Progesterone suppresses podocalyxin partly by up-regulating miR-145 and miR-199 in human endometrial epithelial cells to enhance receptivity in in vitro models.

Establishment of endometrial surface receptivity is crucial for the initiation of embryo implantation yet the molecular mechanisms are not well understood, especially in humans. We have recently discovered that podocalyxin (PODXL) is a critical negative regulator of human endometrial surface receptivity. PODXL is highly expressed in all epithelial and endothelial cells in the non-receptive endometrium, but down-regulated specifically in the luminal epithelium at receptivity. We have further shown that PODXL inhibits embryo implantation, and that PODXL down-regulation is essential for endometrial surface receptivity. Our previous study also indicated that progesterone down-regulates PODXL; however, the exact molecular regulations are unknown. Here, we investigated whether progesterone suppresses PODXL via microRNAs (miRNAs). We first bioinformatically predicted 13 miRNAs that may potentially target human PODXL, then experimentally determined whether any of these 13 miRNAs are altered in primary human endometrial epithelial cells (HEECs) by progesterone, and whether the identified miRNAs can affect PODXL expression in Ishikawa cells without progesterone and alter receptivity to embryo implantation. Progesterone significantly up-regulated miR-145 and miR-199 while suppressing PODXL in HEECs. When these two miRNAs were transfected into Ishikawa cells, both significantly down-regulated PODXL mRNA and protein in the absence of progesterone. Moreover, both miR-145 and miR-199 significantly enhanced receptivity of the Ishikawa monolayer to embryo implantation in in vitro models. This study thus provides in vitro evidence that PODXL is down-regulated by progesterone partly via miR-145 and miR-199 during the development of human endometrial epithelial receptivity. These results also reveal the likely importance of hormonal regulation of miRNAs for embryo implantation.

Podocalyxin molecular characteristics and endometrial expression: high conservation between humans and macaques but divergence in mice†.

Podocalyxin (PODXL) is a newly identified key negative regulator of human endometrial receptivity, specifically down-regulated in the luminal epithelium at receptivity to permit embryo implantation. Here, we bioinformatically compared the molecular characteristics of PODXL among the human, rhesus macaque, and mouse, determined by immunohistochemistry and in situ hybridization (mouse tissues) whether endometrial PODXL expression is conserved across the three species and examined if PODXL inhibits mouse embryo attachment in vitro. The PODXL gene, mRNA, and protein sequences showed greater similarities between humans and macaques than with mice. In all species, PODXL was expressed in endometrial luminal/glandular epithelia and endothelia. In macaques (n = 9), luminal PODXL was significantly down-regulated when receptivity is developed, consistent with the pattern found in women. At receptivity, PODXL was also reduced in shallow glands, whereas endothelial expression was unchanged across the menstrual cycle. In mice, endometrial PODXL did not vary considerably across the estrous cycle (n = 16); however, around embryo attachment on d4.5 of pregnancy (n = 4), luminal PODXL was greatly reduced especially near the site of embryo attachment. Mouse embryos failed to attach or thrive when co-cultured on a monolayer of Ishikawa cells overexpressing PODXL. Thus, endometrial luminal PODXL expression is down-regulated for embryo implantation in all species examined, and PODXL inhibits mouse embryo implantation. Rhesus macaques share greater conservations with humans than mice in PODXL molecular characteristics and regulation, thus represent a better animal model for functional studies of endometrial PODXL for treatment of human fertility.

Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity.

Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

Podocalyxin inhibits human embryo implantation in vitro and luminal podocalyxin in putative receptive endometrium is associated with implantation failure in fertility treatment.

