United States

PCOS confers an increased risk for obesity-related comorbidities. It has been challenging to identify precise risk genes associated with the pathogenesis of PCOS. A novel variant located within MYO10 intron 2 (rs9312937) was identified in a PCOS GWAS in the genetically isolated Finnish and related Estonian populations. We identified PCOS risk variants at the MYO10 locus in additional PCOS populations. We hypothesized that PCOS risk variants at the locus were associated with metabolic traits.
Women from Iceland (n=1347) and Boston (n=591) were diagnosed with PCOS using the Rotterdam criteria and NIH criteria, respectively, with a non-overlapping replication cohort (n=586) identified using an algorithm incorporating ICD codes and natural language processing. Controls included population controls from Iceland (effective n=1200), well-phenotyped controls from Boston (n=441) and a cohort with no PCOS diagnoses or features in the electronic medical record (EMR; n=749). We performed an association study to identify variants in the MYO10 locus associated with PCOS. We also examined the association of variants in MYO10 and 15 independent phenotypic traits in women of European ethnicity from the Boston PCOS cohort. Data were log-normalized and analyzed using linear regression, then corrected for the false discovery rate using the Benjamini-Hochberg procedure.
Two novel risk variants for PCOS were identified within intron 2 of MYO10, rs31506-T (p = 1.6X10-05, OR 0.83 [95% CI 0.77-0.91]) and rs253336-A (p = 2.4X10-06, 0.82 [0.75-0.89]). The rs31506 variant is in moderate linkage disequilibrium with the novel variant rs9312937 (r² ≥ 0.4) and rs253336 (r² ≥ 0.4). The variant rs31506 maps to enhancer sites in pancreas, osteoblast and fetal adrenal cells, whereas rs253336 maps to methylation sites in adipose stem cells and ovary (pubs.broadinstitute.org/mammals/haploreg). Neither variant had eQTLs, and expression was highest in lung, thyroid, neurons, breast and ovary (gtexportal.org).
Among cases and controls, the variant rs253336 was associated with BMI (β=0.015±0.0057; p=0.01), waist circumference (β=0.011±0.0047; p=0.02), hip circumference β=0.010±0.0035; p=0.003) and triglycerides (β=0.028±0.013; p=0.03). The variant rs31506 was also associated with BMI (β=0.0065±0.0027; p=0.02), waist circumference (β=0.0051±0.0022; p=0.02), hip circumference (β=0.0039±0.0017; p=0.02), HDL (β=-0.0083±0.0037; p=0.02) and triglycerides (β=0.018±0.0063; p=0.003). The association between rs253336 and hip circumference, and rs31506 and triglyceride levels remained significant after correction. There was no association between the variants and BMI in the subjects identified through the EMR.
We identified two novel risk variants for PCOS in intron 2 of MYO10 (rs31506 and rs253336), a gene recently implicated within a risk locus for PCOS. We also demonstrated that the variants are associated with hip circumference and triglyceride levels. Previous data demonstrate increased expression of MYO10 in omental adipose of obese type 2 diabetics and an association with lean body mass. The data suggest a potential role for MYO10 in the metabolic etiology of PCOS.

ENDO 2022

Risk Variants at the MYO10 Locus are Associated with Metabolic Traits

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four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
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technical paper

