Nuclear hormone receptor and epigenetic regulation of cancer and metabolic disease

Hormone signalling: how cells, tissues and organs communicate in health and disease.

To maintain the correct functioning of the body, and respond to many different challenges and demands of daily life, the human body needs to communicate and transmit messages over long distances to many different organs, tissues and cells. To do this, humans and animals utilize chemicals known as hormones that are produced and/or released from organs and glands (the endocrine system) into the bloodstream. Organs and tissue in the endocrine system include the thyroid gland, ovaries, testes, and adrenal glands.

The Muscat Group focuses on the steroid hormone superfamily. These hormone signalling molecules traverse the circulatory system identifying target tissues and entering the cell binding to Nuclear Receptors. The Hormone and Nuclear Receptors control the expression of specific genes in time and space.

The effects of the steroid hormone family, for example, estrogen, testosterone, thyroid hormone, Vit A and D, are mediated by the Nuclear Receptor hormone dependent regulatory proteina that bind genes, and acts as hormone inducible modulators of gene expression. The Nuclear Receptor proteins act as a conduit that translates hormonal and physiological signals into gene regulation. There are 48 nuclear receptors (NRs) in humans.

Research overview

Research projects

Nuclear receptor RORγ dependent regulation of breast cancer 

Breast cancer, a heterogeneous disease with >20 subtypes, is one of the most common invasive cancers in women1,2. Diagnosis and treatment of breast cancer involves measuring the expression of two nuclear hormone receptors (NRs), the estrogen and progesterone receptors (ER and PR), and the human epidermal growth factor receptor 2 (HER2) that underpin disease management and prognosis2. However, triple-negative breast cancers (TNBC; ER-/PR-/HER2-) and other subtypes are resistant to hormonal therapy and are associated with poor clinical outcomes. Unfortunately, current therapeutics display adverse effects and inadequate efficacy against TNBC, basal-like and several other subtypes, underscoring the need to identify new targets for subtype-selective pharmaceuticals.

Fig.1: Schematic illustrating the functions of RORγ1 in breast cancer cells

We recently published that expression of another NR, RORγ1 (also denoted as RORC): (i) was decreased in ER negative breast cancer, and the aggressive breast cancer subtypes, (ii) inversely correlated with histological grade (i.e. decreased in advanced cancers), (iii) was associated with improved clinical outcomes, and (iv) negatively regulated pathways of breast carcinogenesis (Fig.1), including transforming growth factor β signalling, epithelial mesenchymal transition, and mammary stem cell growth. Moreover, RORγ1 depletion in cells increased expression: (i) of many pro-metastatic genes (associated with poor clinical outcomes) including c-MET, and (ii) of the non-coding RNA, LINC00511, in TNBC cells, and basal-like breast tumours. Moreover, we identified a significant inverse correlation between the expressions of RORγ1 and pro-metastatic markers in the TCGA cohort.  

The NR ligand dependent transcription factors translate physiological signals into gene regulation. Drugs targeting NRs account for 15% of FDA approvals, underscoring the therapeutic utility of NRs in health. Significantly, we also showed that: (i) decreased RORγ1 expression is associated with tamoxifen resistance and endocrine resistance and (ii) an RORγ1 agonist (SR1078) attenuates (in vitro) breast cancer cell viability, migration, cell invasion and stem cell growth6. These findings suggested pharmacological modulation of RORγ1 activity has curative utility. Our new preliminary research: (i) demonstrated an inverse correlation between RORγ1 and LINC00511 in ER+, Her2+, and TNBC human breast cancer subytpes, (ii) identified other novel non-coding RNAs, increased in human basal-like breast cancers, and significantly correlated with important genes in human cohorts, that regulate mammary stem cells and cell invasion, and (iii) shown an RORγ1 agonist displays efficacy against breast cancer cell induced xenograft tumour growth. In normal breast we have also identified RORγ1 as a biomarker of the luminal lineage, supporting its role in normal biology that is disrupted on RORγ1 decrease in carcinogenesis.

Our data provides compelling evidence that RORγ1 is a promising candidate for ER+ and ER- breast cancer. We are currently focusing on genetic and pharmacological modulation of RORγ1 expression/activity to conduct a preclinical evaluation of the therapeutic utility of the NR, RORγ1, in animal models of breast cancer.

