Michael Moriarty

Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis

Weighted gene coexpression network analysis (WGCNA) is a powerful guilt-by-association-based method to extract coexpressed groups of genes from large heterogeneous messenger RNA expression data sets. We have utilized WGCNA to identify 11 coregulated gene clusters across 2342 breast cancer samples from 13 microarray-based gene expression studies. A number of these transcriptional modules were found to be correlated to clinicopathological variables (e.g. tumor grade), survival endpoints for breast cancer as a whole (disease-free survival, distant disease-free survival and overall survival) and also its molecular subtypes (luminal A, luminal B, HER2+ and basal-like). Examples of findings arising from this work include the identification of a cluster of proliferation-related genes that when upregulated correlated to increased tumor grade and were associated with poor survival in general. The prognostic potential of novel genes, for example, ubiquitin-conjugating enzyme E2S (UBE2S) within this group was confirmed in an independent data set. In addition, gene clusters were also associated with survival for breast cancer molecular subtypes including a cluster of genes that was found to correlate with prognosis exclusively for basal-like breast cancer. The upregulation of several single genes within this coexpression cluster, for example, the potassium channel, subfamily K, member 5 (KCNK5) was associated with poor outcome for the basal-like molecular subtype. We have developed an online database to allow user-friendly access to the coexpression patterns and the survival analysis outputs uncovered in this study (available at http://glados.ucd.ie/Coexpression/).

Monoamine oxidase inhibitors l-deprenyl and clorgyline protect nonmalignant human cells from ionising radiation and chemotherapy toxicity

l-Deprenyl (R-(−)-deprenyl, selegiline) is an inhibitor of monoamine oxidase-B (MAO-B) that is known to protect nerve cells from a variety of chemical and physical insults. As apoptosis is a common mechanism of radiation-induced cell death, the effect of l-deprenyl on the survival of cultured cells and tissue explants was studied following exposure to gamma radiation. The results obtained were compared with the effects of the less-selective MAO-B inhibitor pargyline and the MAO-A inhibitor clorgyline. l-Deprenyl at a concentration of 10−9u2009M protected the nontumorigenic cell line (HaCaT) and normal human urothelial explants from the effects of cobalt-60 gamma radiation, but did not protect tumorigenic human cell lines HaCaT-ras, HPV-transfected human keratinocytes (HPV-G cells), or PC3. Human bladder carcinoma explants were not protected. Clorgyline showed a smaller protective effect of normal cells, whereas pargyline had no effect. Radiation-induced delayed effects (genomic instability measured as delayed cell death) were prevented in normal cells by l-deprenyl but, interestingly, deprenyl appeared to increase the amount of delayed death in the tumorigenic cell lines. Studies using l-deprenyl prior to the exposure of nonmalignant cells to cisplatin showed that cell death due to this agent was also reduced. Treatment of cultures of nontumorigenic cells with l-deprenyl or clorgyline significantly increased the levels of the protein Bcl-2 following irradiation, but there was no such effect on the already-elevated levels of this protein in the tumour samples. Since the Bcl-2 has been shown to be an inhibitor of apoptosis or programmed cell death, this would imply that the protective effects of l-deprenyl and clorgyline involve activation of antiapoptotic pathways within the normal cell. This hypothesis is supported by data showing reduced levels of apoptosis in HaCAT cells and in normal bladder explant cultures following treatment with l-deprenyl.

A case-control study of head and neck cancer in the Republic of Ireland.

A retrospective case-control study of 200 patients with head and neck cancer, and 200 controls matched for age and sex, confirmed the importance of tobacco and alcohol consumption in the aetiology of malignant tumours of the upper gastrointestinal and upper respiratory tracts. A male-female ratio of 3:1 was found, and the association of smoking with laryngeal cancer and of alcohol with cancer of the tongue was particularly strong. A significant excess of alcohol-related occupations was found among the cases. These findings are discussed.

The role of tobacco and alcohol in the aetiology of lung and larynx cancer.

A RECENT STUDY of head and neck cancer in Ireland (Herity et al., 1981) confirmed that tobacco and alcohol consumption were significant risk factors in the development of cancer of the larynx; a heavy smoker had a risk almost 40 times that of a non-smoker and a heavy drinker had a 3-fold increase in risk over a nondrinker. Many other workers have described similar findings (Wynder et al., 1956, 1976; Vincent & Marchetta, 1963; Rothman & Keller, 1972; Feldman & Hazan, 1975; McMichael, 1978; Ward Hinds et al., 1979). Another neoplasm which has been consistently related to heavy smoking is lung cancer; a comparative study of lung and larynx cancer in relation to tobacco and alcohol consumption is thus of interest. Sixty-eight cases of larynx cancer were included in the sample of 200 headand neck-cancer patients in the abovementioned study 59 males and 9 females-and details of age, marital status, occupation, education and tobacco/ alcohol consumption had been recorded for these patients. A presenting sample of 68 lung-cancer patients matched with the larynx-cancer patients for sex but not for age, was interviewed using the same precoded questionnaire as in the earlier study. The control group for the earlier study was again used. Diagnoses of the control group included cancers of the skin, haemopoietic system, gastrointestinal tract, breast, male and female genital tracts, brain, endocrine system, and connective tissue and preAccepted 23 September 1982

A re-appraisal of risk factors for skin carcinoma in Ireland. A case control study.

