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. Author manuscript; available in PMC: 2008 Jun 16.
Published in final edited form as: Int J Cancer. 2008 Apr 1;122(7):1611–1617. doi: 10.1002/ijc.23135

Leptin and leptin receptor genotypes and colon cancer: Gene–gene and gene–lifestyle interactions

Martha L Slattery 1,*, Roger K Wolff 1, Jennifer Herrick 1, Bette J Caan 2, John D Potter 3
PMCID: PMC2430084  NIHMSID: NIHMS50851  PMID: 18059035

Abstract

Leptin may play an important role in colorectal cancer because of its role in energy balance, insulin and inflammation. We evaluated the LEP rs2167270 (19 G > A) and rs7799039 (−2548 G > A) polymorphisms and the leptin receptor, LEPR rs6588147 (located in intron 2), polymorphism with risk of developing colon cancer in a study of 1,567 cases and 1,965 controls. We evaluated the effects of the polymorphisms with body mass index (BMI), recent use of aspirin/NSAIDs and genetic variations in genes related to insulin signaling pathways including insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3), and insulin- related substrates 1 and 2 (IRS1, IRS2) and the vitamin D receptor (VDR). We observed a slight reduction in colon cancer risk with the AA LEP rs2167270 genotype (OR 0.79 95% CI 0.64, 0.98) and although not reaching statistical significance, with the combined GG LEP rs2167270 and GG LEPR rs6588147 (OR 0.70, 95% CI 0.49, 1.02) genotypes. BMI did not interact with any of these polymorphisms to alter colon cancer risk. However, recent aspirin/NSAID use significantly interacted with both LEP polymorphisms. Likewise, variants of IGF1 and IRS2 interacted with the LEP rs2167270 polymorphism. VDR polymorphisms interacted with all LEP and LEPR polymorphisms. These data support an association between LEP and colon cancer. They also suggest that the mechanisms linking leptin to colon cancer may be independent of energy balance.

Keywords: colon cancer, leptin, leptin receptor, polymorphisms, VDR, aspirin, obesity, insulin, inflammation

Leptin is a 16 kDa glycolprotein product of the leptin gene (LEP), which is expressed almost exclusively (>95%) by adipocytes. 1 Initial interest in leptin focused on its role in obesity but it is rapidly becoming evident that its physiological properties extend beyond those associated with energy homeostasis. In addition to regulation of energy balance, leptin, has been associated with reproductive factors, inflammatory response, insulin signaling, bone remodeling and neuroendocrine function. 2 The leptin receptor, a class I pro-inflammatory cytokine, is a member of the cytokine receptor family that includes IL-6. The leptin receptor plays a key role in how leptin functions. When leptin binds to the leptin receptor, Janus kinase (JAK2), a tyrosine kinase is activated to initiate downstream signaling of suppressors of cytokine signaling 1 and 3 (SOCS) and signal transducer and activator of transcription-3 (STAT).2–4 Of particular importance for cancer is the influence of leptin on SOCS which in turn limits insulin signaling; SOCS3 has been shown to limit leptin signaling. Studies have shown that leptin also can stimulate expression of the inflammatory marker CRP (C-reactive protein).5

There is growing evidence that nuclear hormone receptors regulate the action of leptin and other class I cytokines.6 However, data also suggest that hormone receptors, such as the vitamin D receptor, might play an important role in regulating leptin; vitamin D3 has been shown to inhibit leptin secretion in human adipose tissue.7 Leptin has been shown to attenuate expression of renal 25-hydroxyvitamin D(3)-1α-hydroxylase in mice via the leptin receptor.8

Although data directly linking leptin to colon cancer are limited, some studies have shown increased risk of colon and colorectal cancer with high serum leptin levels.9–11 Observations that the leptin receptor is expressed in human colon cancer cells lines and CRC derived human tissue lends additional support to leptin playing a role in CRC.12 Energy balance, adiposity, insulin, inflammation and vitamin D have been associated with colon cancer and also are associated with leptin and its receptor.13–17

Polymorphisms in the leptin and leptin receptor gene have been studied in conjunction with leptin levels and obesity and might also provide insight into associations with cancer. In one study, having an A allele at the −2548 site (rs7799039) in the leptin gene (LEP) was associated with higher expression of the leptin receptor as well as with prostate cancer.18–20 Lower leptin binding to the leptin receptor has been shown with the A allele of the Gln223Arg SNP (rs1137101) of the leptin receptor (LEPR) gene.21 Two other SNPs, Ser343Ser (rs790419) in exon 1 and Lys109arg (rs1137100) in exon 4 of the LEPR gene, have been associated with obesity in women in France.22

To obtain a better understanding of the association between leptin and colon cancer, we evaluated 2 variants of the LEP gene, the more commonly studied rs7799039 as well as rs2167270 and 1 variant of the LEPR gene, rs6588147 with risk of developing colon cancer. These variants were selected because of previous reports of their reported functional importance23 and their being in linkage disequilibrium with other common LEP markers such as rs2167270 and rs3828942. These analyses, therefore, serve as an initial evaluation of the potential importance of LEP and LEPR genes in colon cancer. In addition to evaluating the independent associations between LEP and LEPR polymorphisms and colon cancer, we evaluated the joint effects of LEP and LEPR polymorphisms with factors available in our dataset for which we believe there is a biological basis for an interaction. We evaluated how LEP and LEPR polymorphisms were related to BMI, an indicator of energy balance since leptin has been associated with energy homeostasis. We evaluated recent use of aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs), possible indicators of inflammation, since LEP and LEPR are pro-inflammatory cytokines. We evaluated genes involved in an insulin pathway, including insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein (IGFBP3), insulin-receptor substrates 1 and 2 (IRS1 and IRS2) since leptin levels are associated with insulin. We evaluated the vitamin D receptor (VDR) genotypes given reports of the associations between the nonsteroid nuclear receptor VDR and leptin levels.

