Proteome Profiling of Breast Tumors by Gel Electrophoresis and Nanoscale Electrospray Ionization Mass Spectrometry

The Helen Rollason Research Laboratory, Helen Rollason Heal Cancer Charity, Chelmsford, Essex CM1 1LL, United Kingdom.
Journal of Proteome Research (Impact Factor: 4.25). 05/2008; 7(4):1458-69. DOI: 10.1021/pr7007829
Source: PubMed


We have conducted proteome-wide analysis of fresh surgery specimens derived from breast cancer patients, using an approach that integrates size-based intact protein fractionation, nanoscale liquid separation of peptides, electrospray ion trap mass spectrometry, and bioinformatics. Through this approach, we have acquired a large amount of peptide fragmentation spectra from size-resolved fractions of the proteomes of several breast tumors, tissue peripheral to the tumor, and samples from patients undergoing noncancer surgery. Label-free quantitation was used to generate protein abundance maps for each proteome and perform comparative analyses. The mass spectrometry data revealed distinct qualitative and quantitative patterns distinguishing the tumors from healthy tissue as well as differences between metastatic and non-metastatic human breast cancers including many established and potential novel candidate protein biomarkers. Selected proteins were evaluated by Western blotting using tumors grouped according to histological grade, size, and receptor expression but differing in nodal status. Immunohistochemical analysis of a wide panel of breast tumors was conducted to assess expression in different types of breast cancers and the cellular distribution of the candidate proteins. These experiments provided further insights and an independent validation of the data obtained by mass spectrometry and revealed the potential of this approach for establishing multimodal markers for early metastasis, therapy outcomes, prognosis, and diagnosis in the future.

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    • "As for Oct4, Nanog expression has also been observed in different types of tumors, and its expression has been associated with aggressive tumor progression and poor patient diagnosis. Nanog-expressing tumors have been found in a wide variety of tissues, such as breast (Ezeh et al., 2005; Alldridge et al., 2008), cervix (Ye et al., 2008), kidney (Bussolati et al., 2008), oral cavity (Chiou et al., 2008), lung (Chiou et al., 2010), ovary (Zhang et al., 2008), gastric epithelium (Lin et al., 2012), pancreas (Wen et al., 2010), colon (Meng et al., 2010), and in germ cells (Gillis et al., 2011). A report by Jeter et al. analyzed the knockdown of Nanog in different cancer cell lines and found reduced clonal and clonogenic growth, reduced proliferation, and altered differentiation potential after Nanog ablation (Jeter et al., 2009). "
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    ABSTRACT: Though expression of the homeobox transcription factor Nanog is generally restricted to pluripotent cells and early germ cells, many contradictory reports about Nanog’s involvement in tumorigenesis exist. To address this, a modified Tet-On system was utilized to generate Nanog-inducible mice. Following prolonged Nanog expression, phenotypic alterations were found to be restricted to the intestinal tract, leaving other major organs unaffected. Intestinal and colonic epithelium hyperplasia was observed—intestinal villi had doubled in length and hyperplastic epithelium outgrowths were seen after 7 days. Increased proliferation of crypt cells and downregulation of the tumor suppressors Cdx2 and Klf4 was detected. ChIP analysis showed physical interaction of Nanog with the Cdx2 and Klf4 promoters, indicating a regulatory conservation from embryonic development. Despite downregulation of tumor suppressors and increased proliferation, ectopic Nanog expression did not lead to tumor formation. We conclude that unlike other pluripotency-related transcription factors, Nanog cannot be considered an oncogene.
    Full-text · Article · Sep 2014 · Stem Cell Research
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    • "All tissues were collected under Local Research Ethics Committee (LREC) and National Health Services (NHS) Trust approval as previously described [5] [15]. Tumor tissue was placed on ice immediately in the operating theater. "
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    ABSTRACT: The γ subunit of the major histocompatibility complex (MHC) class II complex, CD74, is overexpressed in a significant proportion of metastatic breast tumors, but the mechanistic foundation and biologic significance of this phenomenon are not fully understood. Here, we show that when CD74 is overexpressed in human cancer and noncancerous epithelial cells, it interacts and interferes with the function of Scribble, a product of a well-known tumor suppressor gene. Furthermore, using epithelial cell lines expressing CD74 under the control of tetracycline-inducible promoter and quantitative high-resolution mass spectrometry, we demonstrate that, as a result of CD74 overexpression, the phosphorylation pattern of the C-terminal part of Scribble undergoes specific changes. This is accompanied with a translocation of the protein from the sites of cell-to-cell contacts at the plasma membrane to the cytoplasm, which is likely to effectively enhance the motility and invasiveness of the cancer cells.
    Full-text · Article · Jun 2013 · Neoplasia (New York, N.Y.)
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    • "Especially, NANOG is a novel homeobox-containing transcription factor and a key regulator of embryonic stem cell self-renewal and pluripotency [6] [7]. NANOG is expressed not only in GCTs, but also in other tumors including carcinomas of the breast, cervix, oral cavity, kidney, and ovary [8] [9] [10] [11] [12] [13] [14]. Moreover, ectopic overexpression of NANOG induced the proliferation and transformation of NIH3T3 and 293T cells; NANOG has also been demonstrated to regulate human tumor development [15] [16] [17]. "
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    ABSTRACT: We investigated transactivation by NANOG in regulating growth and differentiation factor 3 (GDF3) expression in NCCIT cells. GDF3 expression was affected by shRNA-mediated downregulation and by exogenous overexpression of NANOG specifically, as well as by retinoic acid-mediated differentiation. GDF3 transcription was activated by NANOG, and the upstream region (-183 to -1) was sufficient to induce minimal transcriptional activity. Moreover, NANOG binds to the GDF3 minimal promoter in vivo and the transcriptional activity is mediated by NANOG transactivation domain. This study provides the first evidence that NANOG is a transcriptional activator of the expression of the oncogenic growth factor GDF3 in embryonic carcinoma cells.
    Full-text · Article · Aug 2012 · FEBS letters
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