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities. The gut microbiome is responsible for fermentation of indigestible fiber to produce short-chain fatty acids (SCFA), which help maintain metabolic function and reduce gut inflammation. Alteration in the gut microbiome, or dysbiosis, affects overall metabolic homeostasis and contributes to pathogenesis of metabolic diseases. While dysbiosis has been observed in women with PCOS, the molecular mechanisms of hyperandrogenemia-induced dysbiosis and its contribution to SCFA excretion and gut inflammation have not been studied in PCOS.
Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Animals were treated day 90 and the gastrointestinal tract was collected. Expression levels of mRNA were assessed by RT-qPCR. Fecal microbiota was assessed by 16S rRNA gene sequencing and profiled using MicrobiomeAnalyst; SCFA concentrations were assessed by LC-MS.
Results: DHT-treated female mice had significantly increased lean mass (21.35 ± 0.84 vs 19.25 ± 0.55 g, p<0.05) and increased fat mass (5.18 ± 1.45 vs 2.73 ± 0.64 g, p<0.05) compared to the vehicle counterparts. DHT-treated female mice showed increased HbA1C (5.18 ± 1.45 vs 2.73 ± 0.64 %, p<0.05). Hyperandrogenemic females showed altered gastrointestinal morphology, having decreased small intestine (33.03±2.05 vs 28.69±2.604 cm, p<0.05) and colon length (7.88 ± 0.64 vs 6.60 ± 0.55 cm, p<0.05) as well as increased immune cell infiltration assessed by histology. DHT-treated female mice have an increase in the relative percent abundance of Proteobacteria (2.04 ± 0.62 vs 0.35 ± 0.23%, p<0.05) and in the ratio of Firmicutes/Bacteriodetes (1.92 ± 0.47 vs 1.11 ± 0.17, p<0.05). Fecal concentrations of short-chain fatty acids were higher in DHT-females for butyric acid (92.33 vs 404.8 ng/mg feces, p<0.05), acetic acid (571.8 vs 1592 ng/mg feces, p<0.05), and propionic acid (80.59 vs 167.3 ng/mg feces, p<0.05). However, expression of the SCFA receptor Ffar2 in the colon was upregulated by DHT (1.31 ± 0.15-fold, p<0.05), indicating the possibility of a compensation to ameliorate increased SCFA excretion. The mRNA expression of lipopolysaccharide induced inflammation markers were significantly upregulated by DHT in the proximal colon (Litaf, 1.69 ± 0.29-fold; Tlr4 1.27 ± 0.15-fold, p<0.05) compared with vehicles.
Conclusions: Androgen-mediated gut dysbiosis may contribute to altered glucose handling, adiposity, and metabolic homeostasis in PCOS. Increased elimination of SCFA in DHT-treated female mice may indicate less efficient absorption, which can contribute to colonic inflammation, altered gastrointestinal morphology, and increased gut permeability. Together, our results highlight the potential role of androgen-mediated gut dysbiosis to influence metabolic dysfunction.

Contribution of Hyperandrogenemia to Dysbiosis, Gut Inflammation, and Dysregulation of Short-Chain Fatty Acid Homeostasis in Mouse Model of Polycystic Ovary Syndrome

Objective: Adolescent polycystic ovary syndrome (PCOS) is a heterogenous clinical syndrome characterized by hyperandrogenism and irregular menses. Heterogeneous features of the syndrome include the presence/absence of obesity, degree of insulin resistance, and clinical and/or biochemical hyperandrogenism. In adult women with PCOS, discrete metabolic and reproductive subgroups can be identified. A recent study described distinct phenotypes using unsupervised hierarchical cluster analysis: a metabolic subgroup characterized by higher BMI, glucose, and insulin levels, and a reproductive subgroup demonstrating higher LH and SHBG levels. We hypothesized that non-obese and obese adolescent girls with PCOS would exhibit similar distinct phenotypes at the time of diagnosis.

Methods: Data were extracted from a multi-site PCOS registry including seven academic centers in the US with the following inclusion criteria: PCOS confirmed per 2018 international guidelines, diagnosis before age 18 years, and at least one follow-up visit. Data collected included demographics, medical and family history, physical characteristics including hirsutism, acanthosis nigricans, and laboratory measures such as HbA1c, ALT, lipid profiles and reproductive hormones (androgens, LH, FSH, SHBG). Hormone levels were normalized to the upper limit provided by the measuring laboratory. Patients were divided by obesity status, defined as obese (OB) with BMI ≥ 95th percentile for age and sex and non-obese (NO) < 85th percentile for age and sex. Differences in continuous variables were assessed with unpaired t-tests using GraphPad Prism.

