How Estrogen, Testosterone, and Sex Differences Influence Serum Immunoglobulin Isotype Patterns in Mice and Humans.

Females often exhibit superior immune responses compared to males toward vaccines and pathogens such as influenza viruses and SARS-CoV-2. To help explain these differences, we first studied serum immunoglobulin isotype patterns in C57BL/6 male and female mice. We focused on IgG2b, an isotype that lends to virus control and that has been previously shown to be elevated in murine females compared to males. Improvements in IgG2b serum levels, and/or IgG2b ratios with other non-IgM isotypes, were observed when: (i) wildtype (WT) female mice were compared to estrogen receptor knockout mice (IgG2b, IgG2b/IgG3, IgG2b/IgG1, and IgG2b/IgA were all higher in WT mice), (ii) unmanipulated female mice were compared to ovariectomized mice (IgG2b/IgA was higher in unmanipulated animals), (iii) female mice were supplemented with estrogen in the context of an inflammatory insult (IgG2b and IgG2b/IgG3 were improved by estrogen supplementation), and (iv) male mice were supplemented with testosterone, a hormone that can convert to estrogen in vivo (IgG2b, IgG2b/IgG3, IgG2b/IgG1, and IgG2b/IgA were all improved by supplementation). We next examined data from three sets of previously described male and female human blood samples. In each case, there were higher IgG2 levels, and/or ratios of IgG2 with non-IgM isotypes, in human females compared to males. The effects of sex and sex hormones in the mouse and human studies were subtle, but frequent, suggesting that sex hormones represent only a fraction of the factors that influence isotype patterns. Examination of the gene loci suggested that upregulation of murine IgG2b or human IgG2 could be mediated by estrogen receptor binding to estrogen response elements and cytosine-adenine (CA) repeats upstream of respective Cγ genes. Given that murine IgG2b and human IgG2 lend to virus control, the isotype biases in females may be sufficient to improve outcomes following vaccination or infection. Future attention to sex hormone levels, and consequent immunoglobulin isotype patterns, in clinical trials are encouraged to support the optimization of vaccine and drug products for male and female hosts.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

The COVID-19 Pandemic Impact on Breast Cancer Diagnosis: A Retrospective Study.

OBJECTIVE: This study aimed to evaluate the diagnostic profile of breast cancer cases during the coronavirus disease 2019 (COVID-19) pandemic compared with the previous year. METHODS: It is a retrospective study of cases diagnosed by a reference service in the public health system of Campinas, SP, Brazil. Two periods were analyzed: March to October 2019 (preCOVID period) and March to October 2020 (COVID-period). All women diagnosed during the periods were included. The Chi-Squared or Fisher exact and Mann-Whitney tests were used. RESULTS: In the preCOVID and COVID periods, breast cancers were diagnosed, respectively, in 115 vs 59 women, and the mean ages at diagnosis were 55 and 57 years (p = 0.339). In the COVID period, the family history of breast cancer was more observed (9.6% vs 29.8%, p < 0.001), cases were more frequently symptomatic (50.4% vs 79.7%, p < 0.001) and had more frequently palpable masses (56.5% vs 79.7%, p = 0.003). In symptomatic women, the mean number of days from symptom to mammography were 233.6 (458.3) in 2019 and 152.1 (151.5) in 2020 (p = 0.871). Among invasive tumors, the proportion of breast cancers in stages I and II was slightly higher in the COVID period, although not significantly (76.7% vs 82.4%, p = 0.428). Also in the COVID period, the frequency of luminal A-like tumors was lower (29.2% vs 11.8%, p = 0.018), of triple-negative tumors was twice as high (10.1% vs 21.6%, p = 0.062), and of estrogen receptor-positive tumors was lower (82.2% vs 66.0%, p = 0.030). CONCLUSION: During the COVID-19 pandemic, breast cancer diagnoses were reduced. Cases detected were suggestive of a worse prognosis: symptomatic women with palpable masses and more aggressive subtypes. Indolent tumors were those more sensitive to the interruption in screening.

