Molecular Regulation Laboratory

Head Prof Sharad Kumar NHMRC Senior Principal Research Fellow Co-director, Centre for Cancer Biology Address   Centre for Cancer Biology SA Pathology Frome Road, Adelaide SA 5000, Australia Phone +61 8 8222 3738 Fax +61 8 8222 3162 Email sharad.kumar@health.sa.gov.au Affiliations: Affiliate Professor, Department of Medicine, The University of Adelaide Affiliate Professor, School of Molecular and Biomedical Sciences, The University of Adelaide Qualifications: MSc, PhD Staff Postdoctoral Research Scientists Telephone Email Natasha Boase 8222 3604 natasha.boase@health.sa.gov.au Hazel Dalton 8222 3604 hazel.dalton@health.sa.gov.au Donna Denton 8222 3604 donna.denton@health.sa.gov.au (The University of Adelaide Affiliate) Loretta Dorstyn 8222 3604 loretta.dorstyn@health.sa.gov.au (Cancer Council Research Fellow & The University of Adelaide Affiliate) Natalie Foot 8222 3604 natalie.foot@health.sa.gov.au Jantina Manning 8222 3604 jantina.manning@health.sa.gov.au (Mary Overton Early Career Fellow) Sonia Shalini 8222 3604 sonia.shalini@health.sa.gov.au Claire Wilson 8222 3604 claire.wilson2@health.sa.gov.au     Research Assistants     Kathryn Mills 8222 3604 kathryn.mills@health.sa.gov.au Shannon Nicolson 8222 3604 shannon.nicolson@health.sa.gov.au Layla Zhu 8222 3604 wenying.zhu@health.sa.gov.au     PhD Students     Joey Puccini 8222 3604 joey.puccini@health.sa.gov.au

Caspase function in cancer and aging

Loretta Dorstyn, Sonia Shalini, Claire Wilson and Joey Puccini

Caspases are cysteine proteases that act as executioners of apoptosis. Having cloned one of the first caspases (caspase-2), our laboratory has an ongoing program in understanding caspase biology, regulation and function. Our current focus is to delineate the in vivo function of caspase-2. Accumulated data support a critical function for caspase-2 as an initiator caspase which links death signals to mitochondrial outer membrane permeabilization (MOMP). We have found that while mouse embryonic fibroblasts (MEFs) from caspase-2 null mice are normally sensitive to a number of chemotherapeutic drugs, they show significant resistance to killing by drugs that are known to induce apoptosis by disrupting the cytoskeleton. Reduced caspase-2 expression is often associated with many cancers and low caspase-2 levels have been correlated with drug-resistance. Thus it is likely that caspase-2 is required for apoptosis under certain pathological conditions, such as cancer. We are using cell and molecular biology techniques as well as caspase-2 knockout mice and mouse models of cancer to understand how the loss of caspase-2 contributes to tumorigenesis

	Mouse chromosomes from wild type MEFs (blue), showing telomere staining in red (PNA-Cy5 FISH). Normal chromosome number is illustrated in (A) and abnormal chromosome number (42) is shown in (B).

Drosophila as a model to study developmental cell death

Donna Denton, Loretta Dorstyn, Shannon Nicolson and Layla Zhu

Work in our laboratory has taken advantage of the vinegar fly Drosophila melanogaster as a model system to examine the regulation of apoptosis. Many of the apoptotic components and pathways found in mammals are conserved in Drosophila, thus it is a useful model system to study cell death regulation during development. We are particularly interested in studying (1) the regulation and function of caspases, enzymes that mediate apoptosis, during Drosophila development, (2) the role of autophagy and growth arrest in developmental cell death and (3) the transcriptional regulation of cell death genes in response to the steroid hormone, ecdysone.

Current Projects (contact: donna.denton@health.sa.gov.au) (1) Our ongoing studies have led to several seminal findings, including the discovery of the key canonical pathway of programmed cell death (PCD) involving the caspase Dronc, and the adaptor Ark. We have also discovered a novel potential regulator of caspase activation. This project involves the characterisation of the role of this protein in caspase activation and cell death, and to identify other potential regulators of caspase activation.

(2) In recent studies we discovered that the canonical apoptotic pathway, while essential for most PCD, is largely dispensable for developmental PCD in specific tissues. This is most obvious in the larval midgut, which undergoes PCD during metamorphosis. We found that the inhibition of autophagy leads to a delay in midgut removal indicating a potential role for autophagy in midgut PCD. Given that the role of autophagy in cell death is a matter of extensive debate, our discovery that midgut PCD can be delayed by genetically blocking autophagy provides a unique model for delineating this controversy. In this project, we are further exploring this novel mechanism of midgut cell death and the role growth pathways play in regulating it.

	Inhibition of autophagy prevents midgut degradation. Morphology of dying midgut following knockdown of autophagy gene (bottom panels) shows delayed removal of gastric caeca at 4 hr RPF (arrow). Pupal sections at 12 hr RPF reveal an enlarged larval gut (bottom middle panel) compared to control (top middle panel). Autophagy is inhibited as no GFP-Atg5 puncta (an autophagy marker) is detected at 2 hr RPF (bottom right panel) compared to control (top right).

