Oestrogens regulate proliferation in colorectal cancer via GPER and the hippo signalling pathway

The concentration of circulating oestrogens is associated with the incidence and outcomes of colorectal cancer (CRC). Both the physiological and pathological effects of oestrogens are mediated by oestrogen receptors. This project aims to investigate the potential role of G protein-coupled oestrogen receptor 1 (GPER) as an oestrogen-induced mediator in CRC proliferation. To achieve this, colorectal adenoma and carcinoma cell lines were examined for protein expression of oestrogen receptors. Immunoblotting did not report ERa and ERb expression, although GPER was detected. Following this, the GPER-associated gene connective tissue growth factor (CTGF) expression was measured after I7β-estradiol (E2) and GPER agonist (G1) treatment. qRT-PCR results showed no significant increase in CTGF expression levels, 24 and 48 hours after treatment. In addition, GPER interaction with the Hippo pathway in CRC was examined by treating cells with E2 and G1 for 0, 15, 30 minutes, 1, and 2 hours. Alterations in P-Y API expression were unclear after treatment in the cell lines examined. However, addition of GPER antagonist, G15, resulted in significant inhibition in YAP1 phosphorylation in HCT116 cells, 15 and 30 minutes of treatment. Consequently, our data supports that oestrogens and G1 treatment leads to increase in YAP phosphorylation and nucleus-cytoplasmic shuttling via GPER stimulation. YAP1 knock-down studies and pharmacological inhibition followed by proliferation assays established that this early metabolic effect translates into increased cellular proliferation. Collectively, our data propose a novel oestrogen-driven pro-proliferative pathway via GPER through Hippo pathway's key downstream effector, YAP1, in CRC. Further studies are required to 'reveal the detailed signalling cascade by which GPER mediates the increased YAP1 phosphorylation

Estrone sulfate transport and steroid sulfatase activity in colorectal cancer: implications for Hormone Replacement Therapy

Hormone replacement therapy (HRT) affects the incidence and potential progression of colorectal cancer (CRC). As HRT primarily consists of estrone sulfate (E(1)S), understanding whether this conjugated estrogen is transported and metabolized in CRC will define its potential effect in this malignancy. Here, we show that a panel of CRC cell lines (Colo205, Caco2, HCT116, HT-29) have steroid sulfatase (STS) activity, and thus can hydrolyze E(1)S. STS activity is significantly higher in CRC cell lysate, suggesting the importance of E(1)S transport in intracellular STS substrate availability. As E(1)S transport is regulated by the expression pattern of certain solute carrier organic anion transporter polypeptides, we show that in CRC OATP4A1 is the most abundantly expressed transporter. All four CRC cell lines rapidly transported E(1)S into cells, with this effect significantly inhibited by the competitive OATP inhibitor BSP. Transient knockdown of OATP4A1 significantly disrupted E(1)S uptake. Examination of estrogen receptor status showed ERα was present in Colo205 and Caco2 cells. None of the cells expressed ERβ. Intriguingly, HCT116 and HT29 cells strongly expressed the G protein coupled estrogen receptor (GPER), and that stimulation of this receptor with estradiol (E(2)) and G1, a GPER agonist, significantly (p < 0.01) increased STS activity. Furthermore, tamoxifen and fulvestrant, known GPER agonist, also increased CRC STS activity, with this effect inhibited by the GPER antagonist G15. These results suggest that CRC can take up and hydrolyze E(1)S, and that subsequent GPER stimulation increases STS activity in a potentially novel positive feedback loop. As elevated STS expression is associated with poor prognosis in CRC, these results suggest HRT, tamoxifen and fulvestrant may negatively impact CRC patient outcomes

Differential activity and expression of human 5β-reductase (AKR1D1) splice variants

Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5beta-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were over-expressed in HEK293 cells, and successful overexpression confirmed by qPCR and western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following over-expression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action

Estrogen activation by steroid sulfatase increases colorectal cancer proliferation via GPER

Abstract Context Estrogens affect the incidence and progression of colorectal cancer (CRC), although the precise molecular mechanisms remain ill-defined. Objective The present study investigated prereceptor estrogen metabolism through steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase activity and subsequent nongenomic estrogen signaling in human CRC tissue, in The Cancer Genome Atlas colon adenocarcinoma data set, and in in vitro and in vivo CRC models. We aimed to define and therapeutically target pathways through which estrogens alter CRC proliferation and progression. Design, Setting, Patients, and Interventions Human CRC samples with normal tissue-matched controls were collected from postmenopausal female and age-matched male patients. Estrogen metabolism enzymes and nongenomic downstream signaling pathways were determined. CRC cell lines were transfected with STS and cultured for in vitro and in vivo analysis. Estrogen metabolism was determined using an ultra-performance liquid chromatography–tandem mass spectrometry method. Primary Outcome Measure The proliferative effects of estrogen metabolism were evaluated using 5-bromo-2′-deoxyuridine assays and CRC mouse xenograft studies. Results Human CRC exhibits dysregulated estrogen metabolism, favoring estradiol synthesis. The activity of STS, the fundamental enzyme that activates conjugated estrogens, is significantly (P &amp;lt; 0.001) elevated in human CRC compared with matched controls. STS overexpression accelerates CRC proliferation in in vitro and in vivo models, with STS inhibition an effective treatment. We defined a G-protein–coupled estrogen receptor (GPER) proproliferative pathway potentially through increased expression of connective tissue growth factor in CRC. Conclusion Human CRC favors estradiol synthesis to augment proliferation via GPER stimulation. Further research is required regarding whether estrogen replacement therapy should be used with caution in patients at high risk of developing CRC. </jats:sec

