Unexplored functions of sex hormones in glioblastoma cancer stem cells

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 non-reproductive 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, 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. Glioblastoma 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 versus 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.

Human INHBB gene variant (c.1079T>C:p.Met360Thr) alters testis germ cell content, but does not impact fertility in mice

Testicular derived inhibin B (α/βB dimers) acts in an endocrine manner to suppress pituitary production of follicle stimulating hormone (FSH), by blocking the actions of activins (βA/B/βA/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (βB-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate Inhbb M364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male Inhbb M364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area, in Inhbb M364T/M364T males compared to wildtype males. Despite this testis phenotype, male Inhbb M364T/M364T mutant mice retained normal fertility. Serum hormone analyses however, indicated that the Inhbb M364T variant resulted in reduced circulating levels of activin B, but did not affect FSH production. We also examined the effect of this p.Met360Thr, and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man, on inhibin B and activin B in vitro biosynthesis. It was found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males.

Comprehensive sex steroid profiling in multiple tissues reveals novel insights in sex steroid distribution in male mice

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography-tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After ORX, the levels of active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice.

New insights in the interaction of FGF/FGFR and steroid receptor signaling in breast cancer

Luminal breast cancer (BrCa) has a favorable prognosis compared to other tumor subtypes. However, with time tumors may evolve and lead to disease progression. Thus, there is a great interest in unraveling the mechanisms that drive tumor metastasis and endocrine resistance. In this review we focused in one of the many pathways that have been involved in tumor progression, the FGF/FGFR axis. We emphasized in data obtained from in vivo experimental models since we believe that in luminal BrCa, tumor growth relies in a crosstalk with the stromal tissue. We revisited the studies that illustrate the interaction between hormone receptors and FGFR. We also highlighted the most frequent alterations found in BrCa cell lines and we provide a short review on the trials that use FGFR inhibitors in combination with endocrine therapies. The analysis of this data suggests that there are many players involved in this pathway that might be also targeted to decrease FGF signaling in addition to specific FGFR inhibitors that may be exploited to increase their efficacy.

Vasoactive intestinal peptide indirectly elicits pituitary LH secretion independent of GnRH in female zebrafish

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhβ mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild type and gnrh3-/- females at 1 hour post-treatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA’s manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and E2 in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

FOXO transcription factors are required for normal somatotrope function and growth

Understanding the molecular mechanisms underlying pituitary organogenesis and function is essential for improving therapeutics and molecular diagnoses for hypopituitarism. We previously found that deletion of the forkhead factor, Foxo1, in the pituitary gland early in development delays somatotrope differentiation. While these mice grow normally, they have reduced growth hormone expression and free serum IGF1 levels, suggesting a defect in somatotrope function. FOXO factors show functional redundancy in other tissues, so we deleted both Foxo1 and its closely related family member, Foxo3, from the primordial pituitary. We find that this results in a significant reduction in growth. Consistent with this, male and female mice in which both genes have been deleted in the pituitary gland (dKO) exhibit reduced pituitary growth hormone expression and serum IGF1 levels. Expression of the somatotrope differentiation factor, Neurod4, is reduced in these mice. This suggests a mechanism underlying proper somatotrope function is the regulation of Neurod4 expression by FOXO factors. Additionally, dKO mice have reduced Lhb expression and females also have reduced Fshb and Prl expression. These studies reveal FOXO transcription factors as important regulators of pituitary gland function.

Growth hormone-induced STAT5B regulates Star gene expression through a cooperation with cJUN in mouse MA-10 Leydig cells

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star mRNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (EMSA and supershift) and in vivo (ChIP) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

Deletion of PTP 1B from Brain Neurons Partly Protects Mice from Diet-Induced Obesity and Minimally Improves Fertility

Reproductive dysfunction in women has been linked to high calorie diet (HCD)-feeding and obesity. Central resistance to leptin and insulin have been shown to accompany diet-induced infertility in rodent studies, and we have previously shown that deleting suppressor of cytokine signaling 3, which is a negative regulator of leptin signaling, from all forebrain neurons partially protects mice from HCD-induced infertility. In this study, we were interested in exploring the role of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of both leptin and insulin signaling, in the pathophysiology of HCD-induced obesity and infertility. To this end, we generated male and female neuron-specific PTP1B knockout mice and compared their body weight gain, food intake, glucose tolerance and fertility to control littermates under both normal calorie diet-feeding and HCD-feeding conditions. Both male and female mice with neuronal PTP1B deletion exhibited slower body weight gain in response to HCD-feeding, yet only male knockout mice exhibited improved glucose tolerance compared with controls. Neuronal PTP1B deletion improved the time to first litter in HCD-fed mice, but did not protect female mice from eventual HCD-induced infertility. While the mice fed a normal caloric diet remained fertile throughout the 150 day period of assessment, HCD-fed females became infertile after producing only a single litter, regardless of their genotype. These data show that neuronal PTP1B deletion is able to partially protect mice from HCD-induced obesity, but is not a critical mediator of HCD-induced infertility.

Circadian Rhythms in the Neuronal Network Timing the Luteinizing Hormone Surge

For 3.5 billion years before electric light was invented in 1879, life on Earth evolved under the pattern of light during the day and darkness during the night. Through evolution, nearly all organisms internalized the temporal rhythm of Earth’s 24-hour rotation and evolved self-sustaining biological clocks with a ~24-hour rhythm. These internal rhythms are called circadian rhythms, and the molecular constituents that generate them are called molecular circadian clocks. Alignment of molecular clocks with the environmental light-dark rhythms optimizes physiology and behavior. This is particularly true for reproductive function, in which seasonal breeders use day-length information to time yearly changes in fertility. However, it is becoming increasingly clear that light-induced disruption of circadian rhythms can negatively impact fertility in non-seasonal breeders as well. In particular, the luteinizing hormone surge promoting ovulation, is sensitive to circadian disruption. In this review, we will summarize our current understanding of the neuronal networks that underlie circadian rhythms and the luteinizing hormone surge.