Aberrant Methylation of p16 in Anatomic and Gender-specific Subtypes of Sporadic Colorectal Cancer

Colorectal cancer (CRC) occurring in the proximal colon and among women may represent a distinct subtype of the disease. In the present study of 120 sporadic CRCs, we used methylation-specific PCR to test whether methylation of the CpG island in the 5* region of the p16 tumor suppressor gene was associated with anatomical location, gender, or other clinicopathological characteristics. Overall, 18.3% of the tumors had detectable p16 methylation. A marked preponderance of methylated tumors occurred within the proximal colon; cancers occurring proximal to the sigmoid colon were 13.1 times more likely to contain methylated p16 compared with distal tumors. In addition, female patients were 8.8 times more likely than males to have methylation-positive cancers, and p16 methylation was also associated with poorly differentiated tumors. The localization of tumors with p16 methylation within the proximal colon and among female patients specifically adds to a growing database of molecular alterations that define important subtypes of sporadic CRC. The potentially reversible nature of CpG methylation may provide novel therapeutic opportunities for this increasing subtype of the disease, which, due to anatomical location, presents a great challenge for early detection. Introduction CRC is the third most common form of internal malignancy in Westernized countries. In the United States, 143,000 people were diagnosed with CRC in 1995 (1, 2). Epidemiological studies of migrant populations show that environmental factors play an important role in the etiology of this form of cancer. Persons migrating from low to high CRC risk countries acquire the risks of the high-risk population within a single generation (3, 4). The incidence of CRC worldwide is very dynamic; CRC rates are increasing in traditionally low-risk countries that are adopting a globalized Western-style diet high in fat and meat protein and low in fiber, vegetables, and fruit (5) and a more sedentary lifestyle. Within high-risk countries such as the United States, CRC rates are rising rapidly among AfricanAmerican (6), Hispanic, and American Indian populations (7). Whereas a dominant role has been ascribed to diet and lifestyle in the etiology of CRC, a striking and perplexing aspect of the epidemiology of CRC is the variation in risk associated with gender and anatomical location. Numerous studies have implicated hormonal and reproductive influences in the etiology of CRC in women (5). Recent examples include studies that found reduced risks for CRC (8, 9) and adenomatous polyps (10, 11) in women receiving postmenopausal hormone replacement therapy. Moreover, a female excess of right-sided CRC at all ages and an excess of left-sided cancers among males have been documented repeatedly (12–15). These gender associations with anatomical subsite parallel the risk observed in lowversus high-risk countries (16). A notable feature of the abovementioned increase in CRC within United States minority groups is the pronounced increase in right-sided CRC among older women (6, 7). These observations indicate differential environmental influences in women and men and in specific locations in the large bowel. The role of female hormones and their effects on bile acid metabolism were previously proposed to explain gender influences on the anatomical subsite distribution of CRC (17, 18). Creating an etiological model of CRC that integrates these epidemiological features of the disease with the manifold molecular and genetic aspects of CRC that have recently been discovered is a formidable challenge. The well-described model of colorectal carcinogenesis envisions a stepwise accumulation of early and late genetic alterations in the progression of adenoma to carcinoma (19). Epidemiological clues to the etiology of CRC have been sought by studying the mutational patterns within these loci (e.g., the p53 gene; Ref. 20). Identification of mutations within the familial APC (21) and within HNPCC genes (22) has helped to explain the inherited forms of CRC. Interestingly, loci involved in the heritable forms of CRC may Received 12/4/98; revised 3/15/99; accepted 4/7/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Foundation Marato (A. L., M. J. L.) and the National Institutes of Environmental Health Sciences NIH Grant P42-ESO4705 with funds provided by the Environmental Protection Agency (J. K. W., S. Z., C. G.). 2 To whom requests for reprints should be addressed, at Laboratory for Molecular Epidemiology, Department of Epidemiology and Biostatistics, University of California San Francisco, 500 Parnassus Avenue, MU-W 420, San Francisco, CA 94143-0560. 3 The abbreviations used are: CRC, colorectal cancer; MSPCR, methylationspecific PCR; APC, adenomatous polyposis coli; HNPCC, hereditary nonpolyposis colorectal cancer; OR, odds ratio. 501 Vol. 8, 501–506, June 1999 Cancer Epidemiology, Biomarkers & Prevention on October 14, 2017. © 1999 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from also be altered somatically in sporadic CRC (23), providing a mechanistic bridge between familial and sporadic CRC. In addition to mutational changes, epigenetic mechanisms play a prominent role in CRC. Aberrant DNA methylation has long been noted in colorectal carcinogenesis; imbalances in methylation are thought to occur early in the process and are characterized by genome-wide hypomethylation and regional and locus-specific hypermethylation (24). Among the common targets for aberrant DNA methylation is the 59 region of the p16 tumor suppressor gene (25, 26). The p16 protein plays an important role in the passage of cells through the G1 phase of the cell cycle by binding to cyclin-dependent kinases and inhibiting their interaction with cyclin D1 and reducing phosphorylation of the retinoblastoma protein (pRB; Ref. 27). Methylation of cytosine residues at CpG sites in p16 is common in CRC cell lines and is associated with transcriptional silencing of the gene (28, 29). Epigenetic alterations that suppress gene expression are potentially reversible, offer unique opportunities for the development of new therapies, and could also be the focus of preventive strategies. In the present study, we measured the occurrence of p16 methylation in primary CRCs using a recently developed MSPCR