Unexplored Functions of Sex Hormones in Glioblastoma Cancer Stem Cells

Abstract

Biological sex impacts a wide array of molecular and cellular functions that impact organismal development and can influence disease trajectory in a variety of pathophysiological states. In nonreproductive cancers, epidemiological sex differences have been observed in a series of tumors, and recent work has identified previously unappreciated sex differences in molecular genetics and immune response. However, the extent of these sex differences in terms of drivers of tumor growth and therapeutic response is less clear. In glioblastoma (GBM), the most common primary malignant brain tumor, there is a male bias in incidence and outcome, and key genetic and epigenetic differences, as well as differences in immune response driven by immune-suppressive myeloid populations, have recently been revealed. GBM is a prototypic tumor in which cellular heterogeneity is driven by populations of therapeutically resistant cancer stem cells (CSCs) that underlie tumor growth and recurrence. There is emerging evidence that GBM CSCs may show a sex difference, with male tumor cells showing enhanced self-renewal, but how sex differences impact CSC function is not clear. In this mini-review, we focus on how sex hormones may impact CSCs in GBM and implications for other cancers with a pronounced CSC population. We also explore opportunities to leverage new models to better understand the contribution of sex hormones vs sex chromosomes to CSC function. With the rising interest in sex differences in cancer, there is an immediate need to understand the extent to which sex differences impact tumor growth, including effects on CSC function.

Developmental biology principles provide a useful scaffold for the study of cancer biology, including the observation that tumors contain cellular hierarchies and are comprised of heterogeneous cell populations, much like the tissues and organs from which they arise. This paradigm has expanded to include the identification of self-renewing cancer stem cell (CSC) populations within tumors that exhibit plasticity and can contribute to therapeutic resistance, making them a significant barrier to treatment. CSCs have been identified in a variety of different tumor types and, as such, remain a major focus of investigation. The formal definition of a CSC includes 2 functional criteria, namely, self-renewal capacity and the ability to initiate a new tumor that retains the cellular heterogeneity of the original (1). Along with these functional definitions, CSCs are complex, demonstrating a high degree of plasticity by virtue of cell intrinsic and extrinsic interactions with the tumor microenvironment (2,3). Glioblastoma (GBM), the predominant primary brain malignancy in adults, is uniformly fatal, a feature that has been partially attributed to the presence of CSCs in these tumors. GBM CSCs were first identified in patient tumors on the basis of cell-surface enrichment of CD133 (4,5), and follow-up studies revealed a series of additional cell surface enrichment markers (CD15, CD44, L1CAM, CD49f) (6-9) and intracellular regulators, some of which are also present in pluripotent stem cell populations (SOX2, OCT4, NANOG, c-MYC) (10-13). CSC function is further regulated through a series of bidirectional interactions with the surrounding microenvironment that integrates with cell intrinsic factors to ensure CSC maintenance. These extrinsic interactions extend to immune and neural populations, metabolic substrate availability, and hypoxia, along with genetic and epigenetic regulation, and represent active areas of investigation. What remains less clear is how sex differences, which influence both of the abovementioned cell intrinsic and cell extrinsic mechanisms, regulate the CSC state. Here, we will focus on this question, using GBM as a model to explore how sex hormones impact CSCs.

The inclusion of sex as a biological variable in basic and clinical research is a relatively recent development, owing to the long-held belief that the estrous cycle in females introduces intrinsic variability that reasonably justified their exclusion from studies. The National Institutes of Health (NIH) began instituting policies requiring the inclusion of women in NIH-funded clinical research in the 1990s, and sex as a biological variable mandates were introduced by the NIH in 2016 (14). As a result, the study of sexual differences has expanded rapidly and represents a growing body of literature but remains incompletely understood. In mammals, differentiation of the gonads is controlled by genes present on sex chromosomes, including the sex-determining region Y (Sry) gene, which is located on the Y chromosome and induces testis formation, while its absence results in development of the ovaries (15). Sex differences in the mammalian brain are evident early in development through the process of “masculinization,” which is well documented and occurs as a function of embryonic exposure to testosterone, ultimately driving male behavior in adults (16).

