Concise Review: Androgen Receptor Differential Roles in Stem/Progenitor Cells Including Prostate, Embryonic, Stromal, and Hematopoietic Lineages

Authors

  • Chiung-Kuei Huang,

    1. Departments of Pathology, Urology, Radiation Oncology, the George Whipple Lab for Cancer Research, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA
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  • Jie Luo,

    1. Departments of Pathology, Urology, Radiation Oncology, the George Whipple Lab for Cancer Research, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA
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  • Soo Ok Lee,

    1. Departments of Pathology, Urology, Radiation Oncology, the George Whipple Lab for Cancer Research, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA
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  • Chawnshang Chang

    Corresponding author
    1. Departments of Pathology, Urology, Radiation Oncology, the George Whipple Lab for Cancer Research, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA
    2. Sex Hormone Research Center, China Medical University/Hospital, Taichung, Taiwan
    • Correspondence: Chang, Ph.D., George Whipple Lab for Cancer Research, University of Rochester Medical Center, Rochester, New York 14642. Telephone: 585-273-4500; Fax; 585-756-4133; e-mail: chang@urmc.rochester.edu

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Abstract

Stem/progenitor (S/P) cells are special types of cells that have the ability to generate tissues throughout their entire lifetime and play key roles in the developmental process. Androgen and the androgen receptor (AR) signals are the critical determinants in male gender development, suggesting that androgen and AR signals might modulate the behavior of S/P cells. In this review, we summarize the AR effects on the behavior of S/P cells, including self-renewal, proliferation, apoptosis, and differentiation in normal S/P cells, as well as proliferation, invasion, and self-renewal in prostate cancer S/P cells. AR plays a protective role in the oxidative stress-induced apoptosis in embryonic stem cells. AR inhibits the self-renewal of embryonic stem cells, bone marrow stromal cells, and prostate S/P cells, but promotes their differentiation except for adipogenesis. However, AR promotes the proliferation of hematopoietic S/P cells and stimulates hematopoietic lineage differentiation. In prostate cancer S/P cells, AR suppresses their self-renewal, metastasis, and invasion. Together, AR differentially influences the characteristics of normal S/P cells and prostate cancer S/P cells, and targeting AR might improve S/P cell transplantation therapy, especially in embryonic stem cells and bone marrow stromal cells. Stem Cells 2014;32:2299–2308

Introduction

Stem cells were first identified by Ernest A. McCulloch and James E. Till in the 1960s [1]. They represent one special type of cells that have the ability to generate tissues throughout their entire lifetime. Currently, there are two major kinds of normal stem cells: embryonic stem cells (ESCs) and adult stem cells. ESCs are pluripotent stem cells derived from the inner mass of a blastocyst [2]. It has been shown that several organs contain stem cells, including hematopoietic stem progenitor cells (HSPCs), mammary stem cells, intestinal stem cells, bone marrow stromal cells (BMSCs), endothelial stem cells, testicular stem cells, prostate stem cells (PSCs), neural stem cells, etc [3]. Recently, more attention has been given to the stem cell studies due to the potential therapeutic applications of stem cells in treating different kinds of diseases. To better use stem cells in clinical applications, it is important to explore the signaling pathways inside stem cells and determine the best condition to maintain stemness of stem cells in vitro and in vivo.

Numerous studies have revealed that, in addition to normal stem cells, cancer stem/progenitor (S/P) cells, which have self-renewal and tumorigenesis abilities, also exist in tumors [4, 5]. Cancer S/P cells have several characteristics, including high tumorigenesis ability, high metastatic ability, and relatively high resistance to the traditional cancer therapies [6]. Therefore, targeting cancer S/P cells is emerging as a new study area in treating cancers.

Androgen and the androgen receptor (AR) signals play key roles in the male reproductive system [7]. Binding of testosterone and its metabolite, 5α-dihydroxytestosterone (DHT), to AR would induce AR dimerization, translocation into nuclei, and binding to the AR response elements (AREs) to regulate gene expression [8]. The expression of AR has been detected in various tissues other than those of the reproductive system [7, 9-13]. Deficiency of AR causes several diseases [14, 15] and AR is one of the key determinants in prostate cancer (PCa). AR promotes the proliferation of PCa while suppressing its metastasis, suggesting the dual roles of AR in PCa progression [16]. Interestingly, AR is not expressed in PSCs but is in most of the other types of stem cells [17]. Recently, several groups reported that characterization of several stem cells can be influenced by androgen and AR signals either in vitro or in vivo [11, 18-20]. This evidence might suggest that the AR signal participates in the regulation of normal or cancer S/P cells and modulation of the AR signal could be robustly significant in improving current tissue transplantation and cancer therapies. In this review, we will summarize the roles of AR in several kinds of normal stem cells and prostate cancer S/P cells.