 

Nuclear receptor Nur77/NR4A1 dependent regulation of breast cancer 

We have demonstrated differential/aberrant expression of the entire Nuclear Receptor (NR) superfamily (in addition to ER and PR) in breast cancer relative to normal breast, and highlighted the therapeutic and prognostic value of many other NRs. We have identified over- expression of NR4A1 in (ER positive and ER negative) breast cancer, and a novel negative association between NR4A1 and histological grade.

Our new preliminary data in collaboration with Professor Chris Ormandy and Dr David Gallego_Ortega (from The Kinghorn Cancer Centre, GARVAN Institute for Medical Research) and Professor Chrstine Clarke (Westmead Institute for Medical Research, Uni. Of Sydney) demonstrates NR4A1 expression: (i) is associated with improved clinical outcomes in humans, and (ii) is decreased in TNBC cell lines. Moreover, analysis of preclinical genetic mammary tumourigenesis animal models has indicated NR4A1 decreases tumour growth and attenuates metastasis.

Our current studies are directed toward evaluating the potential of experimental drugs that modulate NR4A1 activity to decrease tumour growth, block metastasis and increase survival rates in pre-clincial animal models. The significance of NR4A1 is emphasized by the array of medicines targeting NRs that account for 15% of prescription pharmaceuticals and FDA approvals.

 

Epigenetic signalling and the regulation of breast cancer 

As discussed current therapeutics display adverse effects and inadequate efficacy against many heterogeneous breast cancer subtypes, underscoring the need to identify new targets for subtype-selective pharmaceuticals. Interestingly, Genetic alterations are associated with breast carcinogenesis, but do not provide an encompassing explanation for breast cancer etiology and progression. In this context,  evidence has indicated aberrant epigenetic signalling is an important driver of carcinogenesis. Protein arginine methylation is a common post-translational epigenetic modification catalysed by a family of nine human protein arginine methyltransferases (PRMTs) that have critical roles in steroid/nuclear hormone receptor (NR) dependent breast cancers. In addition, a recent review we have written with important new primary data underscores the therapeutic utility of epigenetic signalling in breast cancer on clinical and survival outcomes. We have also demonstrated that PRMT6 is an NR coregulator necessary for optimal ER-dependent gene expression and proliferation. Moreover, we have shown that: (i)  PRMT6 regulates the expression and splicing of genes that regulate proliferation, tumour suppression and carcinogenesis, and (ii) PRMT6 dependent gene expression controls the probability of metastasis free survival.  Our new preliminary data demonstrates increased PRMT6 expression is associated with decreased probability of relapse free survival, disease free and overall survival in breast and several other cancers. RNA-seq analysis after gain and loss of function analysis in a mammary specific PRMT6 transgenic mouse model and PRMT6 depleted human breast cancer lines, demonstrated  PRMT6 expression modulates tumourigenic processes in breast cancer, including cell cycle signalling, and inflammation. Moreover, we identified molecular crosstalk between steroid hormone signalling and PRMT6 expression by ChIP-seq, ChIP-qPCR and expression profiling analysis. Significantly, PRMT6 expression regulates the expression of the tumour suppressor, Phosphatase and Tensin Homolog (PTEN) gene. Finally, we have also identified PRMT6 dependent mechanisms controlling resistance to current therapeutic regimes in drug resistant cells and patients. In conclusion, aberrant epigenetic PRMT6 signalling drives breast carcinogenesis, and is associated with drug resistance and provides opportunities for the adjuvant treatment of breast cancer subtypes refractory to current pharmaceutical options.

 

Epigenetic signalling and type 2 diabetes: novel targets for pharmaceutical intervention

Type 2 Diabetes is a chronic disease, and a significant health burden in the 21st century. The significance of identifying new targets for intervention in Type2 Diabetes are underscored by the fact that the pandemic affects >350M and has emerged as a worldwide financial burden on health systems.  Existing therapeutics display inadequate efficacy, underscoring the urgent challenge to identify therapeutics that target glycaemic pathophysiology.

