Proteomics of breast cancer: Enhanced expression of cytokeratin19 in human epidermal growth factor receptor type 2 positive breast tumors

Abstract

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.

Cytokeratin19 induced by HER2/ERK binds and stabilizes HER2 on cell membranes

“…A previous study showed that KRT19 was increased in HER2-positive tumors. 26 They concluded that KRT19 might be responsible for driving the phenotypes of HER2-positive tumors; however, they did not provide any mechanistic relationship between HER2 and KRT19.…”

Proteomic identification of down-regulation of oncoprotein DJ-1 and proteasome activator subunit 1 in hepatitis B virus-infected well-differentiated hepatocellular carcinoma

“…The protein spots were manually excised from the gel and digested with trypsin, as described previously (19). After being dehydrated with acetonitrile and dried in a SpeedVac centrifuge, the gels were re-swelled with 20-40 μl digestion solution containing 20 mM ammonium bicarbonate and 20 ng/μl of sequencing grade trypsin (Promega, Madison, WI, USA) and treated at 37˚C overnight.…”

“…After equilibration for 2x15 min in equilibration buffer, IPG strips were applied for SDS-PAGE (12%) using a PROTEAN ® II xi Cell system (Bio-Rad, Hercules, CA, USA). The separated proteins were visualized by silver staining (19).…”

“…2-DE and image analysis were performed as described previously (19). Briefly, equal amounts (120 μg) of proteins from tumour and matched non-tumour tissues were separately mixed with 350 μl of rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer (pH 4-7), and rehydrated for 12 h at 20 V using the Immobiline IPG DryStrips (180 mm, pH 4-7) (GE Healthcare).…”

Metastasis-related Plasma Membrane Proteins of Human Breast Cancer Cells Identified by Comparative Quantitative Mass Spectrometry

“…In this disease, malignant cells often disseminate to regional lymph nodes and establish distant metastases, preferentially in the bone, lung, and liver, resulting in poor outcome and high mortality (2,3).…”