OBJETIVO: Este estudo teve como objetivo avaliar o perfil diagnóstico dos casos de câncer de mama na pandemia de coronavirus disease 2019 (COVID-19) em comparação com o ano anterior. MéTODOS: Este é um estudo retrospectivo de casos diagnosticados em um serviço de referência da rede pública de saúde de Campinas, SP, Brasil. Foram analisados dois períodos: de março a outubro de 2019 (período pré-COVID) e de março a outubro de 2020 (período COVID). Todas as mulheres diagnosticadas durante os períodos foram incluídas. Foram utilizados os testes do qui-quadrado ou exato de Fisher e Mann-Whitney. RESULTADOS: Nos períodos pré-COVID e COVID, o câncer de mama foi diagnosticado, respectivamente, em 115 e 59 mulheres, e a média de idade no diagnóstico foi de 55 e 57 anos (p = 0,339). No período COVID, foram mais frequentes a história familiar de câncer de mama (9,6% vs 29,8%, p < 0,001), casos sintomáticos (50,4% vs 79,7%, p < 0,001) e com massas palpáveis (56,5% vs 79,7%, p = 0,003). Nas mulheres sintomáticas, a média de dias desde os sintomas até a mamografia foi de 233,6 (458,3) no pré-COVID e 152,1 (151,5) no COVID (p = 0,871). Entre os tumores invasivos no período COVID, a proporção de cânceres nos estágios I e II foi ligeiramente maior, porém não significativa (76,7% vs 82,4%, p = 0,428). Ainda no período COVID, a frequência de tumores tipo luminal A-like foi menor (29,2% vs 11,8%, p = 0,018), de tumores triplo-negativos foi duas vezes maior (10,1% vs 21,6%, p = 0,062), e de tumores positivos para receptor de estrogênio foi inferior (82,2% vs 66,0%, p = 0,030). CONCLUSãO: Durante a pandemia de COVID-19, houve uma redução no diagnóstico de câncer de mama. Os casos detectados eram sugestivos de pior prognóstico: mulheres sintomáticas com massas palpáveis e subtipos mais agressivos. Os tumores indolentes foram os mais sensíveis à interrupção do rastreamento.

Impact of Surgical Delay on Tumor Upstaging and Outcomes in Estrogen Receptor-Negative Ductal Carcinoma in Situ Patients.

BACKGROUND: The delay of elective surgeries by the coronavirus 2019 (COVID-19) pandemic prompted concern among surgeons to delay estrogen receptor (ER)-negative ductal carcinoma in situ (DCIS) for fear of missing an ER-negative invasive cancer and compromising survival of patients. STUDY DESIGN: Female patients ≥40 years old diagnosed with ER-negative DCIS from 2004 to 2017 were examined from the National Cancer Database. Multivariable logistic regression, adjusting for patient and tumor factors, was used to determine factors associated with tumor upstage. Multivariable Cox proportional hazards modeling was used to determine if surgical delay impacted overall survival of ER-negative DCIS patients that were upstaged to invasive disease. RESULTS: There were 219,731 patients with DCIS of which 24,338 (11.1%) had tumor upstage. Of these patients, 5,675 (16.2%) of ER-negative and 18,663 (10.1%) of ER-positive DCIS patients were upstaged (p ≤ 0.001). From 2004 to 2017, ER-negative DCIS upstage rates increased from 12.9% to 18.9%. Independent factors associated with tumor upstage were younger age (odds ratio [OR] 0.75 [95% CI 0.69 to 0.81]) and Black race (OR 1.34 [95% CI 1.22 to 1.46]). Compared with patients with ≤30 days between biopsy and surgery, patients with a 31- to 60-day interval (OR 1.13 [95% CI 1.05 to 1.20]) and a >60-day interval (OR 1.12 [95% CI 1.02 to 1.23]) had an increased rate of tumor upstage. Among ER-negative DCIS patients whose tumors were upstaged to invasive disease, Cox proportional hazard regression modeling showed no association between the number of days between biopsy and surgery and overall survival. CONCLUSIONS: Delays in surgery were associated with higher tumor upstage rates but not with worse overall survival.