In the context of GBM, recent studies demonstrate that male and female patients exhibit differences in disease incidence and outcome, as well as other factors such as disease presentation, molecular characteristics, and therapeutic response (17,18). Sex differences in tumor generation have also been revealed, with astrocytes from females demonstrating a greater degree of protection from malignant transformation compared to astrocytes from males due to increased p16 and p21 activity (19). Furthermore, investigators showed that transformed mouse astrocytes from males were more tumorigenic, had enhanced self-renewal (an in vitro surrogate of CSC maintenance) and increased proliferation rates, and exhibited greater inactivation of the RB tumor suppressor when compared with female astrocytes (20). In addition to the effects of sex on cell intrinsic properties of GBM tumor cells, several studies have reported interactions between sex and tumor cell extrinsic factors, including immune interactions and metabolic alterations (21,22). Given these findings, examination of sex differences in the context of GBM CSCs has the potential to reveal new insights into how CSCs are regulated and their unique interactions with the surrounding microenvironment.

Estrogen and Progesterone

Estrogen and progesterone are female steroid hormones that exert regulatory actions by binding to their cognate receptors, classified as nuclear receptors and membrane receptors depending on the location. Hormone binding to nuclear receptors directly regulates DNA transcription, whereas membrane receptors signal through cytoplasmic signal transduction (23).

Estrogen signaling is predominantly mediated via the nuclear receptors estrogen receptors alpha (ERα) and beta (ERβ). Estradiol (E2), the most potent and prevalent estrogen, has 2 isoforms, of which 17β-E2 demonstrates higher potency compared to 17α-E2. As 17β-E2 has higher binding affinity for ERα than ERβ, estrogens primarily exert their function though ERα. Despite decreased hormone affinity, ERβ signaling does play a regulatory role in CSCs. Elevated levels of ERβ in CSCs correlate with high levels of CSC markers such as CD44 and ALDH1, suggesting potential use of ERβ as CSC marker (24). However, ERβ has also been shown to positively correlate with disease-free survival (25) and decreases proliferation and induces apoptosis in breast cancer cells, in addition to decreasing tumor formation in xenograft models (26). Furthermore, an elevated level of ERβ was found in prostate CSCs (27), and selective activation of ERβ induces cell death in castration-resistant stem cells and attenuates self-renewal of stem cells (28).

Nuclear receptors for progesterone are also divided into 2 isoforms, progesterone receptors A (PR-A) and B (PR-B). PR-A and PR-B are present at similar levels in healthy reproductive tissue, whereas PR-A becomes the dominant form in breast cancer cells (29). PR-A signaling has also been found to promote expansion of CSCs and expression of CSC-related genes in a T47D cell model of breast cancer (30).

Whereas progesterone is known to enhance the expansion and activities of CSCs (31), the roles for estrogen in regulating CSC populations have not been clearly defined, and contradictory functions have been reported. In endometrial cancer cells, ERα binds to the promoter region of piwi-like RNA-mediated gene silencing 1 (PIWIL1), a critical gene for stem cell self-renewal, thereby enhancing stem cell properties (32). The estrogen-ERα complex also induces a positive feedback loop of FXYD4/SOX9/SRC expression, which is required for the maintenance of breast CSCs (33). In contrast, estrogen reduces the proportion of stem cells in the normal human mammary gland and breast tumors by downregulating the expression of stem cell genes such as SOX2, NANOG, and OCT4 (34). Anti-estrogen drugs including tamoxifen are widely used in ER^pos breast cancer patients, but de novo or acquired resistance to the hormonal therapy often occurs. Tamoxifen treatment reduces cell proliferation but increases breast CSC activities in a JAG1-NOTCH4 receptor-dependent pathway, suggesting that hormone therapy should be combined with therapies targeting NOTCH4 signaling to treat tamoxifen-resistant tumors (35). Tamoxifen treatment was also shown to increase PR-A expression in breast cancer, leading to tumor recurrence possibly via enhanced CSC activities (36). Another ER antagonist, ICI 182,780, did not suppress tumor growth but increased CSCs activities in breast cancer mouse model and increased tumorsphere self-renewal in vitro (37). Metformin, an antidiabetic drug, reduces stemness of breast CSCs by inhibiting the binding of ER-estrogen complex on the promoter regions of OCT4 (38). In the same way, OCT4 expression can be also regulated by tocopherols (39) and melatonin (40) shown in breast cancer cells.

