Publications

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    ABSTRACT: High levels of vitamin D are hypothesized to reduce the risk of breast cancer. In order to be biologically active, dietary vitamin D must be converted to its biologically active form 1,25(OH)2D3. Cyp2r1 is a major vitamin D hydroxylase that catalyzes the first step of this activation producing 25(OH)D3. Cyp2r1 is located within SuprMam1, a mammary tumour susceptibility locus identified in the BALB/c­Trp53+/­ mouse model of spontaneous breast cancer (Blackburn et al, Am J Path, 2007). We have examined the vitamin D pathway in SM09 congenic mice, which contain the BALB/c SuprMam1 locus on a C57BL/6 background. qPCR and western blotting for Cyp2r1 in tissues from SM09 and control mice revealed a significant 2­3­fold reduction in Cyp2r1 expression in mammary glands and liver (female but not male) of SM09 mice, however differences in plasma 25(OH)D3, calcium or phosphate levels were not found. Instead, 3­fold higher levels of plasma parathyroid hormone (PTH), a major vitamin D / calcium regulator, were present in female (but not male) mice carrying the BALB/c allele of the SuprMam1 locus. Affymetrix expression profiling of mammary glands found differential expression of many genes of the vitamin D pathway, consistent with disruption of the pathway. Increasing dietary calcium or vitamin D returned PTH levels to normal in BALB/c and SM09 mice. We are currently characterizing several polymorphisms in the Cyp2r1 promoter which may alter promoter function. Thus, chronically elevated PTH levels due to an interaction between low calcium / vitamin D intake and reduced Cyp2r1 expression from the BALB/c allele of Cyp2r1 may contribute to increased breast cancer susceptibility. The SM09 congenic mice may serve as a valuable model for studying the role of gene­environment interactions of the vitamin D pathway in cancer and other diseases.
    International Association for Breast Cancer Research Conference; 09/2014
  • Genetics Society of AustralAsia Conference, Sydney, Australia; 07/2014
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    Melissa Rooke, Lucy A Coupland, Thy Truong, Anneke C Blackburn
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    ABSTRACT: Sarcomas are cancers that arise from tissues of mesenchy- mal origin and there has been limited improvement in treatments over the last 30 years. The Warburg effect is a widespread metabolic phenotype of cancer, where glycolysis is favoured despite the presence of oxygen. Dichloroacetate (DCA) is a pyruvate dehydrogenase kinase (PDK) inhibitor in clinical use that can reverse the Warburg effect, inhibiting growth and enhancing apoptosis in a range of cancers. We have investigated its effectiveness against sarcoma cells in vitro and in vivo.
    Metabolism, Diet and Disease 2014: Cancer and metabolism, Washington DC, USA; 05/2014
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    ABSTRACT: There is a continuing need to improve the efficacy of anticancer agents and to reduce the toxicity associated with them. Recently, reversal of the glycolytic phenotype of cancer cells with novel agents such as DCA (dichloroacetate) has been recognised as an important new target for cancer therapy. DCA is currently being assessed as a novel chemotherapeutic in clinical trials. It is anticipated that DCA will be used in combination with other well-established chemotherapeutic drugs. It is therefore critically important to determine the effects of DCA on therapeutic efficacy, and off-target effects of existing frontline anti-cancer drugs. We have recently discovered that DCA induces glutamate cysteine ligase, the rate-limiting step in glutathione (GSH) synthesis, a major cellular antioxidant. We hypothesised that the ability of DCA to stimulate GSH-synthesising capacity could reduce cisplatin-induced nephrotoxicity, which is thought to be mediated in part by oxidative stress. To determine whether DCA can attenuate cisplatin-induced nephrotoxicity, and to determine if DCA influences the anti-cancer properties of cisplatin. 120 Balb/c mice were injected subcutaneously with the syngeneic 4T1 breast cancer cell line and then co-treated with DCA and cisplatin weekly for 1 month. Controls included treatment with cisplatin alone, DCA alone, or normal saline injections. Serum blood urea nitrogen (BUN) and creatinine were measured and kidney damage was assessed histologically. Tumour size was monitored throughout. DCA prevented increases in serum creatinine, BUN, and renal proximal tubule apoptosis evident in cisplatin-only treated mice. The tumour size in mice in the co-treated group decreased at the same rate as the cisplatin-only treated mice. We have thus concluded that DCA largely prevents the development of cisplatin-induced nephrotoxicity, and does not attenuate its anti-cancer properties.
