Molecular Endocrinology - Cell Growth

Steroid Receptor / Immunophilin Research

Our major goal is to understand how steroid hormones regulate cell growth, particularly estrogens and androgens, since they are important in cancers of the breast and prostate. The action of these hormones in the cell is mediated by estrogen and androgen receptors. Resistance to antiestrogen and antiandrogen therapy in breast and prostate cancer is due to adaptations of hormone signalling mechanisms that result in receptor activation causing tumour growth.

In our search for new treatments, we are looking for ways to remove the receptors from the cancer cell. We are addressing this issue by focusing on chaperones present in receptor complexes that are essential for receptor function and fine-tune hormonal responses. Our studies have identified a number of novel agents that target receptor complexes causing receptor depletion. This may have important implications for ligand-independent approaches aimed at controlling estrogen and androgen signalling in breast and prostate cancers.

Calcium-Sensing Receptors

As part of its clinical role, our laboratory established, some ten years ago, a screening service to identify aberrant calcium-sensing receptor alleles. The calcium-sensing receptor is a bio-medically important cell-surface receptor that plays a pivotal role in various disorders of calcium homeostasis, with specific mutations in the receptor causing a number of disease syndromes and low levels of receptor expression in enlarged parathyroid tissue contributing to the severity of primary and secondary hyperparathyroidism. The reason for this decrease is unknown, but is central to the understanding, prevention and treatment of primary hyperparathyroidism.

We have active projects aimed to identify molecules that regulate calcium-sensing receptor trafficking, cell-surface expression and function. Manipulation of these pathways may lead to therapies to alleviate the severity of primary hyperparathyroidism.

Paget's Disease

Paget's disease of bone (PDB) is a chronic, progressive disorder, characterised by focal areas of excessive osteoclastic bone resorption accompanied by a secondary increase in osteoblastic bone formation. This results in bone expansion and structural weakness, causing pain, deformity, and a range of complications. Positional cloning studies have shown that mutations in the sequestosome 1 (SQSTM1) gene are the cause of PDB linked to the 5q35 locus. PDB-causing mutations cluster within the C-terminal ubiquitin-associated (UBA) domain of SQSTM1.

As part of our research program we are screening the C-terminal region of SQSTM1 for mutations associated with both sporadic and familial PDB. Our study aims to provide insight into molecular mechanisms through which different SQSTM1 mutations may cause PDB.

Senior Research Staff

A/Professor Tom Ratajczak Head, Molecular Endocrinology Research: mechanism of steroid hormone action; disorders of calcium metabolism

Dr Bryan Ward Fellow Research: calcium receptors and homeostasis; hormonally controlled cancers

Research Details

Steroid Receptor / Immunophilin Research

In the search for new treatments to counteract resistance to hormone therapy in breast and prostate cancers, we are looking for novel ways to interfere with signalling pathways mediated by estrogen and androgen receptors, respectively. There is an urgent need to determine the molecular profile of tumours from women diagnosed with breast cancer. This will allow the biological mechanisms underlying these tumours to be defined and survival improved.

Our current studies address these two major issues by focusing on heat shock protein 90 (Hsp90) molecular chaperone machinery that is essential for steroid receptor function and in particular immunophilin cochaperones that form part of receptor-Hsp90 complexes and fine-tune receptor responses to hormone. We are working to determine the expression profiles of these immunophilins in tissue microarrays of tumours from women with breast cancer. The immunophilin cochaperones are potentially unique biomarkers that may impact patient outcome.

Inhibitors of Hsp90 function cause the simultaneous depletion of many signalling proteins implicated in malignancy. These novel anticancer agents then form an important component of new strategies for the treatment of breast and prostate cancer. Novobiocin and related coumarin antibiotics bind to a distinct locus within the Hsp90 C terminus, a domain central to Hsp90 function.

We have shown that the coumarin-type inhibitors antagonize Hsp90 function through a unique mode of action by destabilizing Hsp90 dimer formation. Our recent data also shows that novobiocin differentially affects immunophilin-Hsp90 binding and depletes glucocorticoid and estrogen receptor levels in cancer cells. We are now in the process of testing new, high affinity novobiocin analogues to determine which best interfere with estrogen and androgen signalling and to establish whether they can be used in combination with antiestrogens and androgen antagonists in the treatment of breast and prostate cancers.

Cyclophilin 40 (CyP40), an immunophilin cochaperone we have identified in association with the estrogen receptor, and its structurally related partner, FKBP52, share two distinct functional domains - a prolyl isomerase domain and a docking domain for Hsp90. CyP40 and FKBP52 may alter hormone-binding affinity by targeting a proline-containing peptide in the receptor ligand-binding domain. An important aim of our laboratory is to identify this active-site peptide that may effectively block the immunophilin catalytic site and may therefore be useful therapeutically in strategies aimed at controlling receptor function.