PR antagonists such as mifepristone and onapristone have regulatory effects on CSCs and cancer progression. Mifepristone reduces PR activity by competing with progesterone for PRs. In a triple-negative breast cancer model, mifepristone reduced the CSC population by downregulating KLF5, a stem cell transcription factor, and suppressed tumor growth in vitro and in vivo (41). The addition of mifepristone to glucocorticoid and paclitaxel treatment significantly increased apoptosis of chemotherapy-resistant cells (42). Onapristone inhibits phosphorylation of PR-A, which is necessary for its nuclear translocation, thereby inhibiting PR-mediated CSC activities (43). Another study further showed that FOXO1 mediates PR phosphorylation and that inhibition of FOXO1 alone or in combination with onapristone treatment prevented tumorsphere formation of breast cancer cells (30).

Most CSCs do not express nuclear ER or PR (44,45), yet estrogen and progesterone regulate CSC activity via paracrine signaling. Treatment of breast cancer cells with estrogen induced expansion of CSCs, and this was demonstrated to be mediated by soluble factors. Fibroblast growth factor secreted by ER^pos non-CSCs binds to fibroblast growth factor receptors on CSCs and activates Tbx3, a transcription factor critical for expansion of stem cells (46). Harrison et al also showed that the paracrine effect of estrogen is mediated through epidermal growth factor and NOTCH receptor signaling in vitro and in vivo (47). Progesterone-induced paracrine signals regulate PR^neg CSCs in multiple ways. Enhanced expression of CXCR4 on CSCs and CXCL12 produced by PR^pos non-CSCs induce expansion of CSCs in normal mammary cells (48) and in prostate CSCs with a high degree of radiotherapy resistance (49). Furthermore, progesterone stimulates PR^pos cells to produce Wnt4 and receptor activator of nuclear factor kappa-Β ligand, which in turn respond by upregulating their cognate receptors on PR^neg CSCs, thereby enhancing transcription of CSC-related genes (50,51).

In conjunction with paracrine signaling, estrogen and progesterone can affect CSCs via cell membrane-associated receptors (52). The estrogen membrane receptor GPER/GPR30 induces expression of the transcription factor tafazzin, which is highly associated with self-renewal and tumor-initiating properties of CSCs (53,54). GPER/GPR30 also positively regulates CSCs via activation of protein kinase A/BAD signaling in breast CSCs (55). ERα36 is a variant of ERα that is present in the plasma membrane. ERα36 positively regulates CSCs by enhancing stem cell renewal (56,57). Increased ERα36 expression has been shown to be a mechanism of tamoxifen resistance in breast cancer, which suggests ERα36 as a novel therapeutic target (58, 59). Membrane-bound PRs (mPRs) mediate signals via multiple pathways including the extracellular signal-regulated kinase, protein kinase C, and phosphoinositide 3-kinase/protein kinase B pathways in normal tissue (60). Furthermore, progesterone treatment enhanced expression of KLF4, which is necessary for CSC initiation and maintenance, via the mPR-phosphoinositide 3-kinase/protein kinase B pathway in irradiated basal-like mammary cells lacking nuclear PRs (61). While there are some examples of mPRs impacting CSC function, as described in the previous discussion, this is an emerging field and additional studies are needed to determine the function of mPRs on CSCs phenotypes.

Sex hormones also regulate CSCs via micro RNAs (miRNAs). Estrogen induces miR-21 expression, which reduces self-renewal of cancer cells with stem-cell-like properties by inhibiting the translation of the stem cell renewal genes OCT4, c-MYC, NANOG, and SOX2 (62,63). Estrogen negatively regulates miR-140, which targets SOX2 in breast cancer cells (64,65). miR-29 is a regulator of KLF4 expression, a transcription factor critical for stem maintenance. Estrogen and progesterone are opposing regulators in that expression of miR-29 is enhanced by estrogen, whereas progesterone suppresses miR-29 expression (45,66). A screen for regulatory miRNAs revealed multiple miRNAs associated with stemness of breast CSCs, suggesting further targets to control CSCs; however, the relation of these miRNAs with estrogen or progesterone has not been explored (67).

