Structural, biochemical and clinical analyses of a cancer-related protein, C35 (C17orf37)

Identification of cancer-specific biomarkers has enormous potential to enhance detection, treatment, and prognosis. C35 (C17orf37) is a novel gene, which locates at chromosome 17q12-21 and is found to be up-regulated in many cancers. In a previous study, immunohistochemical analysis detected robust and frequent expression of C35 protein in breast cancer tissues. Here, we attempt to characterize the properties of C35 protein as well as to demonstrate a functional association between C35 and tumor progression. We cloned the full length gene of C35 from a breast cancer cell line, SK-BR-3, and expressed it to gain recombinant C35 protein from E. coli for circular dichroism(CD) and nuclear magnetic resonance (NMR) analyses. The CD spectrum of C35 protein revealed a possible alpha-helix secondary conformation existed. To our surprise, we also observed an extremely heat tolerance and a pH stable properties of this protein. Additionally, based on one-dimensional NMR analysis, it also showed a well organize protein structure. Finally, tertiary structure determined by two-dimensional NMR assignment reveals C35 as thioredoxin-like molecule. To warrant C35 as a cancer related gene, we first survey its expression in different cancer cell lines and tissues by using RT-PCR, Western blot and immunohistochemistry, respectively. Immunohistochemistry had showed high relevance of C35 in gastric cancer. Otherwise, survey of different cell lines by Western blotting reveals highly endogenous C35 expression in breast cancer cells, BT474. Furthermore, to investigate the function of C35 in normal tissues, analyzing of ten normal mice’s tissues reveals no detectable C35 protein. Effects of C35 on cellular proliferation, migration and apoptosis are investigating. In a preliminary test using the modified Boyden chamber assay, overexpression of C35 enables promoting cell migration toward fetal bovine serum. Beyond this, we will further establish the role of C35 in tumor progression.