Human papilloma virus 16 E6 oncoprotein associated with p53 inactivation in colorectal cancer

Abstract

Aim: To investigate the association between human papilloma virus (HPV) infection and colorectal cancer.

Methods: Sixty-nine patients with pathologically confirmed primary colorectal cancer including 6 stage I, 24 stage II, 21 stage III, and 18 stage IV patients were enrolled in this study to investigate whether HPV 16 could be involved in colorectal tumorigenesis. Nested-polymerase chain reaction (nested-PCR) was used to detect HPV16 DNA in colorectal tumor tissues and further confirmed by in situ hybridization (ISH). In addition, immunohistochemistry analysis was performed to examine the E6 oncoprotein in colorectal tumors. To verify whether E6 could inactivate the p53 transcriptional function, the levels of p21 and Mdm2 mRNA expression were evaluated by real-time reverse transcription (RT)-PCR.

Results: Of the 69 colorectal tumors, HPV16 DNA was detected in 11 (16%) by nested-PCR, and HPV16 DNA was present in 8 of the 11 (73%) tumors which was confirmed by ISH. The presence of HPV16 DNA in colorectal tumors was not associated with patients' clinical parameters including age, gender, smoking status, tumor site; however, HPV16 infection was more common in stage I patients than in late-stages patients (II, III and IV). We next asked whether HPV16 infection could be linked with colorectal cancer development. Immunohistochemical data indicated that 8 of the 11 HPV16 DNA-positive tumors had E6 oncoprotein expression. Moreover, we also observed that the adjacent normal tissues including endothelial cells, lymphocytes, fibroblasts, and gland cells in E6-positive tumors had E6 oncoprotein expression. In addition, 3 of the 4 (75%) E6-positive tumors carrying p53 wild-type had negative immunostaining, but one tumor had less p53 immunostaining. We further examined whether E6-positive and/or p53 mutated tumors reduce p53 transcriptional activity. Real-time RT-PCR analysis indicated that p21 and mdm2 mRNA expression levels in E6/p53-wildtype tumors were significantly lower than in their adjacent normal tissues; as expected, E6-positive/p53-mutated tumors had lower p21 and mdm2 mRNA expression levels compared with their adjacent normal tissues. These results clearly indicate that the E6 oncoprotein expressed in p53 wildtype tumors may reduce p21 and mdm2 expression via p53 inactivation.

Conclusion: These results suggest that HPV16 infection may be involved in a subset of colorectal cancer, and we suggest that the transmission of HPV to the colon and rectum might occur through peripheral blood lymphocytes.

Keywords: Blood lymphocytes; Colorectal cancer; Human papilloma virus; p21; p53.

Figure 1

Representatives of positive and negative human papilloma virus 16 DNA detected by nested polymerase chain reaction in tumors of colorectal cancer patients. M: 100-bp ladder DNA marker; P: Positive control; DNA of SiHa cell line served as positive control for human papilloma virus 16, respectively; N: Negative control, the DNA template was replaced with distilled water.

Figure 2

The representative reciprocal relationships between human papilloma virus 16 E6 and p53 immunostainings in human papilloma virus 16-infected colorectal (400 ×). HPV: Human papilloma virus.

Figure 3

Immunohistochemical analysis of human papilloma virus 16 E6 protein in colorectal tumors and adjacent normal tissues. A: A negative result of immunostaining in tumor cells (100 ×); B: Human papilloma virus 16 (HPV16) E6 protein expressed in endothelial cells (400 ×); C: HPV16 E6 protein expressed in endothelial cells and lymphocytes (100 ×); D: HPV16 E6 protein expressed in lymphocytes and dysplastic gland (400 ×); E: HPV16 E6 protein expressed in endothelial cells and tumor cells (400 ×); F: HPV16 E6 protein expressed in normal gland (400 ×); G: HPV16 E6 protein in endothelial cells and Fibroblast (400 ×); H: HPV16 E6 protein in endothelial cells, lymphocytes and tumor cells (400 ×). E: Endothelial cells; T: Tumor cells; L: Lymphocytes; DG: Dysplastic gland.