REVIEW URRENT C OPINION
Regulators of prostate cancer stem cells Candace L. Kerr a,c and Arif Hussain a,b,d
Purpose of review Significant advances have been made toward identifying prostate cancer stem cells (CSCs). This review will highlight the latest developments in defining this population and the discovery of mechanisms involved in their survival and metastasis. Recent findings Several groups have identified master regulators of stem cells in prostate cancer. These include genetic and epigenetic factors known to control pluripotency in embryonic stem cells and in highly metastatic prostate tumors. For instance, tumors of patients with poor prognosis demonstrate elevated levels of the pluripotent markers OCT4 and SOX2 as well as the polycomb complex protein Bmi-1 and enhancer of zeste homolog 2. Cells that are derived from these patient tumors provide an opportunity to expand our current knowledge regarding how these cells survive and the mechanisms that regulate their proliferation. Summary Understanding the mechanisms of highly invasive and therapy resistant prostate cancer cells resides in understanding the CSCs, which facilitate cancer recurrence. Some of these factors are just emerging and provide a platform for developing targeted drugs for the future treatment of advanced prostate cancer. Keywords cancer stem cells, metastasis, proliferation, prostate cancer, tumorigenicity
INTRODUCTION Prostate cancer continues to be the most frequently diagnosed cancer in men and a leading cause of cancer-related deaths [1]. A major contributing factor to prostate cancer-related deaths is the eventual development of resistance to androgen deprivation therapy (ADT) amongst men being treated with ADT for their advanced disease. Several mechanisms related to androgen biosynthesis and/or the androgen receptor can occur during the development of ADT resistance. In addition, there is a growing body of evidence that some of this androgen resistance may be because of the presence of prostate cancer stem cells (CSCs), which can persist after chemotherapy, radiation or androgen deprivation. These highly undifferentiated cells represent a small percentage of the heterogeneity seen in metastatic tumors and express markers of normal prostate stem cells. Currently, there are two models that are believed to contribute to tumor development. One model, known as the stochastic model, assumes that all cancer cells have an equal probability of regenerating a tumor and that cancer develops in a randomized fashion from any given cell when it develops the capacity to proliferate and regenerate a www.co-oncology.com
tumor. Another model, which was originally proposed by Rudolf Virchow, a German pathologist in the mid-19th century, is the model of the CSC [2]. There has been increasing experimental evidence to support the existence of CSCs, particularly over the past three decades. The CSCs are a very small subset of cells within the tumor population, which contribute to tumor growth and metastasis [3–14]. These CSCs exhibit properties of normal stem cells, and are unlike the bulk of cancer cells making up a tumor because the latter cells are more differentiated and non-tumorigenic. Despite a growing body of evidence for the existence of prostate CSCs, many questions still
a Department of Biochemistry and Molecular Biology, bDepartment of Medicine and Greenebaum Cancer Center, University of Maryland School of Medicine, cDepartment of Obstetrics and Gynecology, Johns Hopkins University and dBaltimore VA Medical Center, Baltimore, Maryland, USA
Correspondence to Professor Arif Hussain, Bressler Research Building, Room 9–041 22 S Greene St, Baltimore, Maryland 21201, United States. Tel: +1 (410) 328 7225; fax: +1 (410) 328-2578; e-mail: [email protected] Curr Opin Oncol 2014, 26:328–333 DOI:10.1097/CCO.0000000000000080 Volume 26 Number 3 May 2014
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Regulators of prostate cancer stem cells Kerr and Hussain
KEY POINTS Prostate CSCs have been isolated from prostate cancer cell lines and patient tumors which demonstrate selfrenewing and differentiating abilities. Prostate CSCs demonstrate distinguishing features from bulk tumor cells, including expression of embryonic-like stem cell transcription factors Oct4, Nanog, p63 and Sox2. Prostate CSCs can be derived either by spheroid culture or by cell surface marker selection using a combination of stem cell markers such as CD44, CD133, Sca-1 and a2b1. Prostate CSCs rely on a number of recently identified miRNAs and epigenetic regulators such as Bmi-1 and EZH2 to maintain stem cell properties
remain about the nature of these cells and the mechanisms that drive their stem cell-like behavior. Much of the current knowledge about prostate CSCs can be attributed to the recent wealth of information that has sprung from developmental studies characterizing early stem cells and adult stem cells.