    Pathology 02/2014; 46 Suppl 1:S110-1. DOI:10.1097/01.PAT.0000443702.65813.a9 · 2.62 Impact Factor
  • EMBL Cancer Genomics Conference, Heidelberg, Germany; 11/2013
  • Madara Ratnadiwakara, Rohan B. Williams, Anneke C. Blackburn
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    ABSTRACT: Vitamin D is a group of fat-soluble secosteroids. Apart from its main physiological role in calcium homeostasis, several studies have demonstrated a wide range of functions of vitamin D which can be associated with cancer. Even though the exact mechanism underlying the protective action of vitamin D against cancer is not clearly understood, there is strong experimental evidence showing that vitamin D participates in cell growth regulation, apoptosis and proliferation in normal and malignant breast cells. This evidence has lead to the hypothesis that high levels of vitamin D might reduce the risk of breast cancer. In order to be biologically active, vitamin D must be converted to its active form 1,25(OH)2D3. CYP2R1 is a major vitamin D hydroxylase that catalyzes the first step of this activation producing 25(OH)D3. Cyp2r1 is located within the SuprMam1 locus, a mammary tumour susceptibility locus identified in the BALB/c-Trp53+/- mouse model of spontaneous breast cancer (1). Affymetrix data comparing T cell gene expression have identified Cyp2r1 to be differentially expressed between BALB/c and C57BL/6 mouse strains (0.68 fold reduction in BALB/c, the mammary tumour susceptible strain). As lower CYP2R1 levels should result in lower vitamin D levels and may contribute to the higher cancer susceptibility of BALB/c mice, we studied the Cyp2r1 gene expression and plasma 25(OH)D3 levels in SM09 congenic-mice (BALB/c SuprMam loci in C57BL/6 background) in comparison to C57BL/6-controls. A real-time quantitative PCR of Cyp2r1 in different tissues from SM09 and control mice revealed that there is a significant difference in Cyp2r1 expression in mammary glands of the two strains (2.76 fold reduction in SM09, p=0.04). But this difference was not observed in liver or lymph node tissues of the same mice. Further investigation of the major regulators of vitamin D pathway found that differences in plasma 25(OH)D3 levels were not reliably detected, and there was no difference in plasma phosphate or calcium levels between the congenic and control mouse strains. However, 3-fold higher levels of parathyroid hormone (Pth) were measured in female mice carrying the BALB/c allele of the SuprMam1 locus. Further, expression profiling of the major genes involved in the vitamin D pathway by affymetrix exon array on mammary glands from SM09 and control mice indicated many were differentially expressed, indicating disruption of the vitamin D pathway. Further investigations are underway to determine if genetic differences in the Pth or Cyp2R1 gene are responsible for this disruption, and if this is of biological significance for tumour growth in vivo. 1. Blackburn AC, Hill LZ, Roberts AL, et al. Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk. Am J Pathol. 2007; 170:2030–41.
    Molecular Cancer Research 10/2013; DOI:10.1158/1557-3125.ADVBC-A117 · 4.50 Impact Factor
  • Combined Research Meeting and Cancer Biology Forum, Canberra, Australia; 09/2013
  • Cancer Research 08/2013; 73(8 Supplement):5421-5421. DOI:10.1158/1538-7445.AM2013-5421 · 9.28 Impact Factor
  • Familial Aspects of Cancer 2012 Research and Practice: A combined meeting of kConFab,Australian Breast Cancer Family Study,Australian Colorectal Cancer Family Study,Australian Ovarian Cancer Study,Family Cancer Clinics of Australia and New Zealand, Kingscliff, Australia; 08/2012
  • Cancer Research; 06/2012
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    Hereditary Cancer in Clinical Practice 04/2012; 10(2). DOI:10.1186/1897-4287-10-S2-A90 · 1.71 Impact Factor
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    AC Blackburn, M Rooke, Y Li, JE Dahlstrom, PG Board
    Hereditary Cancer in Clinical Practice 04/2012; 10(2). DOI:10.1186/1897-4287-10-S2-A62 · 1.71 Impact Factor
  • Lorne cancer conference, Lorne, Australia; 02/2012
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    ABSTRACT: Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1(-/-) mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1(-/-) mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1(-/-) mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.
