The transcriptional coactivator YAP1 (yes-associated protein 1) is a critical nuclear effector of the Hippo pathway. The serine/threonine protein kinases STK3/4 and LATS1/2, core components of the Hippo pathway, phosphorylate and inhibit YAP1 nuclear localization. Previously, we reported that the interaction of nuclear YAP1 with androgen receptor (AR) might play a critical role in prostate cancer progression and therapeutic relapse (Kuser-Abali et al., Nat. Commun. 2015). Here, we investigated the regulation of YAP1 by androgens in isogenic, androgen-responsive LNCaP and androgen non-responsive C4-2 prostate cancer cell models. We demonstrated that androgen suppressed the inhibitory phospho-Ser127 site on YAP1 in LNCaP cells, but the effects of androgen on phospho-Ser127 was modest in C4-2 cells. In agreement with this observation, androgen increased the presence of nuclear YAP1 in LNCaP cells, whereas regardless of androgen exposure the YAP1 protein was primarily expressed in C4-2 cell nuclei. We also demonstrated that androgen exposure suppressed the levels of phospho-Ser127 induced by okadaic acid, which is a potent inhibitor of the Ser/Thr phosphatases PP1 and PP2A. Moreover, the pharmacological inhibition of androgen receptor (AR) signaling by enzalutamide reversed the inhibitory effects of androgen on phospho-Ser127, which coincided with the inhibition of YAP1 nuclear localization. Similarly, the genetic inhibition of AR signaling by small interfering RNA (siRNA) reduced phospho-Ser127 levels. Additionally, the silencing of the STK3/4 and LATS1/2 signaling by siRNA resulted in increases in YAP1 protein levels. Furthermore, our analysis of the TCGA (The Cancer Genome Atlas) prostate adenocarcinoma data set indicates that the levels of YAP1 and AR mRNA expression were positively correlated in prostate cancer clinical samples. These observations suggest that AR signaling promotes YAP1 nuclear localization by suppressing phospho-Ser127, possibly through the protein phosphatases PP1 and PP2A, and supporting a new mechanism of YAP1 regulation and YAP1-mediated cancer cell growth and survival.
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Abstract 2634: The role of Eph A receptor 3 tyrosine kinase signaling in prostate cancer progression
Dysregulation of the receptor tyrosine kinases (RTKs) by means of mutation, amplification or overexpression plays a crucial role in cell growth, cell survival, cell motility during cancer progression and metastasis. EPHA3 (erythropoietin-producing hepatocellular carcinoma cell surface type A receptor 3) is a member of the RTKs. Evidence indicates that the upregulation of the EPHA3 activity is implicated in the pathobiology of various cancers, including prostate cancer, and thus, it is a prime therapeutic target in cancer. However, the role of EPHA3 signaling in prostate cancer progression remains obscure. Currently, the development of castration-resistant prostate cancer (CRPC) poses a clinical challenge because it is lethal. The molecular mechanisms that contribute to lethal prostate cancer are largely unknown. The objective of this study is to investigate whether EPHA3 signaling plays a critical role in prostate cancer progression and therapeutic relapse. Our analysis of the prostate cancer public datasets revealed that the EPHA3 gene was amplified up to 19% of metastatic CRPC cases with the neuroendocrine phenotype. Our immunological assay confirmed the positive staining of EPHA3 protein in human prostate cancer specimens. Our semi-quantitative and quantitative PCR assays demonstrated that the levels of EPHA3 vary among established prostate cancer cell lines. Nevertheless, we consistently found that the levels of EPHA3 mRNA in CRPC cell line, C4-2, were 3-fold higher than its castration-sensitive parental LNCaP cells. Furthermore, we demonstrated that an increase in expression of EPHA3 mRNA in C4-2 compared with LNCaP cells coincided with the upregulation of the EPHA3 protein, as independently confirmed by western blotting and immunofluorescence imaging. These findings indicate that EPHA3 may confer an aggressive prostate cancer cell phenotype. Because androgen receptor (AR) signaling is a potent mediator of CRPC cell growth and survival, the targeting of EPHA3 signaling alone or together with AR may improve the efficacy of current therapies for patients with advanced prostate cancer.
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- Award ID(s):
- 1832022
- NSF-PAR ID:
- 10112088
- Date Published:
- Journal Name:
- AACR Annual Meeting 2019
- Volume:
- 79
- Issue:
- 13 Supplement
- Page Range / eLocation ID:
- 2634 to 2634
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background NKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1’s levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date.
Methods A chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells.
Results We uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it.
Conclusions NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.
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Multifunctional protein YBX1 upregulation promotes castration-resistant prostate cancer (CRPC). However, YBX1 protein abundance, but not its DNA status or mRNA levels, predicts CRPC recurrence, although the mechanism remains unknown. Similarly, the mechanism by which YBX1 regulates androgen receptor (AR) signaling remains unclear. We uncovered the first molecular mechanism of YBX1 upregulation at a post-translational level. YBX1 was identified as an Aurora Kinase-A (AURKA) substrate using a chemical screen. AURKA phosphorylates YBX1 at two key residues, which stabilizes it and promotes its nuclear translocation. YBX1 reciprocates and stabilizes AURKA, thereby initiating a synergistic loop. Notably, phospho-resistant YBX1 is dominant-negative and fully inhibits epithelial to mesenchymal transition, chemoresistance, drug-resistance and tumorigenesis in vivo. Unexpectedly, we further observed that YBX1 upregulates AR post-translationally by preventing its ubiquitylation, but not by increasing its transcription as reported before. Uncovering YBX1-mediated AR stabilization is highly significant due to AR’s critical role in both androgen-sensitive prostate cancer and CRPC. As YBX1 inhibitors are unknown, AURKA inhibitors provide a potent tool to degrade both YBX1 and AR simultaneously. Finally, this is the first study to show a reciprocal loop between YBX1 and its kinase, indicating that their concomitant inhibition will be act synergistically for CRPC therapy.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1158/1538-7445.AM2019-2634