AR Roles in Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent and capable of differentiating into any kind of cell belonging to germ layers, including endoderm, mesoderm, and ectoderm. Due to their pluripotent differentiation ability, ESCs are potential therapeutic agents in treating several diseases, such as strokes, osteogenesis imperfecta, myocardial infarction, and neurodegenerative diseases that require the replacement of defective cells with functional cells. To apply ESCs as potential therapeutic treatment in those diseases, it is essential to understand how ESCs respond to differentiation signals and whether scientists can deliver ESCs to the right functional cells in the target organs. Recently, it has been demonstrated that AR is detectable in the inner cell mass, blastocysts, and ESCs and androgen levels are around 1–4 nM in pregnant women [20, 21]. Together, it is obvious that AR might be functional in ESCs self-renewal and differentiation. In fact, it has been shown that AR is transcriptionally functional in ESCs. As the ESCs differentiate, the AR levels continue to increase [22], thus suggesting that AR might promote ESCs differentiation but suppress their self-renewal capacity [22, 23].

AR Suppresses ESCs Self-Renewal

It has been shown that androgen treatment has no significant effects on the self-renewal ability of mouse ESCs [20]. However, AR expressions were increased as embryogenesis progressed, suggesting that AR signaling might suppress ESCs self-renewal capacity [20]. This hypothesis was confirmed through treating ESCs with the antiandrogen, nilutamide, which revealed that nilutamide treatment could significantly stimulate the growth of ESCs and inhibition of AR signals could enhance the self-renewal of ESCs. Mechanistically, antiandrogen treatment promotes self-renewal of ESCs through controlling their cell cycle via Akt and p27 [20]. Interestingly, a recent study demonstrated that testosterone treatment could inhibit proliferation of germ-like cells derived from human ESCs [24]. Although this study showed results consistent with the previous finding that antiandrogen promotes self-renewal of mouse ESCs, the testosterone concentration used in the study was above the physiological range (1–30 nM testosterone in human) and germ-like cells might not possess the same characteristics as ESCs. Further investigation might be needed in order to conclusively determine the roles of androgen and AR signaling in human ESCs. However, the results of mouse ESCs studies, at least, suggest that AR might suppress the self-renewal capacity of ESCs through indirect regulation of Akt and p27 signaling.

AR Protects ESCs from Oxidative Stress-Induced Cell Death

Oxidative stress is mainly caused by reactive oxygen species (ROS), which result from incomplete oxygen reduction. Although low levels of ROS play vital roles in maintaining physiological functions, it has been shown that oxidative stress plays a deleterious role in the embryonic development [25]. As previously discussed, AR has been detected in the inner cell mass and ESCs. Therefore, it would be of interest to clarify AR function in oxidative stress-induced cell death during embryonic development. Actually, it has been shown that H2O2-induced oxidative stress in ESCs could be partially protected against by DHT treatment. H2O2 could induce DNA fragmentation and cell death through activating the phosphorylation of p38, SPAK/JNK, and nuclear factor kappa B (NF-κB) in ESCs. Pretreatment with DHT could partially reverse this H2O2-induced cell death through blocking the phosphorylation of p38, SPAK/JNK, and NF-κB [26]. In addition, DHT could protect ESCs from H2O2-induced apoptosis via enhancing the antioxidant enzyme, catalase, and treatment with the antiandrogen, flutamide, could reverse the protective effects of DHT [27]. Since flutamide competes with DHT to bind to the AR, the DHT-mediated protection in H2O2-induced apoptosis is AR dependent. In conclusion, androgen and AR signaling play an indirect protective role in oxidative stress-induced apoptosis in ESCs.

AR Promotes ESCs Differentiation into Cardiomyocytes

Controlling ESCs differentiation is a critical step for the regenerative medical application of ESCs. Although the differentiation of ESCs to the three germ layers has been well investigated [28], only one study investigated androgen effects on ESCs differentiation [23]. This study mainly focused on the effect of androgen on ESCs differentiation into beating cardiomyocytes. Testosterone treatment could enhance cardiomyocytes differentiation from ESCs and flutamide treatment could reverse these effects, suggesting that the effects of androgen on cardiomyocytes differentiation are AR dependent. Interestingly, the study also explored testosterone secretion from ESCs and showed that male mouse ESCs have testosterone secretion rates similar to human Leydig cells but two times higher than female ESCs. Although female ESCs have less androgen production than do male ESCs, androgen stimulation has similar effects on cardiomyocytes differentiation in ESCs of both sexes. This finding indicates that ESCs could partially influence their cardiomyocytes differentiation capacity by controlling their AR signaling through androgen secretion, but this regulation is not gender or androgen dose dependent [23]. In summary, AR plays a promotional role in ESCs differentiation into cardiomyocytes through directly controlling AREs, since the stimulation effects of AR could be reversed by antiandrogen treatment.

AR Roles in Normal Prostate and Prostate Cancer Stem and Progenitor Cells

AR is the key regulator in prostate development and PCa progression. In normal prostate and PCa cells, AR plays a positive role in promoting cell proliferation and differentiation [7, 29]. Since AR is critical and essential in PCa progression, the current gold standard treatment for PCa is to remove the source of androgen production by either surgical or chemical castration. Although castration could inhibit PCa progression, PCa would eventually recur to become castration resistant PCa. At this advanced stage of PCa, castration is no longer effective because there is still around 1 nM androgen inside the tumor and it has been shown that 1 nM androgen could promote PCa proliferation [30, 31]. Due to this reason, the third generation of antiandrogens (i.e. MDV3100) has been developed and shown to extend advanced PCa patients' life for a few months [32]. Unfortunately, advanced PCa patients still cannot survive following treatment with MDV3100, suggesting that there might be other reasons that tumors escape the antiandrogen treatment. Recently, prostate cancer stem cells (PCSCs) have emerged as the potential candidates that could escape the antiandrogen treatment. However, it remains unclear whether PCSCs could really escape the treatment. To help clarify this issue, it is essential to summarize the roles of AR in normal PSCs and PCSCs, since AR is critical in prostate and PCa development.

Normal Prostate Stem/Progenitor Cells

In the prostate, there are two major types of epithelial cells: luminal versus basal epithelial cells [16]. The CK8-positive luminal epithelial cells are the major type and have high AR expression. Beneath the luminal epithelial cells, there are one or two layers of basal epithelial cells, which attach to the basement membrane [33]. The basal epithelial cells express CK5 and CK14, integrin α6β1, CD44, and p63. In the normal prostate, p63 is important for the maintenance and differentiation of S/P cells [34]. CD44 has also been characterized as a PCa S/P cells marker [4]. Currently, scientists believe that prostate S/P cells exist in the basal layers [35]. However, a recent report indicated that one type of mouse luminal epithelial cells, which expresses Nkx3.1 under castration conditions and has self-renewal ability, could form the prostate duct via a single cell [36], suggesting that S/P-like cells might exist in the luminal epithelial part of the prostate in mice. However, there is no direct evidence showing that there are S/P cells among human prostate luminal epithelial cells.

AR is highly expressed in the luminal epithelial cells but has low expression in the basal epithelial cells, suggesting that AR is lowly expressed in normal prostate S/P cells [35]. In the basal epithelial layer of the human prostate, there is a small population of CD133+/integrin α2β1+ cells, around 1% frequency of total cells isolated from prostate, with characteristics similar to S/P cells [37]. Regarding mouse PSCs, CD49f and Sca1 have been used to identify the stem cell population, which is around 0.5% frequency of total cells isolated from prostate. Indeed, CD49f+/Sca1+ cells have 100-fold higher prostate sphere forming capacity than CD49f+ or Sca1+ cells, and also have tissue regenerating abilities [38]. Importantly, those studies found the expression of AR in these types of prostate S/P cells is very low.

Prostate Cancer Stem/Progenitor Cells

The gold standard treatment for advanced PCa is androgen-deprivation therapy with antiandrogens. However, most patients have a recurrence of castration resistant tumors within a few years and die from metastatic PCa. It has been hypothesized that the PCSCs exist in castration resistant PCa and may influence the PCa progression. There are several published markers used to identify/isolate the S/P cells from PCa cells [4, 5]. However, most scientists have used CD133, CD44, Integrin α6, integrin α2β1, and Sca-1 as PCa S/P cell markers [19, 39-41], and found that the AR-negative population of primary human PCa cells has the highest proliferative ability and S/P cell characteristics [41]. Consistently, AR expression is low in PCa S/P cells, and the PCa cell population with low expression of prostate-specific antigen, a direct target gene of AR, exhibit higher self-renewal ability after castration, indicating that low AR activity may increase the PCa S/P cell population [42]. Importantly, PCa S/P cell population increased after androgen deprivation [43]. In addition, knockout of AR in the epithelial cells resulted in the expansion of the CD44-positive PCa S/P cells (also viewed as CK5/CK8 positive basal intermediate cells) in TRAMP mice [44]. In human PCa samples, the expression of CD133, CD44, and integrin α2β1 increased significantly after castration, and addition of AR-siRNA in CD133-positive PCa S/P cells led to the increased S/P cell population [19]. These findings suggested that the AR in PCa S/P cells plays important yet differential roles as compared to its roles in PCa non-S/P cells. The roles of AR in the PCa S/P cells self-renewal capacity and high tumorigenesis or metastatic abilities will be further discussed in the next sections.