    International journal of obesity (2005) 01/2012; 36(10):1366-9. DOI:10.1038/ijo.2011.267 · 5.39 Impact Factor
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    ABSTRACT: Dichloroacetic acid (DCA) has potential for use in cancer therapy and the treatment of metabolic acidosis. However, DCA can create a deficiency of glutathione transferase Zeta (GSTZ1-1). Gstz1 knockout mice have elevated oxidative stress and low glutathione levels that increases their sensitivity to acetaminophen toxicity. As it is highly likely that patients that are treated with DCA will develop drug induced GSTZ1-1 deficiency we considered they could be at risk of elevated toxicity if they are exposed to other drugs that cause oxidative stress or consume glutathione (GSH). To test this hypothesis we treated mice with DCA and acetaminophen (APAP). Surprisingly, the mice pre-treated with DCA suffered less APAP-mediated hepatotoxicity than untreated mice. This protection is most likely due to an increased capacity for the liver to synthesize GSH, since DCA increased the expression and activity of glutamate-cysteine ligase GCL, the rate-limiting enzyme of GSH synthesis. Other pathways for acetaminophen disposal were unchanged or diminished by DCA. Pre-treatment with DCA may be of use in other settings where the maintenance of protective levels of GSH are required. However, DCA may lower the efficacy of drugs that rely on oxidative stress and the depletion of GSH to enhance their cytotoxicity or of drugs that are detoxified by GSH conjugation. Consequently, as the use of DCA in the clinic is likely to increase, it will be critical to evaluate the interactions of DCA with other drugs to ensure the combinations retain their efficacy and do not cause enhanced toxicity.
    Biochemical pharmacology 11/2011; 83(3):427-33. DOI:10.1016/j.bcp.2011.11.012 · 4.65 Impact Factor
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    Ramon C Sun, Philip G Board, Anneke C Blackburn
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    ABSTRACT: Cancer cells have a different metabolic profile compared to normal cells. The Warburg effect (increased aerobic glycolysis) and glutaminolysis (increased mitochondrial activity from glutamine catabolism) are well known hallmarks of cancer and are accompanied by increased lactate production, hyperpolarized mitochondrial membrane and increased production of reactive oxygen species. In this study we target the Warburg effect with dichloroacetate (DCA) and the increased mitochondrial activity of glutaminolysis with arsenic trioxide (ATO) in breast cancer cells, measuring cell proliferation, cell death and mitochondrial characteristics. The combination of DCA and ATO was more effective at inhibiting cell proliferation and inducing cell death than either drug alone. We examined the effect of these treatments on mitochondrial membrane potential, reactive oxygen species production and ATP levels and have identified new molecular mechanisms within the mitochondria for both ATO and DCA: ATO reduces mitochondrial function through the inhibition of cytochrome C oxidase (complex IV of the electron transport chain) while DCA up-regulates ATP synthase β subunit expression. The potentiation of ATO cytotoxicity by DCA is correlated with strong suppression of the expression of c-Myc and HIF-1α, and decreased expression of the survival protein Bcl-2. This study is the first to demonstrate that targeting two key metabolic hallmarks of cancer is an effective anti-cancer strategy with therapeutic potential.
