Affiliations of authors: Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu; Unit of Hormones and Cancer, International Agency for Research on Cancer, Lyon, France.
Correspondence to: Loïc Le Marchand, M.D., Ph.D., Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, 1236 Lauhala St., Suite 407, Honolulu, HI 96813 (e-mail: loic{at}crch.Hawaii.edu).
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ABSTRACT |
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INTRODUCTION |
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Evidence that the GH/IGF-I axis is involved in colorectal cancer is, thus, accumulating. However, it is yet unclear whether (physiologically) increased levels of IGF-I play a direct role in colorectal cancer or are mere correlates of more relevant mechanisms, such as exposure to chemical carcinogens via smoking or a diet high in well-done red meat (11). The association of colorectal cancer with conditions that affect the level of IGF-I but are not related to lifestyle, such as an inherited genetic variant, would contribute evidence in favor of causality. We tested the hypothesis that a recently identified polymorphism in the human GH1 gene, encoding GH, that appears to result in lower levels of plasma GH and IGF-I (12) is inversely associated with colorectal neoplasia in a large, population-based, casecontrol study of colorectal cancer and in preliminary results from a sigmoidoscopy-screening-based casecontrol study of adenoma in Hawaii. We focused on this gene because GH is the main determinant of circulating levels of IGF-I and its main binding protein, IGFBP-3 (1).
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SUBJECTS AND METHODS |
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The protocols for these two studies were approved by the Committee on Human Studies of the University of Hawaii and by the institutional review board of each participating hospital. We also obtained written informed consent from all participants in both studies.
Subjects
The subjects for the first study have been described in detail elsewhere (13). Patients with colorectal cancer were identified through the rapid reporting system of the Hawaii Surveillance, Epidemiology, and End Results (SEER)1 Program registry and consisted of all Japanese, Caucasian, and Native Hawaiian residents of Oahu who were newly diagnosed with an adenocarcinoma of the colon or rectum between January 1994 and August 1998. Control subjects were selected from participants in an ongoing population-based health survey conducted by the Hawaii State Department of Health and from Health Care Financing Administration participants. One control subject was matched to each case patient by sex, ethnicity, and age (±2 years). Personal interviews were obtained from 768 matched pairs, resulting in a participation rate of 58.2% for case patients (from a total of 1320 eligible case patients identified, including 770 Japanese, 343 Caucasians, and 207 Native Hawaiians) and 53.2% for control subjects (from a total of 1444 control subjects identified as eligible). If a control subject declined to participate, he or she was replaced. The reasons for nonparticipation were as follows: in case patients, refusal (22.5%), death before contact (10.9%), severe disease (6.7%), and inability to locate (1.7%); in control subjects, refusal (34.8%), inability to locate (9.2%), and major illness or death (2.8%). Compared with noninterviewed case patients, interviewed case patients had a similar ethnic distribution, were less likely to have a regional or distant metastasis (46% versus 55%), and were younger by an average of 1.8 years. A blood sample was obtained from 548 (71%) of interviewed case patients and 656 (85%) of interviewed control subjects. Case patients and control subjects who donated blood were similar to all interviewed subjects with regard to age, sex, and ethnicity, as well as the variables found to be associated with the risk of colorectal cancer in this study (see below). Stock of extracted DNA was depleted for 13 case patients and six control subjects. Consequently, 535 case patients with adenocarcinoma and 650 control subjects were studied.
The subjects for the second study were identified among individuals who underwent flexible sigmoidoscopy screening as part of the baseline examination for the Hawaii component of the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial between July 1996 and February 2000 or in the Colorectal Screening Clinic of Kaiser Permanente, Hawaii, between December 1995 and April 2000. For all case patients, the first diagnosis of an adenomatous polyp was confirmed histologically. Control subjects had a normal sigmoidoscopy and were matched to case patients in a 2 : 1 ratio by age, sex, ethnicity, endoscopy date, and screening center. The participation rate was 70.3% for case patients (196 case patients were interviewed of 279 identified as eligible) and 70.8% for control subjects (303 control subjects interviewed of 428 identified as eligible). The reasons for nonparticipation among case patients were refusal (24.4%), physician refusal (3.2%), and inability to locate (2.1%). The reasons for nonparticipation among control subjects were refusal (22.9%), physician refusal (4.0%), and inability to locate (2.3%). A blood sample was obtained from 94.3% of interviewed case patients and 91.0% of interviewed control subjects. The present analysis was performed on the first 139 case patients and 202 control subjects genotyped for human GH1.