NORMAL PROSTATE STEM CELLS AND THE ORIGIN OF CANCER STEM CELLS Prostate CSCs are similar to normal prostate stem cells in that they are long lived, slow cycling cells that remain quiescent in an undifferentiated or embryonic-like state. As such, prostate CSCs express many early developmental markers of normal stem cells. The mature prostate is made up of a stratified layer of immature cuboidal basal cells attached to the basement membrane with differentiated luminal cells projecting into the lumina. There are also neuroendocrine cells scattered throughout (reviewed in [15 ]). Luminal cells represent the majority of cells in the normal and hyperplastic prostate, they depend on androgens for survival, and they secrete PSA and prostatic acid phosphatase (PAP) into the lumina. Unlike luminal cells, basal cells are less differentiated, express low or undetectable levels of androgen receptor and do not depend on androgens for survival. A distinct population of basal cells have been identified as the stem cells of the prostate in both mouse and human [16,17]. Although they share similar marker expression, the location of normal stem cells within the basal layer is different in mouse and human. In the mouse, these cells are located in the proximal region of the ducts and are believed to give rise to the proliferating transit-amplifying cells. In humans, prostate stem cells appear intermingled throughout &
the acini and ducts. Normal prostate stem cells express cell surface markers integrin a2b1 [16,18] and CD133 [19] in addition to epithelial stem cell markers p63, CK5 and CK18 [20]. The cellular origins of prostate cancer remain somewhat controversial. It was originally assumed that the terminally differentiated luminal cells were the source of all tumorigenicity because they represent the bulk of the tumor. However, several studies have lent evidence to the possibility that CSCs derived from either a normal basal stem cell or oncogenic progenitor are involved. For instance, studies have shown that a very small number of cells (0.1%) with basal cell-like properties are also found at metastatic sites [20]. This is further supported by the fact that advanced prostate cancers are androgen-independent, may have low to undetectable androgen receptor and metastases contain cells expressing basal stem cell markers [21]. For example, the basal stem cell marker p63 is a transcription factor specifically required for basal cell and not luminal cell development, and is frequently expressed by prostate CSCs [22,23]. As p63 has not been seen in all prostate CSCs, and other basal stem cell markers such as CK5 and CK14 are rarely seen, an intermediate progenitor for CSCs has also been proposed as a cancer source [24]. Lineage tracking experiments have also demonstrated that only basal cells appear to give rise to a tumor [25]. Taken together, these studies support the theory that such CSCs originate from stem cells of the basal layer.
MARKERS OF PROSTATE CANCER STEM CELLS Evidence that supports prostate CSCs are derived from normal stem cells includes the detailed characterization of markers shared among these two cell types (summarized in Tables 1 and 2) [13,14,19,22– 24,26–29,30 ,31–33]. These include transcription factors, enzymes and cell surface markers that are expressed by CSCs and normal prostate stem cells. For instance, both prostate CSCs and normal stem cells express the cell surface protein CD44. CD44þ CSCs from prostate xenograft tumors and cell lines demonstrate higher proliferation and tumor-initiating ability in vivo compared with CD44 cells [14]. CD44þ cells also express other markers of embryonic stem cells, including Oct4, B-catenin and Bmi-1 (B cell-specific Moloney murine leukemia virus integration site 1 protein), and are androgen receptor [14]. In another study, telomerase reverse transcriptase, an enzyme elevated in normal stem cells, was used to generate stable cell lines from normal prostate cancer epithelial cells. In this study, clonally derived CD44þ human prostate CSCs were
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Genitourinary system Table 1. List of cell surface markers used to isolate prostate cancer stem cells Marker
Reference
a2b1highCD133þCD44þ
Collins et al. [13]
CD44þ/CD133þ
Li et al. [26]
Sca-1þ
Xin et al. [27], Burger et al. [28]
Lin();Sca1(þ);CD49f(hi)
Jiao et al. [29]
CD166þ
Jiao et al. [29]
CD44þ
Liu et al. [30 ], Patrawala et al. [14] &&
isolated that could regenerate tumors in mice [33]. Moreover, the tumors formed from these CSCs contained luminal, basal and neuroendocrine cells, demonstrating their differentiating capability, which is a hallmark of stem cells [33]. This study further confirmed the existence of prostate CSCs, which expressed markers similar to normal prostate stem cells from the basal layer, including Oct4, Nanog, Sox2, c-Kit and CD133, and were androgen receptor [33]. a2b1 marker expression has also been used in combination with CD133 and CD44 to identify CSCs. For instance, CD44þ/a2b1high/CD133þCSCs
