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Association between Phosphatidylinositol 3-Kinase Regulatory Subunit p85 Met326Ile Genetic Polymorphism and Colon Cancer Risk

Authors' Affiliations: ¹ Departments of Family Medicine-Research Division, Epidemiology, and Biostatistics, Case Western Reserve University/University Hospitals of Cleveland; ² Case Center for Transdisciplinary Research on Energetics and Cancer, Case Comprehensive Cancer Center; ³ Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio; and ⁴ Cancer Control Program, University of Kentucky, Lexington, Kentucky

Requests for reprints: Li Li, Department of Family Medicine-Research Division, Case Western Reserve University, 11001 Cedar Avenue, Suite 306, Cleveland, OH 44106-7136. Phone: 216-368-5437; E-mail: lxl62{at}cwru.edu.

	Abstract

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Purpose: The phosphatidylinositol 3-kinase signaling pathway is frequently activated in cancer. Emerging evidence supports the p85 regulatory subunit gene, PIK3R1, as a novel oncogene.

Experimental Design: We examined the association of a functional missense polymorphism (Met326Ile) of PIK3R1 with colon cancer risk in a population-based case-control study of 421 incident cases and 483 controls.

Results: In our base unconditional logistic regression model controlling for age, gender, and race, we observed a 47% increase in risk among those carrying one or two copies of the 326Ile variant (P = 0.01). Further adjustment for family history of colorectal cancer, body mass index, nonsteroidal anti-inflammatory drugs, smoking, alcohol consumption, and physical activity strengthened the association [odds ratio (OR), 1.73; 95% confidence interval (CI), 1.24-2.42, P = 0.001]. The association was more pronounced among those older than 64 years (OR, 2.10; 95% CI, 1.19-3.70, P = 0.01). Evaluation of the genotypes assuming an additive mode of inheritance showed a significant trend for gene-dose response, where compared with Met/Met, the OR estimates for Ile/Met and Ile/Ile were 1.68 (95% CI, 1.19-2.37) and 2.27 (95% CI, 0.98-5.29), respectively (P for trend = 0.001).

Conclusions: This study is the first to describe a significant association between a germ line functional variant in PIK3R1 and cancer, providing new evidence supporting a role for PIK3R1 in the development of colon cancer.

A number of genetic and functional studies have established an important role for the PI3K signaling pathway in the development of cancer (5, 7). It has been shown that activation of the PI3K pathway occurs in most tumor types through a variety of mechanisms (8). The heterodimeric PI3Ks are composed of catalytic and regulatory subunit variants encoded by separate genes and alternative splicing. Genomic amplification, deletions, and more recently, somatic missense mutations in the PIK3CA gene, which code for the p110 catalytic subunit of PI3K, have been reported in a number of different tumor types (9–12), including a large series of colorectal cancers which revealed a high frequency (31.6%) of somatic mutations in PIK3CA (13).

Somatic mutations in the PIK3R1 gene, which encodes the p85 regulatory subunit, have also been reported in primary colon tumors (14). Expression of one of these mutant proteins led to constitutive activation of the PI3K complex, providing the first evidence that PIK3R1 could act as an oncogene in colon tumor cells (14). Mouse models containing a heterozygous deletion of the PTEN gene show a near doubling of the incidence of intestinal polyps when at least one allele of the PIK3R1 gene is also deleted (15).

A single nucleotide missense polymorphism in codon 326 of PIK3R1 results in the substitution of methionine (Met) by isoleucine (Ile) near the NH₂-terminal SH2 domain coding region that is crucial for the binding of receptor tyrosine kinases. Functional studies show that the PIK3R1 Met326Ile polymorphism results in reduced p85 protein expression and increased binding to IRS-1 (16). The regulatory subunit p85 is an important component of the PI3K pathway, and it has been proposed that a reduction of p85 protein would lead to reduced negative regulation of PI3K and hyperactivation of the PI3K pathway (17). The only epidemiologic study of human cancer reported thus far found no association of the PIK3R1 Met326Ile polymorphism and risk of prostate cancer (18). The potential role of this genetic variant in the development of colon cancer remains unexplored.

