Coexistence of K-ras mutations and HPV infection in colon cancer

Nur Buyru 1 Ayda Tezol 1 Nejat Dalay 0 0 Oncology Institute, Istanbul University , Istanbul , Turkey 1 Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University , Istanbul , Turkey Background : Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods : K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results : HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion : Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype. - Background Colorectal carcinogenesis is a complex, multistep process involving environmental and lifestyle factors, sequential genetic changes and possibly viral components in discrete geographical areas. Genetic changes inactivating tumor suppressor and mismatch repair genes or activation of oncogenes which are involved in cell growth, proliferation and differentiation are implicated in the development of colon carcinoma. The target genes for these alterations are APC (Adenomatous Polyposis Coli), the ras family and p53 [1]. The ras family of oncogenes (Nras, H-ras and K-ras) encode a small 21-kD protein (p21 ras) involved in the transduction of external stimuli to effector molecules across plasma membranes [2]. This protein has an intrinsic GTPase activity allowing inactivation following signal transduction in the normal cellular environment [2,3]. Activation of the K-ras protooncogene by point mutation is one of the most frequent genetic alterations associated with human cancers [4,5]. Mutated ras p21 has a structure that disfavors its ability to bind the GTPase activating protein (GAP), thus keeping the p21 in the GTP-bound, activated state [6]. Approximately, 90 % of these activating mutations occur in codons 12 and 13 of exon 1 identifying these codons as hot-spot targets [7]. The incidence of ras gene mutations, however, varies considerably among different types of cancer and the profile of ras oncogene activation is often specific for each tumor type. For example, K-ras mutations are the predominant ras mutation found in pancreatic cancer [8,9], N-ras mutations predominate in acute myeloid leukemias [10], and H-ras mutations, which are generally rare, are most frequently observed in bladder cancer [11]. Oncogenic mutations of K-ras are involved in 2050 % of sporadic colorectal carcinomas [12-14]. The association between human papillomavirus (HPV) infection and the development of cervical and anogenital tumors is widely accepted. However, the relationship between human papilloma viruses and malignant diesases at various body sites, including the upper respiratory and digestive tracts and the breast is still not clear [15-17]. HPV types 16 and 18 have been associated with a higher oncogenic potential and are considered as "high risk" types. The "low risk" HPV types HPV-6 and HPV-11 are predominantly associated with benign mucosal lesions of the genital tract and rarely result in invasive tumors [15,18]. The oncogenic HPV gene product E6 promotes degredation of the p53 tumor suppressor protein, whereas the E7 protein inactivates the Rb protein and related pocket proteins [19,20]. However, the tumorigenic properties of the E6 and E7 proteins may not necessarily be limited only to the Rb and p53-related pathways [21,22]. The presence of HPV infection alone also is not sufficient to cause tumorigenesis and requires additional cellular modifications such as alterations in the p53 and K-ras genes. Although the role and distribution of K-ras mutations in colon cancer has been studied extensively there are no reports in the literature investigating the K-ras mutation status in HPV-associated colorectal cancer. The present study was undertaken to investigate the role of K-ras codon 12 and codon 13 mutations in HPV-associated colon tumors. Methods Tumor samples were obtained at the time of surgery from 53 patients with colon cancer. The corresponding normal tissues surrounding the tumors were also analyzed. The study was approved by the Institutional Ethics Committee. Genomic DNA was extracted from the tumors and corresponding normal colon tissue samples by phenol/ chloroform extraction. To detect K-ras codon 12 and 13 mutations, DNA was amplified by a mutagenic PCR assay. A mismatched upstream primer for codon 12 and a mismatched downstream primer for codon 13 which introduced a Bst N1 (codon 12) and a HaeIII (codon 13) restriction site in the wild type allele, respectively, were used for amplification as described previously [23]. The PCR reactions were carried out in a total volume of 25 l containing 200 ng of genomic DNA, 50 mM KCl, 1.5 mM MgCl2, 10 pmol each of the forward and reverse primers, 1U Taq polymerase (MBI, Fermentas, Lithuania), 200 M dNTP mix and 20 mM Tris-HCl, pH 8.3. The reaction mixture was heated to 95C for 5 min. for initial denaturation, followed by 35 cycles of denaturation at 95C for 1 min., annealing at 60C for 1 min. and extension at 72C for 1 min. Final extension was allowed to proceed for 6 min. at 72C. Digestion was performed in a total volume of 25 l containing 10 l PCR product, 2.5 l 10x digestion buffer, 5 g BSA and 10 U of Bst N1 or HaeIII by overnight incubation at 60C or 37C, respectively. The digested products were separated by 8% non-denaturing polyacrylamide gel electrophoresis at 120 V for 3 h, the gel was stained with ethidium bromide and the genotypes were determined using a video gel documentation system (Vilber-Lourmat, Marne-La-Valle, France). To investigate the HPV infection specific regions from the HPV genome were amplified by the MY 09 and MY 11 consensus primers which amplify a region of about 450 bp from the L1 open reading frame. DNA from a HPVpositive patient with cervix cancer was used as the positive control and DNA from lymphocytes were used as the negative control. The PCR react (...truncated)