Chemoprevention with the metabolism modifying drugs dichloroacetate and metformin in the Li-Fraumeni Syndrome model, Trp53+/- mice

BACKGROUND: While genetic testing for familial cancer has excelled, the prevention options for those carrying high risk alleles have not. Altered bioenergetics is now acknowledged as a hallmark of cancer, and several very safe drugs are available that can target this phentoype. Dichloroacetate (DCA) inactivates pyruvate dehydrogenase kinase, resulting in activation of pyruvate dehydrogenase, reduced lactic acid production and increased mitochondrial activity. Metformin, a type 2 diabetes treatment which activates AMPK, thereby inhibiting mTOR, has unambiguously been demonstrated to reduce the risk of many cancer types in diabetics. We have tested these drugs as chemopreventive agents against the mammary tumours that occur in the BALB/c-Trp53+/- mouse spontaneous tumour model. MATERIALS and METHODS Breast cancer cell lines were examined for cell viability after DCA and/or metformin treatment in vitro (neutral red uptake assay). Four groups of female BALB/c-Trp53+/- mice were given distilled water (n=75), DCA (1.5 g/L in drinking water, ~180 mg/ kg/day, n=53), metformin (0.25 g/L in drinking water, ~30 mg/kg/day, n=61) or DCA +metformin (n=51) from 8 weeks of age, and monitored for tumour development over 78 weeks, and Kaplan-Meier survival analysis was performed. RESULTS In vitro, DCA (1-5 mM) and metformin (30-300 uM), alone or combined, significantly inhibited breast cancer cell growth. In vivo, the overall tumour-free survival curves for BALB/c-Trp53+/- mice were not significantly different between treatment groups, suggesting that metformin does not reduce cancer risk in non-diabetics. However, analysis of mammary tumours alone found that DCA reduced the number and increased their latency (28.0% vs 20.8% of mice with mean latency of 55.0 vs 63.8 weeks, untreated vs DCA respectively), whereas metformin had no effect (26.2% of mice, mean latency 54.7 weeks). DCA appeared to eliminate the early onset mammary tumours (latency <52 weeks, p=0.02), while not affecting the occurrence of longer latency tumours. In contrast, the two drug combination had worse outcomes for tumour development, (35.3% of mice, latency 48.8 weeks, p<0.02 compared to DCA alone). Preliminary western blotting results in MDA-MB-468 breast cancer cells found that DCA could block the activation of AMPK by metformin, indicating the potential for drug interactions.Supported by NHMRC Career Development Award, National Breast Cancer Foundation Novel Concept Award, and Cancer Australia

Passenger or Driver: Can Gene Expression Profiling Tell Us Anything about LINE-1 in Cancer?

LINE-1 retrotransposons are expressed in epithelial cancers but not normal adult tissues. Previously, we demonstrated repression of cell proliferation, migration, and invasion genes in L1-reverse transcriptase-inhibited T47D cells, while genes involved in cell projection, formation of vacuolar membranes, and intercellular junctions were upregulated. Extending this, we examined microarray data from L1-silenced and Efavirenz-treated T47D cells by Weighted Gene Correlation Network Analysis and literature mining. Hub genes in the most significant module comparing L1-silenced and untreated controls included HSP90AB2p, DDX39A, PANK2, MT1M, and LIMK2. HSP90AB2p is related to HSP90, a master regulator of cancer, cancer evolvability and chemo-resistance. DDX39A is a known cancer driver gene while PANK2 and MT1M affect multiple pathways. LIMK2 and SYBL1 impact actin cytoskeletal dynamics and the cofilin pathway, cancer cell motility, and the epithelial-to-mesenchymal transition. Also affected were signal transduction, HIF1 pathways, iron/redox metabolism, stress granules and cancer stem cell-related metabolic reprogramming and the eIF4F translation initiation complex. Hub genes in other modules, including BTRC, MDM2, and FBXW7, stabilize oncoproteins like MYC, p53, and NOTCH1 or reflect CXCL12–CXCR4 signalling. Our findings support mounting evidence that L1 activity is a cause, rather than a consequence of oncogenesis, with L1 affecting the formation of cancer stem cells

