C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancer

C35 protein sequence is conserved among species. National Center for Biotechnology Information accession numbers are as follows: Homo sapiens, AY508814; Rhesus monkey Macaca mulatta, XP_001091012; Mus musculus, NP_079835; Rattus norvegicus, XP_001081367; Bos taurus, XP_582041; Canis familiaris, XP_537653; Xenopus tropicalis, NP_001015996. A predicted prenylation site (CVIL) is bolded and italicized. The immunoreceptor tyrosine-based activation consensus sequence is in bold.

Chromosomal location of C35. The C35 gene is located on chromosome 17q12, 7402 nucleotides from the start of GRB7 and 505 nucleotides from the end of ERBB2. Direction of transcription is denoted by arrows, and positions of each gene on the chromosome are identified based on Human Genome build 36.1. The full-length C35 transcript is 730 nucleotides in length, consisting of four exons (black boxes).

A, anti-C35 antibody specifically binds endogenous C35 protein in Western blot analysis. C35 protein was detected using rabbit polyclonal antiserum B78-2 in lysates of C35^hi tumor 21MT-1 but not in C35^lo “normal” breast cell line H16N2. Binding can be competed by preincubation of the antiserum with purified C35 protein, showing specificity of the antibody in the assay. Corresponding fast green stains of the blots show equal sample loading and transfer. Molecular weight markers (kDa) are indicated with arrows. B, immunohistochemistry of cell lines derived from metastatic breast carcinoma and normal breast epithelium. Sections of formalin-fixed, paraffin-embedded cell pellets were stained with polyclonal rabbit anti-C35 or isotype control rabbit IgG antibodies. Tumor stains intensely with a punctate pattern. Magnification, ×100.

C35 transcript regulation. Cell lines were analyzed for abundance of C35 gene or transcript by real-time PCR to compare genomic amplification with expression of mRNA. All genomic amplification data are relative to H16N2, which is set to reflect one copy. The C35/ERBB2 locus is amplified in 21MT-1, corresponding with overexpression of both transcripts in this line. In contrast, C35 transcript is overexpressed in Colau without amplification of the gene locus. Furthermore, luciferase activity driven by C35 promoter region mimics cDNA levels and contrasts with genomic copy number in C35⁺ cell lines, such as Colau and Mel1700.

Immunohistochemistry of intraductal carcinoma of breast. A, sections of formalin-fixed, paraffin-embedded tissue were stained with polyclonal rabbit anti-C35, anti-c-erb-B2, or isotype control antibodies. Top, example of a C35⁺/ERBB2⁺ tumor (scores of 4+ and 3+, respectively); bottom, example of a C35⁺/ERBB2⁻ tumor (scores 2+ and 0, respectively; faint cytoplasmic stain is negative according to HercepTest guidelines). ERBB2⁺ tumor has intense plasma membrane staining, whereas C35⁺ tumors show intense, homogeneous, cytoplasmic staining. Magnification, ×40. B, comparison of immunohistochemistry scores for HER-2/neu and C35 in number of IDC cases. Dotted lines indicate delineation between positive and negative scores.

Normal human tissues stained with rabbit anti-C35 polyclonal antibodies (top) and control rabbit IgG (bottom). Magnification, 40×. A, breast tissue from a non-cancer patient. B, breast tissue from cancer patient containing tumor (4+, red arrows) and adjacent morphologically “normal” epithelium (2+, black arrows). Inset, magnification, ×100. C, testes, Leydig cells are C35⁺ (blue arrows). D, heart. E, liver (polymer detection). F, lung. G, kidney. H, ovary. I, uterus. J, prostate. K, salivary gland.

C35 positivity correlates with age in ILC. C35 is expressed at a higher frequency in young patients with ILC. Expression in IDC does not correlate with patient age.