Publications by authors named "Christina A Mitchell"

96 Publications

A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer.

Mol Cell Oncol 2021 9;8(4):1954470. Epub 2021 Sep 9.

Cancer Program, Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

AKT is the central phosphoinositide 3-kinase (PI3K) signaling effector, however, (p110α subunit of PI3Kα)-mutant estrogen receptor-positive (ER) breast cancers exhibit minimal AKT activation and the downstream signaling is poorly characterized. We discovered that a subset of -mutant ER breast cancers exhibit increased inositol polyphosphate 4-phosphatase type II (INPP4B) expression, which promotes late endosome formation and glycogen synthase kinase 3 beta (GSK3β) trafficking, leading to enhanced Wingless-related integration site (WNT)/catenin beta 1 (β-catenin) activation.
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http://dx.doi.org/10.1080/23723556.2021.1954470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489923PMC
September 2021

Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

Cell Rep 2021 Sep;36(11):109689

The Garvan Institute of Medical Research, St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2010, Australia.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.
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http://dx.doi.org/10.1016/j.celrep.2021.109689DOI Listing
September 2021

The INPP4B paradox: Like PTEN, but different.

Adv Biol Regul 2021 Jun 16;82:100817. Epub 2021 Jun 16.

Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, 3800, Australia. Electronic address:

Cancer is a complex and heterogeneous disease marked by the dysregulation of cancer driver genes historically classified as oncogenes or tumour suppressors according to their ability to promote or inhibit tumour development and growth, respectively. Certain genes display both oncogenic and tumour suppressor functions depending on the biological context, and as such have been termed dual-role cancer driver genes. However, because of their context-dependent behaviour, the tumourigenic mechanism of many dual-role genes is elusive and remains a significant knowledge gap in our effort to understand and treat cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) is an emerging dual-role cancer driver gene, primarily known for its role as a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. In response to growth factor stimulation, class I PI3K generates PtdIns(3,4,5)P at the plasma membrane. PtdIns(3,4,5)P can be hydrolysed by inositol polyphosphate 5-phosphatases to generate PtdIns(3,4)P, which, together with PtdIns(3,4,5)P, facilitates the activation of AKT to promote cell proliferation, survival, migration, and metabolism. Phosphatase and tensin homology on chromosome 10 (PTEN) and INPP4B are dual-specificity phosphatases that hydrolyse PtdIns(3,4,5)P and PtdIns(3,4)P, respectively, and thus negatively regulate PI3K/AKT signalling. PTEN is a bona fide tumour suppressor that is frequently lost in human tumours. INPP4B was initially characterised as a tumour suppressor akin to PTEN, and has been implicated as such in a number of cancers, including prostate, thyroid, and basal-like breast cancers. However, evidence has since emerged revealing INPP4B as a paradoxical oncogene in several malignancies, with increased INPP4B expression reported in AML, melanoma and colon cancers among others. Although the tumour suppressive function of INPP4B has been mostly ascribed to its ability to negatively regulate PI3K/AKT signalling, its oncogenic function remains less clear, with proposed mechanisms including promotion of PtdIns(3)P-dependent SGK3 signalling, inhibition of PTEN-dependent AKT activation, and enhancing DNA repair mechanisms to confer chemoresistance. Nevertheless, research is ongoing to identify the factors that dictate the tumourigenic output of INPP4B in different human cancers. In this review we discuss the dualistic role that INPP4B plays in the context of cancer development, progression and treatment, drawing comparisons to PTEN to explore how their similarities and, importantly, their differences may account for their diverging roles in tumourigenesis.
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http://dx.doi.org/10.1016/j.jbior.2021.100817DOI Listing
June 2021

INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/β-catenin signaling in breast cancer.

Nat Commun 2021 05 25;12(1):3140. Epub 2021 May 25.

Cancer Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER breast cancer via PI(3,4)P to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies.
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http://dx.doi.org/10.1038/s41467-021-23241-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149851PMC
May 2021

Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone.

Front Cell Dev Biol 2021 30;9:634649. Epub 2021 Apr 30.

Cancer Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.