CSCs isolated from human tumors demonstrated self-renewing properties in vitro and generated androgen receptorþ/PAPþ/CK18þ luminal cells under differentiating conditions [13]. In patient samples, approximately 0.1% of CD44þ/a2b1high/CD133þ cells were found in all tumors, but there was no correlation between the number of these cells and tumor grade. Although Sca-1 expression has been found in normal prostate epithelial stem cells, it may not be required for their stemness, as there is some inconsistency among laboratories on its role in prostate stem cells. Quiescent Sca-1þ stem cells isolated from normal mouse prostate can differentiate into basal and luminal cells and can generate tumors in mice [27,29]. In contrast, other studies have shown similar prostate-regenerating activity in both Sca-1þ and Sca-1 cells isolated from the prostate [28]. One of the reports extends the characterization of Sca1þ cells by isolating Sca1(þ)/CD49f(hi)/Lin() cells from normal prostate and demonstrating their significantly increased ability to form spheres in vitro and initiate cancer in a PTEN-null prostate cancer model [29]. Further, the same group also identified CD166 as a potential marker of normal Sca1(þ)/ CD49f(hi)/Lin() prostate stem progenitors and showed that although CD166 was not directly involved in their metastasis, it was a marker of
Table 2. List of stem cell markers in normal stem cells and cancer stem cells Marker
Normal stem cells
Cancer stem cells
Reference
a2b1high
H
H
Collins et al. [13]
CD133þ
H
H
Li et al. [26], Richardson et al. [19]
CD49f(hi)
H
H
Jiao et al. [29]
CD166þ
H
H
Jiao et al. [29]
CD44þ
H
H
Liu et al. [30 ], Patrawala et al. [14]
Sca-1
H
H
Xin et al. [27], Burger et al. [28]
CK5
H
van Leenders et al. [24]
CK14
H
van Leenders et al. [24]
CK18
H
van Leenders et al. [24], Liao et al. [31]
P63
H
Oct4
H
H
Lee et al. [32], Gu et al. [33]
Sox2
H
H
Lee et al. [32], Gu et al. [33]
Nanog
H
H
Lee et al. [32], Gu et al. [33]
Nestin
H
H
Gu et al. [33]
EZH2
H
H
Li et al. [26]
Bmi-1
H
H
Patrawala et al. [14]
&&
Signoretti et al. [22], Kurita et al. [23]
B-catenin
H
H
Patrawala et al. [14]
C-kit
H
H
Gu et al. [33]
Runx2
H
H
Liao et al. [31]
Survivin
H
H
Liao et al. [31]
Rex-1
H
H
Lee et al. [32]
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Regulators of prostate cancer stem cells Kerr and Hussain
castration-resistant prostate CSCs [29]. Together, these studies support the notion that prostate CSCs can be derived from normal stem cells.
EPIGENETIC REGULATION IN PROSTATE CANCER STEM CELLS The Polycomb group transcriptional repressors Bmi-1 and enhancer of zeste homolog 2 (EZH2) have been recently implicated in regulating the prostate CSC phenotype. These two proteins belong to two distinct protein complexes (PRC2 and PRC1, respectively) that act sequentially to regulate gene expression. Initial steps in stem cell reprogramming involve the recruitment of histone deacetylase by activity of the PRC2 complex containing EZH2, which causes local deacetylation of chromatin and subsequent methylation of the histone H3 component K27. The result of this methylation is recruitment of the Bmi-1 PRC1 complex to the site leading to the monoubiquitination of Lys119 histone H2A and suppression of gene expression [34]. The coordinate action of these two complexes plays an important role in the regulation and maintenance of gene expression during development and contributes to the epigenetic memory of stem cells [35,36]. Loss and gain-of-function analysis in prostate stem cells indicates that Bmi-1 expression is required for self-renewal activity and maintenance of normal p63(þ) stem cells and prostate CSCs [37,38,39 ]. As such, Bmi-1 has also been considered a marker for normal prostate stem cells and CSCs. More recently, regulators of Bmi-1 have been found, including miRNA-128 and the small molecule Smoothened (Smo) antagonist Erismodegib, that inhibit prostate CSC growth by targeting Bmi-1 expression [35]. Like Bmi-1, EZH2 has also recently been shown to play a role in prostate CSCs. This is not surprising, as several studies have demonstrated an important role of EZH2 in maintaining stemness in embryonic stem cells [40]. EZH2 regulates the expression of genes that promote proliferation and suppress differentiation [40,41]. For example, EZH2 has been shown to interact with the embryonic stem cell genes Oct4, Sox2 and Nanog to maintain stem cell pluripotency. CD44þ/CD133þ prostate CSCs have also demonstrated elevated levels of EZH2 in addition to Oct4, Sox2 and Nanog. Moreover, siRNA-mediated knockdown of EZH2 in these cells inhibited cell growth and G1/S arrest and induced apoptosis, suggesting a regulatory role of EZH2 in defining stemness [26]. Additionally, microRNAs (miRNAs) (miR-10 and Let-7) that downregulate EZH2 in prostate CSCs reduce survival of the CSCs [26,42]. &&
MICRORNA REGULATION IN PROSTATE CANCER STEM CELLS miRNAs are classes of small noncoding RNAs (22 nt), which normally function as negative regulators of target mRNA expression, although in some cases they can also function as positive regulators of gene expression. It is estimated that 1–4% of the genes in the human genome are miRNAs. They have been shown to play critical roles in diverse cellular processes, including cell growth and proliferation, tissue differentiation, embryonic development and apoptosis [43,44]. Specific miRNAs are also known to regulate properties of both normal stem cells and CSCs, and their dysregulation has been implicated in tumorigenesis, including in prostate cancer. The majority of the miRNAs discovered to date in prostate cancer act as or target tumor suppressor genes. In general, these miRNAs exhibit lower expression in the prostate CSCs compared with non-stem cancer cells, and when their