ATM acts downstream of ATR in the DNA damage response signaling of bystander cells.

This study identifies ataxia-telangiectasia mutated (ATM) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, ATM, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/ATM/DNA-PK–proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and ATM but not DNA-PK–deficient bystander cells. ATM activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, ATM-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to ?H2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Impact of PSA testing and prostatic biopsy on cancer incidence and mortality: comparative study between the Republic of Ireland and Northern Ireland

Objectives: To investigate the impact of different PSA testing policies and health-care systems on prostate cancer incidence and mortality in two countries with similar populations, the Republic of Ireland (RoI) and Northern Ireland (NI).

Methods: Population-level data on PSA tests, prostate biopsies and prostate cancer cases 1993–2005 and prostate cancer deaths 1979–2006 were compiled. Annual percentage change (APC) was estimated by joinpoint regression.

Results: Prostate cancer rates were similar in both areas in 1994 but increased rapidly in RoI compared to NI. The PSA testing rate increased sharply in RoI (APC = +23.3%), and to a lesser degree in NI (APC = +9.7%) to reach 412 and 177 tests per 1,000 men in 2004, respectively. Prostatic biopsy rates rose in both countries, but were twofold higher in RoI. Cancer incidence rates rose significantly, mirroring biopsy trends, in both countries reaching 440 per 100,000 men in RoI in 2004 compared to 294 in NI. Median age at diagnosis was lower in RoI (71 years) compared to NI (73 years) (p < 0.01) and decreased significantly over time in both countries. Mortality rates declined from 1995 in both countries (APC = -1.5% in RoI, -1.3% in NI) at a time when PSA testing was not widespread.

Conclusions: Prostatic biopsy rates, rather than PSA testing per se, were the main driver of prostate cancer incidence. Because mortality decreases started before screening became widespread in RoI, and mortality remained low in NI, PSA testing is unlikely to be the explanation for declining mortality.

Novel in vitro assays for the characterization of EMT in tumourigenesis

Background Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-ß1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis. Methods AY-27 rat bladder cancer cells incubated with 3ng/ml TGF-ß1 or control media for 24 or 48h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed. Results TGF-ß1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48h, the mean Spindle Index of TGF-ß1 treated cells was significantly higher than Mock (p=0.02 Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p = 0.02, Chi-Square). TGF-ß1 significantly increased matrigel invasion. Conclusion The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system. Oncogene (2010) 29, 752-762; doi: 10.1038/onc.2009.379; published online 9 November 2009

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

Nox2 NADPH Oxidase Promotes Pathologic Cardiac Remodeling Associated with Doxorubicin Chemotherapy

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2(-/-)) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2(-/-) versus WT mice (fractional shortening: 29.5 +/- 1.4 versus 25.7 +/- 1.0%; P

Cancer incidence and mortality in patients with insulin-treated diabetes: a UK cohort study

Raised risks of several cancers have been found in patients with type II diabetes, but there are few data on cancer risk in type I diabetes. We conducted a cohort study of 28 900 UK patients with insulin-treated diabetes followed for 520 517 person-years, and compared their cancer incidence and mortality with national expectations. To analyse by diabetes type, we examined risks separately in 23 834 patients diagnosed with diabetes under the age of 30 years, who will almost all have had type I diabetes, and 5066 patients diagnosed at ages 30 - 49 years, who probably mainly had type II. Relative risks of cancer overall were close to unity, but ovarian cancer risk was highly significantly raised in patients with diabetes diagnosed under age 30 years ( standardised incidence ratio ( SIR) = 2.14; 95% confidence interval (CI) 1.22 - 3.48; standardised mortality ratio (SMR) = 2.90; 95% CI 1.45 - 5.19), with greatest risks for those with diabetes diagnosed at ages 10 - 19 years. Risks of cancer at other major sites were not substantially raised for type I patients. The excesses of obesity- and alcohol-related cancers in type II diabetes may be due to confounding rather than diabetes per se.