AR Role in PCSC Self-Renewal

One of the gold standards to identify S/P cells is the self-renewal ability. The AR-negative PCa cell lines have higher sphere formation ability compared to the AR-positive PCa cells [45]. Using AR-siRNA to reduce AR expression led to increase the sphere numbers of PCa cells [19], indicating that AR inhibits the self-renewal ability of PCa cells. Consistently, overexpressing AR in the CD133+ cells, which are isolated from PCa LNCaP or C4-2 cell lines, dramatically reduces the number of spheres [19]. This is the first direct evidence to confirm that AR could suppress the self-renewal ability of PCa S/P cells. Intriguingly, the use of the demethylation agent, 5-Azacytidine (5-AZA), to treat PCa S/P cells, demonstrated that the sphere numbers were significantly reduced, suggesting that induction of AR expression, through demethylating the AR promoter region, suppressed the self-renewal ability of PCa S/P cells [17].

There are several important proteins regulating the self-renewal ability of S/P cells. The canonical Wnt signaling pathway could upregulate the self-renewal ability of PCa S/P cells in the PCa cell lines, LNCaP and C4-2 [46]. AKT/PI3K signaling plays an important role in the sphere formation ability of PCa cells [47]. Knocking-down of the AR in the PCa cells could induce the expression of OCT4, NANOG, and SOX2 genes, which are important S/P cell factors in regulating self-renewal ability (Chang et al. unpublished data and [48-50]). In contrast, overexpression of AR can suppress the AKT and Wnt signaling pathways to attenuate the self-renewal ability of LNCaP and C4-2 cells S/P populations [19]. Taken together, AR negatively modulates the self-renewal ability of PCSCs either through direct transcriptional regulation, such as SOX2 gene, or indirectly modulating OCT4.

AR Role in PCSC Invasion

Cancer S/P cells are highly associated with metastatic tumors [51], and cancer S/P cells possess the characteristic of epithelial-mesenchymal transition (EMT) that plays key roles in the PCa metastasis [52]. Actually, the PCa epithelial AR is a suppressor for PCa metastasis and addition of functional AR-cDNA in the AR negative PC3 cells suppressed PCa metastasis [53]. In addition, suppression of AR signals via the cytokine CCL5 resulted in increased PCa metastasis [54]. Another study further suggested that AR signaling is linked to the alteration of EMT, and androgen deprivation might induce EMT that resulted in induction of zinc-finger E-box binding homeobox 1 expression to enhance metastasis [55]. Furthermore, infiltrating BMSCs also could suppress AR signals in PCa cells resulting in increased PCa S/P cell population, which in turn enhanced PCa metastasis [54]. Consistently, addition of the antiandrogens, Casodex and MDV3100, in PCa S/P cells could increase S/P cell invasion ability [56], whereas overexpression of AR in PCa S/P cells suppresses invasion.

In addition to the EMT process, AR also controls cancer metastasis through regulating microRNA (miR) expression. Chang et al. found that the CD133+ PCa S/P cells have relatively lower miR331 expression compared to non-S/P cells (unpublished data). miR331 is an important suppressor of ERBB2, which is a key proto-oncogene [57]. Addition of functional AR-cDNA in the CD133+ PCSCs increased the miR331 expression that might suppress the invasion ability of the PCSCs. Together, these data suggest that AR may play a suppressor role in regulating invasion ability of the PCa S/P cells.

AR Roles in BMSCs

BMSCs possess self-renewal and multilineage differentiation capacities. Due to these characteristics, BMSCs have been widely used in clinical trials of regenerative therapy to treat diseases, including osteogenesis imperfecta, myocardial infarction, and liver cirrhosis [58-60]. Moreover, it has been demonstrated that BMSCs could be used to treat inflammatory diseases through anti-inflammation characteristics via a paracrine manner [61]. Although BMSCs are very popular due to their therapeutic potentials, long-term clinical trials displayed unexciting outcomes with diseases recurring after a few years of BMSCs transplantation [62]. Therefore, further enhancing the therapeutic potential of BMSCs is an important issue before clinicians can fully and effectively use BMSCs in clinics.

Differential outcomes caused by gender differences have been observed in BMSCs regarding their activation, as well as response to oxidative stress, injury, and inflammatory stimuli [63]. It also has been suggested that AR might be involved in osteogenesis and adipogenesis of BMSCs. AR basically promotes osteogenesis but suppresses adipogenesis in BMSCs [9]. Since AR is extensively involved in cellular behaviors of BMSCs, characterizing the AR function in the self-renewal and differentiation of BMSCs would be important in using the transplantation therapy of BMSCs in clinics. We summarize current progress regarding AR roles in BMSCs self-renewal and differentiation in the following sections.

