Cancer cell membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, cellular secretion or cell lysis, and represent promising candidates for interrogation. Because many known disease biomarkers are both glycoproteins and membrane bound, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins from breast cancer cell membrane fractions using the LTQ Orbitrap mass spectrometer. Our initial goal was to select membrane proteins from breast cancer cell lines and then to use the hydrazide method to identify the N-linked proteome as a prelude to evaluation of plasma/serum proteins from cancer patients. A combination of steps facilitated identification of the glycopeptides and also defined the glycosylation sites. In MCF-7, MDA-MB-453 and MDA-MB-468 cell membrane fractions, use of the hydrazide method facilitated an initial enrichment and site mapping of 27 N-linked glycosylation sites in 25 different proteins. However, only three N-linked glycosylated proteins, galectin-3 binding protein, lysosome associated membrane glycoprotein 1, and oxygen regulated protein, were identified in all three breast cancer cell lines. In addition, MCF-7 cells shared an additional 3 proteins with MDA-MB-453. Interestingly, the hydrazide method isolated a number of other N-linked glycoproteins also known to be involved in breast cancer including, epidermal growth factor receptor (EGFR), CD44, and the breast cancer 1, and early onset isoform 1 (BRCA1) biomarker. Analyzing the N-glycoproteins from membranes of breast cancer cell lines highlights the usefulness of the procedure for generating a practical set of potential biomarkers.

The immunogenicity of gangliosides found on human melanoma cells was determined from sera of 26 melanoma patients who were immunized every 1-4 weeks for 4 months with tumor-cell vaccine (TCV) prepared from cultured melanoma cells. Total lipid-bound sialic acid in the gangliosides isolated from TCV was 0.38 mumol/10(8) cells, and was distributed as follows: 44.8% to GM3, 44.2% to GD3, 5.6% to GM2, and 4.6% to GD2. Sera were tested at monthly intervals for antibodies to each ganglioside by ELISA with purified gangliosides as the antigen source. The immunologic specificity of the antibody was confirmed by absorption tests. None of the 26 patients had detectable anti-GM3, anti-GD3, or anti-GD2 antibodies before immunization, although anti-GM2 antibody was detected in 3 patients. After immunization, 2 patients developed IgM anti-GD2, 10 developed IgM anti-GM2, and 2 developed IgG anti-GM2 antibodies. No patient developed detectable anti-GM3 or anti-GD3 antibodies. These results indicate that both GD2 and GM2 expressed on human melanoma cells are immunogenic in humans, although GM2 appears to be more immunogenic. The other two gangliosides, GM3 and GD3, are present in human sera and in human normal tissues, and thus immunologic tolerance may have been established against these gangliosides. Alternatively, circulating GM3 and GD3 may have neutralized anti-GM3 and anti-GD3 antibodies, if any were induced by TCV immunization.

Human melanoma synthesizes a large quantity of gangliosides, glycosphingolipids containing sialic acid. The authors previously have demonstrated that the ganglioside profile differs among individual melanomas and is widely heterogeneous. In the current study, a retrospective study was performed to compare the relationship between the quantity of five major gangliosides of human melanoma (GM3, GM2, GD3, GD2, and alkali-labile ganglioside) and nine clinical factors (sex, age, site, stage, tumor size, pigmentation, histopathologic type of primary tumor, chemosensitivity, and prognosis). Melanoma specimens studied were obtained from patients of our clinic and included 52 biopsy specimens and 28 cultured cell lines. Analysis of melanoma biopsy specimens have shown a differential ganglioside expression among different sites of tumor, pigmentation, and histopathologic types. Results of cultured melanoma cell lines differed from those of biopsy specimens, but ganglioside expression also differed among the site of tumor, tumor size, histopathologic types, and chemosensitivity. GM3 positively correlated with a good prognosis in both biopsy and cultured melanomas.

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein. Mol Cancer Ther; 10(8); 1500-8. Ó2011 AACR.