Cancer Letters

Cancer Letters

Volume 408, 1 November 2017, Pages 155-163
Cancer Letters

Original Article
Androgen-deprivation therapy with enzalutamide enhances prostate cancer metastasis via decreasing the EPHB6 suppressor expression

https://doi.org/10.1016/j.canlet.2017.08.014Get rights and content

Highlights

  • Loss of EPHB6 expression is associated with poor prognosis in prostate cancer patients.

  • Overexpression of EPHB6 decreases PCa invasion in vitro.

  • Enz could decrease EPHB6 expression to induce the activation of JNK pathway to increase CRPC cell invasion.

Abstract

Early studies suggested that using ADT with the recently developed anti-androgen Enzalutamide (Enz, also named as MDV3100 could extent castration resistant prostate cancer (CRPC) patients' survival an extra 4.8 months. Yet the therapy in most patients might eventually fail due to development of Enz-resistance. Here we found Enz might also increase some unwanted side-effects via increasing the CRPC cell invasion that might involve altering the Enz-mediated androgen receptor (AR)/EPHB6 suppressor/JNK signaling. Results from multiple clinical surveys also indicated that EPHP6 might function as a suppressor of PCa metastasis. Mechanism dissection revealed that Enz-mediated AR might function via binding to the androgen-response-element (ARE) on the EPHB6 promoter to decrease EPHB6 suppressor expression, which might then activate the phosphorylation of JNK signals to increase the CRPC cell invasion. Targeting this newly identified AR/EPHB6/JNK signaling with JNK inhibitor (SP600125) may then block/reverse the Enz-increased CRPC cell invasion. Collectively, our results suggest that Enz may increase CRPC cell invasion via altering the AR/EPHB6/JNK/MMP9 signaling and targeting this newly identified signaling may help us to increase the Enz efficacy to better suppress the CRPC at the later metastatic stage.

Introduction

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in men [1], [2], which may involve the development of PCa metastasis [3]. Since the androgen receptor (AR), a classical nuclear receptor that binds androgens to activate gene transcription, plays very important roles in PCa progress [4], targeting these androgen/AR signals with the androgen deprivation therapy (ADT) with various antiandrogens remains as an effective therapy to suppress the PCa progression. However, most of PCa patients will progress into metastatic castration resistant prostate cancer (CRPC) inevitably after an average treatment of 12–18 months [5], [6].

Enzalutamide (Enz, also named MDV3100) is a recently developed powerful antiandrogen with a better capacity to prevent the androgens from binding to the AR and the AR nuclear translocation that results in better suppression of the AR target genes [7]. Enz has been approved recently by the FDA after demonstration of improving the overall survival of 4.8 months in men with metastatic CRPC [8], [9]. However, recent preclinical studies using multiple CRPC cells and in vivo mouse models also indicated that ADT with Enz (ADT-Enz) might have the unwanted side-effects of increasing PCa cell invasion that involves multiple mechanisms [10], [11], [12]. The tumor suppressor EPHB6, is a member of erythropoietin-producing hepatocyte (Eph) tyrosine kinase receptor family that involves migration during embryonic development [13], [14] and cancer invasiveness [15], [16]. However, the linkage of Enz-increased CRPC cell invasion to altering the expression of tumor suppressors, remains unclear.

Recent studies indicated that EPHB6 downregulation was consistently correlated with enhanced aggressiveness and invasiveness in colorectal cancers, non-small cell lung carcinoma, melanoma, and breast cancers [17], [18], [19], [20], [21]. EPHB6 could inhibit the JNK activation following the stimulation with ephrinB1 [22], and EPHB6 could also suppress cancer cells' aggressiveness through interacting with EPHB4 [23]. However, the role of EPHB6 in the progression of CRPC, especially the Enz-increased CRPC cell invasion, has been poorly investigated.

Here, we found Enz could promote CRPC cell invasion via regulating the AR/EPHB6/JNK signals, and targeting this newly identified signaling with the small molecule JNK inhibitor may help us to increase the Enz efficacy to better suppress the metastatic PCa at the castration-resistant stage.

Section snippets

Cell culture

The C4-2 cell line was from Dr. Jer-Tsong Hsieh, UT Southwestern Medical Center, Dallas and CWR22Rv1 cell lines were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 media containing 10% fetal bovine serum (FBS), antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin), and 2 mM glutamine (Invitrogen, Grand Island, NY) in a humidified 5% CO2 environment at 37 °C.

Reagents and materials

GAPDH (6c5), AR (N-20) antibodies were purchased from Santa Cruz Biotechnology. EPHB6 antibody

Clinical surveys indicated that EPHB6 expression is negatively correlated with PCa metastasis and progression

Early studies of EPHP6 suggested that it might function as suppressor in several cancers [17], [18], [19], [20]. The role of EPHB6 in PCa metastasis, especially the impacts on ADT, however, remains unclear. We first performed clinical surveys using the TCGA databases to analyze the linkage of EPHP6 expression to the PCa progression. The results revealed that EPHP6 expression was negatively correlated with PCa Gleason score showing lower EPHP6 expression in patients with higher Gleason score (

Discussion

For PCa patients, metastasis is the primary underlying cause of fatality and only one-third of metastatic PCa patients survive for five years after diagnosis [26]. The detailed mechanisms of PCa metastasis, however, remain to be further elucidated.

ADT-Enz has been reported to improve the survival and quality-of-life of CRPC patients [8], [27]. However, recent clinical and preclinical studies indicated that ADT-Enz might also induce some unwanted side-effects including inducing the

Acknowledgements

We thank Karen Wolf for help with manuscript preparation. This work was supported by NIH grant CA156700 and George Whipple Professorship Endowment, Taiwan Department of Health Clinical Trial and Research Center of Excellence grant DOH99-TD-B-111-004 and the National Natural Science Foundation of China (81072107, 81130041, 81773206, 81472679) and the National Key Research and Development Program (2016YFC0902603).

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