Overexpression of estrogen receptor GPER1 and G1 treatment reduces SARS-CoV-2 infection in BEAS-2B bronchial cells.

Gender-bias in COVID-19 severity has been suggested by clinical data. Experimental data in cell and animal models have demonstrated the role of sex hormones, particularly estrogens, in viral infections such as in COVID-19. SARS-CoV-2 uses ACE2 as a receptor to recognize host cells, and the protease TMPRSS2 for priming the Spike protein, facilitating virus entry into cells. However, the involvement of estrogenic receptors in SARS-CoV-2 infection are still being explored. Thus, in order to investigate the role of estrogen and its receptors in COVID-19, the estrogen receptors ERα, ERß and GPER1 were overexpressed in bronchial BEAS-2B cell, and then infected with SARS-CoV-2. Interestingly, the levels of ACE2 and TMPRSS2 mRNA were higher in SARS-CoV-2-infected cells, but no difference was observed in cells with estrogen receptors overexpression. GPER1 can be involved in virus infection or replication, since its higher levels reduces SARS-CoV-2 load. On the other hand, pharmacological antagonism of GPER1 enhanced viral load. Those data suggest that GPER1 has an important role in SARS-CoV-2 infection.

Relationship between ENaC Regulators and SARS-CoV-2 Virus Receptor (ACE2) Expression in Cultured Adult Human Fungiform (HBO) Taste Cells.

In addition to the α, ß, and γ subunits of ENaC, human salt-sensing taste receptor cells (TRCs) also express the δ-subunit. At present, it is not clear if the expression and function of the ENaC δ-subunit in human salt-sensing TRCs is also modulated by the ENaC regulatory hormones and intracellular signaling effectors known to modulate salt responses in rodent TRCs. Here, we used molecular techniques to demonstrate that the G-protein-coupled estrogen receptor (GPER1), the transient receptor potential cation channel subfamily V member 1 (TRPV1), and components of the renin-angiotensin-aldosterone system (RAAS) are expressed in δ-ENaC-positive cultured adult human fungiform (HBO) taste cells. Our results suggest that RAAS components function in a complex with ENaC and TRPV1 to modulate salt sensing and thus salt intake in humans. Early, but often prolonged, symptoms of COVID-19 infection are the loss of taste, smell, and chemesthesis. The SARS-CoV-2 spike protein contains two subunits, S1 and S2. S1 contains a receptor-binding domain, which is responsible for recognizing and binding to the ACE2 receptor, a component of RAAS. Our results show that the binding of a mutated S1 protein to ACE2 decreases ACE2 expression in HBO cells. We hypothesize that changes in ACE2 receptor expression can alter the balance between the two major RAAS pathways, ACE1/Ang II/AT1R and ACE2/Ang-(1-7)/MASR1, leading to changes in ENaC expression and responses to NaCl in salt-sensing human fungiform taste cells.

Estrogen and Estrogen Receptor Modulators: Potential Therapeutic Strategies for COVID-19 and Breast Cancer.

Owing to the ongoing coronavirus disease 2019 (COVID-19) pandemic, we need to pay a particular focus on the impact of coronavirus infection on breast cancer patients. Approximately 70% of breast cancer patients express estrogen receptor (ER), and intervention therapy for ER has been the primary treatment strategy to prevent the development and metastasis of breast cancer. Recent studies have suggested that selective estrogen receptor modulators (SERMs) are a potential therapeutic strategy for COVID-19. With its anti-ER and anti-viral combined functions, SERMs may be an effective treatment for COVID-19 in patients with breast cancer. In this review, we explore the latent effect of SERMs, especially tamoxifen, and the mechanism between ER and virus susceptibility.

Prevention and therapy of COVID-19 via exogenous estrogen treatment for both male and female patients.

