Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

<div><p>DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G₂/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G₂/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G₂/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G₂/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.</p></div

Biological processes and molecular functions enriched with cadmium-responsive genes

We used 286 genes that were significantly changed following a 24 h exposure to 100 μM cadmium and 86 genes that were significantly changed following a 4 h exposure in the GO analysis. GO terms with < 0.05, and ≥4 changed genes in at least one of four conditions (up or down regulated after 4 or 24 h cadmium exposures) are displayed (Additional data files 3 and 4). The brighter the color, the more significant the enrichment of the pathway.<p><b>Copyright information:</b></p><p>Taken from "Toxicogenomic analysis of reveals novel genes and pathways involved in the resistance to cadmium toxicity"</p><p>http://genomebiology.com/2007/8/6/R122</p><p>Genome Biology 2007;8(6):R122-R122.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC2394766.</p><p></p

Differential expression of 259 present miRNAs in both wild type and ATM-deficient HME-CCs.

<p>Differential miRNA expression between wild type and ATM-deficient HME-CCs obtained from three independent replicates of each. The <i>y</i>-axis displays the ATM-deficient to WT expression ratio, the <i>x</i>-axis displays the average expression of each miRNA; both axes are in logarithmic scale. Differentially expressed miRNAs of <i>p</i>-value ≤0.05 and at least 1.5 fold change are blue. Representative significant miRNAs are labelled. Each sample had a separately generated sequencing library and was run in an individual sequencing lane.</p

Genome-Wide Small RNA Sequencing and Gene Expression Analysis Reveals a microRNA Profile of Cancer Susceptibility in ATM-Deficient Human Mammary Epithelial Cells

<div><p>Deficiencies in the ATM gene are the underlying cause for ataxia telangiectasia, a syndrome characterized by neurological, motor and immunological defects, and a predisposition to cancer. MicroRNAs (miRNAs) are useful tools for cancer profiling and prediction of therapeutic responses to clinical regimens. We investigated the consequences of ATM deficiency on miRNA expression and associated gene expression in normal human mammary epithelial cells (HME-CCs). We identified 81 significantly differentially expressed miRNAs in ATM-deficient HME-CCs using small RNA sequencing. Many of these have been implicated in tumorigenesis and proliferation and include down-regulated tumor suppressor miRNAs, such as hsa-miR-29c and hsa-miR-16, as well as over-expressed pro-oncogenic miRNAs, such as hsa-miR-93 and hsa-miR-221. MicroRNA changes were integrated with genome wide gene expression profiles to investigate possible miRNA targets. Predicted mRNA targets of the miRNAs significantly regulated after ATM depletion included many genes associated with cancer formation and progression, such as SOCS1 and the proto-oncogene MAF. While a number of miRNAs have been reported as altered in cancerous cells, there is little understanding as to how these small RNAs might be driving cancer formation or how they might be used as biomarkers for cancer susceptibility. This study provides preliminary data for defining miRNA profiles that may be used as prognostic or predictive biomarkers for breast cancer. Our integrated analysis of miRNA and mRNA expression allows us to gain a better understanding of the signaling involved in breast cancer predisposition and suggests a mechanism for the breast cancer-prone phenotype seen in ATM-deficient patients.</p> </div

Depletion of ATM leads to deregulation of miRNAs important in cancer formation.

<p>A) List of 4 known tumor suppressors and 8 oncomirs with significant expression changes after the depletion of ATM. B) Expression levels, tags per million (TpM), of four examples of deregulated miRNAs. Dark gray bars represent wild type expression and light gray bars represent expression in ATM-deficient cells. Error bars are standard error from the expression of 3 independent replicates of each genotype.</p

Depletion of ATM in non-cancerous cells reveals effects on miRNAs and target mRNAs that suggest an early event in transformation to cancer.

<p>A) Gene Families representing the 202 significantly regulated genes determined using GSEA to give a functional overview of the types of genes affected by changes in miRNA expression. B) Top Functions analysis of ATM-dependent correlated miRNAs and possible mRNA targets by IPA. Only selected significant functional groups are depicted. The dashed line indicates a <i>p</i>-value of 0.01.</p

Process Map and Summary of Next Gen Sequencing data.

<p>A) Small RNA Sequencing pipeline overview. B) Summary statistics of Small RNA sequencing data at different stages of data analysis.</p