Self-reported risk of obstructive sleep apnea syndrome, and awareness about it in the community of 4 insular complexes comprising 41 Greek Islands

Obstructive Sleep Apnea Syndrome (OSAS) is a chronic disease that significantly increases morbidity and mortality of the affected population. There is lack of data concerning the OSAS prevalence in the insular part of Greece. The purpose of this study was to investigate the self-reported prevalence of OSAS in 4 Greek insular complexes comprising 41 islands, and to assess the awareness of the population regarding OSAS and its diagnosis. Our study comprised 700 participants from 41 islands of the Ionian, Cyclades, Dodecanese and Northeast Aegean island complexes that were studied by means of questionnaires via a telephone randomized survey (responsiveness rate of 25.74%). Participants were assessed by the Berlin Questionnaire (BQ) for evaluation of OSA risk, by the Epworth Sleepiness Scale (ESS) for evaluation of excessive daytime sleepiness, and by 3 questions regarding the knowledge and diagnosis of OSAS. The percentage of participants at high risk according to BQ was 27.29% and the percentage of people who were at high risk according to ESS was 15.43%. A percentage of 6.29% of the population was at high risk for OSAS (high risk both in BQ and ESS). A high percentage of 73.43%, were aware of OSAS as a syndrome however a significantly less percentage (28.00%) was aware of how a diagnosis of OSAS is established. The community prevalence of OSAS in Greek islands in combination with the low-level awareness of the OSAS diagnostic methods highlights the need for development of health promotion programs aiming at increasing the detection of patients at risk while increasing the awareness of OSAS

11β-HSD1 inhibition in men mitigates prednisolone-induced adverse effects in a proof-of-concept randomised double-blind placebo-controlled trial

Glucocorticoids prescribed to limit inflammation, have significant adverse effects. As 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates active glucocorticoid, we investigated whether 11β-HSD1 inhibition with AZD4017 could mitigate adverse glucocorticoid effects without compromising their anti-inflammatory actions. We conducted a proof-of-concept, randomized, double-blind, placebo-controlled study at Research Unit, Churchill Hospital, Oxford, UK (NCT03111810). 32 healthy male volunteers were randomized to AZD4017 or placebo, alongside prednisolone treatment. Although the primary endpoint of the study (change in glucose disposal during a two-step hyperinsulinemic, normoglycemic clamp) wasn’t met, hepatic insulin sensitivity worsened in the placebo-treated but not in the AZD4017-treated group. Protective effects of AZD4017 on markers of lipid metabolism and bone turnover were observed. Night-time blood pressure was higher in the placebo-treated but not in the AZD4017-treated group. Urinary (5aTHF+THF)/THE ratio was lower in the AZD4017-treated but remained the same in the placebo-treated group. Most anti-inflammatory actions of prednisolone persisted with AZD4017 co-treatment. Four adverse events were reported with AZD4017 and no serious adverse events. Here we show that co-administration of AZD4017 with prednisolone in men is a potential strategy to limit adverse glucocorticoid effects

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Follow your gut: Does AKR1D1 impact gastrointestinal health?

Dysfunction of the gut-liver axis contributes to non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Bile acid homeostasis is disrupted in NAFLD, and this has been suggested to increase cytotoxic bile acids species in the intestine, disrupt cytoprotective intestinal bile acid signalling and drive gut dysbiosis, impairing intestinal barrier function and inducing hepatic inflammation. We have previously shown that expression of the bile acid synthesis enzyme 5β-reductase (AKR1D1) is decreased in patients with NAFLD. In this thesis, I have examined the impact of AKR1D1 deletion on intestinal barrier function and hepatic inflammation in mice. Female and male wild type (WT) and AKR1D1 knockout (KO) mice were maintained on a control diet until 12 months of age. Suggesting increased bacterial translocation, liver bacterial load was increased in AKR1D1 KO mice. Toll-like receptor 4 (TLR4) signalling was upregulated in the female mice, as was the pan-macrophage marker, F4/80, at mRNA and protein level. Suggestive of impaired barrier function, intestinal morphology of the AKR1D1 KO mice was modified, key ileal and colonic tight junction mRNA and protein levels were altered, goblet cell number and mRNA levels of key antimicrobial peptides were reduced. To examine the mechanisms that underpin these changes, caecal bile acid levels were examined. AKR1D1 KO mice had reduced total bile acid levels and altered bile acid composition, with decreased hydrophobicity and reduced farnesoid X receptor (FXR) affinity. Accordingly, RNAseq analysis revealed a decrease in the mRNA levels of key antioxidant and FXR target genes in the ileum. Although caecal microbiome analysis showed no change in total phylum bacterial counts and key markers of dysbiosis (ratio of Firmicutes to Bacteroidetes and species diversity), bacterial composition was altered suggesting that microbiota could play a role in the intestinal barrier dysfunction. To examine the role of AKR1D1 in NAFLD, AKR1D1 KO mice were challenged for 12 months with a NAFLD inducing diet (American lifestyle-induced obesity syndrome (ALIOS)). Although the ALIOS diet impaired intestinal barrier function and activated hepatic TLR4 signalling in WT mice, the deletion of AKR1D1 did not worsen the effects. In summary, AKR1D1 deletion and disruption of bile acid synthesis impairs intestinal barrier function and drives hepatic inflammation in female mice by reducing intestinal FXR signalling in the ileum and altering caecal microbiome composition. This is the first study that demonstrates the impact of disrupting bile acid synthesis on gastrointestinal health