Results: Complete diagnostic data from 390 patients from 7 different sites were included for analysis, with 284 OB (Age 15.3±1.5 years, BMI 36.1±5.6 kg/m²) and 68 NO (Age 15.8±1.3 years, BMI 22.6±3.4 kg/m²). OB presented with more severe acanthosis (p<0.0001) and higher HbA1c (OB 5.5±0.5, NO 5.2±0.3; %, p<0.0001), ALT (OB 35±30, NO 22±17; IU/L, p=0.0006), and triglycerides (OB 145±90, NO 110,±88; mg/dL, p=0.002). OB also presented with higher free testosterone (OB 219±188% vs NO 158±125% above upper limit of normal; p=0.003), while NO presented with higher total testosterone (OB 121±68 %, NO 152±88 % above upper limit of normal; p=0.003), androstenedione (OB 87±37 %, NO 125±62 above upper limit of normal; p=0.002), and LH (OB 9.7±4.9, NO 15.2±10.4; mIU/mL, p=0.0001). No differences in Ferriman Gallwey score or number of menses in the 6 months prior to diagnosis were detected.

Conclusions: Metabolic and reproductive phenotypes differed in OB and NO adolescent girls at the time of PCOS diagnosis. The reproductive phenotype in NO was characterized by increased LH, total testosterone and androstenedione levels. In contrast, OB girls had a phenotype characterized by insulin resistance-driven hyperandrogenemia established by lower SHBG and higher free testosterone levels. Our data are consistent with findings reported for adult women with two divergent phenotypes: an insulin resistance-driven metabolic subtype and a primarily reproductive subtype, potentially driven by neuroendocrine mechanisms.

Heterogeneity of Clinical Presentation of Adolescent PCOS Is Attributable to Distinct Subtypes

During the SARS-COV2 pandemic (COVID-19) many studies have examined changes in women's stress, depression/anxiety, reproduction, plus post-vaccination cycle/flow changes¹. Although there are retrospective basal temperature data², we lack menstrual cycle and ovulation information during the pandemic. This analysis compared two independent, similar-design, convenience/community-based, single-cycle cohort studies 13 years apart: Menstruation Ovulation Study (MOS³, n=301, 2007-8) and MOS2 (n=112) during the pandemic. With MOS as a control, and given evidence subclinicalovulatory disturbances (SOD; short luteal phase/anovulatory menstrual cycles of normal lengths) occur before changed cycle lengths4 5, we hypothesized MOS2 would have increased SOD but similar cycle lengths as MOS. Methods: In both studies, recruitment of menstruating women ages 19-35 years, not using systemic or combined hormonal contraceptives (CHC) used posters/eblasts/social media. In MOS, ovulation was assessed by 3-fold increased follicular (FP)-to-luteal (LP/premenstrual) urinary progesterone (PdG); in MOS2, by validated Quantitative Basal Temperature© (QBT)6 7 (normal LP=10+ days). We performed the same interviewer-administered (CaMos©) questionnaire for demographics, SES, and reproduction, measured anthropomorphic variables, plus collected daily Menstrual Cycle Diary© (Diary) for all. FP and LP/premenstrual PdG or salivary progesterone (Ps) samples were respectively collected. Participants in MOS2 were not different from MOS in: average age 29, menarche age 12.5, BMI 24, living situation and education (≥75% university graduates). Cohorts also differed: MOS2 women were less likely to to be White (56% vs 76%), work fulltime, ever use CHC (68% vs 79%) or to be parous (8% vs 20%); they were younger at starting CHC (17.9 vs 18.6 years). Results: MOS2 and MOS had similar cycle (30.3 vs 29.9 days, P =.306) and flow lengths (median 6.0 days; P=.055). MOS2 recorded significantly more SOD cycles (>50% anovulatory) vs MOS³ (63% vs 10%; P<.001). MOS2 Diary analyses by Principal Components Analysis showed significantly increased anxiety/depression/frustration (negative moods) and "outside stresses" plus sleep problems and headaches vs MOS (all P<.001). Discussion: This is the first evidence that ovulatory disturbances without cycle length changes may be associated with the multidimensional stresses women experience during the pandemic. Increased SOD may also relate to greater nulliparity, younger CHC teen use and more non-White women in MOS2, as well higher prevalence of negative moods, outside stresses and sleep problems. Salivary progesterone, cortisol and estradiol levels remain pending. Prevalent SOD cycles, if persistent/recurrent, risk increased infertility, bone loss⁵, early heart attacks, and breast and endometrial cancers⁸. In this 'experiment of nature,' the pandemic was associated with disturbed ovulation rather than menstrual cycles. ¹Edelman AB Ob Gyn 2022; ²Nguyen JK PLOS ONE 2021; ³Prior JC IJERPH 2018; ⁴Prior JC NEJM 1990; ⁵Li D Epidiol Rev 2014; ⁶Prior JC Clin Invest. Med 1990; ⁷Bedford JL Eur J ObGyn Reprod Biol 2009; ⁸Prior JC DDDM 2020.