Androgens

The role of androgens in the proliferation of CSCs has been examined in several cancers, with tumor-specific effects observed. Specifically, androgen depletion seems to drive stemness in prostate cancer (68-71), but the opposite effect has been observed in GBM (72), breast cancer (73), ovarian cancer (74), and bladder cancer (75). What underlies the divergent effects of androgens on CSCs between different cancers has yet to be elucidated, but this topic merits further clinical attention.

Not surprisingly, prostate cancer is the most frequently studied cancer with respect to androgenic effects on CSCs, and its androgen-mediated effects appear to be unique relative to other cancers that have been investigated to date. Prostate cancer has received significant clinical attention for its potential reliance on androgens for tumor cell survival and proliferation, giving credence to the use of androgen deprivation therapy as a viable therapeutic measure to be used in conjunction with radiotherapy. However, this strategy of androgen depletion may in fact select for prostate CSCs and encourage their proliferation. Indeed, while androgen deprivation proves highly efficacious for many patients in the short term, many patients develop aggressive, castration-resistant prostate cancer (76). The development of castration-resistant prostate cancer is putatively driven by prostate CSCs, which in turn have been demonstrated to proliferate following androgen deprivation therapy (76,77). Hypoxia-inducible factor signaling has been shown to increase stemness in prostate cancer cells, and this signaling is upregulated in the absence of androgens, providing a mechanism for androgen depletion to drive CSC proliferation (76). It is possible that the absence of androgens and androgen signaling is favorable for prostate CSCs, a hypothesis that is supported by the reported lack of androgen receptor (AR) in self-renewing prostate CSCs due to its targeting by MDM2 for ubiquitination and degradation (78). The role of NANOG in prostate CSC maintenance and proliferation is somewhat more controversial, as opposite effects have been reported—both that NANOG is upregulated in the presence of androgen depletion (70,71) and that NANOG is upregulated by AR activity through direct binding of the NANOG promoter (79). Overall, the majority of available evidence indicates that, in the case of prostate cancer, androgen depletion drives CSC formation and proliferation. Further investigation is warranted to ensure that androgen depletion therapy does not unintentionally select for CSCs.

The role of androgens in GBM, bladder cancer, ovarian cancer, and breast cancer CSCs appears to be the opposite of that observed in prostate cancer. Specifically, AR activity in GBM, urothelial cell carcinoma, triple-negative breast cancer, and ovarian cancer has been demonstrated to drive CSC proliferation and increase CSC frequency. Whereas androgen deprivation therapy may inadvertently select for prostate CSCs, the same therapy may actually be effective at mitigating the proliferation of CSCs in the aforementioned cancers. In a urothelial cell carcinoma model, AR activity was shown to increase CSC formation and proliferation (75). The mechanism of androgen-mediated CSC formation and proliferation in ovarian CSCs has been shown to rely on NANOG upregulation as a function of AR binding directly to the NANOG promoter (74). In triple-negative breast cancer, AR has been shown to bind similar consensus DNA response elements as PR and ER, thereby facilitating formation and proliferation of CSCs (73). A study evaluating the impact of anti-androgens found that enzalutamide improved survival and reduced GBM CSC proliferation (72). Specifically, NANOG and OCT4 expression decreased following enzalutamide treatment. Whether these mechanisms are generalizable across different cancers remains to be seem, and it is worth noting that studies of androgen-mediated effects on CSCs in the aforementioned cancers are still limited; further investigation is necessary to definitively qualify these effects.

Future Directions—Sex Hormones, CSCs, and GBM

GBM cells often express AR, raising the question of how androgens are involved in regulating the formation and proliferation of GBM CSCs (80). The role of androgens in GBM has been investigated extensively due to the apparent sex differences in GBM incidence and survival. Many studies report that androgens promote GBM proliferation, survival, and resistance to treatment, with enzalutamide (81), cedrol (82), and flutamide (83) showing promise as anti-androgenic antitumor agents, especially when used alongside standard chemotherapeutics such as temozolomide. The reliance of GBM on androgen-mediated activity for proliferation is given credence by the finding that AR is overexpressed in GBM relative to healthy brain tissue in both men and women (80,81). In contrast, ER is not expressed widely, if at all, in GBM (84). As such, anti-androgen therapy has emerged as a potential treatment option for AR-expressing GBM that merits further investigation.