There is recent evidence that infertility of both males and females in the FKBP52 mutant mouse line traces to androgen insensitivity in the prostate and progesterone insensitivity in the uterus. The results are highly relevant to the hormonal control of breast and prostate cancer and argue against redundancy among the immunophilins. From our current understanding of the role of CyP40 in estrogen signalling, we expect estrogen-regulated processes to be altered in a CyP40 knockout mouse line. Such an animal would be valuable for our pursuit of new therapies. The targeting of chaperone-receptor interactions may provide novel ligand-independent approaches to manipulate signalling pathways linked to estrogen and androgen receptors in hormone-refractory breast and prostate cancers.

Calcium-Sensing Receptor Research

The calcium-sensing receptor (CaR), a member of the G-protein coupled receptor (GPCR) superfamily, is expressed in many tissues, including neurons, kidney, intestine, parathyroid and bone. Binding of Ca²⁺ favours receptor activation and G-protein coupling, inducing signalling pathways that result in increased cytoplasmic Ca²⁺. The CaR has a very large (>600 residues) extracellular domain and is unusual in that it has a rather long intracellular tail (~200 amino acids). We have identified several novel inactivating and gain-of-function mutations in the CaR associated with heritable calcium disorders. Structure-function studies of these naturally occurring CaR mutations provide insight into receptor domains important for cell-surface expression, ligand binding and G-protein coupling. GPCRs have been found to interact with scaffold proteins within protein networks that facilitate the physical linkage of receptor to various effectors, resulting in enhanced efficiency and/or specificity of cellular signalling pathways. In a recent yeast two-hybrid screen with the CaR intracellular tail we have identified interacting proteins that link the CaR to cell surface focal adhesion complexes, the actin cytoskeleton (filamin), cell regulatory processes and trafficking. The significance of these interactions in CaR function is currently being examined.

Paget's Disease

Also known as p62, the most well-described and extensively studied function of SQSTM1/p62 is its role in the selective modulation of the transcription factor NF-κB. SQSTM1/p62 is dramatically induced during osteoclastogenesis and recent evidence suggests that the protein plays an important role in osteoclast differentiation and NF-κB activation in response to the TNFα-like cytokine, RANKL. Although the precise mechanisms that affect SQSTM1/p62 function remain to be clarified, the UBA mutations might alter interactions with regulatory components having a critical role in modulating bone turnover. Investigation of p62 mutations in our cohort of familial PDB patients has revealed a novel mutation (K378X) that eliminates 63 amino acids from the C-terminus of p62, including the UBA domain. The resulting truncation is the largest thus far identified for p62 and is associated with a severe disease phenotype, consistent with observations that patients with truncating mutations of p62 are prone to more extensive disease. Our finding provides us with a unique opportunity to make comparisons between this naturally occurring p62 truncated protein and wild type p62 in functional assays of osteoclastogenesis and RANKL-signalling pathways and in their interaction with specific proteins that regulate p62 function. We are preparing to perform a yeast two-hybrid screen to identify specific p62 interactors.

Notable Achievements

Steroid Receptor/Immunophilin Research

• Foundation paper on the isolation and identification of CyP40 as a component of estrogen receptor complexes. CyP40 and its partner immunophilin, FKBP52, identified as tetratricopeptide repeat (TPR)-containing proteins. [J Biol Chem 268:13187-13192, 1993]
• Evidence that CyP40 competes with FKBP52 for binding to Hsp90. [J Biol Chem 271:2961-2965, 1996]
• The C-terminal MEEVD sequence in Hsp90 identified as the common TPR acceptor site for steroid receptor-associated immunophilins. [J Biol Chem 274:2682-2689, 1999]
• Crystal structure of bovine CyP40 solved by collaborator, Malcolm Walkinshaw, University of Edinburgh. The TPR domain shown to provide a binding surface for the Hsp90 C-terminal MEEVD sequence. CyP40 shown to exist in two crystal forms - one in which the TPR domain consists of seven helices (normal conformation) and the other forming an extended 'jackknife' structure. [Structure 9:431-438, 2001]
• A structure-based mutational analysis of CyP40 identifies key residues in the core TPR domain mediating interaction with Hsp90. [J Biol Chem 277:40799-40809, 2002]
• Evidence provided by David Smith, Mayo Clinic Scottsdale that FKBP52 potentiates glucocorticoid signalling by increasing glucocorticoid receptor (GR) hormone-binding affinity. Potentiation requires both the Hsp90-binding ability and the prolyl isomerase domain of FKBP52. TR goes on sabbatical to David Smith's lab to study the yeast model used to demonstrate GR potentiation by FKBP52. [EMBO J 22:1158-1167, 2003]

Calcium-sensing Receptor Research

• We report on a functional deletion of the calcium-sensing receptor (CaR) in a case of neonatal severe hyperparathyroidism. We detected point mutations on separate alleles of the receptor, resulting in premature stop codon substitutions at G94 and R648, both leading to severely truncated receptors and an effective so-called knockout of functional CaR. [J Clin Endocrinol Metab 89:3721-3730, 2004]

Genetic Studies in Paget's Disease of Bone

• We identify a novel mutation (K378X) that is the first to be reported within exon 7 of the SQSTM1/p62 gene and is the only mutation to date resulting in elimination of the entire UBA domain. The mutation was found to significantly potentiate osteoclast formation and bone resorption in assays using monocytic precursors from the affected patient. [J Bone Min Res 21:1136-1145, 2006]