OBJECTIVE: To study whether endometrial epithelial podocalyxin (PCX) inhibits implantation of human embryos in vitro and in patients undergoing in vitro fertilization (IVF). DESIGN: We have recently identified PCX as a key negative regulator of endometrial epithelial receptivity. Podocalyxin is expressed in all epithelial cells in the nonreceptive endometrium, but is selectively downregulated in the luminal epithelium (LE) for receptivity. In the current study, we first investigated whether high levels of PCX in Ishikawa monolayer inhibit attachment and/or penetration of human blastocysts in in vitro models. We then examined PCX by immunohistochemistry in putative receptive endometrial tissues biopsied from 81 IVF patients who underwent frozen embryo transfer in the next natural cycle and retrospectively analyzed the association between PCX staining in LE and clinical pregnancy as a proxy of successful implantation. SETTING: RMIT University, Australia; Vrije Universiteit Brussel, Belgium. PATIENT(S): In vitro fertilization patients undergoing frozen/thawed embryo transfer. INTERVENTION(S): N/A. MAIN OUTCOME MEASURE(S): Endometrial epithelial PCX inhibits implantation of human embryos in vitro and in IVF patients. RESULT(S): High levels of PCX in Ishikawa monolayer significantly inhibited blastocyst attachment and penetration. Among the 81 putative receptive tissues, 73% were negative, but 27% were heterogeneously positive for PCX in LE. The clinical pregnancy rate was 53% in those with a PCX-negative LE but only 18% in those with a PCX-positive LE. If LE was positive for PCX, the odds ratio of no clinical pregnancy was 4.95 (95% Confidence interval, 1.48-14.63). CONCLUSION(S): Podocalyxin inhibits embryo implantation. Assessment of PCX may aid the evaluation and optimization of endometrial receptivity in fertility treatment.

Podocalyxin is a key negative regulator of human endometrial epithelial receptivity for embryo implantation.

STUDY QUESTION: How is endometrial epithelial receptivity, particularly adhesiveness, regulated at the luminal epithelial surface for embryo implantation in the human? SUMMARY ANSWER: Podocalyxin (PCX), a transmembrane protein, was identified as a key negative regulator of endometrial epithelial receptivity; specific downregulation of PCX in the luminal epithelium in the mid-secretory phase, likely mediated by progesterone, may act as a critical step in converting endometrial surface from a non-receptive to an implantation-permitting state. WHAT IS KNOWN ALREADY: The human endometrium must undergo major molecular and cellular changes to transform from a non-receptive to a receptive state to accommodate embryo implantation. However, the fundamental mechanisms governing receptivity, particularly at the luminal surface where the embryo first interacts with, are not well understood. A widely held view is that upregulation of adhesion-promoting molecules is important, but the details are not well characterized. STUDY DESIGN, SIZE, DURATION: This study first aimed to identify novel adhesion-related membrane proteins with potential roles in receptivity in primary human endometrial epithelial cells (HEECs). Further experiments were then conducted to determine candidates' in vivo expression pattern in the human endometrium across the menstrual cycle, regulation by progesterone using cell culture, and functional importance in receptivity using in vitro human embryo attachment and invasion models. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary HEECs (n = 9) were isolated from the proliferative phase endometrial tissue, combined into three pools, subjected to plasma membrane protein enrichment by ultracentrifugation followed by proteomics analysis, which led to the discovery of PCX as a novel candidate of interest. Immunohistochemical analysis determined the in vivo expression pattern and cellular localization of PCX in the human endometrium across the menstrual cycle (n = 23). To investigate whether PCX is regulated by progesterone, the master driver of endometrial differentiation, primary HEECs were treated in culture with estradiol and progesterone and analyzed by RT-PCR (n = 5) and western blot (n = 4). To demonstrate that PCX acts as a negative regulator of receptivity, PCX was overexpressed in Ishikawa cells (a receptive line) and the impact on receptivity was determined using in vitro attachment (n = 3-5) and invasion models (n = 4-6), in which an Ishikawa monolayer mimicked the endometrial surface and primary human trophoblast spheroids mimicked embryos. Mann-Whitney U-test and ANOVA analyses established statistical significance at *P ≤ 0.05 and **P ≤ 0.01. MAIN RESULTS AND THE ROLE OF CHANCE: PCX was expressed on the apical surface of all epithelial and endothelial cells in the non-receptive endometrium, but selectively downregulated in the luminal epithelium from the mid-secretory phase coinciding with the establishment of receptivity. Progesterone was confirmed to be able to suppress PCX in primary HEECs, suggesting this hormone likely mediates the downregulation of luminal PCX in vivo for receptivity. Overexpression of PCX in Ishikawa monolayer inhibited not only the attachment but also the penetration of human embryo surrogates, demonstrating that PCX acts as an important negative regulator of epithelial receptivity for implantation. LIMITATIONS, REASONS FOR CAUTION: Primary HEECs isolated from the human endometrial tissue contained a mixture of luminal and glandular epithelial cells, as further purification into subtypes was not possible due to the lack of specific markers. Future study would need to investigate how progesterone differentially regulates PCX in endometrial epithelial subtypes. In addition, this study used primary human trophoblast spheroids as human embryo mimics and Ishikawa as endometrial epithelial cells in functional models, future studies with human blastocysts and primary epithelial cells would further validate the findings. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study add important new knowledge to the understanding of human endometrial remodeling for receptivity. The identification of PCX as a negative regulator of epithelial receptivity and the knowledge that its specific downregulation in the luminal epithelium coincides with receptivity development may provide new avenues to assess endometrial receptivity and individualize endometrial preparation protocols in assisted reproductive technology (ART). The study also discovered PCX as progesterone target in HEECs, identifying a potentially useful functional biomarker to monitor progesterone action, such as in the optimization of progesterone type/dose/route of administration for luteal support. STUDY FUNDING/COMPETING INTEREST(S): Study funding was obtained from ESHRE, Monash IVF and NHMRC. LR reports potential conflict of interests (received grants from Ferring Australia; personal fees from Monash IVF Group and Ferring Australia; and non-financial support from Merck Serono, MSD, and Guerbet outside the submitted work. LR is also a minority shareholder and the Group Medical Director for Monash IVF Group, a provider of fertility preservation services). The remaining authors have no potential conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Overexpression of aromatase associated with loss of heterozygosity of the STK11 gene accounts for prepubertal gynecomastia in boys with Peutz-Jeghers syndrome.