The role of androgen-mediated activity on GBM CSCs is less clear, with preliminary data showing an effect similar to that seen in urothelial cell carcinoma, triple-negative breast cancer, and ovarian cancer. Enzalutamide was effective at reducing GBM CSC proliferation in both a murine-derived GBM cell line and a patient-derived xenograft model (72). This study observed that AR expression positively correlated with GBM CSC genes, with decreased AR nuclear translocation associated with decreased CSC gene marker expression. Although this study was limited in terms of the number of GBM cell lines assessed (as GBM is highly heterogeneous), it provides assurance that the role of androgens in promoting the survival and proliferation of GBM CSCs is a promising avenue for inquiry.

A critical barrier to the study of sex differences is the ability to independently investigate sex hormones and sex chromosomes. The 4 core genotype (FCG) mouse model is a unique tool that helps overcome these challenges by allowing for the separation of sex hormone effects from those mediated by sex chromosomes (85). FCG animals are generated through deletion of the Sry gene from the Y chromosome and its subsequent integration into a given autosome in the same mouse (86). Resulting animals are therefore XY-Sry with female gonads and, when mated with XX females, give rise to the 4 “core” genotypes, namely XX and XY− with ovaries and XXSry and XYSry with testes. The FCG model has previously been leveraged to investigate sex differences as they relate to brain development and differentiation as well as adult brain function and will undoubtedly be useful in the study of GBM CSCs (87,88).

In addition to the direct regulation of CSC signaling and phenotype as described in the previous discussion, sex hormones can indirectly affect CSC behavior by regulating immune cells in the tumor microenvironment. In general, females have more active immune systems, which often correlates with lower incidence of cancer and better prognosis and disease progression (89). Sex hormone receptors are highly expressed by various immune cells, and the effect of sex hormones on multiple immune cell subsets has been well studied (90). As CSCs and immune cells can interact in the tumor microenvironment (91), further investigation will be needed to elucidate how sex hormones regulate GBM CSCs via altered immune cell function.

Conclusions

Targeting CSCs in GBM remains a barrier to the development of efficacious therapies, and a more complete understanding of factors influencing the CSC state is therefore critical. CSC heterogeneity is becoming well recognized, with distinct molecular alterations clustering with specific CSC populations (92). However, it is unclear whether there are sex difference between CSC populations, and this could help inform overall sex difference in GBM progression. Moreover, there is an accumulating amount of genomics data from GBM and CSCs, both bulk and single cell RNA-sequencing, and little has been annotated with biological sex of the patient, and this is another priority for the field, as outlined recently (93). In addition, there is limited information about the impact of sex hormones and chromosome-dependent gene expression on CSC function constitutes a significant gap in our understanding of GBM. While the effects of estrogens and androgens are well described and are commonly targeted in other cancer types, it is not currently appreciated how these hormones may regulate GBM CSCs or, for that matter, CSC populations in other nonreproductive cancers, whether directly by interacting with receptors expressed by these cells or indirectly by affecting the tumor microenvironment (Fig. 1). This is further confounded by the reduction in sex hormones as a result of aging and the general increase in cancer onset at older age, especially for GBM. The development of new modeling systems and investigation into the crosstalk between the tumor microenvironment, sex hormones, and GBM CSCs may be the key to promising new therapeutic targets. Collectively, the previous work on sex differences in GBM underscores the importance of prioritizing the study of these differences in the CSC state.

Abbreviations

Abbreviations
 
• AR

  androgen receptor

 
• CSC

  cancer stem cell

 
• E2

  estradiol

 
• ERα

  estrogen receptor alpha

 
• ERβ

  estrogen receptor beta

 
• FCG

  4 core genotype

 
• GBM

  glioblastoma

 
• miRNA

  micro RNA

 
• mPR

  membrane-bound progesterone receptor

 
• PR-A

  progesterone receptor A

 
• PR-B

  progesterone receptor B.

Acknowledgments

We would like to thank Dr. Erin Mulkearns-Hubert for editorial assistance.

Financial Support

Work in the Lathia laboratory on sex differences is supported by the Cleveland Clinic, Case Comprehensive Cancer Center, the American Brain Tumor Association, National Brain Tumor Society, and National Institutes of Health P01 CA245705.

Disclosures

The authors have nothing to disclose.

Data Availability

Data sharing is not applicable to this article because no data sets were generated or analyzed during the present study.

References

Author notes