In this population-based case-control study, we seek to investigate the relationship between this functional genetic polymorphism and colon cancer. We hypothesize that the PIK3R1 326Ile variant is associated with an increased risk of colon cancer.

	Materials and Methods

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Study population. The study population was comprised of 421 incident colon cancer cases and 483 population controls. All subjects were recruited between July 2003 and April 2006. Eligible cases were identified through the population-based Surveillance, Epidemiology, and End Results Kentucky Cancer Registry, covering all residents living in the State of Kentucky at the time of diagnosis. We queried the cancer registry database every 3 months and identified all histopathologically confirmed incident primary colon cancer cases reported within 6 months of diagnosis preceding the recruitment.

We first sent an introductory letter explaining the study to potentially eligible cases. After 3 weeks, or when the subject initiated contact, we phoned each case subject for a screening interview to determine eligibility and their willingness to participate in the study. We collected information on demographics, family history of colorectal cancer, and personal history of cancer at the recruitment phone call. We then used a two-step approach to collect blood samples and lifestyle questionnaire data. First, we sent a prepacked phlebotomy kit with detailed written instructions for blood sample collection and written consent forms to each consenting case subject. The subjects were instructed to go to their physician's offices or adjacent medical facilities for blood draw after overnight fasting. The samples were collected in Purple Top (K₃EDTA) blood collection tubes and shipped overnight on frozen ice packs. Upon receipt, the blood tubes were spun for 15 min at 600 x g and aliquots of plasma and concentrated buffy coat were prepared and frozen at –80°C. We then sent each subject a self-administered lifestyle risk factor questionnaire developed by the National Cancer Institute Colon Cancer Family Cancer Registry⁵ to collect detailed information on family history of colorectal cancer, lifestyle, and behavioral risk factors.

We used random digit dialing to recruit population controls following a protocol similar to that described above for cases. As a proxy for frequency matching, the residential locations between the cases and the controls, the area codes and exchanges of the phone numbers of potential cases were used, along with randomly generated four-digit numbers, to produce the list of phone numbers for control recruitment. Recruitment was not conducted when a business was reached. We included as controls subjects who were 40 years or older and free of personal history of cancer other than skin cancer. We excluded those with known inflammatory bowel diseases, family history of familial adenomatous polyposis, and hereditary nonpolyposis colorectal cancer. As with case subjects, each control first donated a sample of blood after fasting overnight and then was asked to complete the self-administered risk factor questionnaire.

The participation rates were 72.2% for case subjects. Due to the nature of random digit dialing, the response rate for controls cannot be precisely assessed as we could not determine the eligibility of those subjects that were reached, but refused to disclose personal information. Nevertheless, among those individuals that allowed us to explain the study and were determined to be eligible, the participation rate was 62.5%. The overall participation rate among potential controls reached by random digit dialing, regardless of their eligibility, was 33.3%. The completion proportions—i.e., the percentage of participants that returned blood samples and completed the risk factor questionnaire—were 86.5% for cases and 91.3% for control subjects, respectively. All participants provided written informed consent. The study was approved by the Institutional Review Boards of the University of Kentucky (Lexington, KY), and Case Western Reserve University/University Hospitals of Cleveland (Cleveland, OH).

Genotypes. The PIK3R1 Met326Ile variant (rs3730089) was genotyped using the TaqMan allelic discrimination assay with a predesigned primer/probe set (C_25474527_10; Applied Biosystems). Assays were done on 2.5 ng of genomic DNA extracted from buffy coat fractions (Biorobot EZ1; Qiagen) which were quantitated with Quant-It picogreen reagent (Invitrogen). Assays were read and genotypes assigned using a 7900HT Sequence Detection System with SDS 2.2 software (Applied Biosystems). The genotyping failure rate was <0.1%. Two percent of the replicate samples were sequenced for quality control, and had a concordance rate of 100%. Laboratory personnel were blinded to case-control status.