AR Suppresses the Self-Renewal of BMSCs

Early studies have suggested that androgen and AR signals negatively regulate the self-renewal process of ESCs [20]. AR knockout (ARKO) mice, generated with agenesis of vas deferens, epididymis, seminal vesicle, testis, and prostate organs, were used to study the self-renewal of BMSCs and the results demonstrated that AR deficiency enhanced the self-renewal of BMSCs [11, 18]. As expected, AR overexpressed transgenic mice exhibit reduced self-renewal potential in BMSCs [64]. Interestingly, studies related to the gender effects on the self-renewal potential of BMSCs remain unclear with one study showing gender has little effects on the BMSCs self-renewal [65], yet another study was able to show that the male gender has suppressive effects on the generation of adipocyte-derived stromal cells (ADSCs), which have been shown to possess characteristics similar to BMSCs [66]. Importantly, ADSCs and BMSCs isolated from ARKO mice exhibit better self-renewal potential than those from wild-type (WT) mice. Further mechanism dissection studies suggest that AR deficiency can promote proliferation of BMSCs through stimulating the activation of AKT and Erk1/2 via elevating epidermal growth factor receptor expression [18]. Collectively, all evidences point to the fact that androgen and AR signals can inhibit the self-renewal potential of BMSCs through indirect regulation of AKT and Erk1/2.

Gender-induced differences have been shown in adipose accumulation with the observation that males have less adipose tissue than females, thus suggesting that androgen and AR signals may inhibit adipogenesis. However, white adipocytes are different from fatty marrow and fatty marrow has been suggested to be involved in osteoporosis [67]. To further address the effects of androgen and AR on BMSC associated adipogenesis and osteoporosis, the effects of androgen and AR on BMSCs differentiation will be discussed in the next section.

Differential AR Roles in BMSCs Differentiation

In earlier decades, BMSCs were recognized by their therapeutic potential through differentiating into osteogenic and chondrogenic cell lineages [68]. Recently, BMSCs differentiation into cardiomyocytes and hepatocytes has also been suggested for the potential treatment of myocardial infarction and liver fibrosis [69, 70] based on the evidence that BMSCs have been shown to colocalize with functional cardiomyocytes and hepatocytes [69, 71]. For the osteogenic and chondrogenic differentiation, BMSCs transplantation therapy has been shown to improve bone-related diseases with new bone formation through osteoblast and chondrocyte differentiation [58]. Therefore, the differentiation potential of BMSCs is crucial for their therapeutic applications.

Androgen and AR signals have been extensively studied in BMSCs regarding their differentiation capacities into myocytes, adipocytes, and osteoblasts. It has been shown that androgen treatments have little effects on alkaline phosphatase activity and calcium deposition, two important indications for osteoblast differentiation in BMSCs, implying that androgen treatment has no effects on osteoblast differentiation. However, ARKO mice display reduced bone formation through controlling osteoblast differentiation and mineralization [72]. With microarray analysis, the osteoblast differentiation-related category genes are downregulated in ARKO mice-derived BMSCs as compared to WT mice-derived BMSCs [9]. For the question of why androgen could not promote BMSCs osteogenesis in vitro, one possible explanation is that the ARs in BMSCs might already be occupied and activated by androgens because the current in vitro culture conditions contain comparable amounts of testosterone (around 1.1 nM in charcoal stripped fetal bovine serum and 2.7 nM in regular fetal bovine serum) in the media [23], and as low as 0.1 nM androgen has been shown to promote AR transactivation activity [73]. Therefore, treatments using androgens might not be able to further promote AR function in osteogenesis of BMSCs. Alternatively, it is also possible that androgen effects may not be equal to AR effects on osteogenesis, which also occurred in several other tissues or cell types [12, 19].

Androgen has been shown to promote myogenesis in BMSCs derived cell lines and castrated rats [74]. In addition, androgen has promotional effects in muscle mass when it is used to treat hypogonadal men, and the effects of androgen in enhancing muscle mass were proved through modulating myogenesis of mesenchymal stem cells in vitro [75]. Mechanistically, testosterone promotes myogenesis of mesenchymal stem cells through crosstalk between AR/β-catenin and follistan/transforming growth factor β signaling pathways. Knockdown of β-catenin could block androgen-promoted myogenesis in mesenchymal stem cells, suggesting that β-catenin is crucial in AR-mediated myogenesis [76]. Collectively, androgen and AR signals play a stimulatory role in myogenesis of BMSCs.