Interviews
For both studies, in-person interviews were conducted at the subject's home by trained interviewers, using the same questionnaire. This questionnaire included detailed information on demographics; a quantitative food-frequency questionnaire; a lifetime history of the use of tobacco, alcohol, and nonsteroidal anti-inflammatory drugs; a history of recreational sports activities since age 18 years; a personal history of various relevant medical conditions; a family history of colorectal cancer in parents and siblings; information on height and weight at different ages; and for women, a history of reproductive events and hormone use. The diet questionnaire assessed the frequencies and amounts consumed during the year before the onset of symptoms for case patients with colorectal cancer or the year before interview for case patients with adenoma and all control subjects for 268 food items or categories. Participants also reported their intakes and dosages of vitamin and mineral supplements during the reference period.
Laboratory Assays
In the adenoma study, all subjects were asked to fast for 10 hours before blood was drawn. In both studies, the samples were processed within 4 hours of collection and kept frozen at 80 °C until analysis. All laboratory assays were performed blinded to the casecontrol status and ethnicity of the subjects. DNA was extracted from buffy coat cells. The polymerase chain reaction assay used to detect the T-to-A variant at position 1663 (T1663A) in intron 4 of the GH1 gene was a two-step method because of the close homologies that exist between GH1 and the other related genes in the GH cluster. The first amplification involved denaturation at 94 °C for 5 minutes, followed by 25 cycles of 94 °C for 30 seconds, 64 °C for 30 seconds, and 72 °C for 1 minute 30 seconds, and a final extension at 72 °C for 10 minutes, using the primers F4 (5`-GGCTGACCCAGGAGTCC-3`) and R1 (5`-AGAAGGACACCTAGTCAGACA-3`) to produce a 2176-base-pair (bp) product. One microliter of this product was further amplified with the mutagenic forward primer GH1MF2 (5`-GAGAAACACTGCTGCCCTCTTTTTAGACG-3`, modified nucleotides are underlined) and GH1R2 (5`-AAGAGAAGGAGAGGCCAAGC-3`) to produce a 180-bp product. The T allele was digested with AatII to fragments of 149 bp and 31 bp, whereas the A allele was not digested with AatII (Fig. 1). The authenticity of this assay was confirmed by DNA sequencing.
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Data Analysis
The statistical analysis used unconditional logistic regression (14) to adjust odds ratios (ORs) for the matching variables (age, sex, and ethnicity), as well as for the variables found to be associated with risk (see Table 2), entered as continuous variables. Nutrient intakes were adjusted for total calories by the method of residuals (15). The likelihood ratio test was used to determine the statistical significance of interaction between certain variables with respect to risk for colorectal cancer. The test compares a main-effects, no-interaction model with a fully parameterized model containing all possible interaction terms for the variables of interest. Genedosage effects were modeled by assigning a value of 1, 2, or 3 to the genotype variable according to the subject's number of variant A alleles (zero, one, and two variant alleles, respectively). The
2 test was used to compare the observed genotype distributions with those expected by the Hardy-Weinberg equilibrium. Multiple covariance analysis was used to compute adjusted means for plasma hormones. All statistical tests are two-sided.
