Professor Christina A. Mitchell, MB BS (University of Melbourne), FRACP, FRCPA, PhD (Monash University)
Dean of Medicine, Nursing and Health Sciences
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Department of Biochemistry and Molecular Biology,
Faculty of Medicine, Nursing & Health Sciences
Building 64, Level 1
Monash University, Victoria 3800 Australia
Telephone: +61 3 9905 4318
Facsimile: +61 3 9544 0183
Email: christina.mitchell@monash.edu
One of the most common signalling pathways that is altered in human cancer is the proto-oncogene phosphoinositide 3-kinase (PI3-kinase) pathway, which generates critical signalling molecules including PtdIns(3,4,5)P3 and PtdIns(3,4)P2. These signalling molecules bind the Pleckstrin Homology (PH) domain of the proto-oncogene, Akt, a serine/threonine kinase which promotes cell survival; proteins that regulates Rac and Rho and thereby actin architecture and cell migration; ARF GEFs/GAPs and thereby vesicular trafficking (Figure 1).
PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are virtually undetectable in quiescent cells, however upon extracellular stimuli they are rapidly synthesised. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are rapidly metabolized by specific lipid phosphatases that dephosphorylate the 5-, 4-, or 3-position phosphate. PTEN is a tumour suppressor gene that acts as a lipid 3-phosphatase to regulate PtdIns(3,4,5)P3 levels. A family of enzymes termed 5-phosphatases specifically remove the 5-position phosphate from PtdIns(3,4,5)P3, generating PtdIns(3,4)P2 and thereby regulate PI3-kinase signalling (Figure 1).
Figure 1 PI3-kinase, 5-phosphatases and human disease/cancer
Many components of the PI3-kinase pathway, including PI3-kinase itself, Akt, PTEN and 5-phosphatases exhibit altered expression or mutations in human disease and cancer. There are 10 mammalian 5-phosphatases most of which are associated with human disease or cancer. These include breast cancer, ciliopathy syndromes, diabetes/insulin signalling, neuronal disorders, leukaemia and developmental disorders.
Our laboratory has cloned many of the 5-phosphatases. Majority of our current research concentrates on generating and characterising 5-phosphatase knockout mouse models as well as determining the role of these enzymes in human disease. In addition, our research also involves characterisation of specific 3- and 4-phosphatases in innate immunity and breast cancer, respectively, and characterisation of the FHL family of LIM proteins in muscle disorders (see below).
1) The role of inositol polyphosphate phosphatases in cancer development.
The PI3K pathway promotes cell growth and survival and is the most common signalling pathway hyper-activated in human cancer. Activation of the pathway occurs through several different mechanisms including oncogenic mutation of PIK3CA, the catalytic subunit of PI3K, or loss of function of negative regulators of PI3K signalling such as PTEN or the inositol polyphosphate phosphatases. Oncogenic mutation of PIK3CA has been reported in a variety of solid tumours while the inositol polyphosphate phosphatases have been implicated in breast, prostate, cervical and stomach cancer as well as melanoma, metastatic adenocarcinoma and squamous cell carcinoma. Regulation of the PI3K signalling pathway has emerged as a significant target for cancer therapy but an understanding of the molecular mechanisms underlying the disease is critical to improve patient outcome.
The Mitchell laboratory is utilising knockout and transgenic mouse models to study the role of inositol polyphosphate phosphatase proteins in the development of breast, prostate and brain cancer. The laboratory also analyses inositol polyphosphate phosphatase protein expression in human cancer specimens to determine whether these proteins represent potential prognostic markers.
Figure 2
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Mammary fat pad from a breast cancer model mouse showing ductal hyperplasia (arrows).
Human mammary tissue showing inositol polyphosphate phosphatase expression (brown staining indicated by arrows) in the luminal epithelial cells.
The laboratory utilises a range of techniques to analyse cancer development including:
- Generation and analysis of cancer cell lines with over- or under-expression of inositol polyphosphate phosphatases
- Cell proliferation, apoptosis and migration assays
- Immunohistochemistry/immunofluorescence
- Xenografts
- Knockout of inositol polyphosphate phosphatases in cancer model mice
2) PI3-kinase and development.
A precise role for PI3K in embryonic development is yet to be defined; however, genetic deletion of multiple components within this pathway results in embryonic lethality in mice. Targeted deletion of Pik3ca, which encodes p110α, is embryonically lethal prior to E12.5 as a result of defective angiogenesis. Deletion of Pik3cb, encoding p110β, results in early embryonic lethality at the blastocyst stage of development. Pten knock-out mice die between E6.5 and E9.5. Genetic deletion of Inpp5k is embryonically lethal prior to E10.5 due to unknown causes. Combined deletion of Ocrl1 and Inpp5b is embryonically lethal at E8.5. Homozygous deletion of Inpp5e results in embryonic lethality after E18.5, with embryos exhibiting exencephaly, polydactyly, and kidney cysts, physiological features of ciliopathies. Indeed, INPP5E is mutated in two human cilipoathy syndromes, MORM and Joubert syndrome. The Mitchell group is currently investigating the role of 5-ptases in embryonic development in numerous cell and tissue types utilising conditional knock-out mouse strategies.
Figure 3 Bright-field microscopic image of E10.5 wild-type embryo
3) Mechanism of skeletal muscle disease and identification of novel therapies.