In humans, asymmetrical dimethyl arginine (ADMA) plasma levels are significantly associated with insulin resistance, obesity and type 2 diabetes. ADMA is produced by the catabolic turnover of (arginine) methylated chromatin and proteins (i.e products of epigenetic activity). Methylation of proteins is driven by protein arginine methyl transferase (PRMT) enzymes that drive the methylation of arginine residues (and ADMA levels) that impacts on human health. PRMTs are involved in epigenetic regulation, i.e. changes in genome function that transpire without alterations in DNA sequence, associated with chemical modification of chromatin.  There is an increasing body of literature indicating aberrant PRMT activity drives human disease. Significantly, PRMTs can be therapeutically exploited by medicinal chemistry, providing a new opportunity to (pharmacologically) modulate ADMA levels and treat type 2 diabetes. 

We previously showed PRMT4 (denoted as CARM1 in humans) is preferentially expressed in mouse muscle (in vitro and in vivo) and that PRMT4-signalling selectively modulates gene expression involved in glucose homeostasis. Pilot studies also revealed improved glucose tolerance in animal models with ‘muscle-specific’ expression of human PRMT4 enzymes with impaired catalytic activity. Moreover, preliminary analysis identified genetic polymorphisms/SNPs in CARM1 in a swedish cohort of normal patients (that drive increased PRMT activity) associated with increased fasting glucose, and decreased insulin sensitivity. SNPs in CARM1  were associated with increased fasting glucose, higher HOMA-IR and lower Matsuda index (i.e. decreased insulin sensitivity). This novel evidence, coupled to the observation that PRMTs are tractable drug targets provides a compelling argument for evaluating the genetic basis and pharmacological utility (of this pathway) in the context of type 2 diabetes.

We have initiated a a strategic collaboration between UQ (IMB)-, and the Lee Kong Chian School of Medicine,Nanyang Technological University & Imperial College London to enable enable the utilisation and mining of large pre-diabetic and diabetic clinical  human cohorts (associated with genetic and 10 yr follow up data) to examine the association between polymorphisms in CARM1 (a pharmacologically tractable enzyme) and susceptibility and predisposition to type 2 diabetes. The strategic collaborations will involve two high profile academic leaders from NTU;  Professor’s  Boehm (Director of Diabetes , NTU) who has >400 manuscripts, and Professor Walter Wahli who has >330 manuscripts (on metabolism) at LKC School of Medicine, NTU & Imperial College London.

Research training opportunities

Research title: Nuclear receptors and metabolism

Summary of research interests: Our research focuses on elucidating the functional role of nuclear hormone receptors (NRs) in the regulation of metabolism in the context of metabolic disease (e.g. diabetes and obesity) and breast cancer. NRs belong to a superfamily of hormone-dependent DNA binding factors that translate pathophysiological, metabolic, and nutritional signals into gene regulation. Dysfunctional NR signalling results in obesity, type 2 diabetes and cancer. The significance of NRs in human health is emphasised by the array of prescription pharmaceuticals that target NRs in the context of reproduction, inflammation, cancer and metabolic and endocrine diseases. Our current research aims to examine the role of NRs and epigenetic NR coregulators in metabolic disease and breast cancer. We are testing the hypothesis that the orphan NRs, for example RORs and NR4As, control the pathophysiological process in metabolic disease and cancer.

Traineeships, honours and PhD projects include

  • Elucidating the role of RORα and RORγ in the resistance to diet-induced obesity and fatty liver disease.
  • Understanding the role of the NR4A and ROR subgroup in breast cancer survival outcomes
  • Analysing the role of epigenetic regulators (histone methyltransferases) in (i) glycaemic control and (ii) breast cancer clinical outcomes.

Contact: Professor George Muscat

+61 7 33346 2039
g.muscat@imb.uq.edu.au


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Engagement and impact

Nuclear hormone receptors (NRs) are hormone dependent regulatory proteins that translate endocrine, metabolic and pathophysiological signals into gene regulation. Drugs targeting dysfunctional NR signalling account for 15% of prescription pharmaceuticals and FDA approvals, displaying utility against inflammation, osteoporosis, many endocrine and reproductive disorders, cancer etc. Our research utilises human patient cohorts, cell culture and tissue specific transgenic animal models and focuses on understanding the molecular role of NRs, and NR-associated epigenetic enzymes in breast, muscle, fat and liver in the context of breast cancer, diabetes, obesity and exercise. Epidemiological evidence points to associations between cancer and metabolic disease.

