Original ArticlesSEC-induced activation of ANXA7 GTPase suppresses prostate cancer metastasis
Introduction
Cancer metastasis is the major cause of cancer mortality, accounting for approximately 90% of deaths. Once cancer cells leave and spread beyond their primary site, metastatic cancers are highly incurable and fatal, whereas most primary tumors are manageable or curable [1]. Prostate cancer is the most frequently diagnosed cancer in men with an estimated 180,000 new cases in U.S. in 2016. Five-year relative survival rate of localized and regional prostate cancer reaches approximately 99%; however, only about 27% of metastatic prostate cancer patients survive in 5 years [2]. Cancer metastasis is a complicated progress that involves four essential steps: detachment, migration, invasion and adhesion [3]. Whereas our understanding of the biology behind mechanisms of androgen receptor pathway activation, tumor-microenvironment interaction, and anti-tumor immunotherapy has advanced therapies for metastatic prostate cancer [4], a better understanding of molecular and cellular mechanisms of suppressor gene and associated signaling pathway in prostate cancer metastasis is critical and in great need to identify new therapeutic targets and agents.
Annexin A7 (ANXA7), a member of annexin superfamily, is a Ca2+- and phospholipid-binding protein and possesses GTPase activity [5,6]. The ANXA7 gene is located on human chromosome 10q21, where multiple potential tumor suppressor genes exist. Homozygous ANXA7 (−/−) knockout mice showed a lethal phenotype at embryonic day 10 [7]. Heterozygous ANXA7 (±) knockout mice have a high frequency of spontaneous tumors (about 20–50%) in multiple organs, including liver, prostate, endometrium, salivery gland and thymus [8]. Moreover, tumor metastasis was observed in 42% of the mutant mice developing spontaneous tumor [8]. These findings suggest that ANXA7 may play an important role in inhibiting tumorigenesis and metastasis. Compared with that in normal and healthy prostate tissue, ANXA7 expression is reduced by approximately 50% in primary prostate tumors and even more downregulated in metastatic prostate cancers [7,9]. However, the mechanism of ANXA7 GTPase in the regulation of metastasis has not been established. Our recent work has demonstrated that activation of ANXA7 GTPase by a small molecule SEC ((S)-ethyl 1-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3-(4-methoxyphenyl)-1 H-pyrazole-5 -carboxylate) induces prostate cancer cell apoptosis [10]. We wondered whether activated ANXA7 GTPase could inhibit prostate cancer metastasis.
Raf kinase inhibitory protein (RKIP) has been associated with natural anti-metastasis property in multiple tumor types [11]. The metastatic suppression role of RKIP was initially identified in prostate cancer [12]. RKIP expression is low in metastatic prostate cancer cell lines PC3 and DU145, while RKIP is relatively high expressed in non-invasive prostate cancer cell line LNCaP [13]. Analysis of clinical samples showed that metastases of human prostate cancer exhibited dramatically decreased RKIP level compared with benign prostate hyperplasia and localized prostate cancer samples [12,13]. It has been proposed that low RKIP expression is a good prognostic marker for the pathogenesis of human prostate cancer [14].
Accumulating evidence suggests that RKIP associates with GTP-binding proteins [15]. RKIP associates with Raf-1 and inhibits the Raf/MEK/ERK/Myc signaling, leading to the inhibition of Snail and TET activity and decreased pro-metastatic gene expression [16]. RKIP interferes with NF-κB signaling by the interaction with several upstream kinases TAK1, NIK and IKK, contributing to metastasis inhibition [17]. The binding of RKIP and its target proteins could inhibit the interactive proteins activation by their activators, leading to the inversion of related signaling and the inhibition of tumor metastasis [18]. In addition, it was reported that nonylphenol induced the upregulation of RKIP in testicular Sertoli cells, accompanied with decreased ANXA7 protein level [19]. However, the role and mechanism of RKIP in the regulation of ANXA7 signaling pathway remains unknown so far. We seek to determine whether RKIP binds to ANXA7 and antagonizes the activation of ANXA7 GTPase by SEC.
In the current study, we used metastatic prostate cancer cell PC3 as well as non-metastatic LNCaP, and constructed HEK293T RKIP−/− cell line, together with HEK293T RKIP+/+ cell line as the cell model, to study the role and mechanism of ANXA7 GTPase in prostate cancer metastasis. Our substantial evidence suggests that SEC treatment increases AMPK phosphorylation via activating ANXA7 GTPase, and that activated AMPK inhibits STAT3 nuclear translocation via the mTORC1/STAT3 pathway, leading to downregulation of pro-metastatic genes and inhibition of prostate cancer metastasis. Notably, RKIP interacts with and compromises the inhibitory effect of SEC on prostate cancer metastasis. Together, our findings indicate that activation of ANXA7 by a small molecule SEC antagonizes prostate cancer metastasis with low RKIP expression via the AMPK/mTORC1/STAT3 signaling pathway, providing a novel insight into how prostate cancer metastasis is suppressed by a small molecule SEC via previously uncharacterized but essential interplay between the two metastasis suppressors ANXA7 and RKIP.
Section snippets
Antibodies
Antibodies to RKIP (CST#5291), AMPKα (CST#5832), phospho-AMPKα (T172) (CST#8208), p70S6K (CST#2708), phospho-p70S6K (CST#9208), 4EBP1 (CST#9644), phospho-4EBP1 (CST#9459), STAT3 (CST#9132), phospho-STAT3 (Tyr705) (CST#9131) and phospho-STAT3 (Ser727) (CST#9134) were purchased from Cell Signaling Technology. Antibodies to E-Cadherin (20874-1-AP) and vimentin (10366-1-AP) were obtained from Proteintech Group. Anti-ANXA7 antibody (sc-17815) used for co-immunoprecipitation and anti-p-Thr antibody
SEC inhibited migration in HEK293T RKIP−/- cells
It is well established that RKIP has an anti-metastatic property. To get an in-depth understanding of underlying mechanism, we constructed HEK293T cell lines carrying RKIP knockout (RKIP−/−) and wild-type RKIP expression (RKIP+/+) (Fig. 1A). RKIP-null HEK293T cells showed higher migration ability than wild-type RKIP expressing cells (Fig. 1B). The small molecule SEC dramatically suppressed HEK293T RKIP−/− cell migration while had no effect on HEK293T RKIP+/+ cells (Fig. 1B). Moreover, SEC
Discussion
Directly modulating specific target proteins and signaling pathways with small-molecule drugs or agonistic antibodies is widely used for cancer metastasis therapies [[39], [40], [41]]. ANXA7 acts as a metastasis suppressor in prostate cancer. However, the mechanism of ANXA7 on inhibiting prostate cancer metastasis is not clear. In the current study, we revealed for the first time that activation of ANXA7 GTPase by SEC increases AMPK phosphorylation to suppress mTORC1/STAT3 signal pathway and to
Disclosure of potential conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 91313303, 31570834, 81502948, 31270877), the Major Project of Science and Technology of Shandong Province (NO. 2015ZDJS04001) and Shandong Excellent Young Scientist Award Fund (No. BS2015YY031, BS2013SW001). The Yan lab was supported in part by funds provided by University of North Carolina at Charlotte and grants from the NIH/NIGMS (R15GM101571 and R15GM114713).
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