CONTEXT: Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder that arises as a consequence of mutations in the STK11 gene that encodes LKB1. PJS males often have estrogen excess manifesting as gynecomastia and advanced bone age. We and others have previously described an increase in testicular aromatase expression in PJS patients. However, the underlying mechanism has not yet been explored. OBJECTIVE: The aim of this study was to characterize the role of LKB1 in regulating the expression of aromatase in boys with PJS via signaling pathways involving AMP-activated protein kinase (AMPK) and cyclic AMP-responsive element binding protein-regulated transcription coactivators (CRTCs). PATIENTS: We studied testicular biopsies from two boys with STK11 mutations: a 13-year-old boy and an unrelated 4-year-old boy with prepubertal gynecomastia and advanced bone age, as well as breast tissue from the 13-year-old boy. RESULTS: Loss of heterozygosity of STK11, measured by the absence of LKB1 immunofluorescence, was observed in Sertoli cells of abnormal cords of testis samples from affected individuals. This was associated with loss of p21 expression and decreased phosphorylation of AMPK, known downstream targets of LKB1, as well as the increased expression of aromatase. Similar results of low LKB1 expression in cells expressing aromatase were observed in the mammary epithelium from one of these individuals. Nuclear expression of the CRTC proteins, potent stimulators of aromatase and known to be inhibited by AMPK, was significantly correlated with aromatase. CONCLUSIONS: Loss of heterozygosity of the STK11 gene leads to an increase in aromatase expression associated with an increase in CRTC nuclear localization, thereby providing a mechanism whereby PJS results in increased endogenous estrogens in affected males.

HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma.

INTRODUCTION: The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E2 (PGE2), stimulate CREB1 binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to the increased expression of aromatase and biosynthesis of estrogens within human breast adipose stromal cells (ASCs). Hypoxia inducible factor-1α (HIF-1α), a key mediator of cellular adaptation to low oxygen levels, is emerging as a novel prognostic marker in breast cancer. We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1α in the activation of aromatase PII. METHODS: HIF-1α expression and localization were examined in human breast ASCs using quantitative PCR (QPCR), Western blotting, immunofluorescence and high content screening. QPCR and tritiated water-release assays were performed to assess the effect of HIF-1α on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer). Double immunohistochemistry for HIF-1α and aromatase was performed on tissues obtained from breast cancer and cancer-free patients. RESULTS: Results indicate that PGE2 increases HIF-1α transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1α and aromatase expression. CONCLUSIONS: This study is the first to identify HIF-1α as a modulator of PII-driven aromatase expression in human breast tumor-associated stroma and provides a novel mechanism for estrogen regulation in obesity-related, post-menopausal breast cancer. Together with our on-going studies on the role of AMP-activated protein kinase (AMPK) in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.

CREB-regulated transcription co-activator family stimulates promoter II-driven aromatase expression in preadipocytes.