Primary cilia are evolutionary conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Phosphoinositides (PI) are membrane-associated signaling lipids that regulate numerous cellular events via the recruitment of lipid-binding effectors. The temporal and spatial membrane distribution of phosphoinositides is regulated by phosphoinositide kinases and phosphatases. Recently phosphoinositide signaling and turnover has been observed at primary cilia. However, the precise localization of the phosphoinositides to specific ciliary subdomains remains undefined. Here we use superresolution microscopy (2D stimulated emission depletion microscopy) to map phosphoinositide distribution at the cilia transition zone. PI(3,4,5)P and PI(4,5)P localized to distinct subregions of the transition zone in a ring-shape at the inner transition zone membrane. Interestingly, the PI(3,4,5)P subdomain was more distal within the transition zone relative to PtdIns(4,5)P. The phosphoinositide effector kinase pAKT(S473) localized in close proximity to these phosphoinositides. The inositol polyphosphate 5-phosphatase, INPP5E, degrades transition zone phosphoinositides, however, studies of fixed cells have reported recombinant INPP5E localizes to the ciliary axoneme, distant from its substrates. Notably, here using live cell imaging and optimized fixation/permeabilization protocols INPP5E was found concentrated at the cilia base, in a distribution characteristic of the transition zone in a ring-shaped domain of similar dimensions to the phosphoinositides. Collectively, this superresolution map places the phosphoinositides in situ with the transition zone proteins and reveals that INPP5E also likely localizes to a subdomain of the transition zone membrane, where it is optimally situated to control local phosphoinositide metabolism.
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http://dx.doi.org/10.3389/fcell.2021.634649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8120242PMC
April 2021

Structural analysis of the PTEN:P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1.

Sci Signal 2021 May 4;14(681). Epub 2021 May 4.

Biomedicine Discovery Institute, Monash University, Clayton, 3800 Victoria, Australia.

The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P to PI(4,5)P to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gβγ subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gβγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.
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http://dx.doi.org/10.1126/scisignal.abc4078DOI Listing
May 2021

Bidirectional interconversion between PtdIns4P and PtdIns(4,5)P is required for autophagic lysosome reformation and protection from skeletal muscle disease.

Autophagy 2021 05 20;17(5):1287-1289. Epub 2021 Apr 20.

Cancer Program, and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia.

Autophagic lysosome reformation (ALR) recycles autolysosome membranes formed during autophagy, to make lysosomes and is essential for continued autophagy function. Localized membrane remodeling on autolysosomes leads to the extension of reformation tubules, which undergo scission to form new lysosomes. The phosphoinositides phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P) induce this remodeling by recruiting protein effectors to membranes. We identified the inositol polyphosphate 5-phosphatase INPP5K, which converts PtdIns(4,5)P to PtdIns4P is essential for ALR in skeletal muscle. mutations that reduce its 5-phosphatase activity are known to cause muscular dystrophy, via an undefined mechanism. We generated skeletal muscle-specific knockout mice which exhibited severe muscle disease, with lysosome depletion and marked autophagy inhibition. This was due to decreased PtdIns4P and increased PtdIns(4,5)P on autolysosomes, causing reduced scission of reformation tubules. ALR was restored in cells with loss of INPP5K by expression of wild-type INPP5K, but not muscle-disease causing mutants. Therefore on autolysosomes, both PtdIns(4,5)P generation and its removal by INPP5K is required for completion of ALR. Furthermore, skeletal muscle shows a dependence on the membrane recycling ALR pathway to maintain lysosome homeostasis and ensure the protective role of autophagy against disease.
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http://dx.doi.org/10.1080/15548627.2021.1916195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143231PMC
May 2021

PTEN and Other PtdIns(3,4,5)P Lipid Phosphatases in Breast Cancer.

Int J Mol Sci 2020 Dec 2;21(23). Epub 2020 Dec 2.

Cancer Program, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P to form PtdIns(4,5)P. PtdIns(3,4,5)P can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.
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http://dx.doi.org/10.3390/ijms21239189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730566PMC
December 2020

Defective lysosome reformation during autophagy causes skeletal muscle disease.