expression is forced, inhibition of various stem cell properties like clonogenic expansion, tumor regeneration and metastasis occurs. For example, forced expression of miR-34a, a p53 target, has been shown to inhibit clonogenic expansion and tumor regeneration in bulk and purified CD44þ prostate cancer cells, and reduced expression using miR-34a antagomirs (or antimiRNA) in CD44 prostate cancer cells promotes tumor development and metastasis. Moreover, injection of miR-34a into mice inhibits metastasis and prolongs survival of tumor-bearing mice [30 ]. Reduced expression of miR-34a and let-7b was also identified in CD44þ/CD133þ/ a2b1þ CSC [45]. In this study, overexpression of let-7 and miR-34a exerted differential inhibitory effects in cells, with miR-34a inducing G1 phase cell-cycle arrest accompanied by cell senescence, whereas let-7 induced G2-M phase cell-cycle arrest without senescence. Reduced expressions of miR-143 and miR-145 have also been linked to metastasis of the bone in prostate cancer cell lines. In these studies, overexpression of miR-143 and miR-145 inhibited cell viability, colony formation, tumor sphere formation, expression of stem cell markers, bone invasion and tumorigenicity of PC-3 cells [46]. In a similar fashion, downregulation of miR200b has been demonstrated in clinical prostatic tumors and prostate cancer cell lines. Forced expression of miR-200b specifically increased EMT and invasive properties via PDFG-D signaling in prostate cancer cells [47], in addition to increasing expression of stem cell markers Bmi-1, E cadherin, P19, CD44 and Oct4, and reducing expression of vimentin[48] – i.e. the hallmarks of the CSC phenotype. Moreover,
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re-expression of miR200b reverses the EMT phenotype and downregulates EMT-associated proteins ZEB2, ZEB3 and Snail2 [47]. A potential mechanism of action for miR200b is Bmi-1. Forced miR200b expression suppresses sensitivity of prostate CSCs to docetaxel by targeting Bmi-1. Reduced expressions of miR-200c and miR-205 have also been discovered in docetaxel-resistant cell lines, and knockdown of these miRNAs led to reduced adhesion and increased invasion, migration and stem cell marker expression [49,50 ]. Finally, miR-708 is another miRNA identified as a tumor suppressor in prostate CSCs. Intratumoral delivery of synthetic miR-708 oligonucleotides can trigger regression in xenograft tumors, whereas silencing its expression promotes tumor growth [51]. &
CANCER STEM CELL NICHE More recently, studies have begun to identify factors in the niche that regulate prostate stem cell maintenance. For instance, upregulation of focal adhesion pathways and extracellular matrix–integrin signaling, including integrin av, collagen type 5 a1 chains, TGFb and Annexin 1 have been found in CSCs, similar to that in normal prostate stem cells [52,53,54 ]. Collectively, these factors can promote prostate CSCs through stem cell–cell niche interactions.
expression, aldehyde dehydrogenase activity, B-cell lymphoma-2-related chemoresistance and enhanced DNA damage response have been reported (reviewed by [57]). By understanding these mechanisms, potentially new strategies can be identified and tested for therapeutic effect in prostate cancer.
CONCLUSION To date, several laboratories collectively lend proof to the existence for a CSC model in prostate cancer. From these studies, it is clear that prostate CSCs exhibit markers similar to both normal prostate stem cells and markers that are expressed by embryonic-like stem cells. In addition, a number of novel mechanisms that regulate the stem cell properties of the prostate CSCs have been recently discovered. These mechanisms involve miRNAs and epigenetic regulators that are required to maintain the selfrenewing and differentiation capacities of the CSCs. This information is critical for finding new targets that will selectively kill CSCs and possibly reduce a major contributing source of recurrent and metastatic disease.
&&
PROSTATE CANCER STEM CELLS AS THERAPEUTIC TARGETS Current treatment for prostate cancer includes androgen ablation, which eradicates the bulk of the androgen-responsive cancer cells within a tumor. However, the initial ADT (and additional second or third-line ‘hormonal’ therapies) eventually fails in the majority of patients, resulting in recurrent and progressive disease. A number of mechanisms related to the androgen and androgen receptor axis have been implicated in the development of recurrent androgen-insensitive disease. Another possible suggested mechanism is that androgen ablation may activate normally quiescent CSCs to repopulate the tumor with androgen-independent cells. Under this scenario, targeting CSCs rather than the more ‘differentiated’ bulk cancer cells would be more effective in controlling disease. A challenge in this regard is that like normal stem cells, the CSCs often possess treatment-resistance mechanisms [55]. These mechanisms include increased levels of DNA repair enzymes, antiapoptotic proteins and expression of drug-resistance proteins, such as MDRI and other ABC transporters [56]. Specific to prostate cancer, ABC transporter 332
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Acknowledgements This work was supported in part by a Merit Review Award, Dept of Veterans Affairs (A.H.) Conflicts of interest There are no conflicts of interest.