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RESULTS |
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Ethnic-specific analyses suggested differences in the strength of the association of the GH1 A allele with adenocarcinoma among ethnic groups. The association was strong in Caucasians and Native Hawaiians but was not observed in Japanese (Table 2). Caucasians with T/T, T/A, and A/A genotypes had adjusted ORs for colorectal cancer of 1.00, 0.85 (95% CI = 0.50 to 1.43), and 0.44 (95% CI = 0.21 to 0.93) (Ptrend = .05). The corresponding ORs for Hawaiians were 1.00, 0.39 (95% CI = 0.16 to 0.94), and 0.20 (95% CI = 0.07 to 0.59) (Ptrend = .003). In contrast, those for Japanese were 1.00, 0.79 (95% CI = 0.56 to 1.12), and 0.85 (95% CI = 0.53 to 1.36) (Ptrend = .34). The results for adenoma were consistent with those ethnic patterns (Table 2
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The association of the A allele with a lower risk of colorectal cancer was found to be stronger for subjects aged 66 years or younger (median). In this group of individuals with T/T, A/T, and A/A genotypes, the adjusted ORs for colorectal cancer were 1.0, 0.74 (95% CI = 0.52 to 1.07), and 0.48 (95% CI = 0.29 to 0.81), respectively (Ptrend = .005). The corresponding ORs for subjects older than 66 years were 1.00, 0.79 (95% CI = 0.52 to 1.20), and 0.79 (95% CI = 0.47 to 1.34) (Ptrend = .32). However, the test for interaction was not statistically significant (P = .54). This modifying effect of age for colorectal cancer was observed in Caucasians and Native Hawaiians but not in Japanese. No clear modifying effect of age was observed for adenoma, although the statistical power to detect differences was limited by the small sample size of these subset analyses. Interactions of the GH1 genotype with height, body mass index, total calories, total fat, saturated fat, and polyunsaturated fat were also investigated in both studies. No suggestions of interaction were detected.
Plasma levels of IGF-I and IGFBP-3 were also explored across GH1 genotypes among 293 subjects (135 case patients and 158 control subjects) in the adenoma study (Table 3). The adjusted means for the level of plasma IGF-I and the IGF-I/IGFBP-3 ratio were lower for the A/A genotype than for the T/T genotype (for both, P = .01). The mean level of plasma IGFBP-1 was higher for individuals with the A/A genotype than for those with the T/T genotype (P = .03). Although limited by the small number of subjects examined, an ethnic-specific comparison found that the pattern of a lower ratio of IGF-I/IGFBP-3 and higher level of IGFBP-1 was associated with the A/A genotype. This pattern was consistent across ethnic groups and was particularly strong in Native Hawaiians. The mean IGF-I/IGFBP-3 ratio for the T/T, T/A, and A/A genotypes (and the P value for differences between T/T and A/A genotypes) were as follows: for Japanese, 0.21, 0.21, and 0.19 (P = .14); for Caucasians, 0.22, 0.22, and 0.21 (P = .57); and for Hawaiians, 0.28, 0.21, and 0.21 (P = .02). Similarly, an analysis stratified on age (less than or equal to the median age of 70 years or greater than the median age) suggested that the differences in IGF-I/IGFBP-3 ratios between the GH1 genotypes were not limited to younger subjects. No differences were found among GH1 genotypes for height, body mass index, or caloric intake (Table 3
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DISCUSSION |
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GH1 is located in the human GH gene cluster on chromosome 17q and is expressed only in the anterior pituitary gland. GH secretion and mean diurnal levels of plasma GH peak around puberty and decline progressively with age. Although the T1663A substitution studied herein is located in a nontranscribed region, it has been shown by Hasegawa et al. (12) to be in close linkage disequilibrium with two GH1 promoter variants (G218T and G439T). The 1663A allele might also be in linkage disequilibrium with a mutation in one of the intronic splice enhancers identified in GH1 (16). Thus, this polymorphism may be related to gene expression or protein dysfunction. Indeed, in the same Japanese study, the 1663A allele was associated with decreased levels of plasma GH and IGF-I and height among prepubertal short children with mild or no GH insufficiency (12). A similar but weaker association was also observed in adults for IGF-I levels but not for height (12). We also found that the AA genotype was associated with lower levels of plasma IGF-I and was unrelated to attained height in adults. Because GH is the main determinant of circulating levels of IGF-I (1), these data are consistent with lower GH secretion for individuals with the A allele.