method and explored the relationship of methylation to demographic and clinicopathological characteristics of the patients. Because of the known epidemiological correlates of gender and anatomical location, we stratified our population by gender and location to test whether p16 methylation is involved in a unique pathway for CRC development. Materials and Methods Patient Population and Clinicopathological Information. Patients were ascertained through the University of Barcelona Hospital Clinic. The Hospital Clinic is a 900-bed institution with about 33,000 patient admissions per year and serves a source population of approximately 600,000 inhabitants of Catalonia, Spain. The Hospital Clinic is a teaching institution and tertiary care facility. During 1996–1997, 275 consecutive CRC patients undergoing surgery were identified through the clinic’s two surgical services as potential subjects for a hospital-based case-control study of CRC. Patients who were $35 years of age and ,90 years were invited to participate in the study. Eighteen subjects refused to participate, leaving a total of 249 enrolled subjects (132 men and 117 women). A consecutive case-series subset of 120 patients within one surgical service also provided tissue specimens (uninvolved colon and tumor) for the current molecular study. Patients provided signed informed consent; all procedures were approved by the Hospital Clinic’s institutional review board (Comite Etico de Investigacion Clinica). Primary tumors were surgically dissected and immediately frozen at 280°C. A questionnaire was administered to each patient by an interviewer; the questionnaire elicited demographic information and data on occupation, diet, and personal medical history. Clinicopathological data were collected and merged with patient questionnaires. A modified version of the Dukes’ staging system was used. Surgical records identified eight subsites: (a) cecum; (b) ascending colon; (c) transverse colon; (d) descending colon; (e) sigmoid colon; (f) rectosigmoid junction; (g) rectum; and (h) anal canal. Proximal tumors were defined as including the right side of the colon and descending colon up to the sigmoid colon. Additional analyses were carried out that included the transverse and descending colon tumors with the distal category of CRCs. DNA was isolated from tumor specimens using standard methods involving RNase, proteinase K, chloroform/isoamyl alcohol extraction, and ethanol precipitation. DNA was quantitated by Hoescht 33258 fluorometry (Hoeffer Scientific). MSPCR. Detection of methylated CpG sites within the 59 region of the p16 gene was carried out using MSPCR (30, 31). Briefly, 1.0 mg of purified DNA was diluted in 36 ml of H20 to which 4 ml of 3.0 M NaOH were added, and DNA was denatured at 37°C for 15 min. The sample was then treated with 416 ml of 3.6 M sodium bisulfite solution (pH 5.0) and 24 ml of 10 mM hydroquinone. Both bisulfite and hydroquinone solutions were prepared fresh for each analysis. Samples were incubated at 55°C for 16 h; 100 ml of mineral oil were layered on top of the solution to prevent evaporation. After incubation, the solution was cooled to 280°C for 10 min, after which the unfrozen mineral oil was removed without disturbing the bisulfite-DNA solution. Bisulfite-modified DNA was purified with the Wizard DNA Clean-UP System and Vacuum Manifold (Promega), according to the manufacturer’s instructions. DNA was eluted twice with a total volume of 100 ml of 10 mM Tris-1 mM EDTA buffer (pH 7.8). The final step of the cytosine to uracil conversion reaction was achieved with alkali treatment (NaOH, final concentration 5 0.3 M) at 37°C for 15 min followed by ammonium acetate (pH 7.0; final concentration, 3.0 M) neutralization and ethanol precipitation. For each set of tumors assayed, two cell line DNA samples were also treated with bisulfite and amplified by MSPCR. Both a methylation-positive CRC cell line (SW-480; American Type Culture Collection) and a methylation-negative cell line (SK-N-SH; American Type Culture Collection) served as controls for the bisulfite conversion, DNA recovery, and PCR reactions. For each tumor, a multiplex PCR amplification of bisulfite-treated DNA was carried out with primers (Life Technologies, Inc.) specific for methylated (M primer) and nonmethylated (U primer) CpG sites within the p16 promoter region (31); (M primer, 59-TTATTAGAGGGTGGGGCGGATCGC and 59-GACCCCGAACCGCGACCGTAA), and nonmethylated (U2 primer, 59-TTATTAGAGGGTGGGGTGGATTGT and 59-CCACCTAAATCAACCTCCAACCA). The PCR mixture contained GeneAmp PCR buffer (Perkin-Elmer Corp.), MgCl2 (1.5 mM), deoxynucleotide triphosphates (200 mM each), primers (0.4 mM for each primer), modified DNA templates (50 ng), and 2.5 Units of AmpliTaq (Perkin-Elmer Corp.) in a total volume of 50 ml. The PCR reaction was repeated for 35 cycles on a GeneAmp 9600 thermal cycler (Perkin-Elmer Corp.) under the following conditions: preheat at 94°C for 1 min, 94°C for 30 s, 65°C for 10 s, 72°C for 30 s, and a final extension at 72°C for 10 min. Aliquots (15 ml) of PCR products were loaded onto 2.5% agarose gels, stained with ethidium bromide, and visualized under UV light. To confirm the specificity of the method, PCR product from the positive and negative controls and several CRC tumor specimens were sequenced using purified PCR product and an ABI 377 automated sequencer with dye primer chemistry. To assess the sensitivity of the MSPCR method, methylated and unmethylated cell line DNA samples were mixed in different ratios; a single unambiguous p16 methylated band was detectable when methylated template was present at greater than 1:32 (3%) of the total DNA. All analyses were carried out on coded samples without knowledge of the patient’s clinical status. Statistical Analysis. Data were analyzed using statistical analysis software. Means and SDs were computed for continuous variables. The ORs and 95% confidence intervals were calculated to detect associations of variables with the p16 methylation status of patients’ tumors. Multivariate analyses (logistic regression) were used to estimate the association of 502 p16 Methylation in Colorectal Cancer on October 14, 2017. © 1999 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from variables that were identified in univariate analyses with p16 methylation. Significance levels of 0.05 were used for hypothesis testing.