The presented work summarizes the results of studies underlining the crucial role of estrogen receptor (ER) signaling in both innate and adaptive immune responses as well as in tissue repairing processes during respiratory virus infection. Experimental studies justify that among respiratory virus infected mice, a weaker ER signaling leads to increased morbidity and mortality in both males and females. In animal experiments, estrogen treatment silences the inflammatory reactions and decreases virus titers leading to improved survival rate; it seems to be an ideal prevention and therapy against COVID-19. We should overcome the widespread reluctance to estrogen therapy as we have a unique estrogen formula; conjugated estrogens, or conjugated equine estrogens available under the brand name of Premarin deriving from natural sources. Premarin can exert similar ER upregulative and gene repairing power like endogenous estrogen without any risk for adverse reactions. Premarin is capable of stopping the COVID-19 pandemic.

BC-N102 suppress breast cancer tumorigenesis by interfering with cell cycle regulatory proteins and hormonal signaling, and induction of time-course arrest of cell cycle at G1/G0 phase.

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.

Delays in operative management of early-stage, estrogen receptor-positive breast cancer during the COVID-19 pandemic: A multi-institutional matched historical cohort study.

BACKGROUND: During the COVID-19 pandemic, guidelines recommended that breast cancer centers delay estrogen receptor-positive breast cancer surgeries with neoadjuvant endocrine therapy. We aimed to evaluate pathologic upstaging of breast cancer patients affected by these guidelines. METHODS: Female patients with stage I/II breast cancer receiving neoadjuvant endocrine therapy were prospectively identified and were matched to a historical cohort of stage I/II estrogen receptor-positive breast cancer patients treated with upfront surgery ≤35 days. Primary outcomes were pathologic T and N upstaging versus clinical staging. RESULTS: After matching, 28 neoadjuvant endocrine therapy and 48 control patients remained. Median age in each group was 65 (P = .68). Most patients (78.6% and 79.2%) had invasive ductal carcinoma with a clinical tumor size of 0.9 cm vs 1.7 cm (P = .056). Time to surgery was 68 days in the neoadjuvant endocrine therapy group and 26.5 days in the control (P < .001). A total of 23 neoadjuvant endocrine therapy patients (82.1%) had the same or lower pT-stage compared with 31 (64.5%) control patients (P = .115). Only 3 (10.7%) neoadjuvant endocrine therapy patients had increased pN-stage vs 14 (29.2%) control patients (P = .063). CONCLUSION: Despite 2.5-times longer delays, patients with early-stage estrogen receptor-positive breast cancer receiving neoadjuvant endocrine therapy did not experience pathologic upstaging during the COVID-19 pandemic. These findings may support the use of neoadjuvant endocrine therapy in similar patients if delays to surgery are projected.

Bridging Endocrine Therapy for HR+/HER2- Resectable Breast Cancer: Is it Safe?

BACKGROUND: The COVID-19 pandemic has required new treatment paradigms to limit exposures and optimize hospital resources, including the use of neoadjuvant endocrine therapy (NAET) as bridging therapy for HR+/HER2-invasive tumors and DCIS. While this approach has been used in locally advanced disease, it is unclear how it may affect outcomes in resectable HR+/HER2- tumors. METHODS: Women ≥18 years diagnosed with in situ (Tis) or non-metastatic HR+/HER2- breast cancer from March-May 2019 and 2020 were included. Fisher's exact test and two-sample t test were used to compare baseline characteristics and surgical outcomes between strata. Sub-analysis was performed between patients who received primary surgery vs a bridging NAET approach. RESULTS: Despite similar clinical characteristics, patients in 2019 were more likely to have a surgery-first approach (75% vs 42%, P-value = .0007), receive surgery sooner (22 vs 29 days, P-value < .001), and within 60 days from diagnosis date (100% vs 85%, P-value = .0301). Neoadjuvant endocrine therapy was a more prevalent approach in 2020 (48% vs 7%, P-value < .0001). Rates of clinical to pathologic up-staging remained consistent across primary surgery vs bridging NAET subgroups (P-value = .9253). DISCUSSION: Pandemic-driven treatment protocols provide a unique opportunity to assess the utility of bridging endocrine therapy for resectable HR+/HER2- tumors. Differences in clinical and pathologic staging were similar across groups and did not appear to be affected by receipt of NAET. Our limited cohort demonstrates this strategic therapeutic avenue can optimize health care utilization and may be a reasonable approach when delaying surgery is preferred.