If it is confirmed that the 1663A allele confers lower levels of GH and IGF-I, the association with lower risk of colorectal cancer found in this study could be related to the mitotic and antiapoptotic function of IGF-I (1). Normal and malignant cells of the large bowel have IGF-I receptors; when activated by IGF-I, these receptors stimulate cell proliferation (17,18). IGF-I may also promote angiogenesis by increasing the production of vascular endothelial growth factor (19). Consistent with these experimental data, high but normal plasma IGF-I levels were found to increase the risk of colorectal cancer (68), especially when combined with low levels of IGFBP-3 (6,7). IGFBP-3 may also exert an independent effect because it not only decreases the amount of bioactive IGF-I but also induces apoptosis (20). Because GH increases circulating levels of both IGF-I and IGFBP-3 (1), the net effect of GH on cancer development should reflect the differential increase in the levels of IGF-I relative to those of IGFBP-3. There is evidence that excessive GH secretion in acromegalic patients results in a net increase in the IGF-I/IGFBP-3 ratio and that a GH deficiency results in an increased level of IGFBP-1 (21,22). Our finding that the GH1 1663 A allele is related inversely to the IGF-I/IGFBP-3 ratio and directly to the level of IGFBP-1 indicates that this may also be true in the physiologic range of GH secretion.
GH may also act locally in colorectal tissue, independently of the endocrine effect of IGF-I. GH receptors are expressed at high levels in colorectal carcinomas, especially in advanced tumors and at lower levels in normal colon tissue (23). GH can directly stimulate mitogenesis by rapid induction of c-myc expression (24). Finally, elevated levels of IGF-I or GH in transgenic mice result in different patterns of abnormalities, suggesting that the pathogenesis of excess GH is not mediated solely by IGF-I (25). Indeed, in acromegaly and during adolescence, high levels of GH serum induce insulin resistance and have been correlated with elevated levels of serum insulin in humans (26,27). In prospective studies, hyperinsulinemia is independently associated with the incidence of colorectal cancer (8,28).
The exact mechanisms notwithstanding, the best evidence for a role of GH in colorectal neoplasia comes from studies of acromegaly patients in whom an increased risk for colon adenoma and adenocarcinoma and an increased proliferation of colonic epithelial cells have been well documented (25,29). Inhibition of GH secretion by administrating a synthetic somatostatin analogue to patients with colorectal cancer also reduces the proliferative activity of colorectal cancer cells (30). Finally, indirect evidence for a possible role of childhood and adolescence GH production in colon cancer comes from reports of an association between height and colon cancer, because GH plays a central role in regulating somatic longitudinal growth. Four cohort studies have found that an increased risk of colorectal cancer was associated with greater attained height (12).
The frequency of the 1663A variant allele in Japanese in Hawaii (42%) was the same as that found in Japan by Hasegawa et al. (12) and in Caucasians in our study. The high frequency (56%) of this allele and the stronger associated risk reduction observed in Native Hawaiians are of interest because this group, like other Polynesians, has unexpectedly low rates of colon cancer (31). It is unclear why the association with colorectal cancer was not observed in Japanese because the A allele seems to affect circulating levels of IGF-I in this ethnic group as well. However, animal studies have shown that nutritional factors influence synthesis and action of various growth hormones. For example, fasting decreases GH binding in the liver, and IGF-I receptor expression in colonocytes is reduced in rats receiving a low-fat diet (32,33). Thus, the lack of association between GH1 and colorectal cancer in Japanese may be due to their low-fat diet (34) or other luminal or circulating factors. Nevertheless, in our data, there was no suggestion of an interaction between GH1 and recent caloric and fat intakes. Alternatively, other downstream, unidentified genetic risk factors over-represented in Japanese could also explain the lack of association in this group.
Our data indicate that further examination of the associations of genetic polymorphisms, growth hormones, and the risk of colorectal cancer is warranted. If the relationships observed in this study are confirmed, they should justify the investigation of approaches to reduce the bioactivity of growth hormones in groups at high risk for colorectal cancer.
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NOTES |
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1 Editor's note: SEER is a set of geographically defined, population-based, central cancer registries in the United States, operated by local nonprofit organizations under contract to the National Cancer Institute (NCI). Registry data are submitted electronically without personal identifiers to the NCI on a biannual basis, and the NCI makes the data available to the public for scientific research.
We thank the Hawaii Tumor Registry, Castle Medical Center, Kaiser-Permanente Medical Center, Kuakini Medical Center, Queen's Medical Center, the Pacific Health Research Institute, Straub Clinic and Hospital, St. Francis Medical Center, and Wahiawa General Hospital for their collaboration. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views or policies of these institutions. We also thank Aleli Vinoya and Lucy Shen from our program for assistance with data analysis.
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Manuscript received August 28, 2001; revised January 17, 2002; accepted January 22, 2002.
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