N E W S
A N D
V I E W S
Unzipping Androgen Action Through ZIP9: A Novel Membrane Androgen Receptor Laura E. Pascal and Zhou Wang Departments of Urology (L.E.P., Z.W.) and Pharmacology and Chemical Biology (Z.W.) and University of Pittsburgh Cancer Institute (Z.W.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15232
N
early 5 decades ago, the existence of an androgen receptor (AR) molecule was originally identified by Shutsung Liao at the University of Chicago with concordant findings published by Nicholas Bruchovsky and Jean Wilson (1–3). These seminal studies used rat prostate tissue and specifically described a protein with high affinity for 5␣-dihydrotestosterone (DHT) that accumulated in the nucleus upon binding, initiating RNA synthesis and explaining the mode of action for androgens within target cells. Subsequent studies by multiple laboratories determined that AR regulates the transcription of androgen-responsive genes and plays a pivotal role in male reproductive tract development, sexually dimorphic brain activities, and multiple other target tissues. Importantly, AR has an essential role in prostate carcinogenesis and progression, with blockade of its activity used as a primary therapeutic target. Classical nuclear AR signaling (also known as genomic actions) in target cells involves the diffusion of testosterone through the cell membrane and into the cytoplasm, where it or its metabolite DHT binds to the AR, triggering conformational changes, dissociation from heat shock protein 90, dimerization, and translocation to the cell nucleus. There, the AR complex binds to DNA at androgen-response elements, recruits coregulators, and activates transcription of genes that are then translated into proteins. This classic or genomic mechanism of androgen action is relatively slow, requiring approximately 1– 6 hours for alterations in gene transcriptional activity. It is noteworthy that as early as 1975, Liao also described rapid androgen actions occurring within 10 minutes of DHT administration that were independent of RNA synthesis, speculating that “androgens may affect known protein factors or others that
are yet to be identified” (4). While research attention remain focused on nuclear steroid receptor signaling pathways, culminating in the cloning of steroid receptor genes in the 1980s, including that for AR (5, 6), evidence began to emerge in the 1990s that androgens are also capable of exerting rapid effects by interacting with AR in the cytoplasm or at the plasma membrane. Membrane-associated ARs were initially described in cell types such as T lymphocytes, monocytes, and osteoblasts to effect influx of calcium (7–9). Subsequent studies found that similar to membrane estrogen receptors, membrane ARs can also interact with signaling molecules to rapidly activate signaling pathways, including the MAPK/ERK (10) and focal adhesion kinase 1 (FAK) /phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways (11, 12). ARs along the cell surface have also been shown more recently to regulate actin cytoskeletal reorganization and prostate-specific antigen secretion in prostate cancer cells (13). The genomic AR signaling pathway has long been the standard therapeutic target for prostate cancer, and initially blockade of nuclear AR activity is very effective in slowing prostate tumor growth and progression. However, tumors eventually become resistant to nuclear AR antagonists and resume growth and rapid metastatic progression. Nongenomic androgen signaling represents an alternative focus for the development of potential treatment strategies that could enhance tumor response to antiandrogen treatment, particularly in delaying the development of castrate-resistant prostate cancer (14). Progress on studies of nongenomic androgen signaling in prostate cancer cells has been hampered by the challenges associated with identification of novel membrane ARs in these
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received September 9, 2014. Accepted September 19, 2014.
Abbreviations: AR, androgen receptor; DHT, 5␣-dihydrotestosterone; FAK, focal adhesion kinase 1; ZIP9, zinc-regulated transporter, iron-regulated transporter-like protein 9.