670nm Photobiomodulation as a Novel Protection against Retinopathy of Prematurity: Evidence from Oxygen Induced Retinopathy Models

INTRODUCTION To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR). MATERIALS AND METHODS During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670 nm light for 3 minutes a day (9J/cm²). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed. RESULTS 670 nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670 nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670 nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670 nm light-exposed animals, and no adverse effects of exposure to 670 nm light were detected. DISCUSSION Low levels of exposure to 670 nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.Australian Research Council Centres of Excellence Program Grant (CE0561903); Canberra Hospital Private Practice Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

670nm Photobiomodulation as a Novel Protection against Retinopathy of Prematurity:Evidence from Oxygen Induced Retinopathy Models

Introduction:To investigate the validity of using 670nm red light as a preventative treatment for Retinopathy of Prematurity in two animal models of oxygen-induced retinopathy (OIR).Materials and Methods:During and post exposure to hyperoxia, C57BL/6J mice or Sprague-Dawley rats were exposed to 670nm light for 3 minutes a day (9J/cm2). Whole mounted retinas were investigated for evidence of vascular abnormalities, while sections of neural retina were used to quantify levels of cell death using the TUNEL technique. Organs were removed, weighed and independent histopathology examination performed.Results:670nm light reduced neovascularisation, vaso-obliteration and abnormal peripheral branching patterns of retinal vessels in OIR. The neural retina was also protected against OIR by 670nm light exposure. OIR-exposed animals had severe lung pathology, including haemorrhage and oedema, that was significantly reduced in 670nm+OIR light-exposed animals. There were no significance differences in the organ weights of animals in the 670nm light-exposed animals, and no adverse effects of exposure to 670nm light were detected. Discussion: Low levels of exposure to 670nm light protects against OIR and lung damage associated with exposure to high levels of oxygen, and may prove to be a non-invasive and inexpensive preventative treatment for ROP and chronic lung disease associated with prematurity.</p

Optimizing poly (ADP-ribose) polymerase inhibition through combined epigenetic and immunotherapy

Triple‐negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor survival outcomes. Currently, there are no targeted therapies available for TNBCs despite remarkable progress in targeted and immune‐directed therapies for other solid organ malignancies. Poly (ADP‐ribose) polymerase inhibitors (PARPi) are effective anticancer drugs that produce good initial clinical responses, especially in homologous recombination DNA repair‐deficient cancers. However, resistance is the rule rather than the exception, and recurrent tumors tend to have an aggressive phenotype associated with poor survival. Many efforts have been made to overcome PARPi resistance, mostly by targeting genes and effector proteins participating in homologous recombination that are overexpressed during PARPi therapy. Due to many known and unknown compensatory pathways, genes, and effector proteins, overlap and shared resistance are common. Overexpression of programmed cell death‐ligand 1 (PD‐L1) and cancer stem cell (CSC) sparing are novel PARPi resistance hypotheses. Although adding programmed cell death‐1 (PD‐1)/PD‐L1 inhibitors to PARPi might improve immunogenic cell death and be crucial for durable responses, they are less likely to target the CSC population that drives recurrent tumor growth. Lysine‐specific histone demethylase‐1A and histone deacetylase inhibitors have shown promising activity against CSCs. Combining epigenetic drugs such as lysine‐specific histone demethylase‐1A inhibitors or histone deacetylase inhibitors with PARPi/anti‐PD‐1/PD‐L1 is a novel, potentially synergistic strategy for priming tumors and overcoming resistance. Furthermore, such an approach could pave the way for the identification of new upstream epigenetic and genetic signatures.This work was supported by the National Health and Medical Research Council (Grant ID APP1068065) and the UC Deepwater Scholarship (TP)

Role of CD44 + Stem Cells in Mural Cell Formation in the Human Choroid: Evidence of Vascular Instability Due to Limited Pericyte Ensheathment