The dramatically increased prevalence of breast cancer after menopause is of great concern and is correlated with elevated local levels of estrogens. This is mainly due to an increase in aromatase expression driven by its proximal promoter II (PII). We have previously demonstrated that the CREB co-activator CRTC2 binds directly to PII and stimulates its activity via mechanisms involving LKB1-AMPK in response to prostaglandin E(2) (PGE(2)). There are three members of the CRTC family (CRTC1-3) and this study aimed to characterize the role of other CRTCs in the activation of aromatase PII. The expression and subcellular localization of CRTCs were examined in preadipocytes using qPCR and immunofluorescence. Under basal conditions, CRTC1 expression was the lowest, whereas CRTC3 transcripts were present at higher levels. Basally, CRTC2 and CRTC3 were mainly cytoplasmic and PGE(2) caused their nuclear translocation. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of CRTCs on PII activity and binding. Basal PII activity was significantly increased with all CRTCs. Forskolin (FSK)/phorbol 12-myristate 13-acetate (PMA), to mimic PGE(2), resulted in a further significant increase in PII activity with all CRTCs, with CRTC2 and CRTC3 having greater effects. This was consistent with ChIP data showing an increased binding of CRTCs to PII with FSK/PMA. Moreover, gene silencing of CRTC2 and CRTC3 significantly reduced the FSK/PMA-mediated stimulation of aromatase activity. Interestingly, CRTCs acted cooperatively with CREB1 to increase PII activity, and both CREs were found to be essential for the maximal induction of PII activity by CRTCs. Phosphorylation of CRTC2 at its AMPK target site, Ser 171, dictated its subcellular localization, and the activation of aromatase PII in preadipocytes. In conclusion, this study demonstrates that aromatase regulation in primary human breast preadipocytes involves more than one CRTC.

Endocrine-related cancers and the role of AMPK.

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis involved in the regulation of a number of physiological processes including ß-oxidation of fatty acids, lipogenesis, protein and cholesterol synthesis, as well as cell cycle inhibition and apoptosis. Important changes to these processes are known to occur in cancer due to changes in AMPK activity within cancer cells and in the periphery. This review aims to present findings relating to the role and regulation of AMPK in endocrine-related cancers. Obesity is a known risk factor for many types of cancers and a number of endocrine factors, including adipokines and steroid hormones, are regulated by and regulate AMPK. A clear role for AMPK in breast cancer is evident from the already impressive body of work published to date. However, information pertaining to its role in prostate cancer is still contentious, and future work should unravel the intricacies behind its role to inhibit, in some cases, and stimulate cancer growth in others. This review also presents data relating to the role of AMPK in cancers of the endometrium, ovary and colon, and discusses the possible use of AMPK-activating drugs including metformin for the treatment of all endocrine-related cancers.

Promoter-specific effects of metformin on aromatase transcript expression.

Phase III aromatase inhibitors (AIs) are proving successful in the treatment of hormone-dependent postmenopausal breast cancer. Side-effects associated with total body aromatase inhibition have prompted new research into the development of breast-specific AIs. The identification of tissue- and disease-specific usage of aromatase promoters has made the inhibition of aromatase at the transcriptional level an interesting approach. We have previously demonstrated that AMPK-activating drugs, including metformin, were potent inhibitors of aromatase expression in primary human breast adipose stromal cells (hASCs). This study examines the promoter-specific effects of metformin on inhibiting aromatase expression in hASCs. Tumour-associated promoters PII/PI.3 were activated using forskolin (FSK)/phorbol ester (PMA), whereas normal adipose associated promoter PI.4 was activated using dexamethasone (DEX)/tumour necrosis factor-α (TNFα). Results demonstrate that metformin significantly decreased the FSK/PMA-, but not the DEX/TNFα-mediated expression of total aromatase at concentrations of 10, 20, and 50 µM (P ≤ 0.05). Using PCR to amplify promoter-specific transcripts of aromatase, it appears that the inhibition of the FSK/PMA-mediated expression of aromatase is due to decreases in PII/PI.3-specific transcripts, whereas no effect of metformin is observed on any promoter-specific transcript, including PI.4, in DEX/TNFα-treated hASCs. This report therefore supports the hypothesis that metformin would act as a breast-specific inhibitor of aromatase expression in the context of postmenopausal breast cancer.