J Clin Invest 2021 01;131(1)

Cancer Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Melbourne, Victoria, Australia.

The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.
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http://dx.doi.org/10.1172/JCI135124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773396PMC
January 2021

PtdIns(3,4,5)P-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.

Proc Natl Acad Sci U S A 2020 11 23;117(45):28056-28067. Epub 2020 Oct 23.

Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia;

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher mRNA in ER/luminal tumors was associated with poor outcome in luminal B cancers. deletion in MMTV- or MMTV- mice reduced Rac1 activation in vivo and improved survival. High level MMTVdriven transgenic expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV- expression in MMTV- mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.
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http://dx.doi.org/10.1073/pnas.2006445117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668035PMC
November 2020

Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression.

Mol Cell 2020 10;80(2):279-295.e8

Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC 3800, Australia. Electronic address:

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.
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http://dx.doi.org/10.1016/j.molcel.2020.09.027DOI Listing
October 2020

Caveolae Control Contractile Tension for Epithelia to Eliminate Tumor Cells.

Dev Cell 2020 07 1;54(1):75-91.e7. Epub 2020 Jun 1.

Division of Cell and Developmental Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia. Electronic address:

Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P, or disabling the interaction between FMNL2 and PtdIns(4,5)P. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.
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http://dx.doi.org/10.1016/j.devcel.2020.05.002DOI Listing
July 2020

AKT signaling promotes DNA damage accumulation and proliferation in polycystic kidney disease.

Hum Mol Genet 2020 01;29(1):31-48

Cancer Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia.

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD, but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3-kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD; however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here, we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD.
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http://dx.doi.org/10.1093/hmg/ddz232DOI Listing
January 2020

The myotubularin MTMR4 regulates phagosomal phosphatidylinositol 3-phosphate turnover and phagocytosis.

J Biol Chem 2019 11 22;294(45):16684-16697. Epub 2019 Sep 22.

Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated silencing increased levels of cell-surface immunoglobulin receptors ( Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and -specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with -specific siRNA were more resistant to -induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the -knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.
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http://dx.doi.org/10.1074/jbc.RA119.009133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851315PMC
November 2019

Intranasal Targeting of Hypothalamic PTP1B and TCPTP Reinstates Leptin and Insulin Sensitivity and Promotes Weight Loss in Obesity.

Cell Rep 2019 Sep;28(11):2905-2922.e5

Metabolism, Diabetes and Obesity Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; Monash Metabolic Phenotyping Facility, Monash University, VIC, Australia; Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia. Electronic address:

The importance of hypothalamic leptin and insulin resistance in the development and maintenance of obesity remains unclear. The tyrosine phosphatases protein tyrosine phosphatase 1B (PTP1B) and T cell protein tyrosine phosphatase (TCPTP) attenuate leptin and insulin signaling and are elevated in the hypothalami of obese mice. We report that elevated PTP1B and TCPTP antagonize hypothalamic leptin and insulin signaling and contribute to the maintenance of obesity. Deletion of PTP1B and TCPTP in the hypothalami of obese mice enhances CNS leptin and insulin sensitivity, represses feeding, and increases browning, to decrease adiposity and improve glucose metabolism. The daily intranasal administration of a PTP1B inhibitor, plus the glucocorticoid antagonist RU486 that decreases TCPTP expression, represses feeding, increases browning, promotes weight loss, and improves glucose metabolism in obese mice. Our findings causally link heightened hypothalamic PTP1B and TCPTP with leptin and insulin resistance and the maintenance of obesity and define a viable pharmacological approach by which to promote weight loss in obesity.
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http://dx.doi.org/10.1016/j.celrep.2019.08.019DOI Listing
September 2019

Women in cancer research.

Nat Rev Cancer 2019 10 15;19(10):547-552. Epub 2019 Aug 15.

Department of Biochemistry and Molecular Biology, John Hopkins School of Public Health, Baltimore, MD, USA.