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Regulators of prostate cancer stem cells Candace L. Kerr a,c and Arif Hussain a,b,d
Purpose of review Significant advances have been made toward identifying prostate cancer stem cells (CSCs). This review will highlight the latest developments in defining this population and the discovery of mechanisms involved in their survival and metastasis. Recent findings Several groups have identified master regulators of stem cells in prostate cancer. These include genetic and epigenetic factors known to control pluripotency in embryonic stem cells and in highly metastatic prostate tumors. For instance, tumors of patients with poor prognosis demonstrate elevated levels of the pluripotent markers OCT4 and SOX2 as well as the polycomb complex protein Bmi-1 and enhancer of zeste homolog 2. Cells that are derived from these patient tumors provide an opportunity to expand our current knowledge regarding how these cells survive and the mechanisms that regulate their proliferation. Summary Understanding the mechanisms of highly invasive and therapy resistant prostate cancer cells resides in understanding the CSCs, which facilitate cancer recurrence. Some of these factors are just emerging and provide a platform for developing targeted drugs for the future treatment of advanced prostate cancer. Keywords cancer stem cells, metastasis, proliferation, prostate cancer, tumorigenicity
INTRODUCTION Prostate cancer continues to be the most frequently diagnosed cancer in men and a leading cause of cancer-related deaths [1]. A major contributing factor to prostate cancer-related deaths is the eventual development of resistance to androgen deprivation therapy (ADT) amongst men being treated with ADT for their advanced disease. Several mechanisms related to androgen biosynthesis and/or the androgen receptor can occur during the development of ADT resistance. In addition, there is a growing body of evidence that some of this androgen resistance may be because of the presence of prostate cancer stem cells (CSCs), which can persist after chemotherapy, radiation or androgen deprivation. These highly undifferentiated cells represent a small percentage of the heterogeneity seen in metastatic tumors and express markers of normal prostate stem cells. Currently, there are two models that are believed to contribute to tumor development. One model, known as the stochastic model, assumes that all cancer cells have an equal probability of regenerating a tumor and that cancer develops in a randomized fashion from any given cell when it develops the capacity to proliferate and regenerate a www.co-oncology.com
tumor. Another model, which was originally proposed by Rudolf Virchow, a German pathologist in the mid-19th century, is the model of the CSC [2]. There has been increasing experimental evidence to support the existence of CSCs, particularly over the past three decades. The CSCs are a very small subset of cells within the tumor population, which contribute to tumor growth and metastasis [3–14]. These CSCs exhibit properties of normal stem cells, and are unlike the bulk of cancer cells making up a tumor because the latter cells are more differentiated and non-tumorigenic. Despite a growing body of evidence for the existence of prostate CSCs, many questions still
a Department of Biochemistry and Molecular Biology, bDepartment of Medicine and Greenebaum Cancer Center, University of Maryland School of Medicine, cDepartment of Obstetrics and Gynecology, Johns Hopkins University and dBaltimore VA Medical Center, Baltimore, Maryland, USA
Correspondence to Professor Arif Hussain, Bressler Research Building, Room 9–041 22 S Greene St, Baltimore, Maryland 21201, United States. Tel: +1 (410) 328 7225; fax: +1 (410) 328-2578; e-mail: [email protected] Curr Opin Oncol 2014, 26:328–333 DOI:10.1097/CCO.0000000000000080 Volume 26 Number 3 May 2014
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Regulators of prostate cancer stem cells Kerr and Hussain
KEY POINTS Prostate CSCs have been isolated from prostate cancer cell lines and patient tumors which demonstrate selfrenewing and differentiating abilities. Prostate CSCs demonstrate distinguishing features from bulk tumor cells, including expression of embryonic-like stem cell transcription factors Oct4, Nanog, p63 and Sox2. Prostate CSCs can be derived either by spheroid culture or by cell surface marker selection using a combination of stem cell markers such as CD44, CD133, Sca-1 and a2b1. Prostate CSCs rely on a number of recently identified miRNAs and epigenetic regulators such as Bmi-1 and EZH2 to maintain stem cell properties
remain about the nature of these cells and the mechanisms that drive their stem cell-like behavior. Much of the current knowledge about prostate CSCs can be attributed to the recent wealth of information that has sprung from developmental studies characterizing early stem cells and adult stem cells.