Figure 4
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Dystrophies and myopathies are a debilitating group of diseases which affect skeletal muscle. They lead to progressive loss of muscle mass and strength due to muscle degeneration. Whilst the genetic cause of some muscle diseases are known, for many the molecular mechanism(s) underlying muscle damage remains to be determined and there are no effective treatments. Our research focus is to (1) identify the molecular basis of muscle diseases and (2) investigate novel therapies to alleviate muscle damage/wasting, with particular emphasis on the FHL protein family and the phosphoinositide signalling pathway. In this regard, we are also interested in the characterization of proteins within these pathways which regulate muscle homeostasis, myoblast fusion and muscle regeneration. We employ a range of strategies including transgenic and knockout mouse models, zebrafish models, histology, immunofluorescence, primary and C2C12 myoblast cell culture and transfection (human and mouse), and analysis of gene transcription and cell signalling pathways.
Funding:
National Health and Medical Research Council of Australia (NHMRC)
Australian Research Council (ARC)
Diabetes Australia Research Trust
Muscular Dystrophy Association (MDA) (USA)
Facioscapulohumeral Dystrophy (FSHD) Global Research Foundation (Australia)
Laboratory personnel:
9 PhD students
9 Postdocs
2 Honours students
3 Technical Assistants
1 Scientific Assistant
Selected Publications:
1. Dyson J, Fedele C, Davies M, Becanovic J, and Mitchell CA. (2012) Phosphoinositide
phosphatases: just as important as the kinases. In "Phosphoinositides I: Enzymes of Synthesis and Degradation" by Springer, Subcell Biochem 58:215-279.
2. Davies EM, Sheffield DA, Tibarewal P, Fedele CG, Mitchell CA and Leslie NR. (2012) The PTEN and myotubularin phosphoinositide 3-phosphatases: linking lipid signalling to human disease. In "Phosphoinositides I: Enzymes of Synthesis and Degradation" by Springer, Subcell Biochem 58:281-336.
3. Astle M.V., Ooms L.M., Cole A.R., Binge L.C., Dyson J.M., Layton M.J., Petratos S., Sutherland C., Mitchell C.A. (2011) Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP): collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation. J. Biol. Chem. 286(26):23407-18 (I.F. 5.520)
4. Cowling B.S., Cottle D.L., Wilding B.R., D'Arcy C.E., Mitchell C.A., McGrath M.J. (2011) Four and a half LIM protein 1 gene mutations cause four distinct human myopathies: A comprehensive review of the clinical, histological and pathological features. Neuromuscul. Disord. 21(4): 237-51.
5. Fedele C.G., Ooms L.M., Ho M., Vieusseux J., O'Toole S.A., Millar E.K., Lopez-Knowles E., Sriratana A., Gurung R., Baglietto L., Giles G.G., Bailey C.G., Rasko J.E., Shields B.J., Price J.T., Majerus P.W., Sutherland R.L., Tiganis T., McLean C.A., Mitchell C.A. (2010) Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers. Proc. Nat. Acad. Sci. USA 107(51): 22231-36. (I.F. 9.4)
6. Ivetac, I., Gurung, R., Hakim, S., Horan, K.A., Sheffield, D.A., Binge, L.C., Majerus, P.W., Tiganis, T., Mitchell, C.A. (2009) Regulation of PI3-kinase/Akt signalling and cellular transformation by inositol polyphosphate 4-phosphatase-1 EMBO Rep. 10(5):487-93 (I.F. 7.8)
7. Cowling B.S., McGrath M.J., Nguyen M.A., Cottle D.L., Kee A.J., Brown S., Schessl J., Zou Y., Joya J., Bönnemann C.G., Hardeman E.C., Mitchell C.A . (2008) Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy. J. Cell Biol. (2008) 183:1033-48. (I.F. 10)
8. Waters, J.E., Astle M.V., Ooms L.M., Balamatsias, D., Gurung, R., Mitchell C.A. (2008) P-Rex1, a multidomain protein that regulates neurite differentiation. J. Cell Sci. 121(17):2892-903 (I.F. 6.45)
9. Horan K.A., Watanabe K., Kong A.M., Bailey C.G., Rasko J.E., Sasaki T., Mitchell C.A. (2007) Regulation of Fc{gamma}R-stimulated phagocytosis by the 72-kDa inositol polyphosphate 5-phosphatase: SHIP1, but not the 72-kDa 5-phosphatase, regulates complement receptor 3 mediated phagocytosis by differential recruitment of these 5-phosphatases to the phagocytic cup. Blood. 110:4480-91 (I.F. 10.5)
10. Kong, A.M., Horan, KA., Sriratana, A., Bailey, C.G., Collyer, L.J., Nandurkar, H.H., Shisheva, A., Layton, M.J., Rasko, J.E.J., Rowe, T. and Mitchell, C.A. (2006) Phosphatidylinositol 3-phosphate [PtdIns3P] is generated at the plasma membrane by an inositol polyphosphate 5-phosphatase: endogenous PtdIns3P can promote GLUT4 translocation to the plasma membrane. Mol. Cell. Biol. 26:6065-81 (I.F. 6.3)
11. McGrath, M.J., Cottle, D.L., Nguyen, M.A., Dyson, J.M., Coghill, I.D., Robinson, P.A., Holdsworth, M., Cowling, B.S., Hardeman, E.C., Mitchell, C.A. and Brown, S. (2006) Four and half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly. J. Biol. Chem. 281(11); 7666-83 (I.F. 5.520)
12. Dyson JM, Munday AD, Kong AM, Huysmans RD, Matzaris M, Layton MJ, Nandurkar HH, Berndt MC, Mitchell CA (2003). SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V. Localization of SHIP-2 to the activated platelet actin cytoskeleton. Blood 102: 940-948 (I.F. 10.5)
13. Dyson JM, O'Malley CJ, Becanovic J, Munday AD, Berndt MC, Coghill ID, Nandurkar HH, Ooms LM and Mitchell CA (2001). The SH2 containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin. J Cell Biol. 155: 1065-1079 (I.F. 10)