Epidemic of Subclinical Ovulatory Disturbances During SARS-COV2 Pandemic—an Experiment of Nature

In orthotopic xenografts and a PDX, our anticancer drug ErSO eradicates primary and metastatic therapy-resistant estrogen receptor alpha (ERα) positive breast cancer, and induces near-complete regression of ovarian cancer. The mechanism by which ErSO induces necrosis and kills ER positive cancer cells was unknown. From genome-wide CRISPR-Cas9 screens in MCF-7 and T47D cells with negative selection against first-generation BHPI and second-generation ErSO, and follow-on experiments, we identified the Ca2+ activated, plasma membrane Na+ channel TRPM4 as the executioner protein that BHPI and ErSO use to induce necrosis. Notably, in 6 ERα+ breast and ovarian cancer cell lines, knockout of TRPM4 completely abolished the ability of ErSO to induce death of cancer cells. Moreover, TRPM4 mRNA and protein were dramatically down-regulated in breast cancer cells selected for resistance to BHPI and ErSO. Furthermore, in a mouse xenograft, while ErSO induced near complete regression of orthotopic MCF-7-ERY537S-luciferase tumors, ErSO had no effect on the TRPM4 knockout tumors, which continued their robust growth. Since necrosis, but not most other death pathways, activates immune cells, inducing immunogenic cell death, this provides a new avenue for enhancing cancer immunotherapy. Importantly, medium from ErSO-treated wild type MCF-7 cells, but not medium from TRPM4 knockout cells, robustly activates human THP-1 monocytes and greatly increases their migration. BHPI and ErSO-induced initial anticipatory unfolded protein response (a-UPR) activation results in elevated cytosolic Ca2+, opening the plasma membrane TRPM4 channel, eliciting a rapid influx of external Na+, accompanying Cl- to balance the charge, and water to maintain osmolality. This swells the cells, causing osmotic stress. Importantly, it is the osmotic stress that sustains UPR hyperactivation, leading to ATP depletion, which contributes to membrane rupture and rapid necrotic cell death and to near complete inhibition of protein synthesis that ultimately kills any surviving cancer cells . Suggesting a broad role of TRPM4 in the actions of necrosis inducing anticancer drugs, TRPM4 knockout also inhibited necrosis induced by unrelated anticancer therapies, the mitochondrial targeting oncolytic peptide, LTX-315 and the Ca2+ channel targeting agent, Englerin A. Since increasing expression TRPM4 by viral transduction results in progressively increased sensitivity of ER positive breast cancer cells to killing by ErSO, this enables identification of breast cancer patients whose elevated TRPM4 levels make them most likely to benefit from this novel therapy. The TRPM4 pathway is a new mechanism for sustained lethal activation of the UPR and for targeting ER positive breast and ovarian cancer.

How Strong and Sustained Activation of the Estrogen Receptor-mediated Anticipatory Unfolded Protein Response Kills Breast and Ovarian Cancer Cells

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the survival and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in recurrent tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. We previously showed that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. PELP1 interacts with PFKFB proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB inhibitors in combination with ER targeted therapies blocked proliferation and tumorsphere formation in multiple models of advanced breast cancer, including paclitaxel (TaxR) and tamoxifen (TamR) resistant models and ER+ patient-derived organoids (PDxO). Chemotherapy and endocrine therapy resistant models contained increased levels of cytoplasmic PELP1, PELP1/SRC-3 complexes, and phenocopied PELP1/SRC-3/PFKFB biology and CSC behavior. To better understand CSC-mediated pathways in the context of therapy resistance, we performed RNA-seq on 3D-cultured TaxR tumorspheres to enrich for CSCs. Pathway analysis revealed that TaxR tumorspheres upregulate stem (Sox2-Oct4-Nanog), HIF, and progesterone mediated pathways. Progesterone receptor (PR) is a known contributor to CSC populations, and we observed increased PR mRNA and protein expression in TaxR cells grown as tumorspheres. Additionally, in vivo emergence of circulating tumor cell (CTC) populations was observed in ER+ TaxR mammary intraductal (MIND) xenografts. Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Our findings are applicable to therapy resistant models, indicating that SR co-activator complexes are a key mediator of resistance in ER+ breast cancer and may cooperate with PR to promote therapy resistant CSCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer.