    Molecular Cancer 11/2011; 10:142. DOI:10.1186/1476-4598-10-142 · 5.40 Impact Factor
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    Anneke C Blackburn
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    ABSTRACT: Advances in genotyping technology have provided us with a large number of genetic loci associated with cancer susceptibility; however, our ability to understand the functional effects of the genetic variants of these loci remains limited. In the previous issue, Smits and colleagues demonstrate the use of congenic rat strains to discover that the Mcs5a breast cancer susceptibility locus is most likely acting through the immune system, via novel transcriptional regulatory mechanisms. This challenges our conventional thinking of cancer susceptibility and gene regulation pathways, and illustrates the potential for rodent models to help us functionally characterize polymorphisms of cancer-associated loci.
    Breast cancer research: BCR 10/2011; 13(5):112. DOI:10.1186/bcr2939 · 5.88 Impact Factor
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    ABSTRACT: Glutathione transferase kappa (GSTK1-1) is a highly conserved, mitochondrial enzyme potentially involved in redox reactions. GSTK1-1-deficient mice were generated to further study the enzyme's biological role. Reduced and total glutathione levels in liver and kidney were unchanged by GSTK1-1 deficiency and NADPH quinone oxidoreductase 1 expression was not elevated indicating that there is no general underlying oxidative stress in Gstk1(-/-) mice. Electron microscopy of liver and kidney showed no changes in mitochondrial morphology with GSTK1-1 deficiency. The death of a number of Gstk1(-/-) males with urinary tract problems prompted close examination of the kidneys. Electron microscopy revealed glomerular basement membrane changes at 3 months, accompanied by detectable microalbuminuria in male mice (albumin:creatinine ratio of 2.66±0.83 vs 1.13±0.20 mg/mmol for Gstk1(-/-) and wild-type (WT), respectively, P=0.001). This was followed by significant foot process effacement (40-55% vs 10% for Gstk1(-/-) and WT, respectively) at 6 months of age in all Gstk1(-/-) mice examined. Kidney tubules were ultrastructurally normal. Compared with human disease, the Gstk1(-/-) kidneys show changes seen in glomerulopathies causing nephrotic syndrome. Gstk1(-/-) mice may offer insights into the early development of glomerular nephropathies.
    Laboratory Investigation 08/2011; 91(11):1572-83. DOI:10.1038/labinvest.2011.107 · 3.83 Impact Factor
  • Familial Aspects of Cancer 2011 Research and Practice: A combined meeting of kConFab, Australian Breast Cancer Family Study,Australian Colorectal Cancer Family Study, Australian Ovarian Cancer Study,Family Cancer Clinics of Australia and New Zealand, Kingscliff, Australia; 08/2011
  • Anneke C Blackburn, D Joseph Jerry
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    ABSTRACT: Genetic factors play an important role in determining risk and resistance to increased breast cancer. Recent technological advances have made it possible to analyze hundreds of thousands of single nucleotide polymorphisms in large-scale association studies in humans and have resulted in identification of alleles in over 20 genes that influence breast cancer risk. Despite these advances, the challenge remains in identifying what the functional polymorphisms are that confer the increased risk, and how these genetic variants interact with each other and with environmental factors. In rodents, the incidence of mammary tumors varies among strains, such that they can provide alternate ideas for candidate pathways involved in humans. Mapping studies in animals have unearthed numerous loci for breast cancer susceptibility that have been validated in human populations. In a reciprocal manner, knockin and knockout mice have been used to validate the tumorigenicity of risk alleles found in population studies. Rodent studies also underscore the complexity of interactions among alleles. The fact that genes affecting risk and resistance to mammary tumors in rodents depend greatly upon the carcinogenic challenge emphasizes the importance of gene x environment interactions. The challenge to rodent geneticists now is to capitalize on the ability to control the genetics and environment in rodent models of tumorigenesis to better understand the biology of breast cancer development, to identify those polymorphisms most relevant to human susceptibility and to identify compensatory pathways that can be targeted for improved prevention in women at highest risk of developing breast cancer.
    Journal of Mammary Gland Biology and Neoplasia 03/2011; 16(1):57-64. DOI:10.1007/s10911-011-9201-9 · 5.00 Impact Factor

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