Gender inequality in science is a real issue, and without frank and open discussions leading to positive action it is likely to remain so. In February 2019, Nature Reviews Cancer was kindly invited to be part of a 'Women in Science Mentoring' panel discussion, which took place at the Lorne Cancer Conference in Victoria, Australia. Inspired by the scientific career paths and experiences of the women on the panel, we decided to share their stories with our readers in this Viewpoint article, along with their opinions on how men and women must equally take responsibility for supporting and empowering female scientists. To this end, we hope we might contribute in some small way to highlighting a few of the issues surrounding gender bias in cancer research, as well as science more generally, and show our commitment to ensuring gender diversity within the journal.
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http://dx.doi.org/10.1038/s41568-019-0176-yDOI Listing
October 2019

Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation.

Nat Commun 2019 01 17;10(1):296. Epub 2019 Jan 17.

Cancer Program, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia.

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-018-08154-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336867PMC
January 2019

β-catenin ablation exacerbates polycystic kidney disease progression.

Hum Mol Genet 2019 01;28(2):230-244

Cancer Program, Monash Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

Polycystic kidney disease (PKD) results from excessive renal epithelial cell proliferation, leading to the formation of large fluid filled cysts which impair renal function and frequently lead to renal failure. Hyperactivation of numerous signaling pathways is hypothesized to promote renal epithelial cell hyperproliferation including mTORC1, extracellular signal-regulated kinase (ERK) and WNT signaling. β-catenin and its target genes are overexpressed in some PKD models and expression of activated β-catenin induces cysts in mice; however, β-catenin murine knockout studies indicate it may also inhibit cystogenesis. Therefore, it remains unclear whether β-catenin is pro- or anti-cystogenic and whether its role is canonical WNT signaling-dependent. Here, we investigate whether β-catenin deletion in a PKD model with hyperactived β-catenin signaling affects disease progression to address whether increased β-catenin drives PKD. We used renal epithelial cell specific Inpp5e-null PKD mice which we report exhibit increased β-catenin and target gene expression in the cystic kidneys. Surprisingly, co-deletion of β-catenin with Inpp5e in renal epithelial cells exacerbated polycystic kidney disease and renal failure compared to Inpp5e deletion alone, but did not normalize β-catenin target gene expression. β-catenin/Inpp5e double-knockout kidneys exhibited increased cyst initiation, cell proliferation and MEK/ERK signaling compared to Inpp5e-null, associated with increased fibrosis, which may collectively contribute to accelerated disease. Therefore, increased β-catenin and WNT target gene expression are not necessarily cyst promoting. Rather β-catenin may play a dual and context-dependent role in PKD and in the presence of other cyst-inducing mutations (Inpp5e-deletion); β-catenin loss may exacerbate disease in a WNT target gene-independent manner.
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http://dx.doi.org/10.1093/hmg/ddy309DOI Listing
January 2019

P-Rex1 and P-Rex2 RacGEFs and cancer.

Biochem Soc Trans 2017 08 14;45(4):963-77. Epub 2017 Jul 14.

Cancer Program, Monash Biomedicine Discovery Institute, and Department of Biochemistry and Molecular Biology, Monash University, 23 Innovation Walk, Clayton, VIC 3800, Australia

Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins are RacGEFs that are synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and Gβγ subunits of G-protein-coupled receptors. P-Rex1 and P-Rex2 share similar amino acid sequence homology, domain structure, and catalytic function. Recent evidence suggests that both P-Rex proteins may play oncogenic roles in human cancers. P-Rex1 and P-Rex2 are altered predominantly via overexpression and mutation, respectively, in various cancer types, including breast cancer, prostate cancer, and melanoma. This review compares the similarities and differences between P-Rex1 and P-Rex2 functions in human cancers in terms of cellular effects and signalling mechanisms. Emerging clinical data predict that changes in expression or mutation of P-Rex1 and P-Rex2 may lead to changes in tumour outcome, particularly in breast cancer and melanoma.
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http://dx.doi.org/10.1042/BST20160269DOI Listing
August 2017

Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing.

J Cell Biol 2017 07 31;216(7):1949-1957. Epub 2017 May 31.

Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well understood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival.
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http://dx.doi.org/10.1083/jcb.201610113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496614PMC
July 2017

Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases.

Biosci Rep 2017 02 10;37(1). Epub 2017 Feb 10.

Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia

Class I phosphoinositide 3-kinase (PI3K) generates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)) at the plasma membrane in response to growth factors, activating a signalling cascade that regulates many cellular functions including cell growth, proliferation, survival, migration and metabolism. The PI3K pathway is commonly dysregulated in human cancer, and drives tumorigenesis by promoting aberrant cell growth and transformation. PtdIns(3,4,5) facilitates the activation of many pleckstrin homology (PH) domain-containing proteins including the serine/threonine kinase AKT. There are three AKT isoforms that are frequently hyperactivated in cancer through mutation, amplification or dysregulation of upstream regulatory proteins. AKT isoforms have converging and opposing functions in tumorigenesis. PtdIns(3,4,5) signalling is degraded and terminated by phosphoinositide phosphatases such as phosphatase and tensin homologue (PTEN), proline-rich inositol polyphosphate 5-phosphatase (PIPP) (INPP5J) and inositol polyphosphate 4-phosphatase type II (INPP4B). PtdIns(3,4,5) is rapidly hydrolysed by PIPP to generate phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)), which is further hydrolysed by INPP4B to form phosphatidylinositol 3-phosphate (PtdIns3). PtdIns(3,4) and PtdIns3 are also important signalling molecules; PtdIns(3,4) together with PtdIns(3,4,5) are required for maximal AKT activation and PtdIns3 activates PI3K-dependent serum and glucocorticoid-regulated kinase (SGK3) signalling. Loss of or expression or function promotes tumour growth in murine cancer models through enhanced AKT isoform-specific signalling. INPP4B inhibits PtdIns(3,4)-mediated AKT activation in breast and prostate cancer; however, INPP4B expression is increased in acute myeloid leukaemia (AML), melanoma and colon cancer where it paradoxically promotes cell proliferation, transformation and/or drug resistance. This review will discuss how PTEN, PIPP and INPP4B distinctly regulate PtdIns(3,4,5) signalling downstream of PI3K and how dysregulation of these phosphatases affects cancer outcomes.
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http://dx.doi.org/10.1042/BSR20160432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301276PMC
February 2017

INPP5E regulates phosphoinositide-dependent cilia transition zone function.

J Cell Biol 2017 Jan 20;216(1):247-263. Epub 2016 Dec 20.

Cancer Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P and PI(3,4,5)P accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.
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http://dx.doi.org/10.1083/jcb.201511055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223597PMC
January 2017

Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling.

Hum Mol Genet 2016 06 7;25(11):2295-2313. Epub 2016 Apr 7.

Cancer Program, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia

Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase (PI3K)-generated PI(3,4,5)P and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression.
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http://dx.doi.org/10.1093/hmg/ddw097DOI Listing
June 2016

PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling.

J Biol Chem 2016 08 29;291(33):17258-70. Epub 2016 Jun 29.

From the Cancer Program, Monash Biomedicine Discovery Institute, and Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia,

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression.
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http://dx.doi.org/10.1074/jbc.M116.743401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016125PMC
August 2016

Regulation of PtdIns(3,4,5)P3/Akt signalling by inositol polyphosphate 5-phosphatases.

Biochem Soc Trans 2016 Feb;44(1):240-52

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia

The phosphoinositide 3-kinase (PI3K) generated lipid signals, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, are both required for the maximal activation of the serine/threonine kinase proto-oncogene Akt. The inositol polyphosphate 5-phosphatases (5-phosphatases) hydrolyse the 5-position phosphate from the inositol head group of PtdIns(3,4,5)P3 to yield PtdIns(3,4)P2. Extensive work has revealed several 5-phosphatases inhibit PI3K-driven Akt signalling, by decreasing PtdIns(3,4,5)P3 despite increasing cellular levels of PtdIns(3,4)P2. The roles that 5-phosphatases play in suppressing cell proliferation and transformation are slow to emerge; however, the 5-phosphatase PIPP [proline-rich inositol polyphosphate 5-phosphatase; inositol polyphosphate 5-phosphatase (INPP5J)] has recently been identified as a putative tumour suppressor in melanoma and breast cancer and SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase 1] inhibits haematopoietic cell proliferation. INPP5E regulates cilia stability and INPP5E mutations have been implicated ciliopathy syndromes. This review will examine 5-phosphatase regulation of PI3K/Akt signalling, focussing on the role PtdIns(3,4,5)P3 5-phosphatases play in developmental diseases and cancer.
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http://dx.doi.org/10.1042/BST20150214DOI Listing
February 2016

The Inositol Polyphosphate 5-Phosphatase PIPP Regulates AKT1-Dependent Breast Cancer Growth and Metastasis.