Steroid receptor co-activator complexes regulate metabolic PFKFB enzymes to drive therapy resistant ER+ breast cancer

Emerging studies show that testis-enriched genes play critical roles in several biological processes. The expression of these genes is tightly regulated and limited to immune-privileged organs; however, many of them escape regulation and are aberrantly expressed in several diseases such as cancer. Intriguingly, our genomic analysis revealed that many of them are transcribed from previously considered gene deserts. We also found that a significant number of them are robustly regulated by estrogen and other peptide hormones such as Tumor Necrosis Factor-alpha (TNF alpha). However, their direct role in estrogen-dependent transcriptional regulation and biological outcomes remains unstudied. In this regard, our integrated genomic approach, including RNA with 4-thiouridine (4sU) coupled with RNA-sequencing and CRISPR-based genetic screening, has identified key estrogen-regulated testis-enriched genes that may modulate estrogen-signaling. Through a series of molecular and phenotypic assays, we selected a potential but understudied transcript, which is predominantly localized to the nucleus. Furthermore, in the current study, we have interrogated the functional mechanisms by which selected transcript controls estrogen-dependent gene regulation in estrogen receptor-positive (ER+) breast cancer. We found that the transcript controls estrogen-dependent cell and tumor growth. Additionally, we have manipulated the levels of the transcript in estrogen-receptor-positive (ER+) breast cancer cells employing gain- and loss-of-function studies and performed 4sU-RNA-seq. Interestingly, the transcript modulates the expression of estrogen-driven transcripts that are transcribed from ER-alpha (ERa) binding sites. Mechanistically, the transcript is localized in the nucleus and interacts with chromatin-associated proteins. In addition, our integrated genomic analyses comprising 'Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)' and 'chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing ChIP-seq' (H3K27ac, H3K27me3) identified a critical role for the transcript in controlling the epigenetic landscape at target gene promoters and enhancers in ER+ breast cancer. Collectively, our data support a functional role for testis-enriched genes in enhancer transcription and chromatin organization. This study sheds light on the molecular functions of testis-enriched genes and presents an opportunity to identify new diagnostic and develop treatment strategies in ER+ breast cancer.
S.S.G. is supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT; RR170020). S.S.G. is also supported by a grant from Lizanell and Colbert Coldwell foundation.

Analysis of testis-enriched genes in estrogen-dependent genome regulation in breast cancer

Although studies have shown beneficial effects of the phytoestrogen genistein on reducing ovarian cancer risk, little is known at the molecular level regarding how genistein inhibits ovarian cancer growth, with estrogen receptor beta (ERb), a hormone-regulated transcription factor, receiving the bulk of the attention. When endogenous and exogenous ligands engage ERb, the receptor binds to regulatory regions in the genome known as enhancers, where it associates with coregulatory proteins, increases histone modifications associated with active enhancers, promotes chromatin looping, stimulates enhancer transcription and the production of enhancer RNAs, and ultimately regulates target gene transcription. Since signaling pathways converge at the transcriptome level, an unbiased approach is needed to examine how genistein acts to suppress ovarian cancer growth beyond ERb.  We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. To determine the genomic responses upon genistein treatment in ovarian cancer cells, we have used PRO-seq to characterize the enhancer landscape in OVCAR3 and OVCAR4 ovarian cancer cell lines treated with genistein. The advantage of this method is the lack of a priori assumptions needed to determine which signaling pathways are regulated by genistein, since PRO-seq allows pathway determination based on the universe of active enhancers.  Ultimately, this approach will allow us to uncover signaling pathways beyond ERb and determine how genistein acts as a signaling ligand to change the transcriptome in ovarian cancer cells.