Gender differences have been shown in adipose accumulation [77]. It has been demonstrated that androgen treatments inhibit adipogenesis of preadipocytes and BMSCs [78-80]. These results might partially account for the gender differences in obesity. Several molecular pathways have been proposed to be involved in androgen and AR signals mediated adipogenesis. Androgen treatments could inhibit expressions of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma and CCAAT-enhancer-binding protein alpha, through forming a protein complex consisting of AR, β-catenin, and T-cell factor 4. In addition, androgen treatments could suppress the expression of insulin-like growth factor (IGF) receptor that has been shown to participate in adipocyte differentiation [80]. Moreover, androgen and AR signals could promote expressions of IGF binding protein 3 (IGFBP3) to inhibit the binding ability of IGF and result in less adipogenesis [9]. Taken together, androgen and AR signaling negatively regulate adipogenesis of BMSCs through indirectly inhibiting IGF signaling.

Targeting AR in BMSCs to Enhance Transplantation Therapy of BMSCs in Liver Fibrosis

BMSCs have been applied in treating different kinds of diseases, including myocardial infarction, osteogenesis imperfecta, liver fibrosis, etc, due to their multilineage differentiation capacity, anti-inflammation characteristics, and relatively higher safety as compared to induced pluripotent stem cells, which require several manipulations [58, 60, 81]. Although BMSCs transplantation therapy shows disease relief at the beginning of treatment, eventually the symptoms recur. In addition, it has been suggested that transplanted BMSCs have high apoptosis and low delivery rates due to microischemia [82], suggesting the need to improve the therapeutic efficacy of BMSCs transplantation via enhancing their delivery rate or reducing apoptosis. Consistent with the ESCs studies, knockout of AR in BMSCs increases their self-renewal potential and also stimulates the migration ability of BMSCs, which in turn leads to better delivery rate. Furthermore, knockout of AR can enhance anti-inflammatory and antifibrosis effects of BMSCs through paracrine secretion to suppress liver fibrosis. Although knockout of AR in BMSCs reduces their osteogenesis potential to cause osteoporosis in ARKO mice, AR-deficient BMSCs do have better therapeutic effects on liver fibrosis without influencing the risk of osteoporosis in the treated animals [11]. Taken together, androgen and AR signaling play a deleterious role in the transplantation therapy of BMSCs.

AR Roles in HSCs

Over the past decades, studies have shown that hematopoietic stem cells (HSCs) are useful transplantation resources for treating patients with leukemia. Understanding the proliferation and lineage differentiation of HSCs would help their therapeutic values in treating leukemia. It has been well documented that androgen treatments have stimulatory effects on colony formation units (CFU) of various HSCs, including primitive erythroid progenitor cells, erythrocytes, and granulocyte-macrophage progenitor cells [83-85]. In addition, androgen treatments could enhance the G1-S transit rate in the cell cycle and survival of HSCs [83], and androgen administration could enhance erythropoiesis [86]. Due to these characteristics, androgen treatment was proposed as a therapeutic approach for anemic patients in earlier eras. The clinical trials and experimental results demonstrated that androgen treatment improved erythropoiesis, which is required for anemic patients [87]. However, it is not clear whether AR is essential for androgen-promoted erythropoiesis and how androgen stimulates the CFU of erythroid progenitor cells. The study using testicular feminized (Tfm) mice, which exhibit an X-linked AR mutation resulting in no binding of androgen, shows that androgen has less effectiveness in the erythroid colony-forming cell response of Tfm mice than mice with intact AR [86]. However, the study of ARKO mice displays that there might be other factors contributing to erythropoiesis because there are no differences in hematocrit levels between WT and ARKO mice [12]. Collectively, androgen mediates erythropoiesis through AR-dependent pathways.

One of the recent studies of androgen effects on HSCs showed that androgen increases human telomerase reverse transcriptase gene expression to enhance telomerase activity in human primary hematopoietic cells through estrogen receptor alpha rather than AR. Furthermore, bone marrows isolated from WT and ARKO mice also showed little difference in the expression of the HSCs markers, CD34 and sca1, suggesting that AR may not be required in the androgen-promoted cell cycle and CFU formation of HSCs [18, 88]. In summary, androgen promotes cell cycle and CFU formation of HSCs in an AR-independent manner, whereas androgen stimulates erythropoiesis via an AR-dependent pathway.

AR Role in HSCs Differentiation to B Cells

An early study showed that castration of normal male mice leads to increase of B cells population and enlargement of the spleen [89]. Consistent observations have also been obtained in castrated rats and Tfm mice [90, 91]. Based on immunohistochemical analyses, AR is mainly expressed in BMSCs and B cells, but not in the spleen, suggesting that androgen and AR signaling effects in B cell development might not be through affecting spleen functions [89, 91]. This conclusion is further supported by the study of castrated WT and ARKO mice. Castrated WT mice display more B cells than normal WT mice; and androgen restoration could reverse the increased B cell numbers. ARKO mice also have elevated B cells, however restoring androgen levels in ARKO mice failed to reverse the ARKO effects, suggesting that androgen-mediated B cell maturation is AR dependent. A study with the B cells AR-specific knockout (B-ARKO) mice further concluded that AR is critical in androgen-mediated B cells differentiation, since B-ARKO mice with normal androgen levels also have more B cells than WT mice [10]. However, ARKO mice still have more B cells than do B-ARKO mice, indicating that AR in other cell types must be involved in the population regulation of B cells. In another study, the conditioned medium generated from DHT-treated BMSCs showed suppressive effects on B-cell colony formation, but the conditioned medium collected from DHT-treated BMSCs of Tfm mice could not affect B cell colony formation, suggesting that the AR in BMSCs is responsible for the observed difference of B-cell numbers between ARKO and B-ARKO mice [92]. Lymphocyte analysis suggests that loss of androgen and AR signals causes an increase of B cell progenitors and precursors [91]. Taken together, loss of androgen and AR signals promote B cell development from HSCs.