Breast cancer is one of the most common invasive cancers in women worldwide. It accounts for 25% of new cancers in women and for 15% of cancer related mortality in women. Like prostate cancer, the incidence of breast cancer is rising with increased life expectancy. The Muscat group examined the molecular role of NRs in breast tissue from normal, estrogen receptor-positive and estrogen negative breast cancer patients. These studies identified NR targets for diagnosis, prognosis, and therapeutic exploitation.

The Muscat group have collaboratively identified new NRs with prognostic and therapeutic utility for breast cancer (ER+ and ER-) and new epigenetic pathways/markers for metastasis free survival. For example, the Muscat Group have identified a nuclear receptor, ROR Gamma, that is influential in breast cancer. They found that the higher the expression of RORGamma in patients the higher the chance they’ll be relapse free and survive. RORGamma therefore, will be a very useful prognosis tool.

Further, when the group uses drugs that activate RORGamma, it blocks the proliferation and growth of cancer cells and stops their ability to migrate and move and metastasize. They are currently in the process of testing these drugs in animal models. 

Genetic alterations are associated with breast cancer, but do not provide an encompassing understanding of breast cancer. Accumulating evidence has indicated aberrant epigenetic activity (influenced by environmental/lifestyle factors) drives human disease, including cancer2,7-13.  Epigenetic activity chemically alters the chromatin. The epigenetic changes in genome function transpire without alterations in DNA sequence. Epigenetic modifications are driven by enzymes including the methyltransferases that have emerged as tractable pharmacological targets in cancer. The Muscat group have shown two epigenetic enzymes, the protein arginine methyltransferases, PRMT2 and PRMT6, are epigenetic markers for breast cancer and metastasis free survival.

We also investigated the role of NRs in obesity, type 2 diabetes and exercise endurance. Specifically, we explored the role of the NR4A3 and RORα in the regulation of metabolism (in muscle and adipose tissue) in the context of obesity, type II diabetes and endurance. We produced transgenic mouse lines with muscle-specific expression of an activated form of the nuclear receptor NR4A3/NOR1. We then demonstrated that NOR1 signalling controls skeletal muscle reprogramming, glucose tolerance, metabolic capacity, physical exercise endurance, and resistance to diet-induced obesity. These findings were the first of their kind and were published as several cover and feature articles in the leading international journal, Molecular Endocrinology between 2012-2016. Our future research will identify novel muscle-specific agonists targeting these NRs for therapeutic uses in obesity, type 2 diabetes and improving exercise capacity. Furthermore, we demonstrated using animal models, that another NR, RORα, controls fat deposition, glucose tolerance and insulin sensitivity in skeletal muscle, and fat tissue. Moreover, the expression of this NR, is associated with resistance to diet induced obesity and the development of fatty liver disease/hepatic steatosis.

In collaboration with IMB’s Parton Lab, we demonstrated that caveolin-1 (CAV1)—the main structural protein of caveolae—regulates liver lipid accumulation, and that this process involves regulation of bile acids and signalling by the nuclear receptor FXR. This provides new targets for the treatment of obesity and hepatic steatosis, also known as fatty liver disease. Recently, with IMB’s Stow Lab, we demonstrated that an NR called RORα controls the expression of cholesterol 25-hydroxylase in macrophages, an important gene (and cell type) controlling responses to infection and immunity.

In conclusion, insights gained from studies in breast cancer patients, and obese and diabetic murine models are helping our team to identify and profile novel NR targets in human disease, that will enable translation of this basic research into improved health outcomes.

Partners and collaborators

Professor Muscat is in collaboration with Professor Chris Ormandy and Dr David Gallego_Ortega (from The Kinghorn Cancer Centre, GARVAN Institute for Medical Research) and Professor Chrstine Clarke (Westmead Institute for Medical Research, Uni. Of Sydney) on Nuclear Receptor Nur77/NR4A1 dependent regulation of Breast Cancer; and strategic collaborations for Epigenetic Signalling and Type 2 Diabetes: novel targets for pharmaceutical intervention will involve two high profile academic leaders from NTU;  Professor’s  Boehm (Director of Diabetes , NTU) who has >400 manuscripts, and Professor Walter Wahli who has >330 manuscripts (on metabolism) at LKC School of Medicine, NTU & Imperial College London.

 

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Professor George Muscat
Group Leader, Genomics of Development and Disease Division

  +61 7 3346 2039  
  g.muscat@imb.uq.edu.au
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