Genomic Interrogation of the Phytoestrogen Genistein in Ovarian Cancer Cells

Approximately 75% of breast cancers are classified as hormone receptor-positive, most of which are estrogen receptor alpha-positive (ER+), which is the primary driver of growth in these tumors. Consequently, endocrine or anti-estrogen therapy is used for treatment. However, despite the benefits of endocrine therapeutics, treatment-resistant relapse is a significant problem that affects 30-50 % of patients who initially respond to endocrine therapy. Therefore, studying resistance to endocrine therapy is critical for breast cancer research.We have shown that invasive lobular breast cancer (ILC) cells resistant to the anti-estrogen tamoxifen increase their expression of metabotropic glutamate receptors (GRMs/mGluRs). GRMs are well-known to play important roles in learning and memory in the brain and protect neurons from excitotoxicity (cell death caused by prolonged exposure to neurotransmitters). It is now appreciated that this pro-survival function of GRMs can be highjacked by cancer cells, including breast cancer. Thus, targeting GRM signaling could prove a valuable therapeutic strategy. We chose to target the GRM signaling pathway with the FDA-approved drug Riluzole currently being used to treat amyotrophic lateral sclerosis.In this study, we test the ability of Riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated, ILC-derived patient-derived xenograft model HCI-013EI. In addition to measuring tumor growth rate and size, primary tumors and organs were collected for immunohistochemistry analysis.Riluzole as a single agent suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing differential histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). In tamoxifen-resistant ILC cells, Riluzole induced apoptotic and ferroptotic cell death, and inhibited phosphorylation of focal adhesion kinase. Riluzole combined with either fulvestrant or 4-hydroxytamoxifen additively or synergistically suppressed ER+ breast cancer cell growth in vitro. Using proliferating cell nuclear antigen (PCNA) staining as a proxy for cell proliferation, the combination of Riluzole plus Fulvestrant significantly reduced PCNA in a patient-derived explant model (t-test p = 0.013). The in vivo experiment showed reduced tumor size and growth between the control and combination treatments. However, unlike in the in vitro experiment, there was little difference between the single-agent fulvestrant and the combination groups. The observed difference between the in vitro and in vivo study may be attributed to the bioavailability of Riluzole in mice.In conclusion, our results show that Riluzole enhances response to endocrine therapy in ER+ breast cancer.

Riluzole suppresses growth and enhances response to endocrine therapy in ER+ breast cancer

Background and Objectives: Human fertility is attained following puberty due to finely orchestrated events driven by hypothalamic release of the excitatory neuropeptide neurokinin B (NKB), which in turn stimulates the release of kisspeptin and then GnRH to activate the downstream pituitary-gonadal axis. Our group has identified that loss-of-function mutations in Makorin Ring Finger Protein 3 (MKRN3) cause central precocious puberty (CPP). Mkrn3 is highly expressed in the hypothalamus of both male and female mice in early postnatal life, then declines prior to the onset of puberty. Mutations in MKRN3 E3 ubiquitin ligase domain, identified in patients with CPP, result in reduced auto-ubiquitination. However, the mechanisms of action of MKRN3 and its targets remain largely unknown. We hypothesized that MKRN3 inhibits the reproductive axis through interactions with hypothalamic substrates, targeting them to degradation pathways.
Methods and Results: Using interactome analysis and candidate approaches in vitro, we identified poly(A) binding protein cytoplasmic 1 and 4 (PABPC1 and 4) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as MKRN3 interactors, consistent with reports from other research groups. Additionally, our proteomic analysis revealed that Igf2bp1 levels are increased in the hypothalamus of Mkrn3 knockout (KO) mice versus wild type animals. Remarkably, we found that MKRN3-IGF2BP1 interaction is mediated by RNA, as their interaction was abrogated by RNase treatment. However, an MKRN3 missense mutation associated with CPP, p.C364F, within the E3 ubiquitin ligase domain, did not affect the RNA mediated interaction. Preliminary data from RNA co-IP, aiming to identify putative RNA targets, suggested that MKRN3 and IGF2BP1 bind to TAC3 mRNA. We further identified an increase in Nkb protein levels in the hypothalamus of Mkrn3 KO mice. We also showed that NKB is a substrate of MKRN3 E3 ubiquitin ligase activity in vitro by demonstrating increased ubiquitination of NKB in cells co-transfected with expression vectors encoding MKRN3 and NKB and treated with proteasome inhibitor. Additionally, MKRN3 overexpression resulted in degradation of NKB protein by western blot analysis, and alteration of NKB intracellular localization by immunofluorescence. These effects were impaired by MKRN3 missense mutations (p.C340G and p.C364F) in the E3 ubiquitin ligase motif, suggesting a pathophysiological mechanism of MKRN3 mutations in CPP.
Conclusions: We identified that MKRN3 interacts with PABPC1, PABPC4 and IGF2BP1 and targets NKB to degradation. We showed that NKB degradation was mediated by the MKRN3 E3 ubiquitin ligase domain, as mutations within this motif abrogated NKB degradation. However, this mutation did not affect MKRN3 RNA-mediated binding to IGF2BP1, highlighting that multiple domains of MKRN3 may contribute to its actions. Here, we propose a mechanism by which MKRN3, interacting with PABPCs and IGF2BP1 and targeting NKB for degradation, may act to inhibit the reproductive axis.

