In treating patients with castration-resistant prostate cancer, enzalutamide, the second-generation androgen receptor 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. The goal of the present proposal is to understand the link between enzalutamide resistance and neuroendocrine differentiation. In our preliminary RNA sequence data, it was found that the neuroendocrine markers such as chromogranin A and parathyroid hormone-related peptide (PTHrP) were highly expressed in an enzalutamide-resistant human prostate cancer cell line compared to the parental cells. After analyzing the promoters of these neuroendocrine differentiation-related genes, it was found that the transcription factor TCF4 mediated neuroendocrine differentiation in response to enzalutamide treatment and was elevated in the enzalutamide-resistant prostate cancer cell line. Importantly, the neuroendocrine differentiation marker PTHrP mediated enzalutamide resistance in tissue culture. When tissues obtained from men who died of metastatic prostate cancer 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 models. Furthermore, enzalutamide stimulated the expression of the androgen receptor splice variant AR-V7, which then removed the suppressive effect of androgen receptor on TCF4 and increased its expression levels. TCF4 inhibitor PKF118-310 and the PTHrP antagonist decreased enzalutamide resistance in vitro cell culture system. Collectively, we hypothesize that TCF4 is induced by AR-V7 and mediates, in part, Enz resistance in prostate cancer cells via the neuroendocrine marker PTHrP. To test this hypothesis, we propose to further investigate the mechanism of enzalutamide resistance induced by the AR-V7/TCF4/PTHrP axis via tissue culture, xenograft mouse models, and human tissues. Briefly, in Aim 1, the mechanism of enzalutamide resistance mediated by TCF4 in vitro culture system and the study of AR:AR-V7 heterodimer for TCF4 gene expression will be studied. Aim 2 contains studies on the clinical implication of TCF4 for enzalutamide resistance. Specifically, the potential therapeutic effect of TCF4 inhibitor PKF118-310 and PTHrP inhibitor PTHrP(7-34) will be assessed in mouse xenograft models. Our preliminary data revealed that castration-resistant prostate cancer tissues have detectable levels of TCF4 and PTHrP. Therefore, simultaneously, we plan to measure AR-V7, TCF4, and PTHrP levels in enzalutamide-resistant prostate cancer tissues obtained from the University of Michigan's rapid autopsy program.