SummaryA CASE control study of risk factors in patients with histologically proven skin carcinoma was undertaken. One hundred and twenty-one consecutive patients with skin carcinoma were compared with 121 patients with other malignancies individually matched for age and sex. The results indicated that patients with skin carcinoma when compared to controls did not have excess exposure to ultra violet radiation, did not show increased sensitivity to the effects of ultra violet radiation and did not exhibit differences in skin type, eye or hair colouring.These results indicate that in Ireland, which has a particularly high incidence of skin carcinoma, other possible risk factors should be investigated in future studies of skin carcinoma.

Transferrin-bound proteins as potential biomarkers for advanced breast cancer patients

Background Serum profiling using mass spectrometry-based proteomic techniques has great potential to detect biomarkers that might improve the management for advanced breast cancer patients. The albuminome has previously been investigated as a tool in biomarker discovery, however other high abundant blood proteins are also likely to sequester potentially interesting molecules. Methods Affinity resin purified and isolated Transferrin and associated bound proteins from normal control and breast cancer patient serum samples were analysed by label-free mass spectrometry during the discovery phase. Results 21 significant proteins were identified with Fibrinogen and Fibronectin selected for further analysis in an independent sample set, with significant difference found when comparing the controls groups (normal healthy control, inflammatory bowel disease and benign breast disease) to stage IV breast cancer. Conclusions The area under the curve value for Fibrinogen compared favourably with cancer antigen 15-3, an established breast cancer tumour marker. A combination of all three biomarkers improved accuracy when comparing control/benign to stage IV breast cancer patient groups. General significance Mass spectrometry profiling of Transferrin-bound proteins has revealed serum proteins that can distinguish between serum from advanced breast cancer patients and healthy control subjects with high confidence.

Absence of correlation between chemo- and radioresistance in a range of human tumour cell lines

The correlation between cellular resistance to radiation and to chemotherapeutic drugs has been investigated in a number of solid tumour cell lines, and preliminary results indicate no direct relationship. The acquisition of a multidrug resistance (MDR) profile by adriamycin-selected variants of a human squamous lung carcinoma, an ovarian carcinoma, a cervical carcinoma and by a colchicine-selected variant of a Chinese hamster ovarian carcinoma resulted in alterations to their radiosensitivity. However, the degree of change in the radiosensitivity of the MDR cell lines could not be predicted from their level of resistance to adriamycin. Clonal populations derived from DLKP-A, an adriamycin-selected MDR variant of the human lung carcinoma cell line DLKP, exhibited individual radiosensitivity profiles, which did not correlate with their chemoresistance. Exposure of DLKP to consecutive increasing doses of radiation did not confer cross-resistance to chemotherapeutic drugs.

Metabolomic and proteomic analysis of breast cancer patient samples suggests that glutamate and 12-HETE in combination with CA15-3 may be useful biomarkers reflecting tumour burden

Evaluation of protein and metabolite expression patterns in blood using mass spectrometry and high-throughput antibody-based screening platforms has potential for the discovery of new biomarkers for managing breast cancer patient treatment. Previously identified blood-based breast cancer biomarkers, including cancer antigen 15.3 (CA15-3) are useful in combination with imaging (computed tomography scans, magnetic resonance imaging, X-rays) and physical examination for monitoring tumour burden in advanced breast cancer patients. However, these biomarkers suffer from insufficient levels of accuracy and with new therapies available for the treatment of breast cancer, there is an urgent need for reliable, non-invasive biomarkers that measure tumour burden with high sensitivity and specificity so as to provide early warning of the need to switch to an alternative treatment. The aim of this study was to identify a biomarker signature of tumour burden using cancer and non-cancer (healthy controls/non-malignant breast disease) patient samples. Results demonstrate that combinations of three candidate biomarkers from Glutamate, 12-Hydroxyeicosatetraenoic acid, Beta-hydroxybutyrate, Factor V and Matrix metalloproteinase-1 with CA15-3, an established biomarker for breast cancer, were found to mirror tumour burden, with AUC values ranging from 0.71 to 0.98 when comparing non-malignant breast disease to the different stages of breast cancer. Further validation of these biomarker panels could potentially facilitate the management of breast cancer patients, especially to assess changes in tumour burden in combination with imaging and physical examination.

7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

The radiobiological response of the thyroid. Part II: Response of sheep thyroid cell in vitro to single doses of X rays

The response of sheep thyroid cells in culture to single doses of X or gamma rays is described. In the absence of cellular proliferation the cells are unusually radioresistant, showing little sign of interphase death at doses up to 9 krad. The follicular morphology characteristic of thyroid cells in vivo is also very radioresistant. Iodide trapping is reduced to 50% of the control value by doses of the order of 2 krad. When proliferation is induced the cells may be assayed for postirradiation survival using a clonogenic endpoint. The survival curves are sigmoid with a Do of 410 rad and a very low extrapolation number.