Lifestyle risk factors. Body mass index (BMI) was calculated based on self-reported current weight (kg) divided by height in meters squared (kg/m²). A positive family history of colorectal cancer was defined as reporting colorectal cancer in one or more first-degree relatives. Regular nonsteroidal anti-inflammatory drug (NSAID) use was defined as reporting usage at least twice a week for 6 or more months. Recreational physical activity, as assessed by metabolic equivalents of energy expenditure units (METS) for different life periods (in their 20s, 30s and 40s, and since turning 50) were grouped into three categories as "light" (METS < 3.0), "moderate" (METS = 3-6), and "vigorous" (METS > 6). We chose to use recreational physical activity in the 20s in the regression analysis as this is the variable that showed the strongest association with risk of colon cancer in our study population, and seemed to be closely correlated with overall adult physical activity. Smoking and alcohol were both grouped into "never" and "ever" based on self-reported usage. Ever smoking was defined as smoking at least one cigarette a day for 3 months or longer. Ever use of alcohol was defined as consuming any alcoholic beverages at least once a week for 6 months or longer.

Statistical analysis. We first examined the Hardy-Weinberg equilibrium of allelic distribution separately for the cases and controls, and compared the allele frequencies between cases and controls. We then evaluated the association of PIK3R1 Met326Ile genotypes with colon cancer in multivariate unconditional logistic regression models assuming dominant (Ile/Ile and Met/Ile versus Met/Met), additive (Ile/Ile, Met/Ile, versus Met/Met) or recessive (Ile/Ile versus Met/Met and Met/Ile) modes of inheritance, respectively. In our base models, we adjusted for age (at diagnosis for the cases and at recruitment for the controls), gender, and race. In our full model, we further controlled for family history of colorectal cancer, BMI, NSAIDs, alcohol use, smoking, and recreational physical activity in the 20s. We further explored potential effect modification by age in analyses stratified by the cases' median age (64 or >64 years) at diagnosis, and tested multiplicative interactions with BMI (centered at the mean), NSAIDs, and family history of colorectal cancer by including the main effects and cross-product terms in a logistic regression model. Statistical significance was assessed via both Wald test and likelihood ratio test, comparing full and reduced models (i.e., with and without the cross-product term). All P values were two-sided, and all analyses were undertaken with SAS software (version 9.1; SAS Institute, Inc.).

	Results

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The majority (93.4%) of the participants were Caucasians (Table 1 ), consistent with the general population in Kentucky. Cases were older at diagnosis than the controls at recruitment, were more likely to have a positive family history of colorectal cancer, had higher BMI, and reported less regular NSAID use. The Ile variant allele was more prevalent among cases (20.1%) than among controls (14.3%). The allele frequency distribution in our control population is comparable with reports by others (18), and conforms to Hardy-Weinberg proportions in both case and control groups (P > 0.1).

	Discussion

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In this population-based case-control study, we found a statistically significant association between the PIK3Rl Met326Ile polymorphism and risk of colon cancer. Those carrying two copies of the Ile variant had a >2-fold increase of risk, and there was a linear trend for gene-dose response relationship. The risk associated with the Ile variant was more pronounced among those older than 64 years. To our knowledge, this is the first study to show a significant association of this functional genetic variant with colon or any type of cancers.

One previous report from the Physician's Health Study found no overall association for the PIK3R1 Met326Ile polymorphism with risk of prostate cancer, although stratified analyses were suggestive of a weak protective effect against high grade/stage diseases for carriers of two copies of the 326Ile variant (OR, 0.61; 95% CI, 0.17-2.17; ref. 18). To this end, it is noteworthy that although somatic mutations of PI3KCA, the gene coding for the catalytic unit of PI3K, have been detected in a number of solid tumors, with the highest frequency in colorectal cancer, there is a lack of evidence of somatic mutations of PI3KCA in prostate cancer (19).

Recent studies provide insight into the mechanisms by which the p85 subunit can negatively regulate PI3K signaling (17). The p85 regulatory subunit of PI3K is necessary for the stability and membrane recruitment of the p110 catalytic subunit. However, in its unbound, monomeric form, p85 can limit the extent of PI3K signaling by competing with the p85-p110 dimer for binding to IRS-1 leading to its sequestration in the cytosol. IRS-1 is a major adaptor molecule that mediates PI3K activation downstream of insulin and insulin-like growth factor-I receptors. A reduction in the gene dosage of p85 is postulated to compromise this dominant negative regulation of IRS-1/PI3K signaling and therefore lead to the hyperactivation of this pathway. The tumor suppressor PTEN, functions through inactivation of the PI3K pathway, and p85 has been shown to regulate PTEN activity directly (20). Furthermore, in the PTEN+/– mouse model, p85 heterozygosity leads to a 2-fold increase in the incidence of intestinal polyps (15). It is therefore consistent that a functional genetic polymorphism of PIK3R1 that results in reduced p85 protein expression and increased IRS-1 binding would lead to enhanced PI3K signaling and hence cancer development.