NORMAL PROSTATE STEM CELLS AND THE ORIGIN OF CANCER STEM CELLS Prostate CSCs are similar to normal prostate stem cells in that they are long lived, slow cycling cells that remain quiescent in an undifferentiated or embryonic-like state. As such, prostate CSCs express many early developmental markers of normal stem cells. The mature prostate is made up of a stratified layer of immature cuboidal basal cells attached to the basement membrane with differentiated luminal cells projecting into the lumina. There are also neuroendocrine cells scattered throughout (reviewed in [15 ]). Luminal cells represent the majority of cells in the normal and hyperplastic prostate, they depend on androgens for survival, and they secrete PSA and prostatic acid phosphatase (PAP) into the lumina. Unlike luminal cells, basal cells are less differentiated, express low or undetectable levels of androgen receptor and do not depend on androgens for survival. A distinct population of basal cells have been identified as the stem cells of the prostate in both mouse and human [16,17]. Although they share similar marker expression, the location of normal stem cells within the basal layer is different in mouse and human. In the mouse, these cells are located in the proximal region of the ducts and are believed to give rise to the proliferating transit-amplifying cells. In humans, prostate stem cells appear intermingled throughout &
the acini and ducts. Normal prostate stem cells express cell surface markers integrin a2b1 [16,18] and CD133 [19] in addition to epithelial stem cell markers p63, CK5 and CK18 [20]. The cellular origins of prostate cancer remain somewhat controversial. It was originally assumed that the terminally differentiated luminal cells were the source of all tumorigenicity because they represent the bulk of the tumor. However, several studies have lent evidence to the possibility that CSCs derived from either a normal basal stem cell or oncogenic progenitor are involved. For instance, studies have shown that a very small number of cells (0.1%) with basal cell-like properties are also found at metastatic sites [20]. This is further supported by the fact that advanced prostate cancers are androgen-independent, may have low to undetectable androgen receptor and metastases contain cells expressing basal stem cell markers [21]. For example, the basal stem cell marker p63 is a transcription factor specifically required for basal cell and not luminal cell development, and is frequently expressed by prostate CSCs [22,23]. As p63 has not been seen in all prostate CSCs, and other basal stem cell markers such as CK5 and CK14 are rarely seen, an intermediate progenitor for CSCs has also been proposed as a cancer source [24]. Lineage tracking experiments have also demonstrated that only basal cells appear to give rise to a tumor [25]. Taken together, these studies support the theory that such CSCs originate from stem cells of the basal layer.
MARKERS OF PROSTATE CANCER STEM CELLS Evidence that supports prostate CSCs are derived from normal stem cells includes the detailed characterization of markers shared among these two cell types (summarized in Tables 1 and 2) [13,14,19,22– 24,26–29,30 ,31–33]. These include transcription factors, enzymes and cell surface markers that are expressed by CSCs and normal prostate stem cells. For instance, both prostate CSCs and normal stem cells express the cell surface protein CD44. CD44þ CSCs from prostate xenograft tumors and cell lines demonstrate higher proliferation and tumor-initiating ability in vivo compared with CD44 cells [14]. CD44þ cells also express other markers of embryonic stem cells, including Oct4, B-catenin and Bmi-1 (B cell-specific Moloney murine leukemia virus integration site 1 protein), and are androgen receptor [14]. In another study, telomerase reverse transcriptase, an enzyme elevated in normal stem cells, was used to generate stable cell lines from normal prostate cancer epithelial cells. In this study, clonally derived CD44þ human prostate CSCs were
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Genitourinary system Table 1. List of cell surface markers used to isolate prostate cancer stem cells Marker
Reference
a2b1highCD133þCD44þ
Collins et al. [13]
CD44þ/CD133þ
Li et al. [26]
Sca-1þ
Xin et al. [27], Burger et al. [28]
Lin();Sca1(þ);CD49f(hi)
Jiao et al. [29]
CD166þ
Jiao et al. [29]
CD44þ
Liu et al. [30 ], Patrawala et al. [14] &&
isolated that could regenerate tumors in mice [33]. Moreover, the tumors formed from these CSCs contained luminal, basal and neuroendocrine cells, demonstrating their differentiating capability, which is a hallmark of stem cells [33]. This study further confirmed the existence of prostate CSCs, which expressed markers similar to normal prostate stem cells from the basal layer, including Oct4, Nanog, Sox2, c-Kit and CD133, and were androgen receptor [33]. a2b1 marker expression has also been used in combination with CD133 and CD44 to identify CSCs. For instance, CD44þ/a2b1high/CD133þCSCs
