Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.

Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (Gsα) in addition to NESP55 and XLαs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally-inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through GPCRs requiring Gsα (eg., PTH, TSH, GHRH, calcitonin) and severe obesity. Paternally-inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue-specific reduction of paternal Gsα expression. Previous reports in mice have shown loss of Gsα causes osteopenia due to impaired osteoblast number and function and suggest AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal-increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized an AHO mouse model generated in our laboratory to address whether Gsα heterozygous inactivation by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (Gnas E1+/-p) and maternally-inherited (Gnas E1+/-m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. Gnas E1+/-p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. Gnas E1+/-m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between Gnas E1+/-p and Gnas E1+/-m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within Gnas E1+/-m osteoclasts and therefore warrant further studies into understanding how Gsα influences osteoblast-osteoclast coupling interactions.

Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.

P514 Influence of the frequency of glucocorticosteroid therapy courses on the hormonal resistance in patients with ulcerative colitis

Background One of the most effective anti-inflammatory agents in ulcerative colitis (UC) are glucocorticosteroids (GCS), which block the release of arachidonic acid, prevent the formation of inflammatory mediators, have an immunomodulatory effect, affect tissue fibrinolysis, which leads to a decrease in inflammation and bleeding of the mucous membrane. However, in 9–16% of patients UC, hormone resistance develops. Objective to establish the dependence of hormonal resistance on the frequency of GCS courses for 12 months without taking into account the starting dosage of GCS. Methods 96 patients with moderate to severe UC (Truelove-Witts) who received several courses of GCS. Males 51 (53.1%), females 45 (46.9%). The median age was 34±4.2 years. The duration of the anamnesis was from 3 to 7 years (Me 4 years). Patients were divided into three groups depending on the frequency of GCS courses for 12 months without taking into account the starting dosage of GCS. Group 1-st (n=30) received ≤1 course of GCS for 12 months for UC exacerbation, group 2-nd (n=34)-1–2 courses of GCS, group 3-rd (n=32)-3 ≥ courses of GCS. The frequency of hormone resistance (HR) was compared in groups of patients. Results Among 96 patients with UC, HR was established in 12 (12.5%) patients. In group 1 of UC patients who received less than 1 course of GCS for 12 months for UC exacerbation, patients with HR were 1 (3.3%), in group 2-2 (5.9%) patients with HR, in group 3–9 (28.1%) patients with HR. When comparing groups 1-st and 2-nd, it was found that the incidence of HR in both patients who received 1–2 courses of GCS for 12 months did not differ (OR-1.813; 95% CI 0.156 - 21.057; p>0.05). When comparing the 1-st and 3-rd groups showed that the incidence of HR in patients who received 3 or more courses of corticosteroids within 12 months is significantly higher than in patients who received ≤1 course of corticosteroids within 12 months (OR-0.088; 95% CI, 0.010–0.747; p<0.05). When comparing the 2-nd and 3-rd groups showed that the incidence of HR in patients who received more than 3 courses of corticosteroids within 12 months also has a tendency to a higher risk of development of HR than the patients receiving 2 or fewer courses of corticosteroids within 12 months (OR-0.160; 95% CI, 0.032–0.810; p<0.05). Conclusion The frequency of courses of glucocorticosteroid therapy for exacerbation of the disease for 12 months affects the risk of developing hormone resistance in patients with ulcerative colitis.

A novel variant in the PDE4D gene is the cause of Acrodysostosis type 2 in a Lithuanian patient: a case report

Background Acrodysostosis is a rare hereditary disorder described as a primary bone dysplasia with or without hormonal resistance. Pathogenic variants in the PRKAR1A and PDE4D genes are known genetic causes of this condition. The latter gene variants are more frequently identified in patients with midfacial and nasal hypoplasia and neurological involvement. The aim of our study was to analyse and confirm a genetic cause of acrodysostosis in a male patient. Case presentation We report on a 29-year-old Lithuanian man diagnosed with acrodysostosis type 2. The characteristic phenotype includes specific skeletal abnormalities, facial dysostosis, mild intellectual disability and metabolic syndrome. Using patient’s DNA extracted from peripheral blood sample, the novel, likely pathogenic, heterozygous de novo variant NM_001104631.2:c.581G > C was identified in the gene PDE4D via Sanger sequencing. This variant causes amino acid change (NP_001098101.1:p.(Arg194Pro)) in the functionally relevant upstream conserved region 1 domain of PDE4D. Conclusions This report further expands the knowledge of the consequences of missense variants in PDE4D that affect the upstream conserved region 1 regulatory domain and indicates that pathogenic variants of the gene PDE4D play an important role in the pathogenesis mechanism of acrodysostosis type 2 without significant hormonal resistance.