Estradiol Inhibits Human Brain Vascular Pericyte Migration Activity: A Functional and Transcriptomic Analysis.

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood-brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-ß and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-ß agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-ß antagonists, respectively, confirming the role of ER-α and ER-ß in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.

Roles of steroid receptors in the lung and COVID-19.

COVID-19 symptoms and mortality are largely due to its devastating effects in the lungs. The disease is caused by the SARS (Severe Acute Respiratory Syndrome)-CoV-2 coronavirus, which requires host cell proteins such as ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) for infection of lung epithelia. The expression and function of the steroid hormone receptor family is important in many aspects that impact on COVID-19 effects in the lung - notably lung development and function, the immune system, and expression of TMPRSS2 and ACE2. This review provides a brief summary of current knowledge on the roles of the steroid hormone receptors [androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), mineralocorticoid receptor (MR) and oestrogen receptor (ER)] in the lung, their effects on host cell proteins that facilitate SARS-CoV-2 uptake, and provides a snapshot of current clinical trials investigating the use of steroid receptor (SR) ligands to treat COVID-19.

Estrogen Receptor Modulators in Viral Infections Such as SARS-CoV-2: Therapeutic Consequences.

COVID-19 is a pandemic respiratory disease caused by the SARS-CoV-2 coronavirus. The worldwide epidemiologic data showed higher mortality in males compared to females, suggesting a hypothesis about the protective effect of estrogens against severe disease progression with the ultimate end being patient's death. This article summarizes the current knowledge regarding the potential effect of estrogens and other modulators of estrogen receptors on COVID-19. While estrogen receptor activation shows complex effects on the patient's organism, such as an influence on the cardiovascular/pulmonary/immune system which includes lower production of cytokines responsible for the cytokine storm, the receptor-independent effects directly inhibits viral replication. Furthermore, it inhibits the interaction of IL-6 with its receptor complex. Interestingly, in addition to natural hormones, phytestrogens and even synthetic molecules are able to interact with the estrogen receptor and exhibit some anti-COVID-19 activity. From this point of view, estrogen receptor modulators have the potential to be included in the anti-COVID-19 therapeutic arsenal.

Breast Cancer Clinical Trials: The Landscape at the Uganda Cancer Institute and Lessons Learned.

The Uganda Cancer Institute, the sole national comprehensive cancer center in Uganda, has a long and rich history of clinical investigation and locally relevant cancer research. Given the increasing burden of breast cancer in Uganda and elsewhere in sub-Saharan Africa (SSA) and driven by the limited availability of immunohistochemistry (IHC), we launched a clinical trial aimed at evaluating locally available diagnostics to detect the presence of hormone receptors (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2. Preliminary data from 32 women in the diagnostic component of the study reveal high sensitivity and specificity for estrogen receptor and progesterone receptor and high specificity for human epidermal growth factor receptor 2 when comparing reverse transcriptase polymerase chain reaction with the gold standard (IHC). Innovative diagnostic and treatment strategies are required to address the burden of breast cancer that is increasing throughout SSA. Given the costs, infrastructure, and trained personnel associated with IHC, alternative testing options (including reverse transcriptase polymerase chain reaction as tested in our study) may provide an expedited and cost-effective method to determine receptor testing in breast cancer. Clinical trials conducted in the local setting are critical to determining optimal strategies for effective breast cancer management in SSA.

Antiviral drugs targeting endosomal membrane proteins inhibit distant animal and human pathogenic viruses.

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds.