No evidence for association of ataxia-telangiectasia mutated gene T2119C and C3161G amino acid substitution variants with risk of breast cancer

BACKGROUND: There is evidence that certain mutations in the double-strand break repair pathway ataxia-telangiectasia mutated gene act in a dominant-negative manner to increase the risk of breast cancer. There are also some reports to suggest that the amino acid substitution variants T2119C Ser707Pro and C3161G Pro1054Arg may be associated with breast cancer risk. We investigate the breast cancer risk associated with these two nonconservative amino acid substitution variants using a large Australian population-based case–control study. METHODS: The polymorphisms were genotyped in more than 1300 cases and 600 controls using 5' exonuclease assays. Case–control analyses and genotype distributions were compared by logistic regression. RESULTS: The 2119C variant was rare, occurring at frequencies of 1.4 and 1.3% in cases and controls, respectively (P = 0.8). There was no difference in genotype distribution between cases and controls (P = 0.8), and the TC genotype was not associated with increased risk of breast cancer (adjusted odds ratio = 1.08, 95% confidence interval = 0.59–1.97, P = 0.8). Similarly, the 3161G variant was no more common in cases than in controls (2.9% versus 2.2%, P = 0.2), there was no difference in genotype distribution between cases and controls (P = 0.1), and the CG genotype was not associated with an increased risk of breast cancer (adjusted odds ratio = 1.30, 95% confidence interval = 0.85–1.98, P = 0.2). This lack of evidence for an association persisted within groups defined by the family history of breast cancer or by age. CONCLUSION: The 2119C and 3161G amino acid substitution variants are not associated with moderate or high risks of breast cancer in Australian women

Activation of ATM and Chk2 kinases in relation to the amount of DNA strand breaks

The diverse checkpoint responses to DNA damage may reflect differential sensitivities by molecular components of the damage-signalling network to the type and amount of lesions. Here, we determined the kinetics of activation of the checkpoint kinases ATM and Chk2 (the latter substrate of ATM) in relation to the initial yield of genomic DNA single-strand (SSBs) and double-strand breaks (DSBs). We show that doses of -radiation (IR) as low as 0.25 Gy, which generate vast numbers of SSBs but only a few DSBs per cell (19 DSBs per cell (e.g. 1 Gy), which cause Chk2 autophosphorylation on Thr387, Chk2-dependent accumulation of p21waf1 and checkpoint arrest in the S phase. Our results indicate that, in contrast to ATM, Chk2 activity is triggered by a greater number of DSBs, implying that, below a certain threshold level of lesions

Suppression of tousled-like kinase activity after DNA damage or replication block requires ATM, NBS1 and Chk1

The human Tousled-like kinases 1 and 2 (TLK) have been shown to be active during S phase of the cell cycle. TLK activity is rapidly suppressed by DNA damage and by inhibitors of replication. Here we report that the signal transduction pathway, which leads to transient suppression of TLK activity after the induction of double-strand breaks (DSBs) in the DNA, is dependent on the presence of a functional ataxia-telangiectasia-mutated kinase (ATM). Interestingly, we have discovered that rapid suppression of TLK activity after low doses of ultraviolet (UV) irradiation or aphidicolin-induced replication block is also ATM-dependent. The nature of the signal that triggers ATM-dependent downregulation of TLK activity after UVC and replication block remains unknown, but it is not due exclusively to DSBs in the DNA. We also demonstrate that TLK suppression is dependent on the presence of a functional Nijmegan Breakage Syndrome protein (NBS1). ATM-dependent phosphorylation of NBS1 is required for the suppression of TLK activity, indicating a role for NBS1 as an adaptor or scaffold in the ATM/TLK pathway. ATM does not phosphorylate TLK directly to regulate its activity, but Chk1 does phosphorylate TLK1 GST-fusion proteins in vitro. Using Chk1 siRNAs, we show that Chk1 is essential for the suppression of TLK activity after replication block, but that ATR, Chk2 and BRCA1 are dispensable for TLK suppression. Overall, we propose that ATM activation is not linked solely to DSBs and that ATM participates in initiating signaling pathways in response to replication block and UV-induced DNA damage

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-β signaling pathways in cancer

Abstract Background Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis. Results Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. Conclusions Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers