The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase ( Traditional cancer chemotherapy agents designed to block cell division are toxic to healthy cells as well as to cancer cells. Targeting specific metabolic pathways to stop cancer growth is potentially less toxic to normal cells and can improve tolerability considerably. Thus, anticancer drug discovery has shifted from the traditional empiric random screening approach to a more rational and mechanistic, target-based approach whereby specific abnormalities in cell functioning are modulated in a classical drug (ligand)-receptor fashion. The HER/ErbB family of transmembrane receptors is one of the most exciting targets currently under evaluation.This ErbB family of receptor tyrosine kinases includes four closely related members: HER-1/ErbB1 (also known as the epidermal growth factor receptor), HER-2/ErbB2 (also known as HER-2/neu), 1 HER-3/ErbB3, and HER-4/ErbB4. These receptors initiate signals by forming ligand-induced combinations of homo-and heterodimers (1) and play a critical role in the pathogenesis of breast cancer. HER-2/neu, one of the most well characterized breast cancer oncogenes, is amplified in about 20 -30% of all human breast cancers (2, 3) and is also overexpressed in a variety of other human tumors, including ovarian, lung, gastric, and oral cancers. It appears

The human epidermal growth factor receptor, type 2 (HER-2/neu or c-erbB-2) is a 185 kDa transmembrane protein that is phosphorylated upon ligand binding and dimerization with members of the HER/c-erbB family and regulates cell growth and differentiation. Its overexpression is strongly associated with advanced disease, metastasis and poor clinical outcome. To better understand the mechanisms underlying the poor prognosis of breast tumors with HER-2/neu-positive status, parallel proteomic analyses were performed on estrogen receptor-negative and node-positive breast tumors with or without overexpression of the HER-2/neu oncogene, using laser capture microdissection and two-dimensional gel electrophoresis. The differentially expressed proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Cytokeratin 19 (CK19), one of the identified proteins, was highly expressed in the HER-2/neu-positive breast tumors when compared with HER-2/neu-negative breast tumors. The enhanced overexpression of CK19 in HER-2/neu-positive tumors was further analyzed using semiquantitative reverse-transcription polymerase chain reaction, Western blotting and reverse-phase protein arrays. Immunohistochemical staining of sections from a breast tumor tissue microarray of 97 tumors showed moderate to strong staining against anti-CK19 antibody in 20 (5 with moderate and 15 with strong staining) of the 26 HER-2/neu-positive tumors (76.9%) and in 22 (12 with moderate and 10 with strong staining) of 52 HER-2/neu-negative tumors (48%) (p = 0.002). Our results indicate that CK19, an intermediate fragment of the cytoskeleton, and other proteins showing differential expression, are likely to be intricately involved in intra- and intercellular molecular events driving the more aggressive tumor proliferation, invasion and metastasis associated with HER-2/neu-positive tumors.

The human epidermal growth factor receptor type 2 (HER-2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER-2/neu-modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER-2/neu-driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER-2/neu-positive and -negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up-regulated whereas 15 were down-regulated in the HER-2/neu-positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose-regulated protein (GRP78/Bip) and Raf-1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER-2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER-2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose-dependent manner, providing evidence of crosstalk between HER-2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER-2/neu-driven tumour proliferation, invasion and metastasis.

Our findings suggest a potential link between the expression of CC3/TIP30 gene and the HER-2/neu oncogene-mediated signal pathway.These findings could not have been predicted from previous experimental studies, and suggest that CC3/TIP30 may play a complex role in breast cancer.