CSCs isolated from human tumors demonstrated self-renewing properties in vitro and generated androgen receptorþ/PAPþ/CK18þ luminal cells under differentiating conditions [13]. In patient samples, approximately 0.1% of CD44þ/a2b1high/CD133þ cells were found in all tumors, but there was no correlation between the number of these cells and tumor grade. Although Sca-1 expression has been found in normal prostate epithelial stem cells, it may not be required for their stemness, as there is some inconsistency among laboratories on its role in prostate stem cells. Quiescent Sca-1þ stem cells isolated from normal mouse prostate can differentiate into basal and luminal cells and can generate tumors in mice [27,29]. In contrast, other studies have shown similar prostate-regenerating activity in both Sca-1þ and Sca-1 cells isolated from the prostate [28]. One of the reports extends the characterization of Sca1þ cells by isolating Sca1(þ)/CD49f(hi)/Lin() cells from normal prostate and demonstrating their significantly increased ability to form spheres in vitro and initiate cancer in a PTEN-null prostate cancer model [29]. Further, the same group also identified CD166 as a potential marker of normal Sca1(þ)/ CD49f(hi)/Lin() prostate stem progenitors and showed that although CD166 was not directly involved in their metastasis, it was a marker of
Table 2. List of stem cell markers in normal stem cells and cancer stem cells Marker
Normal stem cells
Cancer stem cells
Reference
a2b1high
H
H
Collins et al. [13]
CD133þ
H
H
Li et al. [26], Richardson et al. [19]
CD49f(hi)
H
H
Jiao et al. [29]
CD166þ
H
H
Jiao et al. [29]
CD44þ
H
H
Liu et al. [30 ], Patrawala et al. [14]
Sca-1
H
H
Xin et al. [27], Burger et al. [28]
CK5
H
van Leenders et al. [24]
CK14
H
van Leenders et al. [24]
CK18
H
van Leenders et al. [24], Liao et al. [31]
P63
H
Oct4
H
H
Lee et al. [32], Gu et al. [33]
Sox2
H
H
Lee et al. [32], Gu et al. [33]
Nanog
H
H
Lee et al. [32], Gu et al. [33]
Nestin
H
H
Gu et al. [33]
EZH2
H
H
Li et al. [26]
Bmi-1
H
H
Patrawala et al. [14]
&&
Signoretti et al. [22], Kurita et al. [23]
B-catenin
H
H
Patrawala et al. [14]
C-kit
H
H
Gu et al. [33]
Runx2
H
H
Liao et al. [31]
Survivin
H
H
Liao et al. [31]
Rex-1
H
H
Lee et al. [32]
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Regulators of prostate cancer stem cells Kerr and Hussain
castration-resistant prostate CSCs [29]. Together, these studies support the notion that prostate CSCs can be derived from normal stem cells.
EPIGENETIC REGULATION IN PROSTATE CANCER STEM CELLS The Polycomb group transcriptional repressors Bmi-1 and enhancer of zeste homolog 2 (EZH2) have been recently implicated in regulating the prostate CSC phenotype. These two proteins belong to two distinct protein complexes (PRC2 and PRC1, respectively) that act sequentially to regulate gene expression. Initial steps in stem cell reprogramming involve the recruitment of histone deacetylase by activity of the PRC2 complex containing EZH2, which causes local deacetylation of chromatin and subsequent methylation of the histone H3 component K27. The result of this methylation is recruitment of the Bmi-1 PRC1 complex to the site leading to the monoubiquitination of Lys119 histone H2A and suppression of gene expression [34]. The coordinate action of these two complexes plays an important role in the regulation and maintenance of gene expression during development and contributes to the epigenetic memory of stem cells [35,36]. Loss and gain-of-function analysis in prostate stem cells indicates that Bmi-1 expression is required for self-renewal activity and maintenance of normal p63(þ) stem cells and prostate CSCs [37,38,39 ]. As such, Bmi-1 has also been considered a marker for normal prostate stem cells and CSCs. More recently, regulators of Bmi-1 have been found, including miRNA-128 and the small molecule Smoothened (Smo) antagonist Erismodegib, that inhibit prostate CSC growth by targeting Bmi-1 expression [35]. Like Bmi-1, EZH2 has also recently been shown to play a role in prostate CSCs. This is not surprising, as several studies have demonstrated an important role of EZH2 in maintaining stemness in embryonic stem cells [40]. EZH2 regulates the expression of genes that promote proliferation and suppress differentiation [40,41]. For example, EZH2 has been shown to interact with the embryonic stem cell genes Oct4, Sox2 and Nanog to maintain stem cell pluripotency. CD44þ/CD133þ prostate CSCs have also demonstrated elevated levels of EZH2 in addition to Oct4, Sox2 and Nanog. Moreover, siRNA-mediated knockdown of EZH2 in these cells inhibited cell growth and G1/S arrest and induced apoptosis, suggesting a regulatory role of EZH2 in defining stemness [26]. Additionally, microRNAs (miRNAs) (miR-10 and Let-7) that downregulate EZH2 in prostate CSCs reduce survival of the CSCs [26,42]. &&
MICRORNA REGULATION IN PROSTATE CANCER STEM CELLS miRNAs are classes of small noncoding RNAs (22 nt), which normally function as negative regulators of target mRNA expression, although in some cases they can also function as positive regulators of gene expression. It is estimated that 1–4% of the genes in the human genome are miRNAs. They have been shown to play critical roles in diverse cellular processes, including cell growth and proliferation, tissue differentiation, embryonic development and apoptosis [43,44]. Specific miRNAs are also known to regulate properties of both normal stem cells and CSCs, and their dysregulation has been implicated in tumorigenesis, including in prostate cancer. The majority of the miRNAs discovered to date in prostate cancer act as or target tumor suppressor genes. In general, these miRNAs exhibit lower expression in the prostate CSCs compared with non-stem cancer cells, and when their expression is forced, inhibition of