C4.4A as a candidate marker in the diagnosis of colorectal cancer

Abstract

C4.4A is a member of the Ly-6 family with restricted expression in non-transformed tissues. C4.4A expression in human cancer has rarely been evaluated. Thus, it became important to explore C4.4A protein expression in human tumour tissue to obtain an estimate on the frequency of expression and the correlation with tumour progression, the study focusing on colorectal cancer. The analysis of C4.4A in human tumour lines by western blot and immunoprecipitation using polyclonal rabbit antibodies that recognize different C4.4A epitopes revealed C4.4A oligomer and heavily glycosylated C4.4A isoform expression that, in some instances, inhibited antibody binding and interaction with the C4.4A ligand galectin-3. In addition, tumour cell lines released C4.4A by vesicle shedding and proteolytic cleavage. C4.4A was expressed in over 80% of primary colorectal cancer and liver metastasis with negligible expression in adjacent colonic mucosa, inflamed colonic tissue and liver. This compares well with EpCAM and CO-029 expression in over 90% of colorectal cancer. C4.4A expression was only observed in about 50% of pancreatic cancer and renal cell carcinoma. By de novo expression in colonic cancer tissue, we consider C4.4A as a candidate diagnostic marker in colorectal cancer, which possibly can be detected in body fluids.

Figure 1

Production of antibodies against hC4.4A. (A) Schematic representation of the structure of hC4.4A. Circles represent the Ly-6 domains. Parts of the molecule recognised by the antibodies are in red. (B) HEK-293 cells were transfected with cDNA coding for hC4.4A (HEK-293-hC4.4A) or with vector alone (HEK-293). Lysates were analysed in WB with anti-hC4.4A-C (left) and anti-hC4.4A-N (middle). The right lane shows HEK-293-hC4.4A lysates blotted with rabbit IgG. (C) Immunohistochemistry of paraffin-embedded, normal healthy skin using rabbit IgG (negative control) and anti-hC4.4A-C at the concentration of 1.5 μg ml⁻¹. Scale bar=50 μm. (D) Western blot of HaCaT cell lysate with anti-hC4.4A-C (left), anti-hC4.4A-N (right) and rabbit IgG (middle) (negative control).

Figure 2

Expression of hC4.4A in cancer cell lines. (A) C4.4A expression was evaluated in colorectal cancer lines, which were cultured in heat-inactivated (grey area) or fresh ABO serum (black area). Overlays with the negative control (normal rabbit IgG/anti-rabbit APC) are presented. (B) Biotinylated lysates of the indicated cell lines were immunoprecipitated using anti-hC4.4A-C. Immunoprecipitates were boiled, separated by SDS–PAGE and blotted with streptavidin-HRP. Depending on the cell line, several bands were recovered (indicated by arrows), which represent different C4.4A isoforms and/or associated proteins (see Results). (C) Biotinylated lysates of Colo357 and MCF-7 cells were immunoprecipitated using anti-hC4.4A-C, and immunoprecipitates were boiled in the presence of 2-ME before SDS–PAGE and blotting with streptavidin-HRP. The high molecular weight band of about 200 kDa was not recovered after 2-ME treatment. (D) Where indicated, MCF-7 cells were treated with tunicamycin (Tun). Lysates were separated by SDS–PAGE and blotted with anti-hC4.4A-N. The antibody recognizes only lysates from Tun-treated cells. (E) Lysates of MCF-7 cells were biotinylated and immunoprecipitated with the anti-hC4.4A-C antibody. Where indicated, immunoprecipitates were treated with N-glycosidase F (N-glycosid.) or with O-glycosidase (O-glycosid.) plus neuraminidase (NA) before SDS–PAGE separation and WB analysis with streptavidin-HRP. O-glycosidase plus NA treatment resulted in a size reduction of C4.4A from 75 to 35 kDa–40 kDa. (F) Cell lysates of the indicated lines were incubated with GST-galectin-3 or GST. Bound proteins were eluted with 100 mM lactose. Eluted proteins were treated with N-glycosidase F before SDS–PAGE and blotting with anti-hC4.4A-N. C4.4A with a molecular weight of about 55 kDa was recovered in the precipitate of 5 out of 7 lines, but only after incubation with GST-galectin-3 and not after incubation with GST.

Figure 3

Expression of hC4.4A and galectin-3 in colorectal cancer and liver metastasis. Immunohistochemistry of the indicated tissues was performed with anti-hC4.4A-C, anti-galectin-3 or rabbit IgG (negative control) at the concentration of 5 μg ml⁻¹ (colon) and 10 μg ml⁻¹ (liver). Representative examples are shown. Open arrow, mucosa epithelium; white-filled arrow, submucosa; black arrowhead, tumour cells; white arrowhead, tumour stroma; thin white arrow, hepatocytes. Anti-hC4.4A-C stains tumour cell membranes, and anti-galectin stains tumour cell membranes and tumour stroma. Scale bar=50 μm.

Figure 4

Release of rat and human C4.4A. (A) Supernatant of the indicated cell lines was subjected to ultracentrifugation. The pellets were separated by SDS–PAGE and blotted with C4.4 (left) or anti-hC4.4A-N (right). For analysis with hC4.4A-N, pellets were treated with N-glycosidase F before SDS–PAGE. C4.4A was recovered in the pellet (vesicles containing uncleaved C4.4A). (B) Supernatant of ASML cells was subjected to IP with C4.4. Bound proteins were eluted and extracted with Triton X-114. The Triton X-114 fraction (left) and the aqueous fraction (right) were separated by SDS–PAGE and blotted with C4.4. The Triton X-114 fraction contained a considerable amount of C4.4A (GPI anchor-containing C4.4A). Two bands of about 80–90 and 40 kDa were recovered from the aqueous fraction, the lower molecular weight band indicating a proteolytic cleavage product of C4.4A. (C) Rat recombinant C4.4A with a C-terminal myc tag was treated for 2 h at 37°C with 0.01 or 0.1% trypsin. After SDS–PAGE of the undigested and trypsin-digested rrC4.4A protein, blots were incubated with C4.4 (left) and anti-myc (right). The undigested 90 kDa recombinant protein was detected with both C4.4 and anti-myc. Only C4.4 recognized a fragment of about 40 kDa.