(3) During Drosophila metamorphosis obsolete larval tissues are removed by ecdysone-mediated PCD. Ecdysone, a steroid hormone, binds to its heterodimeric receptor EcR/Usp to regulate the expression of components of the cell death machinery, including several caspases. EcR/Usp recruits coactivators that are capable of modifying histones and remodelling chromatin structure. The aim of this project is to understand the role of chromatin remodelling in hormone-mediated transcription of cell death genes.

Nedd4 proteins in physiology and disease

Natasha Boase and Jantina Manning

Collaborators: David Cook (University of Sydney), Philip Poronnik (RMIT University), Grigori Rychkov (University of Adelaide), Natasha Harvey (CCB), Queten Schwarz (CCB) and Michelle Grimbaldeston (CCB).

Aberrations in the ubiquitin system underpin the pathogenesis of many diseases including malignancies, neurodegenerative disorders and channelopathies. Ubiquitin-protein ligases (E3s) determine the substrate specificity of the ubiquitination process. The Nedd4 family of E3s is evolutionarily conserved and required for the ubiquitination of numerous cellular targets involved in processes such as transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. Nedd4 is a gene initially identified in our laboratory. Members of the Nedd4-family can ubiquitinate a range of membrane proteins, resulting in their internalisation and degradation. We have shown that Nedd4, and the closely related protein Nedd4-2, interacts with and ubiquitinates the epithelial sodium channel (ENaC). ENaC is required for sodium absorption across a range of epithelial tissues such as the lungs, colon and kidney and is an important regulator of blood sodium concentration. Ubiquitination of ENaC by Nedd4 and Nedd4-2 leads to its internalisation and degradation. Recently we have shown that Nedd4-2 is a critical in vivo regulator of ENaC in the lung, and that the absence of Nedd4-2 leads to perinatal lethality in mice. Our current focus is to characterise the mechanisms of regulation of ENaC and other ion channels (such as voltage-gated sodium channels) by Nedd4 and Nedd4-2.

	In vivo regulation of ENaC by Nedd4-2. (A) Survival curve of Nedd4-2-/- survivor mice (B) Increased ENaC current observed in Nedd4-2-/- embryonic E18.5 lung cells compared to WT as measured by whole cell patch clamping (C, D) E18.5 Nedd4-2-/- lungs have more compact alveolar air-spaces as visualised by HE staining. Scale bar represents 50 μm. (E, F) Increased bENaC expression observed in Nedd4-2-/- E18.5 lung sections as measured by immunochemistry (G, H) Nedd4-2-/- lungs of survivor mice at P21 show fibrosis and inflammatory infiltration of the alveolar spaces. Scale bar represents 100μm.

In a collaborative study we have recently found that the loss of Nedd4 in mice results in reduced IGF-1 and insulin signalling, reduced growth and neonatal lethality. Nedd4-deficient cells show reduced mitogenic activity. This appears to be due to increased levels of the adaptor protein Grb10 resulting in IGF-1R mislocalization and inhibition of IGF-1 and insulin signalling. We are now studying the mechanism of Grb10 regulation by Nedd4.

There is evidence to suggest that Nedd4 has additional cellular targets. Thus, we are analysing additional phenotypes that may be associated with the knockout of Nedd4.

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Student Projects

The Molecular Regulation Laboratory provides an outstanding environment for Honours and Postgraduate studies. Some of the available projects are listed here, however if you are interested in any other areas of our research, please contact individual staff members.

1. The role of caspase-2 in specific pathways of apoptosis and tumorigenesis.

(Contact: Dr Loretta Dorstyn, loretta.dorstyn@health.sa.gov.au

Our ongoing work has made a landmark discovery that the lack of caspase-2 enhances the ability of cells to transform readily and that caspase-2 deficiency increases the potential of tumourigenesis in vivo. Using the mouse Eμ-Myc lymphoma model we found that the loss of even a single allele of caspase-2 resulted in accelerated tumourigenesis, and this was further enhanced in caspase-2^-/- mice. The caspase-2^-/- cells show increased growth rates, a defective apoptotic response to cell cycle checkpoint regulation and show abnormal cycling following γ-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumour suppressor protein. This project involves testing whether the tumour suppressor function of caspase-2 is limited to only Myc-induced lymphomagenesis, or is more general. Cellular and molecular studies will dissect out the mechanism by which caspase-2 acts as a tumour suppressor and identify target(s) of caspase-2 that mediate its tumour suppressor function.

2. Cell death regulation during animal development.

(Contact: Dr Donna Denton, donna.denton@health.sa.gov.au

We have been utilising Drosophila as an in vivo model to dissect out the mechanisms of developmentally programmed cell death (PCD). Our ongoing studies have led to several seminal findings, including the discovery of the key canonical pathway of PCD involving the caspase Dronc, and the adaptor Ark. We have also discovered a novel potential regulator of caspase activation from a Dronc-interaction screen. This project will now characterise the role of the novel protein in caspase activation and cell death, and identify other potential regulators of caspase activation using a range of molecular, cellular, biochemical and genetic approaches.

