Understanding the mechanisms of action of estrogen receptor mutations in metastatic breast cancer

Estrogen receptor (ER) is a key driver of breast cancer development and progression and is the target for endocrine therapies. Use of endocrine agents has contributed greatly to reductions in breast cancer mortality over the last three decades. However, many patients develop resistance to these therapies, for reasons that remain unclear. Recent studies have identified ESR1 (ER) mutations in metastatic breast cancer that are predicted to encode ER proteins that are constitutively active and resistant to anti-estrogens. However, functional studies to determine the action of these mutations is impeded by the lack of breast cancer cell lines encoding ESR1 mutations. Here, I have developed the engineered CRISPR-Cas9 genome editing system to incorporate ESR1 mutations in the genomically encoded gene in the well-studied estrogen responsive and ER-positive MCF7 breast cancer cell line. I show that MCF7 cells with genomically encoded tyrosine 537 to serine (Y537S) ER mutation are estrogen independent for their growth and show global ligand-independent ER recruitment to regulatory regions and frequently elevated expression of estrogen-responsive genes. Ligand-independent expression and growth was also found for fourteen further CRISPR knockin MCF7 cells encoding additional ESR1 mutations, with between two and four individual clones derived for each mutation. Partial resistance to anti-estrogens is also seen with the ESR1 mutations, as higher concentrations of 4-hydroxytamoxifen and Faslodex are required to achieve the same growth inhibition as are required for the parental (wild-type) cells. All ESR1 mutation knockin lines demonstrated sensitivity to CDK7 inhibitors alone and in combination with Faslodex, highlighting the utility of these lines for the assessment of potential therapeutic agents. Finally, the use of CRISPR to revert an ESR1 mutation back to wild-type in a long-term estrogen-deprived MCF7 cell line, led to the restoration of an estrogen-dependent phenotype, confirming the importance of these mutations for ligand-independence.Open Acces

Expression of CDK7, cyclin H and MAT1 is elevated in breast cancer and is prognostic in estrogen receptor- positive breast cancer

Purpose: CDK-activation kinase (CAK) is required for the regulation of the cell-cycle and is a trimeric complex consisting of Cyclin Dependent Kinase 7 (CDK7), Cyclin H and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. Experimental Design: mRNA and protein expression of CDK7 and its essential co-factors cyclinH and MAT1, were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathological features and patient outcome. Results: We show that expression of CDK7, cyclinH and MAT1 are all closely linked at the mRNA and protein level and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumour grade and size and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. Conclusions: Expression of components of the CAK complex, CDK7, MAT1 and Cyclin H are elevated in breast cancer and correlates with ER. Like ERα , CDK7 expression is inversely proportional to poor prognostic factors and survival

APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer.

Estrogen receptor α (ERα) is the key transcriptional driver in a large proportion of breast cancers. We report that APOBEC3B (A3B) is required for regulation of gene expression by ER and acts by causing C-to-U deamination at ER binding regions. We show that these C-to-U changes lead to the generation of DNA strand breaks through activation of base excision repair (BER) and to repair by non-homologous end-joining (NHEJ) pathways. We provide evidence that transient cytidine deamination by A3B aids chromatin modification and remodelling at the regulatory regions of ER target genes that promotes their expression. A3B expression is associated with poor patient survival in ER+ breast cancer, reinforcing the physiological significance of A3B for ER action

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.Cancer Research U

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Mapping outcomes for recovery of consciousness in studies from 1986 to 2020: a scoping review protocol

