Humoral immune response to p16, a cyclin-dependent kinase inhibitor in human malignancies

Abstract

Abstract. The p16 protein is a cyclin-dependent kinase (CDK) inhibitor, which plays an important role in the regulation of the cell cycle by inactivating the cyclin-dependent kinase (CDK) that phosphorylates the retinoblastoma (Rb) protein.Overexpression of p16 protein has been found in many types of human malignancy. Autoantibody response to p16 in cancer has not been reported. This study determined the extent and frequency of autoantibodies to p16 in diverse malignancies. p16 recombinant protein was expressed in E. Coli BL21 (DE3) cells, and purified using GST fusion protein purification system. In further studies, p16 recombinant proteins were used as antigens in enzyme-linked immunoassay (ELISA) and Western blotting. Sera from 479 cancer patients and 82 normal individuals were analyzed. Autoantibodies to p16 were found in 11.7% in cancer, with significant difference from the normal individuals (p<0.05). The results in this study also showed that the frequency of antibodies to p16 is relatively higher in nasopharyngeal cancer (28.6%), breast cancer (17.1%) and hepatocellular carcinoma (HCC, 21.4%). Of the 56 ELISA positive sera with the anti-p16 antibodies, 85.7% (48/56) had positive reactions in Western blotting. The antigen-antibody absorption experiment was also performed to confirm the specificity of the anti-p16 antibody. In order to increase the frequency of antibody detection in cancer, a combination of three tumor-associated antigens (TAAs) p16, p53 and c-myc were used. Increased frequencies at p<0.01 were found for antibodies to p16 in breast, esophageal, and nasopharyngeal cancer as well as HCC. For antibodies to c-myc, increased frequencies at p<0.01 were found in breast, cervical, colorectal and lung cancer. For antibodies to p53, increased frequencies at p<0.01 were only found in breast cancer. With the successive addition of three TAAs, there was a stepwise increase of positive antibody reaction up to 44% in breast cancer and 43% in nasopharyngeal cancer. In summary, the results in this study suggest that the combination of antibodies might acquire higher sensitivity for early cancer diagnosis. It is conceivable that autoantibody profiles involving different panels or arrays of TAAs might be developed in the future and the results could be useful for cancer diagnosis.

Circulating antibodies to p16 protein-derived peptides in breast cancer

“…Overexpression of p16 protein has been reported in several types of cancer, although normal tissues exhibit low or undetectable levels (18,23). This is possibly a feedback reaction to the development of malignancy in the body.…”

“…As this CDK inhibitor mainly plays a role in suppressing CDK4 and CDK6 activity and arresting cells during the G1 phase of the cell cycle (14,16), it is associated with the unrestrained growth that is the hallmark of cancer (17). A number of studies have suggested that the levels of circulating antibodies to the p16 protein are increased in patients with breast cancer (11,18,19). For example, Looi et al developed an in-house enzyme-linked immunoassay (ELISA) using recombinant p16 protein as antigens to detect anti-p16 IgG levels in the plasma of cancer patients (18) and they observed a significant increase in the prevalence of IgG antibodies to the p16 protein in breast cancer.…”

“…A number of studies have suggested that the levels of circulating antibodies to the p16 protein are increased in patients with breast cancer (11,18,19). For example, Looi et al developed an in-house enzyme-linked immunoassay (ELISA) using recombinant p16 protein as antigens to detect anti-p16 IgG levels in the plasma of cancer patients (18) and they observed a significant increase in the prevalence of IgG antibodies to the p16 protein in breast cancer. Based on our recent studies, the application of linear peptides as antigens may be more sensitive for ELISA in studying circulating antibodies against certain TAAs (20)(21)(22).…”

Serologic Autoantibodies as Diagnostic Cancer Biomarkers—A Review

“…An increase to 44% sensitivity and 97.6% specificity in breast cancer detection was achieved through the successive addition of the three TAAs p53, protein 16 (p16), and avian myelocytomatosis viral oncogene homolog (c-myc; ref. 81). SEREX technology was used by Zhong and colleagues (82) to detect three further breast cancer-associated autoantibodies including serine active site containing 1 (SERAC1), receptor expressed in lymphoid tissues (RELT), and ankyrin repeat and suppressor of cytokine signaling (SOCS) box protein 9 (ASB-9).…”

Mini-array of multiple tumor-associated antigens to enhance autoantibody detection for immunodiagnosis of hepatocellular carcinoma

“…With the successive addition of TAAs to a final total of seven antigens, there was stepwise increase of positive antibody reactions up to 56.9 percent in HCC. In our recent study, p16, a cyclin-dependent kinase inhibitor, which has been implicated as an important tumor suppressor [33], was evaluated as a TAA and added into our previously constituted mini-array of seven TAAs [24]. As shown in Table 1, the frequency of antibodies to eight TAAs in sera from patients with chronic hepatitis, liver cirrhosis and HCC using Enzyme-linked immunosorbent assay (ELISA).…”