various stem cell properties like clonogenic expansion, tumor regeneration and metastasis occurs. For example, forced expression of miR-34a, a p53 target, has been shown to inhibit clonogenic expansion and tumor regeneration in bulk and purified CD44þ prostate cancer cells, and reduced expression using miR-34a antagomirs (or antimiRNA) in CD44 prostate cancer cells promotes tumor development and metastasis. Moreover, injection of miR-34a into mice inhibits metastasis and prolongs survival of tumor-bearing mice [30 ]. Reduced expression of miR-34a and let-7b was also identified in CD44þ/CD133þ/ a2b1þ CSC [45]. In this study, overexpression of let-7 and miR-34a exerted differential inhibitory effects in cells, with miR-34a inducing G1 phase cell-cycle arrest accompanied by cell senescence, whereas let-7 induced G2-M phase cell-cycle arrest without senescence. Reduced expressions of miR-143 and miR-145 have also been linked to metastasis of the bone in prostate cancer cell lines. In these studies, overexpression of miR-143 and miR-145 inhibited cell viability, colony formation, tumor sphere formation, expression of stem cell markers, bone invasion and tumorigenicity of PC-3 cells [46]. In a similar fashion, downregulation of miR200b has been demonstrated in clinical prostatic tumors and prostate cancer cell lines. Forced expression of miR-200b specifically increased EMT and invasive properties via PDFG-D signaling in prostate cancer cells [47], in addition to increasing expression of stem cell markers Bmi-1, E cadherin, P19, CD44 and Oct4, and reducing expression of vimentin[48] – i.e. the hallmarks of the CSC phenotype. Moreover,
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re-expression of miR200b reverses the EMT phenotype and downregulates EMT-associated proteins ZEB2, ZEB3 and Snail2 [47]. A potential mechanism of action for miR200b is Bmi-1. Forced miR200b expression suppresses sensitivity of prostate CSCs to docetaxel by targeting Bmi-1. Reduced expressions of miR-200c and miR-205 have also been discovered in docetaxel-resistant cell lines, and knockdown of these miRNAs led to reduced adhesion and increased invasion, migration and stem cell marker expression [49,50 ]. Finally, miR-708 is another miRNA identified as a tumor suppressor in prostate CSCs. Intratumoral delivery of synthetic miR-708 oligonucleotides can trigger regression in xenograft tumors, whereas silencing its expression promotes tumor growth [51]. &
CANCER STEM CELL NICHE More recently, studies have begun to identify factors in the niche that regulate prostate stem cell maintenance. For instance, upregulation of focal adhesion pathways and extracellular matrix–integrin signaling, including integrin av, collagen type 5 a1 chains, TGFb and Annexin 1 have been found in CSCs, similar to that in normal prostate stem cells [52,53,54 ]. Collectively, these factors can promote prostate CSCs through stem cell–cell niche interactions.
expression, aldehyde dehydrogenase activity, B-cell lymphoma-2-related chemoresistance and enhanced DNA damage response have been reported (reviewed by [57]). By understanding these mechanisms, potentially new strategies can be identified and tested for therapeutic effect in prostate cancer.
CONCLUSION To date, several laboratories collectively lend proof to the existence for a CSC model in prostate cancer. From these studies, it is clear that prostate CSCs exhibit markers similar to both normal prostate stem cells and markers that are expressed by embryonic-like stem cells. In addition, a number of novel mechanisms that regulate the stem cell properties of the prostate CSCs have been recently discovered. These mechanisms involve miRNAs and epigenetic regulators that are required to maintain the selfrenewing and differentiation capacities of the CSCs. This information is critical for finding new targets that will selectively kill CSCs and possibly reduce a major contributing source of recurrent and metastatic disease.
&&
PROSTATE CANCER STEM CELLS AS THERAPEUTIC TARGETS Current treatment for prostate cancer includes androgen ablation, which eradicates the bulk of the androgen-responsive cancer cells within a tumor. However, the initial ADT (and additional second or third-line ‘hormonal’ therapies) eventually fails in the majority of patients, resulting in recurrent and progressive disease. A number of mechanisms related to the androgen and androgen receptor axis have been implicated in the development of recurrent androgen-insensitive disease. Another possible suggested mechanism is that androgen ablation may activate normally quiescent CSCs to repopulate the tumor with androgen-independent cells. Under this scenario, targeting CSCs rather than the more ‘differentiated’ bulk cancer cells would be more effective in controlling disease. A challenge in this regard is that like normal stem cells, the CSCs often possess treatment-resistance mechanisms [55]. These mechanisms include increased levels of DNA repair enzymes, antiapoptotic proteins and expression of drug-resistance proteins, such as MDRI and other ABC transporters [56]. Specific to prostate cancer, ABC transporter 332
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Acknowledgements This work was supported in part by a Merit Review Award, Dept of Veterans Affairs (A.H.) Conflicts of interest There are no conflicts of interest.
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