For articles see pages 4237 and 4250
4120
endo.endojournals.org
Endocrinology, November 2014, 155(11):4120 – 4123
doi: 10.1210/en.2014-1749
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
doi: 10.1210/en.2014-1749
endo.endojournals.org
4121
ity (maximum binding Bmax, 2.8nM/mg protein) AR to mediate androgen-induced apoptosis of ovarian follicle cells and to increase intracellular Zn2⫹. In parallel, Thomas et al (17) confirmed that ZIP9 can function similarly in human prostate and breast cancer cells. Knockdown and overexpression studies showed the requirement of ZIP9 in testosterone induction of apoptosis, which was associated with rapid increases in intracellular free zinc concentration and the up-regulation of proapoptotic genes B-cell lymphoma 2-associated x protein (Bax), tumor protein 53 (p53), c-Jun N-terminal kinase (JNK), caspase 3, and cytochrome c, previously unidentified targets of rapid, membrane-initiated androgen signaling. ZIP9 was widely expressed in human tissues and up-regulated in breast and prostate tumor specimens, suggesting that ZIP9 is a potential therapeutic target for breast and prostate cancer. These important findings provide new insights into the mechanisms of androgen action and may lead to new approaches to target androgen signaling. In the healthy prostate, high zinc concentrations serve to inhibit citrate oxidation, thereby promoting the production and secretion of citrate, a major constituent of prostatic fluid (19). Decreased zinc is a hallmark of prostate cancer, and zinc has been postulated to inhibit prostate cancer cell growth through induction of cell cycle arrest and apoptosis (20 –23). Zinc transporters are decreased in prostate cancer, which could contribute to intracellular zinc deficiency and increased proliferation in tumor cells Ca2+ (24 –26). Thomas et al (17) demonZn2+ strate through overexpression and AR knockdown studies that testosterone 2+ Ca stimulates ZIP9-mediated influx of 2+ Ca AR zinc coupled with apoptosis inducSignaling pathways: tion in prostate cancer cells and that 2+ Zn2+ Zn p38 ERK ZIP9 expression is increased in both DHT FAK prostate and breast tumor tissues. In Proapoptoc genes: ZIP9-transfected breast cancer cells, DHT DHT JNK p53 intracellular zinc and cell death was Bax AR AR DHT DHT also increased in response to testosterone (16). Because ZIP9 transfecAR AR tion induced an increase in intracellular Zn2⫹ and in apoptosis, Thomas et al postulate that therapeutic treatment with ZIP9 agonists might seFigure 1. Genomic and nongenomic androgen signaling in prostate. Genomic AR signaling lectively increase zinc levels and involves the diffusion of testosterone (T) into the cytoplasm, where it can be activated to DHT activate nongenomic androgen sigand bind to AR. AR forms a dimer and translocates to the nucleus, where it binds to the naling to induce apoptosis in maligandrogen-response elements (AREs) on promoter/enhancer regions, recruits coregulators, and forms the transcriptional machinery for AR-regulated gene expression. Nongenomic signaling nant cells. However, this might not involves the rapid activation of signaling pathways, including MAPK/ERK, FAK, and p38, leading be equally effective in breast and to cell proliferation through membrane-bound and/or cytoplasmic AR (10, 11, 14, 33). The ZIP9 prostate cancers. Breast cancer is asprotein functions as a membrane-bound high-affinity, low-capacity AR to mediate androgensociated with increased tissue zinc induced apoptosis and to increase intracellular Zn2⫹ (16, 17). cells. Studies focused on comparative endocrinology can facilitate identifying and characterizing the evolutionarily conserved functioning receptors of rapid signaling of steroids, including androgens (15). In 2 exciting reports in the current issue of Endocrinology, a new/novel highaffinity, limited-capacity membrane AR zinc-regulated transporters, iron-regulated transporter-like protein 9 (ZIP9) was identified and characterized first in the female Atlantic croaker and subsequently in human breast and prostate cancer cells (16, 17). In previous studies, the Thomas lab reported the existence of a novel nonnuclear AR in the ovaries of the Atlantic croaker for rapid, cell surface-mediated androgenic inhibition of estradiol secretion (18). Androgens were shown to inhibit estradiol production in croaker ovaries even in the presence of nuclear AR antagonist. BSA-conjugated DHT, which does not diffuse through the cell membrane, also inhibited estradiol production. Actinomycin, which inhibits transcription, did not block this inhibitory response. Taken together, these results suggested the presence of a membrane-bound AR. In the current study, these investigators have identified this membrane AR as a protein with a high sequence identity with vertebrate ZIP9 subfamily members (16). The authors subsequently provided evidence that this croaker zinc transporter ZIP9 protein functions as a membrane-bound high-affinity (dissociation constant Kd, 12.7nM), low-capac-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
4122
Pascal and Wang
ZIP9 Membrane AR
concentration, whereas prostate cancer is associated with decreased tissue zinc (27), suggesting potential differences in the tumor microenvironment and other factors controlling tumor growth and progression. Studies have demonstrated that breast xenograft tumor growth was inhibited by zinc depletion in male rats (28) and decreased dietary zinc suppressed N-methyl-N-nitrosourea-induced rat mammary tumorigenesis (29), whereas the ability to accumulate zinc in prostate cancer cell lines decreases with increasing tumorigenicity (30, 31). In a testosterone-depleted microenvironment, where prostate tumor cells already overexpress ZIP9, activating ZIP9 without activating the genomic AR pathway could disproportionately increase zinc levels and trigger proapoptotic gene expression in cancer cells, thereby selectively inducing apoptosis in these cells. However, breast tumor growth appears to be stimulated by increased zinc levels, and activation of ZIP9 in vivo might have deleterious effects. Future in vivo studies will no doubt shed light on this potential differential response in the development and progression of tumors as well as in the development and maintenance of normal tissue homeostasis. In summary, androgen signaling includes genomic and nongenomic pathways that may act coordinately or independently in healthy prostate maintenance as well as the development and progression of prostatic disease (Figure 1). Genomic or nuclear androgen signaling is slower than nongenomic, membrane-initiated signaling and regulates the transcription of thousands of androgen-responsive genes. Nongenomic androgen signaling can be mediated through membrane-associated AR and/or ZIP9, which is rapid and initiated at the cell surface and/or cytoplasm to regulate several pathways, including MAPK/ERK, FAK, p38, p53, calcium, and zinc (10, 12, 13, 16, 17, 32, 33). Genomic and nongenomic androgen signaling pathways may cross talk and converge to control cell proliferation and/or apoptosis. Identification of ZIP9 as a membranebound AR provides exciting research opportunities to determine the role of nongenomic androgen signaling via AR vs ZIP9 in development, tissue maintenance, and diseases.
Acknowledgments Address all correspondence and requests for reprints to: Zhou Wang, PhD, Department of Urology, University of Pittsburgh School of Medicine-University of Pittsburgh Medical Center Shadyside, 5200 Centre Avenue, Suite G40, Pittsburgh, PA 15232. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.
Endocrinology, November 2014, 155(11):4120 – 4123
References 1. Anderson KM, Liao S. Selective retention of dihydrotestosterone by prostatic nuclei. Nature. 1968;219:277–279. 2. Bruchovsky N, Wilson JD. The intranuclear binding of testosterone and 5␣-androstan-17-ol-3-one by rat prostate. J Biol Chem. 1968; 243:5953–5960. 3. Liao S, Fang S. Receptor-proteins for androgens and the mode of action of androgens on gene transcription in ventral prostate. In: Vitamins and Hormones. New York, NY: Academic Press, Inc; 1969:17–90. 4. Liang T, Liao S. A very rapid effect of androgen on initiation of protein synthesis in prostate. Proc Natl Acad Sci USA. 1975;72: 706 –709. 5. Chang CS, Kokontis J, Liao ST. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988; 240:324 –326. 6. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM. Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science. 1988;240: 327–330. 7. Gorczynska E, Handelsman DJ. Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology. 1995; 136:2052–2059. 8. Lyng FM, Jones GR, Rommerts FF. Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations. Biol Reprod. 2000;63:736 –747. 9. Benten WP, Lieberherr M, Giese G, et al. Functional testosterone receptors in plasma membranes of T cells. FASEB J. 1999;13:123– 133. 10. Peterziel H, Mink S, Schonert A, Becker M, Klocker H, Cato AC. Rapid signalling by androgen receptor in prostate cancer cells. Oncogene. 1999;18:6322– 6329. 11. Gatson JW, Kaur P, Singh M. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology. 2006;147: 2028 –2034. 12. Gu S, Kounenidakis M, Schmidt EM, et al. Rapid activation of FAK/mTOR/p70S6K/PAK1-signaling controls the early testosterone-induced actin reorganization in colon cancer cells. Cell Signal. 2013;25:66 –73. 13. Papakonstanti EA, Kampa M, Castanas E, Stournaras C. A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors. Mol Endocrinol. 2003;17:870 – 881. 14. Liao RS, Ma S, Miao L, Li R, Yin Y, Raj GV. Androgen receptormediated non-genomic regulation of prostate cancer cell proliferation. Transl Androl Urol. 2013;2:187–196. 15. Thomas P. Rapid steroid hormone actions initiated at the cell surface and the receptors that mediate them with an emphasis on recent progress in fish models. Gen Comp Endocrinol. 2012;175:367–383. 16. Berg AH, Rice CD, Rahman MS, Dong J, Thomas P. Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female Atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells. Endocrinology. 2014;155:4237– 4249. 17. Thomas P, Pang Y, Dong J, Berg AH. Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: II. Role of human ZIP9 in testosterone-induced prostate and breast cancer cell apoptosis. Endocrinology. 2014; 4250 – 4265. 18. Braun AM, Thomas P. Androgens inhibit estradiol-17 synthesis in Atlantic croaker (Micropogonias undulatus) ovaries by a nongenomic mechanism initiated at the cell surface. Biol Reprod. 2003; 69:1642–1650.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
doi: 10.1210/en.2014-1749
19. Costello LC, Feng P, Milon B, Tan M, Franklin RB. Role of zinc in the pathogenesis and treatment of prostate cancer: critical issues to resolve. Prostate Cancer Prostatic Dis. 2004;7:111–117. 20. Zaichick VYe, Sviridova TV, Zaichick SV. Zinc in the human prostate gland: normal, hyperplastic and cancerous. Int Urol Nephrol. 1997;29:565–574. 21. Johnson LA, Kanak MA, Kajdacsy-Balla A, Pestaner JP, Bagasra O. Differential zinc accumulation and expression of human zinc transporter 1 (hZIP1) in prostate glands. Methods. 2010;52:316 –321. 22. Franklin RB, Feng P, Milon B, et al. hZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer. Mol Cancer. 2005;4:32. 23. Liang JY, Liu YY, Zou J, Franklin RB, Costello LC, Feng P. Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate. 1999;40:200 –207. 24. Feng P, Li T, Guan Z, Franklin RB, Costello LC. The involvement of Bax in zinc-induced mitochondrial apoptogenesis in malignant prostate cells. Mol Cancer. 2008;7:25. 25. Feng P, Liang JY, Li TL, et al. Zinc induces mitochondria apoptogenesis in prostate cells. Mol Urol. 2000;4:31–36. 26. Ku JH, Seo SY, Kwak C, Kim HH. The role of survivin and Bcl-2 in
endo.endojournals.org
27.
28. 29.
30.
31.
32.
33.
4123
zinc-induced apoptosis in prostate cancer cells. Urol Oncol. 2012; 30:562–568. Gumulec J, Masarik M, Adam V, Eckschlager T, Provaznik I, Kizek R. Serum and tissue zinc in epithelial malignancies: a meta-analysis. PLoS One. 2014;9:e99790. Mills BJ, Broghamer WL, Higgins PJ, Lindeman RD. Inhibition of tumor growth by zinc depletion of rats. J Nutr. 1984;114:746 –752. Lee S, Simpson M, Nimmo M, Xu Z. Low zinc intake suppressed N-methyl-N-nitrosourea-inducedmammarytumorigenesisinSpragueDawley rats. Carcinogenesis. 2004;25:1879 –1885. Costello LC, Franklin RB. Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer. Prostate. 1998;35:285–296. Huang L, Kirschke CP, Zhang Y. Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression. Cancer Cell Int. 2006;6:10. Benten WP, Lieberherr M, Sekeris CE, Wunderlich F. Testosterone induces Ca2⫹ influx via non-genomic surface receptors in activated T cells. FEBS Lett. 1997;407:211–214. Zoubeidi A, Zardan A, Beraldi E, et al. Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity. Cancer Res. 2007;67:10455– 10465.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