Although more than 40 tagging single nucleotide polymorphisms have been reported for the PIK3R1 gene,⁶ in this study, we carried out a focused investigation of the Met326Ile variant as that is the only known nonsynonymous coding single nucleotide polymorphism that has been shown to be functional. Others have attempted to evaluate gene-wide association with insulin resistance–related phenotypes or biomarkers using selected sets of tagging single nucleotide polymorphisms, and reported inconsistent results (21–24). In a large female twin study of eight selected tagging single nucleotide polymorphisms, Jamshidi et al. found associations between other single nucleotide polymorphisms in PIK3R1 with serum leptin levels, body fat, and other measures of insulin resistance, but reported no association for the Met326Ile variant (21). An earlier Danish study of 380 young healthy subjects reported a significant association of this variant with insulin sensitivity (22), but the same research group failed to replicate that association in a subsequent study (23). We did not find an association of the Met326Ile variant with BMI in our sample.

There are some potential limitations to our study. Controls were randomly recruited regardless of their colon screening status, and it is possible that we included some unscreened controls with asymptomatic adenomatous polyps, a precursor for colon cancer. However, it is important to bear in mind that excluding these participants from the analysis would bias the risk estimate away from the unity. As in all case-control studies, we cannot exclude the possibility of bias in recalling questionnaire-based lifestyle risk factors. Another limitation is the relatively modest participation rate among the controls recruited in our study by random digit dialing. However, it is not conceivable that the controls in our study would have been disproportionately enriched with one allele or another. It is also possible that the sickest individuals with colon cancer were less likely to participate in the study. We attempted to minimize this bias by recruiting incident cases within 3 to 6 months of their diagnosis and by allowing the patients to have blood drawn at scheduled follow-up visits to their personal physicians. Also, we found no difference in stage distribution across the three genotypes among the cases (data not shown). It is thus unlikely that selection bias could have potentially accounted for our observed associations. Moreover, our findings of a protective effect of NSAID use (OR, 0.69; 95% CI, 0.50-0.96, P = 0.02) and a positive association with family history of colorectal cancer (OR, 2.13; 95% CI, 1.46-3.11, P < 0.001) are in agreement with their well-documented associations with colon cancer (25), lending credibility to our data. In any study of genetic factors, investigators must be concerned about potential bias due to population stratification. We addressed this issue first by adjusting for ethnicity in our analyses. In addition, when restricting our analysis to Caucasians only, we observed results very similar to those presented here. This suggests that our findings were unlikely to be due to population stratification bias.

In summary, we found that the functional PIK3R1 Met326Ile polymorphism is associated with an increased risk of colon cancer. This observation is consistent with the dominant negative regulation of PI3K signaling by the p85 subunit, providing important new evidence supporting PIK3R1 as a novel susceptibility gene in colon cancer.

	Footnotes

 
Grant support: Damon Runyon Cancer Research Foundation Clinical Investigator Award, CI-8 (L. Li); the Case Center for Transdisciplinary Research on Energetics and Cancer, U54 CA-116867-01 (L. Li); National Cancer Institute K22 Award, K22 CA120545-01 (L. Li); State of Ohio Biomedical Research and Technology Transfer Commission (G. Casey); and National Cancer Institute Award R25 CA094186-06 (C. Thompson).

⁵ http://epi.grants.cancer.gov/CFR/about__questionnaires.html

⁶ http://www.HapMap.org

Received 5/17/07; revised 10/17/07; accepted 11/ 7/07.

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This Article Abstract Full Text (PDF) Correction (v14,p7963) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, L. Articles by Casey, G. Search for Related Content PubMed PubMed Citation Articles by Li, L. Articles by Casey, G. Related Collections Epidemiology and Prevention Epidemiology and Prevention: Genetics and Biomarkers