3. Role of autophagy in PCD.

(Contact: Dr Donna Denton, donna.denton@health.sa.gov.au)

In recent studies we discovered that the Dronc/Ark pathway, while essential for most PCD, is largely dispensable for developmental PCD in specific tissues. This is most obvious in the larval midgut (MG), which undergoes PCD during metamorphosis, and this process is unaffected in dronc and ark mutants. In preliminary studies we have found that the inhibition of autophagy, a caspase-independent mechanism of PCD, leads to a delay in MG removal indicating a potential role for autophagy in MG PCD. Given that the role of autophagy in cell death is a matter of extensive debate, our discovery that MG PCD can be delayed by genetically blocking autophagy provides a unique model for delineating this controversy. We hypothesise that during development PCD utilises caspase-dependent (most tissues), caspase- and autophagy-dependent (e.g. larval salivary glands), and caspase-independent but autophagy-dependent (e.g. midgut) mechanisms. In this project we will delineate the mechanism of midgut cell death by exploring the contribution of caspases and autophagy. The project also aims to define the role of growth signalling in midgut cell death, to determine how growth signals and death signals may be integrated to regulate autophagy. A range of cellular, molecular and genetic approaches will be utilized in these projects. Given the controversial role of autophagy in human disease a better understanding of the regulation of autophagy is important for future treatments of disease.

4. The role of Nedd4 and Nedd4-2 in hypertension and the regulation of sodium channels.

(Contact: Dr Natasha Boase, natasha.boase@health.sa.gov.au or Dr Jantina Manning, Jantina.Manning@health.sa.gov.au)

Aberrations in the ubiquitin system underpin the pathogenesis of many diseases including cancer, neurodegenerative disorders and channelopathies. The Nedd4 and related ubiquitin ligases (E3s) are required for the ubiquitination of numerous cellular targets involved in processes such as transcription, stability and trafficking of plasma membrane proteins, and the degradation of misfolded proteins. We have shown that Nedd4-2 E3 ubiquitinates the epithelial sodium channel (ENaC). ENaC is required for sodium absorption across a range of epithelial tissues such as the lungs, colon and kidney and is an important regulator of blood sodium concentration and blood pressure. Ubiquitination of ENaC by Nedd4-2 leads to its internalisation and degradation. Our current focus is to characterise the mechanisms of regulation of ENaC by Nedd4-2 in vivo by using Nedd4-2 gene knockout mice. We are also studying the role of Nedd4-2 in regulating additional novel targets. In this project the students will use a range of molecular, cellular and animal techniques.

5. Regulation of animal growth by Nedd4.

(Contact: Dr Natasha Boase, natasha.boase@health.sa.gov.au)

In a collaborative study with Prof Yang (University of Iowa) we have recently found that the loss of Nedd4 in mice results in reduced IGF-1 and insulin signalling, reduced growth and neonatal lethality. Nedd4-deficient cells show reduced mitogenic activity. This appears to be due to increased levels of the adaptor protein Grb10 resulting in IGF-1R mislocalization and inhibition of IGF-1 and insulin signalling. We are now studying the mechanism of Grb10 regulation by Nedd4. There is evidence to suggest that Nedd4 has additional cellular targets. Thus, we are analysing additional phenotypes that may be associated with the knockout of Nedd4. This project aims to decipher the mechanisms by which Nedd4 controls animal growth and development.

6. The regulation of iron homeostasis by the Nedd4 binding proteins, Ndfip1 and Ndfip2.

(Contact: Dr Hazel Dalton, hazel.dalton@health.sa.gov.au)

Iron homeostasis is a highly regulated process which, if perturbed, leads to a number of disease states such as haemochromatosis or anaemia. Our recent work shows that Ndfips regulate the divalent metal ion transporter DMT1, the primary non-heme iron transporter in mammals. DMT1 interacts with both Ndfip1 and Ndfip2, and this promotes DMT1 ubiquitination and degradation by the Nedd4-family ubiquitin ligase, WWP2. Furthermore the Ndfip1 knockout mice show increased hepatic iron deposition, indicating an essential function of Ndfip1 in iron homeostasis. Our current focus is to further characterise WWP2 and Ndfip knockout mice to provide additional understanding of this novel mechanism of regulating iron transport. Students will carry out iron feeding experiments, tissue analysis for iron deposition and a range of cellular and molecular studies to delineate the regulation of DMT1 by Ndfips and WWP2.

Scholarships are available through various sources including those listed below:

• University of Adelaide Scholarships

Honours scholarships and PhD top up scholarships are available through the Royal Adelaide Hopital Research Committee.

The Molecular Regulation Laboratory is affiliated with The University of Adelaide through the following:

• Faculty of Health Sciences, School of Medicine
• Faculty of Science, School of Molecular & Biomedical Science, Discipline of Genetics

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