A N D
V I E W S
Unzipping Androgen Action Through ZIP9: A Novel Membrane Androgen Receptor Laura E. Pascal and Zhou Wang Departments of Urology (L.E.P., Z.W.) and Pharmacology and Chemical Biology (Z.W.) and University of Pittsburgh Cancer Institute (Z.W.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15232
N
early 5 decades ago, the existence of an androgen receptor (AR) molecule was originally identified by Shutsung Liao at the University of Chicago with concordant findings published by Nicholas Bruchovsky and Jean Wilson (1–3). These seminal studies used rat prostate tissue and specifically described a protein with high affinity for 5␣-dihydrotestosterone (DHT) that accumulated in the nucleus upon binding, initiating RNA synthesis and explaining the mode of action for androgens within target cells. Subsequent studies by multiple laboratories determined that AR regulates the transcription of androgen-responsive genes and plays a pivotal role in male reproductive tract development, sexually dimorphic brain activities, and multiple other target tissues. Importantly, AR has an essential role in prostate carcinogenesis and progression, with blockade of its activity used as a primary therapeutic target. Classical nuclear AR signaling (also known as genomic actions) in target cells involves the diffusion of testosterone through the cell membrane and into the cytoplasm, where it or its metabolite DHT binds to the AR, triggering conformational changes, dissociation from heat shock protein 90, dimerization, and translocation to the cell nucleus. There, the AR complex binds to DNA at androgen-response elements, recruits coregulators, and activates transcription of genes that are then translated into proteins. This classic or genomic mechanism of androgen action is relatively slow, requiring approximately 1– 6 hours for alterations in gene transcriptional activity. It is noteworthy that as early as 1975, Liao also described rapid androgen actions occurring within 10 minutes of DHT administration that were independent of RNA synthesis, speculating that “androgens may affect known protein factors or others that
are yet to be identified” (4). While research attention remain focused on nuclear steroid receptor signaling pathways, culminating in the cloning of steroid receptor genes in the 1980s, including that for AR (5, 6), evidence began to emerge in the 1990s that androgens are also capable of exerting rapid effects by interacting with AR in the cytoplasm or at the plasma membrane. Membrane-associated ARs were initially described in cell types such as T lymphocytes, monocytes, and osteoblasts to effect influx of calcium (7–9). Subsequent studies found that similar to membrane estrogen receptors, membrane ARs can also interact with signaling molecules to rapidly activate signaling pathways, including the MAPK/ERK (10) and focal adhesion kinase 1 (FAK) /phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways (11, 12). ARs along the cell surface have also been shown more recently to regulate actin cytoskeletal reorganization and prostate-specific antigen secretion in prostate cancer cells (13). The genomic AR signaling pathway has long been the standard therapeutic target for prostate cancer, and initially blockade of nuclear AR activity is very effective in slowing prostate tumor growth and progression. However, tumors eventually become resistant to nuclear AR antagonists and resume growth and rapid metastatic progression. Nongenomic androgen signaling represents an alternative focus for the development of potential treatment strategies that could enhance tumor response to antiandrogen treatment, particularly in delaying the development of castrate-resistant prostate cancer (14). Progress on studies of nongenomic androgen signaling in prostate cancer cells has been hampered by the challenges associated with identification of novel membrane ARs in these
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received September 9, 2014. Accepted September 19, 2014.
Abbreviations: AR, androgen receptor; DHT, 5␣-dihydrotestosterone; FAK, focal adhesion kinase 1; ZIP9, zinc-regulated transporter, iron-regulated transporter-like protein 9.
For articles see pages 4237 and 4250
4120
endo.endojournals.org
Endocrinology, November 2014, 155(11):4120 – 4123
doi: 10.1210/en.2014-1749
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
doi: 10.1210/en.2014-1749
endo.endojournals.org
4121
ity (maximum binding Bmax, 2.8nM/mg protein) AR to mediate androgen-induced apoptosis of ovarian follicle cells and to increase intracellular Zn2⫹. In parallel, Thomas et al (17) confirmed that ZIP9 can function similarly in human prostate and breast cancer cells. Knockdown and overexpression studies showed the requirement of ZIP9 in testosterone induction of apoptosis, which was associated with rapid increases in intracellular free zinc concentration and the up-regulation of proapoptotic genes B-cell lymphoma 2-associated x protein (Bax), tumor protein 53 (p53), c-Jun N-terminal kinase (JNK), caspase 3, and cytochrome c, previously unidentified targets of rapid, membrane-initiated androgen signaling. ZIP9 was widely expressed in human tissues and up-regulated in breast and prostate tumor specimens, suggesting that ZIP9 is a potential therapeutic target for breast and prostate cancer. These important findings provide new insights into the mechanisms of androgen action and may lead to new approaches to target androgen signaling. In the healthy prostate, high zinc concentrations serve to inhibit citrate oxidation, thereby promoting the production and secretion of citrate, a major constituent of prostatic fluid (19). Decreased zinc is a hallmark of prostate cancer, and zinc has been postulated to inhibit prostate cancer cell growth through induction of cell cycle arrest and apoptosis (20 –23). Zinc transporters are decreased in prostate cancer, which could contribute to intracellular zinc deficiency and increased proliferation in tumor cells Ca2+ (24 –26). Thomas et al (17) demonZn2+ strate through overexpression and AR knockdown studies that testosterone 2+ Ca stimulates ZIP9-mediated influx of 2+ Ca AR zinc coupled with apoptosis inducSignaling pathways: tion in prostate cancer cells and that 2+ Zn2+ Zn p38 ERK ZIP9 expression is increased in both DHT FAK prostate and breast tumor tissues. In Proapoptoc genes: ZIP9-transfected breast cancer cells, DHT DHT JNK p53 intracellular zinc and cell death was Bax AR AR DHT DHT also increased in response to testosterone (16). Because ZIP9 transfecAR AR tion induced an increase in intracellular Zn2⫹ and in apoptosis, Thomas et al postulate that therapeutic treatment with ZIP9 agonists might seFigure 1. Genomic and nongenomic androgen signaling in prostate. Genomic AR signaling lectively increase zinc levels and involves the diffusion of testosterone (T) into the cytoplasm, where it can be activated to DHT activate nongenomic androgen sigand bind to AR. AR forms a dimer and translocates to the nucleus, where it binds to the naling to induce apoptosis in maligandrogen-response elements (AREs) on promoter/enhancer regions, recruits coregulators, and forms the transcriptional machinery for AR-regulated gene expression. Nongenomic signaling nant cells. However, this might not involves the rapid activation of signaling pathways, including MAPK/ERK, FAK, and p38, leading be equally effective in breast and to cell proliferation through membrane-bound and/or cytoplasmic AR (10, 11, 14, 33). The ZIP9 prostate cancers. Breast cancer is asprotein functions as a membrane-bound high-affinity, low-capacity AR to mediate androgensociated with increased tissue zinc induced apoptosis and to increase intracellular Zn2⫹ (16, 17). cells. Studies focused on comparative endocrinology can facilitate identifying and characterizing the evolutionarily conserved functioning receptors of rapid signaling of steroids, including androgens (15). In 2 exciting reports in the current issue of Endocrinology, a new/novel highaffinity, limited-capacity membrane AR zinc-regulated transporters, iron-regulated transporter-like protein 9 (ZIP9) was identified and characterized first in the female Atlantic croaker and subsequently in human breast and prostate cancer cells (16, 17). In previous studies, the Thomas lab reported the existence of a novel nonnuclear AR in the ovaries of the Atlantic croaker for rapid, cell surface-mediated androgenic inhibition of estradiol secretion (18). Androgens were shown to inhibit estradiol production in croaker ovaries even in the presence of nuclear AR antagonist. BSA-conjugated DHT, which does not diffuse through the cell membrane, also inhibited estradiol production. Actinomycin, which inhibits transcription, did not block this inhibitory response. Taken together, these results suggested the presence of a membrane-bound AR. In the current study, these investigators have identified this membrane AR as a protein with a high sequence identity with vertebrate ZIP9 subfamily members (16). The authors subsequently provided evidence that this croaker zinc transporter ZIP9 protein functions as a membrane-bound high-affinity (dissociation constant Kd, 12.7nM), low-capac-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
