Identification of Potential Glycan Cancer Markers with Sialic Acid Attached to Sialic Acid and Up-regulated Fucosylated Galactose Structures in Epidermal Growth Factor Receptor Secreted from A431 Cell Line

Abstract

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding ␣-N-acetylneuraminide ␣-2,8-sialyltransferase 2 (ST8SiA2) and ␣-N-acetylneuraminide ␣-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the Cancers are disease-associated with considerable morbidity, such as disease recurrence, anxiety, and side effects of treatment and mortality (1). Early diagnosis often significantly improves survival rates compared with late stage cancer detection, such as for breast, lung, and colon cancers (2-4). Proteins in the blood hold enormous promise for early stage cancer diagnostic tests, but the complexity and dynamic range of blood have confounded the search for cancer biomarkers. Nevertheless, the pressing need for a clinical assay has prompted us to investigate a different approach toward discovering new breast cancer biomarkers circulating in blood (5-7). In addition, the use of a panel of cancer cell lines, representing cancers with different subtypes, could alleviate the difficulty of analyzing the plasma samples directly. Although there is no substitute for the direct study of clinical samples, genetic and molecular aberrations found in cell lines can be translated to similar dysregulations in tumors (8). Cell lines, through the proteins they secrete or shed, should be a complementary model system for the discovery of circulating blood markers. For cancer biomarkers, the change of gene or protein sequence, such as mutation, is often a preclusion for cancers. A similar argument could also be true for the change of protein glycan structures, which re...

PDCD5 regulates cell proliferation, cell cycle progression and apoptosi

“…As a human model epidermoid carcinoma cell line, A431 has been widely used in studies on the cell cycle and tumor related cell signaling pathways because epidermal growth factor receptor (EGFR) is known to be strikingly upregulated in these cells (65)(66)(67)(68). In this study, we found that in the A431 cell, overexpression of PDCD5 inhibits cell proliferation, induces cell cycle arrest at G2/M phase and apoptosis.…”

Genome Wide Proteomics of ERBB2 and EGFR and Other Oncogenic Pathways in Inflammatory Breast Cancer

“…We have previously studied on the role of two driver oncogenes, EGFR, ERBB2, in epithelial cancers 17–18 and have investigated the changes in their glycosylation patterns 2–4, 19 . To further expand on our previous observations, we have performed a comparative study to explore the total lysate proteome of a well-established epithelial breast cancer cell line, SKBR3, which overexpresses ERBB2 and two primary cell lines (SUM149 and SUM190) isolated from patients with inflammatory breast cancer 5 .…”

Functional roles of sialylation in breast cancer progression through miR-26a/26b targeting ST8SIA4

“…In line with our observations, much of the literature showed that ST8SIA4 was implicated as a potential glycan cancer marker. 30 Additionally, ST8SIA4 has also been shown to confer multidrug resistance in human chronic myeloid leukemia. 31 These studies imply that upregulation of ST8SIA4 was breast cancer progression-related.…”