INTRODUCTION: Historically, heterogeneous outcome assessments have been used to measure recovery of consciousness in patients with disorders of consciousness (DoC) following traumatic brain injury (TBI), making it difficult to compare across studies. To date, however, there is no comprehensive review of clinical outcome assessments that are used in intervention studies of adults with DoC. The objective of this scoping review is to develop a comprehensive inventory of clinical outcome assessments for recovery of consciousness that have been used in clinical studies of adults with DoC following TBI. METHODS AND ANALYSIS: The methodological framework for this review is: (1) identify the research questions, (2) identify relevant studies, (3) select studies, (4) chart the data, (5) collate, summarise and report results and (6) consult stakeholders to drive knowledge translation. We will identify relevant studies by searching the following electronic bibliographic databases: PubMed, Scopus, EMBASE, PsycINFO and The Cochrane Library (including Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials and Cochrane Methodology Register). Criteria for article inclusion are published in the English-language, peer-reviewed studies of interventions aimed at facilitating recovery of consciousness among adults (\u3e 18 years) with DoC following a severe TBI, published from January 1986 to December 2020. Articles meeting inclusion criteria at this stage will undergo a full text review. We will chart the data by applying the WHO International Classification of Functioning, Disability and Health Framework to identify the content areas of clinical outcome assessments. To support knowledge translation efforts, we will involve clinicians and researchers experienced in TBI care throughout the project from conceptualisation of the study through dissemination of results. ETHICS AND DISSEMINATION: No ethical approval is required for this study as it is not determined to be human subjects research. Results will be presented at national conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: CRD42017058383

Immunological Response to Single Pathogen Challenge with Agents of the Bovine Respiratory Disease Complex: An RNA-Sequence Analysis of the Bronchial Lymph Node Transcriptome

<div><p>Susceptibility to bovine respiratory disease (BRD) is multi-factorial and is influenced by stress in conjunction with infection by both bacterial and viral pathogens. While vaccination is broadly used in an effort to prevent BRD, it is far from being fully protective and cases diagnosed from a combination of observed clinical signs without any attempt at identifying the causal pathogens are usually treated with antibiotics. Dairy and beef cattle losses from BRD are profound worldwide and genetic studies have now been initiated to elucidate host loci which underlie susceptibility with the objective of enabling molecular breeding to reduce disease prevalence. In this study, we employed RNA sequencing to examine the bronchial lymph node transcriptomes of controls and beef cattle which had individually been experimentally challenged with bovine respiratory syncytial virus, infectious bovine rhinotracheitis, bovine viral diarrhea virus, <i>Pasteurella multocida</i>, <i>Mannheimia haemolytica</i> or <i>Mycoplasma bovis</i> to identify the genes that are involved in the bovine immune response to infection. We found that 142 differentially expressed genes were located in previously described quantitative trait locus regions associated with risk of BRD. Mutations affecting the expression or amino acid composition of these genes may affect disease susceptibility and could be incorporated into molecular breeding programs. Genes involved in innate immunity were generally found to be differentially expressed between the control and pathogen-challenged animals suggesting that variation in these genes may lead to a heritability of susceptibility that is pathogen independent. However, we also found pathogen-specific expression profiles which suggest that host genetic variation for BRD susceptibility is pathogen dependent.</p></div

Hepatitis C virus testing, liver disease assessment and treatment uptake among people who inject drugs pre- and post-universal access to direct-acting antiviral treatment in Australia : The LiveRLife study

Gaps in hepatitis C virus (HCV) testing, diagnosis, liver disease assessment and treatment uptake among people who inject drugs (PWID) persist. We aimed to describe the cascade of HCV care among PWID in Australia, prior to and following unrestricted access to direct-acting antiviral (DAA) treatment. Participants enrolled in an observational cohort study between 2014 and 2018 provided fingerstick whole-blood samples for dried blood spot, Xpert HCV Viral Load and venepuncture samples. Participants underwent transient elastography and clinical assessment by a nurse or general practitioner. Among 839 participants (mean age 43 years), 66% were male (n = 550), 64% (n = 537) injected drugs in the previous month, and 67% (n = 560) reported currently receiving opioid substitution therapy. Overall, 45% (n = 380) had detectable HCV RNA, of whom 23% (n = 86) received HCV treatment within 12 months of enrolment. HCV treatment uptake increased from 2% in the pre-DAA era to 38% in the DAA era. Significant liver fibrosis (F2-F4) was more common in participants with HCV infection (38%) than those without (19%). Age 50 years or older (aOR, 2.88; 95% CI, 1.18-7.04) and attending a clinical follow-up with nurse (aOR, 3.19; 95% CI, 1.61-6.32) or physician (aOR, 11.83; 95% CI, 4.89-28.59) were associated with HCV treatment uptake. Recent injection drug use and unstable housing were not associated with HCV treatment uptake. HCV treatment uptake among PWID has increased markedly in the DAA era. Evaluation of innovative and simplified models of care is required to further enhance treatment uptake