4122
Pascal and Wang
ZIP9 Membrane AR
concentration, whereas prostate cancer is associated with decreased tissue zinc (27), suggesting potential differences in the tumor microenvironment and other factors controlling tumor growth and progression. Studies have demonstrated that breast xenograft tumor growth was inhibited by zinc depletion in male rats (28) and decreased dietary zinc suppressed N-methyl-N-nitrosourea-induced rat mammary tumorigenesis (29), whereas the ability to accumulate zinc in prostate cancer cell lines decreases with increasing tumorigenicity (30, 31). In a testosterone-depleted microenvironment, where prostate tumor cells already overexpress ZIP9, activating ZIP9 without activating the genomic AR pathway could disproportionately increase zinc levels and trigger proapoptotic gene expression in cancer cells, thereby selectively inducing apoptosis in these cells. However, breast tumor growth appears to be stimulated by increased zinc levels, and activation of ZIP9 in vivo might have deleterious effects. Future in vivo studies will no doubt shed light on this potential differential response in the development and progression of tumors as well as in the development and maintenance of normal tissue homeostasis. In summary, androgen signaling includes genomic and nongenomic pathways that may act coordinately or independently in healthy prostate maintenance as well as the development and progression of prostatic disease (Figure 1). Genomic or nuclear androgen signaling is slower than nongenomic, membrane-initiated signaling and regulates the transcription of thousands of androgen-responsive genes. Nongenomic androgen signaling can be mediated through membrane-associated AR and/or ZIP9, which is rapid and initiated at the cell surface and/or cytoplasm to regulate several pathways, including MAPK/ERK, FAK, p38, p53, calcium, and zinc (10, 12, 13, 16, 17, 32, 33). Genomic and nongenomic androgen signaling pathways may cross talk and converge to control cell proliferation and/or apoptosis. Identification of ZIP9 as a membranebound AR provides exciting research opportunities to determine the role of nongenomic androgen signaling via AR vs ZIP9 in development, tissue maintenance, and diseases.
Acknowledgments Address all correspondence and requests for reprints to: Zhou Wang, PhD, Department of Urology, University of Pittsburgh School of Medicine-University of Pittsburgh Medical Center Shadyside, 5200 Centre Avenue, Suite G40, Pittsburgh, PA 15232. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.
Endocrinology, November 2014, 155(11):4120 – 4123
References 1. Anderson KM, Liao S. Selective retention of dihydrotestosterone by prostatic nuclei. Nature. 1968;219:277–279. 2. Bruchovsky N, Wilson JD. The intranuclear binding of testosterone and 5␣-androstan-17-ol-3-one by rat prostate. J Biol Chem. 1968; 243:5953–5960. 3. Liao S, Fang S. Receptor-proteins for androgens and the mode of action of androgens on gene transcription in ventral prostate. In: Vitamins and Hormones. New York, NY: Academic Press, Inc; 1969:17–90. 4. Liang T, Liao S. A very rapid effect of androgen on initiation of protein synthesis in prostate. Proc Natl Acad Sci USA. 1975;72: 706 –709. 5. Chang CS, Kokontis J, Liao ST. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988; 240:324 –326. 6. Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS, Wilson EM. Cloning of human androgen receptor complementary DNA and localization to the X chromosome. Science. 1988;240: 327–330. 7. Gorczynska E, Handelsman DJ. Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology. 1995; 136:2052–2059. 8. Lyng FM, Jones GR, Rommerts FF. Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations. Biol Reprod. 2000;63:736 –747. 9. Benten WP, Lieberherr M, Giese G, et al. Functional testosterone receptors in plasma membranes of T cells. FASEB J. 1999;13:123– 133. 