Cancer Cell 2015 Aug;28(2):155-69

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia. Electronic address:

Metastasis is the major cause of breast cancer mortality. Phosphoinositide 3-kinase (PI3K) generated PtdIns(3,4,5)P3 activates AKT, which promotes breast cancer cell proliferation and regulates migration. To date, none of the inositol polyphosphate 5-phosphatases that inhibit PI3K/AKT signaling have been reported as tumor suppressors in breast cancer. Here, we show depletion of the inositol polyphosphate 5-phosphatase PIPP (INPP5J) increases breast cancer cell transformation, but reduces cell migration and invasion. Pipp ablation accelerates oncogene-driven breast cancer tumor growth in vivo, but paradoxically reduces metastasis by regulating AKT1-dependent tumor cell migration. PIPP mRNA expression is reduced in human ER-negative breast cancers associated with reduced long-term outcome. Collectively, our findings identify PIPP as a suppressor of oncogenic PI3K/AKT signaling in breast cancer.
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http://dx.doi.org/10.1016/j.ccell.2015.07.003DOI Listing
August 2015

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells.

J Biol Chem 2015 Aug 24;290(34):20827-20840. Epub 2015 Jun 24.

Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.
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http://dx.doi.org/10.1074/jbc.M115.660456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4543645PMC
August 2015

Heterozygous expression of the oncogenic Pik3ca(H1047R) mutation during murine development results in fatal embryonic and extraembryonic defects.

Dev Biol 2015 Aug 7;404(1):14-26. Epub 2015 May 7.

Cancer Biology and Surgical Oncology Research Laboratory, Peter MacCallum Cancer Centre, St. Andrew's Place, Melbourne, Victoria 3002, Australia; Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway regulates many cellular functions including proliferation, migration, survival and protein synthesis. Somatic mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K enzyme, are commonly associated with many human cancers as well as recently being implicated in human overgrowth syndromes. However, it is not clear if such mutations can be inherited through the germline. We have used a novel mouse model with Cre recombinase (Cre)-conditional knock-in of the common H1047R mutation into the endogenous Pik3ca gene. Heterozygous expression of the Pik3ca(H1047R) mutation in the developing mouse embryo resulted in failed 'turning' of the embryo and disrupted vascular remodelling within the embryonic and extraembryonic tissues, leading to lethality prior to E10. As vascular endothelial growth factor A (VEGF-A) signalling was disrupted in these embryos, we used Cre under the control of the Tie2 promoter to target the Pik3ca(H1047R) mutation specifically to endothelial cells. In these embryos turning occurred normally but the vascular remodelling defects and embryonic lethality remained, likely as a result of endothelial hyperproliferation. Our results confirm the lethality associated with heterozygous expression of the Pik3ca(H1047R) mutation during development and likely explain the lack of inherited germline PIK3CA mutations in humans.
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http://dx.doi.org/10.1016/j.ydbio.2015.04.022DOI Listing
August 2015

Inositol polyphosphate 4-phosphatase II (INPP4B) is associated with chemoresistance and poor outcome in AML.

Blood 2015 Apr 3;125(18):2815-24. Epub 2015 Mar 3.

Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia; Department of Clinical Haematology, The Alfred Hospital, Melbourne, VIC, Australia;

Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P3 3-phosphatase (PTEN), PtdIns(3,4,5)P3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4- and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function.
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http://dx.doi.org/10.1182/blood-2014-09-603555DOI Listing
April 2015

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

PLoS One 2015 19;10(2):e0117665. Epub 2015 Feb 19.

Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335040PMC
November 2015
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