Abstract Dysregulated mitosis is a hallmark of cancer. Many centrosomal proteins, including the integrin-linked kinase (ILK), that regulate microtubule organization and mitotic spindle assembly have been implicated in cancers. Inhibition or depletion of ILK result in a mitotic spindle defect that has not been well characterized and while ILK is overexpressed in many cancers, the effect of ILK overexpression on mitotic spindle function is unknown. In this study, we have characterized the defective spindle phenotype, and also looked at the effect of ILK overexpression on mitotic spindle function. HeLa cells stably over-expressing wild-type ILK were used for overexpression studies while parental HeLa cells were treated with QLT-0267, an ILK-specific inhibitor, to study the defective spindle phenotype. While ILK inhibition led to an abnormal mitotic spindle phenotype, the spindle contained bona fide kinetochore-microtubules that persisted after cold- or calcium-treatment. ILK inhibition, however, led to higher sister-centromere stretch in chromosomes aligned between two spindle poles, suggesting that kinetochores were under abnormally high tension, likely due to inhibition of microtubule polymerization. Microtubule regrowth in mitotic cells after cold depolymerization was significantly slower with ILK inhibition. In the context of ILK overexpression in cancers, we first measured the duration of mitosis and showed that ILK-overexpressing cells completed mitosis at a faster rate and also required shorter durations between metaphase and anaphase. ILK overexpression also resulted in increased resistance to paclitaxel-induced cell death. Collectively, our data suggest that ILK regulates microtubule dynamics, which is the focus of ongoing investigations in our laboratory, and ILK overexpression has clinical implications by inducing resistance to paclitaxel, possibly through increased microtubule dynamics. This work was supported by funding from the Canadian Institutes of Health Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2011-2989

Background: Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.Methodology/Principal Findings: We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the "Warburg effect"). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.Conclusion/Significance: The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.

Genetic aberration of EGFR is one of the major molecular characteristics of breast cancer. However, the molecular changes associated with EGFR signaling during different stages of breast cancer development have not been studied. In this study, complementary two-dimensional-DIGE and iTRAQ technologies were used to profile the expression level of proteins in 4 isogenic cell lines in the MCF10AT model of breast cancer progression following a time course of EGF stimulation. A total of 80 proteins (67 from iTRAQ, 15 from DIGE, 2 common in both) were identified to be up- or down-regulated by EGF treatment. Following EGF stimulation, the expression level of MIF, a cytokine that has been implicated in many human cancers, was decreased in MCF10A1 normal breast mammary epithelial cells, increased in MCF10AT1k preneoplastic and MCF10CA1h low grade breast cancer cells, but showed no obvious difference in the MCF10CA1a high grade cancer cells. The increase in MIF expression level following EGF treatment could also be observed in A431 cervical cancer cells. EGF-induced increases of MIF expression levels in CA1h breast cancer cells were abrogated when MEK, but not PIK3CA, was knocked down. In addition, silencing of MIF diminished the proliferation of EGF-stimulated CA1h cells when compared to control cells. Taken together, our data suggested an EGFR --> MEK --> MIF proliferative pathway that has never been reported previously and that this pathway "evolves" during disease progression as modeled by the MCF10AT system. Revelation of the novel relationship between MIF and EGF may contribute to an integrated understanding of the roles of these oncogenic factors during breast cancer development.

Aeromonas hydrophila is a ubiquitous Gram-negative bacterium which can cause motile aeromonad septicemia in both fish and humans. A. hydrophila secretes many extracellular proteins associated with pathogenicity and environmental adaptability. In this study, an extracellular proteome map of A. hydrophila AH-1 was constructed. The major extracellular virulence factors were characterized by comparing the proteomes of various deletion mutants with that of the wild type. The results suggested that serine protease was involved in the processing of a toxin and secreted enzymes such as hemolysin, glycerophospholipid-cholesterol acyltransferase and metalloprotease. We also showed that expressions of polar and lateral flagellins were under the control of temperature, FlhA, LafK, and RpoN. In addition, three novel proteins (potential effector proteins including one ExoT-like protein) were revealed to be secreted via t he type III secretion system (TTSS) of A. hydrophila AH-1. Another novel finding was the demonstration of a crosstalk between the lateral flagellar system and the TTSS in A. hydrophila. These results showed that proteomics is a powerful tool for characterizing virulence factors. The construction of proteome maps will provide a valuable means of finding potential candidates for developing suitable diagnostics and therapeutics for this emerging pathogen.

Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could. be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P < 0.001, n = 81). However, no significant correlation of EGFR expression with diseasefree survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from the normal condition through disease progression using in vitro, xenograft, and matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting the EGFR family of proteins in breast cancer.

B64 Analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS phosphoproteomics method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Gefitinib, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; SingaporeB33 Understanding the molecular etiology and heterogeneity of disease has a direct impact on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro , animal model and clinical breast samples, our study revealed marked reduction of EGFR expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro . Fluorescence in situ hybridization demonstrated that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression on 93 matched normal and breast tumor samples showed that i) diminished EGFR expression could be detected as early as in the pre neoplastic lesion (ductal carcinoma in situ ) and this culminated in invasive carcinomas; ii) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (p<0.001, n=81). However, no significant correlation of EGFR expression with disease free survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from normal condition through disease progression using in vitro , xenograft, matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting EGFR family of proteins in breast cancer. (IRESSA is a trademark of the AstraZeneca group of companies).

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.