AR Role in HSCs Differentiation to T Cells

About a century ago, a human study demonstrated that castration causes enlargement of the thymus [93]. In an animal study, castration significantly increased T cell numbers and testosterone replacement dramatically reduced T cells in castrated mice [94]. Androgen might affect T cell numbers through modulating T cell proliferation and apoptosis, since androgen treatments significantly inhibit T cell proliferation [95]. Although Tfm mice also exhibit thymus enlargement, androgen replacement could not reverse this phenotype. Using bone marrow transplantation (BMT) strategy, Tfm mice that received WT BMT developed enlarged thymus regardless of androgen replacement, suggesting that AR in HSCs might not be crucial in T cell development [96]. This conclusion was further supported with the tissue-specific ARKO mice studies, including thymocyte, fibroblast, and thymic epithelial cell-specific ARKO mice. Consistently, ARKO mice develop enlarged thymus and increased T cells. However, these phenotypes were not observed in thymocyte and fibroblast-specific ARKO mice [13]. Using BMT strategy, it has been concluded that the AR in bone marrow cells had no effects on thymus enlargement and thymocytes increase. They reciprocally transplanted ARKO bone marrow into WT mice and WT bone marrow into ARKO mice, and observed no differences between groups. Surprisingly, thymic epithelial cell-specific ARKO mice did develop enlarged thymus. They also found that T cells positive selection is altered in thymic epithelial cell-specific ARKO mice; however, the molecular mechanism by which thymic epithelium AR modulates T cells positive selection was not clear [13]. Collectively, AR might not directly affect T cell differentiation and development in HSCs, but could affect thymic function in positive selection for T cells, or T cell proliferation and apoptosis [13].

AR Role in HSCs Differentiation to Neutrophils

AR has been shown to be expressed in neutrophil progenitors, band neutrophils, and segmented neutrophils [97]. Neutrophils, unlike B and T cells, are decreased after castration, implying that androgen and AR signals could positively regulate neutrophil development. Consistent observations were obtained in ARKO mice and Tfm mice. ARKO mice which developed neutropenia only have one-tenth of the neutrophils of WT mice. Further analyses in ARKO mice of neutrophil lineage differentiation showed that myelocytes/metamyelocytes and mature neutrophils are significantly reduced. Cell cycle analyses and BrdU incorporation results revealed that neutrophils of ARKO mice have reduced S and G2/M phases in the cell cycle as well as reduced proliferation ability. In the molecular mechanism study, neutrophils of ARKO mice were not sensitive to the treatment of granulocyte colony-stimulating factor that was caused by interupting the interaction between AR and E3 SUMO-protein ligase (PIAS3), which is a cosuppressor for Stat3 transcriptional activity [12]. Clinical observations show that castrated PCa patients have fewer neutrophils than intact patients [12]. Young women who suffered from polycystic ovarian syndrome have hyperandrogenism and elevated neutrophil counts, and the increase of neutrophils could be overcome via treatment with the antiandrogen, flutamide, suggesting that androgen and AR signaling promote neutrophil development [98]. This suggestion was further validated by another study demonstrating that androgen treatment accelerates neutrophil precursor's maturation in the in vivo mouse system [99]. Collectively, androgen and AR signals play a stimulatory role in neutrophil differentiation from HSCs.

Conclusion and Future Perspectives

Androgen and AR signals play important roles in male gender development and differentiation of stem cells is one of the most critical steps in development. As expected from the developmental studies, ARKO mice have small testes, no prostate, and are sterile, suggesting the influence of androgen and AR signals or PSCs [7]. In addition, AR is expressed in most S/P cells except in PSCs where AR is lowly expressed [17], and AR is gradually increased as PSCs differentiate [35]. In this regard, AR is obviously important in controlling prostate differentiation. However, AR is silent or has low expression in PSCs until PSCs perceive the differentiation signals, start to express AR, and then initiate the differentiation process. In the molecular mechanism studies, the methylation of AR promoter is one of the causes of AR suppression in PSCs. With the 5-AZA demethylating agent treatment, AR expression was elevated in PSCs. These studies concluded that AR is essential in the differentiation process of PSCs and suppression of AR could maintain the stemness of PSCs.

In addition, there have been more reported phenotypes in ARKO mice than the ones mentioned above [100]. To sum up how androgen and AR signals modulate the behaviors of S/P cells and result in observed phenotypes in ARKO mice, we illustrate the molecular mechanisms by which androgen and AR signals regulate the behaviors of S/P cells in Figure 1 and summarize the studies in ARKO mice, cell lines, castrated animals, and clinical observations, with several conclusions regarding androgen and AR signaling effects on stem cell behaviors in Table 1. Androgen and AR signals suppress self-renewal of ESCs, promote their differentiation into cardiomyocytes, and play a protective role in oxidative stress-induced cell death. Androgen and AR signals suppress self-renewal of PSCs but promote PSCs differentiation into prostate lineages. For PCSCs, androgen and AR signals are mainly involved in suppressing self-renewal of PCSCs, although they also suppress metastasis of PCSCs. In BMSCs, consistent with ESCs, androgen and AR signals suppress self-renewal and adipogenesis, but promote myogenesis and osteogenesis. Regarding HSPCs, although ARKO mice still have an intact immune system with all differentiated hemapoietic lineages, androgen and AR signals can still affect hemapoietic lineages differentially. Androgens promote proliferation of HSPCs. Androgen and AR signals stimulate differentiation of erythropoiesis and neutrophils, but suppress B cell differentiation. Androgen and AR signals affect proliferation and apoptosis of T cells instead of modulating differentiation. Together, androgen and AR signals differentially modulate behaviors of stem cells in the embryonic stage and adult organs.

Figure 1.

Androgen and AR effects on different kinds of stem/progenitor cells. Abbreviations: Akp, alkaline phosphatase; EGFR, epidermal growth factor receptor; EMT, epithelial to mesenchymal transitions; FAS/FASL, Fas antigen/Fas ligand; hTERT, human telomerase reverse transcriptase; IBSP, bone sialoprotein; IGFBP3, insulin-like growth factor receptor binding protein 3; IGF1/IGF1R, insulin-like growth factor 1/insulin-like growth factor receptor 1; IL1Ra, interleukin-1 receptor antagonist; IL1/IL1R, interleukin-1/interleukin-1 receptor; MHC, muscle-specific myosin heavy chain; miR331, microRNA 331; MMP9, matrix metallopeptidase 9; MyoD, myoblast determination protein 1; pErk1/2, phosphor-mitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PIAS, protein inhibitor of activated STAT; pStat3, phosphor-signal transduction and activator of transcription 3; TCF4, Transcription factor 4; TGFβ, transforming growth factor beta; Wnt, Wingless and INT-1; ZEB1, zinc-finger E-box binding homeobox 1.

Table 1. Androgen and AR effects on S/P cell behaviors
Stem/progenitor cell typesAndrogen and AR effectsAssay proceduresReferences
  1. Abbreviations: AR, androgen receptor; S/P, stem/progenitor.

Prostate cancer S/P cellsInhibit self-renewalSphere forming assay; in vivo cancer initiation frequency[19, 42]
Prostate S/P cellsInhibit self-renewal; promote differentiationSphere-forming assay; differentiation markers[35, 37, 38]
Bone marrow stromal cellsInhibit self-renewal; promote myogenic differentiation and osteogenesis; inhibit adipogenesisColony-forming fibroblast unit, in vivo transplantation; differentiation assay[9, 11, 18]
Embryonic stem cellsInhibit self-renewal; promote cardiomyocytes differentiationProliferation assay; differentiation assay[20, 22]
Hematopoietic stem cellsPromote colony formation of hematopoietic stem cells, enhance erythropoiesis and neutrophil differentiation, inhibit B cells development, suppress cell proliferationCell lineage differentiation markers, studies in androgen receptor knockout mice, colony-forming lineage assay, clinical studies[10, 12, 13, 83, 84]

Further studies in exploring androgen and AR effects on S/P cells would not only advance our knowledge and understanding of androgen and AR signals in functions and behaviors of S/P cells but also help with optimizing the best condition in transplantation therapy of S/P cells.

Acknowledgments

We thank Karen Wolf for help with this manuscript preparation and critical review. We also thank Joshua Cho for help with grammatical error corrections. This work was supported by NIH Grants (CA127300 and CA156700) and Taiwan Department of Health Clinical Trial and Research Center of Excellence Grant DOH99-TD-B-111-004 (China Medical University, Taichung, Taiwan).

Author Contributions

C.K.H., J.L., and S.L.: manuscript writing; C.C.: financial support and manuscript writing. C.K.H. and J.L. are co-first authors.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

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