PURPOSE. To examine mural cell differentiation and pericyte ensheathment during human choroidal vascular formation and into adulthood. METHODS. Triple- and double-labeled immunohistochemistry (alpha-smooth muscle actin [αSMA], desmin, NG2, calponin, cal

The biological and clinical significance of stromal-epithelial interactions in breast cancer

There is evidence that an aberrant tumour microenvironment (TME) facilitates cancer development, progression, and responses to treatment. While many of the mechanisms underlying the phenotype and cancer-promoting behaviour of the TME are unknown, epigenetic mechanisms in cancer cells and the TME are thought to play important roles. As a result, cancer profiling strategies for drug and biomarker development require a thorough understanding of both the epithelial tissue compartment and the TME. This review discusses recent advances in our understanding of how cancer epithelial cells interact with their microenvironment and how this knowledge can be exploited clinically.The original research presented in this review was supported under NHMRC Projects funding scheme (project number APP1048065

Development of the Human Fetal Kidney from Mid to Late Gestation in Male and Female Infants

BACKGROUND During normal human kidney development, nephrogenesis (the formation of nephrons) is complete by term birth, with the majority of nephrons formed late in gestation. The aim of this study was to morphologically examine nephrogenesis in fetal human kidneys from 20 to 41weeks of gestation. METHODS Kidney samples were obtained at autopsy from 71 infants that died acutely in utero or within 24h after birth. Using image analysis, nephrogenic zone width, the number of glomerular generations, renal corpuscle cross-sectional area and the cellular composition of glomeruli were examined. Kidneys from female and male infants were analysed separately. FINDINGS The number of glomerular generations formed within the fetal kidneys was directly proportional to gestational age, body weight and kidney weight, with variability between individuals in the ultimate number of generations (8 to 12) and in the timing of the cessation of nephrogenesis (still ongoing at 37weeks gestation in one infant). There was a slight but significant (r2=0.30, P=0.001) increase in renal corpuscle cross-sectional area from mid gestation to term in females, but this was not evident in males. The proportions of podocytes, endothelial and non-epithelial cells within mature glomeruli were stable throughout gestation. INTERPRETATION These findings highlight spatial and temporal variability in nephrogenesis in the developing human kidney, whereas the relative cellular composition of glomeruli does not appear to be influenced by gestational age.This study was supported by funding from the National Health and Medical Research Council (NHMRC) (1011136) of Australia and National Institutes of Health (NIH) USA grant 3U01DK094526-04S1 (PI A P McMahon). Author Danica Ryan was the recipient of the Biomedicine Discovery Scholarship from Monash University and author Megan R. Sutherland was supported by a NHMRC CJ Martin Fellowship

Microsatellite Instability in Mouse Models of Colorectal Cancer

Microsatellite instability (MSI) is caused by DNA mismatch repair deficiency and is an important prognostic and predictive biomarker in colorectal cancer but relatively few studies have exploited mouse models in the study of its clinical utility. Furthermore, most previous studies have looked at MSI in the small intestine rather than the colon of mismatch repair deficient Msh2-knockout (KO) mice. Here we compared Msh2-KO, p53-KO, and wild type (WT) mice that were treated with the carcinogen azoxymethane (AOM) and the nonsteroidal anti-inflammatory drug sulindac or received no treatment. The induced tumors and normal tissue specimens from the colon were analysed with a panel of five mononucleotide repeat markers. MSI was detected throughout the normal colon in untreated Msh2-KO mice and this involved contraction of the repeat sequences compared to WT. The markers with longer mononucleotide repeats (37–59) were the most sensitive for MSI while the markers with shorter repeats (24) showed only minor change. AOM exposure caused further contraction of the Bat37 and Bat59 repeats in the distal colon of Msh2-KO mice which was reversed by sulindac. Thus AOM-induced carcinogenesis is associated with increased instability of mononucleotide repeats in the colon of Msh2-KO mice but not in WT or p53-KO mice. Chemoprevention of these tumors by sulindac treatment reversed or prevented the increased MSI