MKRN3 inhibits the reproductive axis by interacting with key hypothalamic substrates and targeting neurokinin B to degradation pathways.

Psychological stress is linked to infertility and reproductive dysfunction by suppressing the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator and pulsatile luteinising hormone (LH) secretion. The posterodorsal subnucleus of the medial amygdala (MePD) is a key upstream regulator of GnRH pulse generator activity and displays increased neuronal activation in response to psychological stressors. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centres, however, their functional significance has not been determined. We hypothesis that MePD GABAergic signalling is a crucial mediator of psychological stress-induced suppression of pulsatile LH secretion. We used a chemogenetic approach to selectively silence MePD GABA neurones during psychological stress to determine the effect on pulsatile LH secretion. Female ovariectomised Vgat-cre mice (n=7) were virally infected to selectively express inhibitory hM4DGi-DREADDs in MePD GABA neurones. Mice were exposed to either predator odour (PO) or restraint stress and given an intraperitoneal injection of either saline or the DREADD chemical activator clozapine-N-oxide (CNO, 5mg/kg). Statistical significance was determined using a one-way ANOVA and post-hoc Tukey test. Data are presented as mean ± SEM and p<0.05 was considered significant. PO exposure significantly increased LH inter-pulse interval (IPI) in the saline group (prestress: 19.05 ± 1.16 min vs. PO: 29.28 ± 3.16 min, p<0.05, n=7) and this effect was blocked by DREADD-mediated inhibition of MePD GABA neurones (prestress: 20.59 ± 1.12 min vs. PO:  21.31 ± 1.76 min, p>0.05, n=7). Restraint stress dramatically increased LH IPI in the saline group (prestress: 17.78 ± 1.02 min vs. restraint: 45.71 ± vs 5.50 min, p<0.05, n=7) and DREADD-mediated inhibition of MePD GABA neurones blocked this effect (prestress: 18.57 ± 1.97 min vs. restraint: 27.26 ± 4.02 min, p>0.05, n=7). Direct projections from the MePD to the GnRH pulse generator in the hypothalamic arcuate nucleus (ARC) have been identified, however, their phenotype is unknown. In this study, we optogenetically stimulated potential MePD GABAergic projection neurone terminals in the ARC and determined the effect on LH IPI. MePD GABA neurones in female Vgat-cre ovariectomised mice (n=6) were virally infected to express channelrhodopsin 2 protein and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via a chronically implanted fibre-optic cannula in the ARC. Sustained optogenetic stimulation at 10 and 20Hz of MePD GABAergic terminals in the ARC dose-dependently suppressed pulsatile LH secretion (LH IPI; control: 23.34 ± 2.11 min vs. 10Hz: 38.08 ± 3.61 min; 20Hz: 56.67 ± 3.33 min, p<0.05, n=6). These findings confirm a functionally significant MePD GABAergic projection to the ARC and highlight the importance of GABA signalling in the MePD in mediating stress-induced suppression of reproductive function. Collectively, these findings provide novel insight into the amygdala neural circuitry underpinning stress-induced reproductive function.

Risk Variants at the MYO10 Locus are Associated with Metabolic Traits

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