induced by AR-V7 and mediates, in part, enzalutamide resistance in CaP cells via the neuroendocrine marker PTHrP. To test this hypothesis, we propose the following specific aims:Specific Aim 1: To investigate the mechanism of EnzR induced by AR-V7/TCF4/PTHrP axis: Based on the preliminary data, our questions are, 'How does Enz regulate TCF4 expression' and 'What is the role of ß-catenin in induction of NED by TCF4' Enz inhibits AR activity by binding directly to the ligand binding domain of AR. Our preliminary study showed that Enz treatment increased AR-V7 in androgen-sensitive prostate cancer cell lines, such as LNCaP, 22Rv1, and VCaP. Previously, AR-V7 has been linked to enzalutamide resistance. To study the role of AR-V7 for regulating TCF4 expression, immunoprecipitation (IP) assay, TCF4 promoter serial deletion assay will be performed to determine the region of the AR-binding element. Then, chromatin immunoprecipitation (ChIP) analysis using narrowed promoter regions of TCF4 will be performed. To answer the second question, an inducible TCF4 overexpression xenograft model will be used. Specifically, IP studies using TCF4 and ß-catenin antibodies will be performed. Finally, a protein mapping study of TCF4 and ß-catenin will be carried out to determine the region responsible for the protein-protein interaction. Specific Aim 2: To investigate the clinical implications of AR-V7/TCF4/PTHrP axis in CaP.Subaim 2A: To analyze AR-V7, TCF4, and PTHrP expression levels in enzalutamide-resistant human CaP tissues. The goal of this aim is to study AR-V7, PTHrP, and TCF4 expression levels in tissues obtained from patients who were treated with enzalutamide prior to death. Our question is, 'Does enzalutamide-resistant CaP tissue express increased levels of AR-V7, TCF4, and PTHrP' Our preliminary data revealed that CRPC tissues have detectable levels of TCF4 and PTHrP. Therefore, we plan to measure AR-V7, TCF4, and PTHrP levels in enzalutamide CaP tissues obtained from the University of Michigan's rapid autopsy program.Subaim 2B: To investigate the therapeutic potential of blocking TCF4/PTHrP axis in enzalutamide-resistant CaP cells. Here, our question is, 'Does AR-V7/TCF4/PTHrP axis induce enzalutamide resistance in vivo' Our preliminary in vitro data implied that AR-V7 increases the expression levels of TCF4, which, in turn, stimulates the production of PTHrP and results in enzalutamide resistance in human CaP cell lines. Furthermore, the overexpression of TCF4-rendered CaP cells more resistant to enzalutamide in mice. Therefore, we will utilize three enzalutamide-resistant CaP cell lines already established in our laboratory to determine the effect of blocking TCF4 and PTHrP. Neutralization of TCF4 and PTHrP will be carried out with PKF118-310 and PTHrP(7-34), respectively. Simultaneously, we will establish cell lines with inducible knockdown of AR-V7 and examine the effect on NED and enzalutamide resistance.Impact: Due to a relative ease of administration and low toxicity profile, enzalutamide is used widely to treat CRPC. However, treatment resistance is a key limitation. Therefore, understanding the mechanism of resistance to enzalutamide has the potential to improve its efficacy as well as identify new targets of intervention in men with an end-stage CaP. In the present proposal, we have identified TCF4 and PTHrP as important mediators of enzalutamide resistance. Therefore, a successful completion of our proposal will provide the rationale for targeting TCF4 and or PTHrP in men with enzalutamide-resistant CaP.
|Effective start/end date||9/1/17 → 8/31/20|
- Congressionally Directed Medical Research Programs (CDMRP)