Diabetes, obesity, and insulin resistance in COVID-19: molecular interrelationship and therapeutic implications

Background Our understanding of the pathophysiology of the COVID-19 manifestations and evolution has improved over the past 10 months, but the reasons why evolution is more severe in obese and diabetic patients are not yet completely understood. Main text In the present review we discuss the different mechanisms that may contribute to explain the pathophysiology of COVID-19 including viral entrance, direct viral toxicity, endothelial dysfunction, thromboinflammation, dysregulation of the immune response, and the renin–angiotensin–aldosterone system. Conclusions We show that the viral infection activates an integrated stress response, including activations of serine kinases such as PKR and PERK, which induce IRS-1 serine phosphorylation and insulin resistance. In parallel, we correlate and show the synergy of the insulin resistance of COVID-19 with this hormonal resistance of obesity and diabetes, which increase the severity of the disease. Finally, we discuss the potential beneficial effects of drugs used to treat insulin resistance and diabetes in patients with COVID-19.

Molecular Definition of Pseudohypoparathyroidism Variants

Pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) are caused by mutations and/or epigenetic changes at the complex GNAS locus on chromosome 20q13.3 that undergoes parent-specific methylation changes at several differentially methylated regions (DMRs). GNAS encodes the alpha-subunit of the stimulatory G protein (Gsα) and several splice variants thereof. PHP type Ia (PHP1A) is caused by heterozygous inactivating mutations involving the maternal exons 1-13. Heterozygosity of these maternal GNAS mutations cause PTH-resistant hypocalcemia and hyperphosphatemia because paternal Gsα expression is suppressed in certain organs thus leading to little or no Gsα protein in the proximal renal tubules and other tissues. Besides biochemical abnormalities, PHP1A patients show developmental abnormalities, referred to as Albright’s hereditary osteodystrophy (AHO). Some, but not all of these AHO features are encountered also in patients affected by PPHP, who carry paternal Gsα-specific mutations and typically show no laboratory abnormalities. Autosomal dominant PHP type Ib (AD-PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16, which are associated with loss of methylation at the A/B DMR alone or at all maternally methylated GNAS exons. Loss of methylation of exon A/B and the resulting biallelic expression of A/B transcript reduces Gsα expression thus leading to hormonal resistance. Epigenetic changes at all differentially methylated GNAS regions are also observed in sporadic PHP1B, which is the most frequent PHP1B variant. However, this disease variant remains unresolved at the molecular level, except for rare cases with paternal uniparental isodisomy or heterodisomy of chromosome 20q (patUPD20q).

Hypercholesterolemia in Two Siblings with Resistance to Thyroid Hormones Due to Disease-Causing Variant in Thyroid Hormone Receptor (THRB) Gene

Resistance to thyroid hormone beta (RTHβ) is a syndrome characterized by a reduced response of target tissues to thyroid hormones. In 85% of cases, a pathogenic mutation in the thyroid hormone receptor beta (THRB) gene is found. The clinical picture of RTHβ is very diverse; the most common findings are goiter and tachycardia, but the patients might be clinically euthyroid. The laboratory findings are almost pathognomonic with elevated free thyroxin (fT4) levels and high or normal thyrotropin (TSH) levels; free triiodothyronin (fT3) levels may also be elevated. We present three siblings with THRB mutation (heterozygous disease-variant c.727C>T, p.Arg243Trp); two of them also had hypercholesterolemia, while all three had several other clinical characteristics of RTHβ. This is the first description of the known Slovenian cases with RTHβ due to the pathogenic mutation in the THRB gene. Hypercholesterolemia might be etiologically related with RTHβ, since the severity of hormonal resistance varies among different tissues and hypercholesterolemia in patients with THRB variants might indicate the relatively hypothyroid state of the liver. We suggest that cholesterol levels are measured in all RTHβ patients.

Effect of the exosome-mediated suppression of the estrogen signaling: the role in the progression of the hormonal resistance of breast cancer cells

The most effective treatment of the hormone-dependent breast cancer is based on the antiestrogens SERM and aromatase inhibitor treatment, however its efficiency is limited by the acquired resistance to the drugs.Previously we have revealed the effect of the transferring of the hormonal resistance from the resistant to the sensitive cells under in vitro cell co-cultivation, and demonstrated exosomes involvement in this process. Here we have shown that the exosomes of the resistant cells caused the marked inhibition of the estrogen signaling in the recipient cells, and identified microRNAs – ERα suppressors that overexpressed in the resistant exosomes.Taken together, the results obtained demonstrate the important role of the estrogen signaling suppression in the exosome-induced transferring of the hormonal resistance, and revealed the involvement of the exosomal microRNA in the ERα down-regulation.