10. Peterziel H, Mink S, Schonert A, Becker M, Klocker H, Cato AC. Rapid signalling by androgen receptor in prostate cancer cells. Oncogene. 1999;18:6322– 6329. 11. Gatson JW, Kaur P, Singh M. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells. Endocrinology. 2006;147: 2028 –2034. 12. Gu S, Kounenidakis M, Schmidt EM, et al. Rapid activation of FAK/mTOR/p70S6K/PAK1-signaling controls the early testosterone-induced actin reorganization in colon cancer cells. Cell Signal. 2013;25:66 –73. 13. Papakonstanti EA, Kampa M, Castanas E, Stournaras C. A rapid, nongenomic, signaling pathway regulates the actin reorganization induced by activation of membrane testosterone receptors. Mol Endocrinol. 2003;17:870 – 881. 14. Liao RS, Ma S, Miao L, Li R, Yin Y, Raj GV. Androgen receptormediated non-genomic regulation of prostate cancer cell proliferation. Transl Androl Urol. 2013;2:187–196. 15. Thomas P. Rapid steroid hormone actions initiated at the cell surface and the receptors that mediate them with an emphasis on recent progress in fish models. Gen Comp Endocrinol. 2012;175:367–383. 16. Berg AH, Rice CD, Rahman MS, Dong J, Thomas P. Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female Atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells. Endocrinology. 2014;155:4237– 4249. 17. Thomas P, Pang Y, Dong J, Berg AH. Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: II. Role of human ZIP9 in testosterone-induced prostate and breast cancer cell apoptosis. Endocrinology. 2014; 4250 – 4265. 18. Braun AM, Thomas P. Androgens inhibit estradiol-17 synthesis in Atlantic croaker (Micropogonias undulatus) ovaries by a nongenomic mechanism initiated at the cell surface. Biol Reprod. 2003; 69:1642–1650.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
doi: 10.1210/en.2014-1749
19. Costello LC, Feng P, Milon B, Tan M, Franklin RB. Role of zinc in the pathogenesis and treatment of prostate cancer: critical issues to resolve. Prostate Cancer Prostatic Dis. 2004;7:111–117. 20. Zaichick VYe, Sviridova TV, Zaichick SV. Zinc in the human prostate gland: normal, hyperplastic and cancerous. Int Urol Nephrol. 1997;29:565–574. 21. Johnson LA, Kanak MA, Kajdacsy-Balla A, Pestaner JP, Bagasra O. Differential zinc accumulation and expression of human zinc transporter 1 (hZIP1) in prostate glands. Methods. 2010;52:316 –321. 22. Franklin RB, Feng P, Milon B, et al. hZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer. Mol Cancer. 2005;4:32. 23. Liang JY, Liu YY, Zou J, Franklin RB, Costello LC, Feng P. Inhibitory effect of zinc on human prostatic carcinoma cell growth. Prostate. 1999;40:200 –207. 24. Feng P, Li T, Guan Z, Franklin RB, Costello LC. The involvement of Bax in zinc-induced mitochondrial apoptogenesis in malignant prostate cells. Mol Cancer. 2008;7:25. 25. Feng P, Liang JY, Li TL, et al. Zinc induces mitochondria apoptogenesis in prostate cells. Mol Urol. 2000;4:31–36. 26. Ku JH, Seo SY, Kwak C, Kim HH. The role of survivin and Bcl-2 in
endo.endojournals.org
27.
28. 29.
30.
31.
32.
33.
4123
zinc-induced apoptosis in prostate cancer cells. Urol Oncol. 2012; 30:562–568. Gumulec J, Masarik M, Adam V, Eckschlager T, Provaznik I, Kizek R. Serum and tissue zinc in epithelial malignancies: a meta-analysis. PLoS One. 2014;9:e99790. Mills BJ, Broghamer WL, Higgins PJ, Lindeman RD. Inhibition of tumor growth by zinc depletion of rats. J Nutr. 1984;114:746 –752. Lee S, Simpson M, Nimmo M, Xu Z. Low zinc intake suppressed N-methyl-N-nitrosourea-inducedmammarytumorigenesisinSpragueDawley rats. Carcinogenesis. 2004;25:1879 –1885. Costello LC, Franklin RB. Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer. Prostate. 1998;35:285–296. Huang L, Kirschke CP, Zhang Y. Decreased intracellular zinc in human tumorigenic prostate epithelial cells: a possible role in prostate cancer progression. Cancer Cell Int. 2006;6:10. Benten WP, Lieberherr M, Sekeris CE, Wunderlich F. Testosterone induces Ca2⫹ influx via non-genomic surface receptors in activated T cells. FEBS Lett. 1997;407:211–214. Zoubeidi A, Zardan A, Beraldi E, et al. Cooperative interactions between androgen receptor (AR) and heat-shock protein 27 facilitate AR transcriptional activity. Cancer Res. 2007;67:10455– 10465.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 May 2015. at 08:38 For personal use only. No other uses without permission. . All rights reserved.