Methods

Data for the study come from a case-control study of first primary colon cancer (ICD-O 2nd edition codes 18.0, 18.2 to 18.9) diagnosed between October 1, 1991 and September 30, 1994 conducted in the Northern California Kaiser Permanent Medical Care Program (KPMCP), the Wasatch Front area of Utah and the Twin Cities Metropolitan area of Minnesota. The study was approved by the University of Utah Institutional Review Board as well as Institutional Review Boards at the Kaiser Permanente Medical Care Program of Northern California (KPMCP) and the University of Minnesota. Case eligibility was determined by the Surveillance Epidemiology and End Results24 Cancer Registries in Northern California and in Utah and the Minnesota Cancer Surveillance System. Eligibility included being between 30 and 79 years of age at time of diagnosis, English speaking, mentally competent to complete the interview, no previous history of colorectal cancer14 and no known (as indicated on the pathology report) familial adenomatous polyposis, ulcerative colitis or Crohn’s disease. Of cases contacted, 83% participated at KPMCP, 76% in Utah and 67% in Minnesota.

Controls were frequency matched to cases by sex and by 5-year age groups. At the KPMCP, controls were randomly selected from membership lists. In Utah, controls 65 years and older were randomly selected from lists provided by the Centers for Medicare and Medicaid Services (formerly HCFA) and controls younger than 65 were randomly selected from driver’s license lists. In Minnesota, controls were randomly selected from driver’s license lists. Of controls contacted for the colon cancer study, 73% participated at KPMCP, 53% participated from Minnesota and 69% participated from Utah.

Data collection

Trained and certified interviewers collected diet and lifestyle data.25,26 The referent year for the study was the calendar year ~2 years before date of diagnosis (cases) or selection (controls). Information was collected on demographic factors such as age, sex and study center; diet, physical activity, aspirin and nonsteroidal drug use, body size, and other lifestyle factors including medical, family and reproductive history.

Genotyping

DNA was extracted from blood drawn from study participants. TaqMan assays for Leptin markers LEP rs2167270 (19 G > A) and LEP rs7799039 (~2548 G > A), and Leptin Receptor marker LEPR rs6588147 (located in intron 2) were purchased from Applied Biosystems (Foster City, CA). Genotyping was performed on 20 ng of genomic DNA as described by the manufacturer. Data were collected as .fluorescent endpoint measurements of the TaqMan reactions using a 7900HT sequence detection instrument. Control samples representing all 3 possible genotypes were included at 4 positions each in every 384-well tray. In addition, internal replicates representing >1% of the sample set were blinded and included.

The intron 8 Bsm I (rs154410) and Fok1 (rs10735810) VDR polymorphisms were amplified from genomic DNA and digested as described previously.27,28 Presence of the restriction site was scored as allele “b,” and absence of the restriction site was scored as allele “B.” Presence of the restriction site was scored as allele “f,” absence of the restriction site was scored as allele “F.” Genotyping of IGF1, IGFBP3, IRS1, and IRS2 have been described.29,30 The IGF1 CA repeat, the IGFBP3 −202 A>C, the IRS1 G972R and the IRS2 G1057D polymorphisms were evaluated.

Statistical methods

SAS statistical package version 9.1 was used to conduct the analyses. We evaluated the distribution of the LEP and LEPR genotypes and the independent associations of these genetic polymorphisms with colon cancer. Odds ratios (OR) and 95% confidence intervals (CI) are used to report associations obtained from multiple logistic regression models. Associations were evaluated for men and women separately, for proximal and distal colon tumors and by age. Associations were adjusted for age, sex, center and race because these variables were matching variables in the study design. Since over 90% of study participants were non-Hispanic white, it was not feasible to assess associations for different racial and ethnic groups. Factors such as body mass index,31,32 physical activity and family history of colorectal cancer, cigarette smoking and dietary composition did not alter associations and therefore are not included as covariates in the final logistic models. We report the joint effect of LEP and LEPR genotypes and BMI (kg/m2), aspirin/NSAIDs, Bsm1 and Fok1 VDR polymorphisms, and IGF1, IGFBP3, IRS1 and IRS2 polymorphisms. For the IRS1, IRS and Fok1 VDR genes, we evaluated a dominant model because of similar associations across heterozygote and homozygote variant genotypes. For joint effects, multivariate logistic regression models were used to calculate odds ratios for each category of exposure and each genotype. Because we did not observe significant differences in effect by gender for interactions, we present interaction models for the entire population. Effect modification between genotypes and exposure variables were evaluated by likelihood ratio test for a multiplicative-interaction term in the logistic regression model.

Results

The AA genotype of the LEP rs2167270 SNP was associated with reduced risk of colon cancer (OR 0.79 95% CI 0.64, 0.98) (Table I). The rs7799039 LEP SNP was not significantly associated with risk of colon cancer among men, women or everyone combined. Among men, the LEPR rs6588147 SNP reduced risk of colon cancer (OR 0.71 95% CI 0.52, 0.97). Evaluation of both the LEPR and LEP SNPs together, showed the greatest reduction in colon cancer risk for the AA genotype of the LEP rs2167270 SNP and the GG genotype of the rs6588147 LEPR SNP. However, the interactions between LEPR and LEP were not statistically significant. Subsequent analyses are therefore presented for each SNP separately with men and women combined.

TABLE I.

Association between Leptin (LEP) and Leptin Receptor (LEPR) Genotypes and Colon Cancer

LEPR (rs6588147)
AA
 AG
 GG
Controls (n) Cases (n) OR1 95% CI Controls (n) Cases (n) OR 95% CI Controls (n) Cases (n) OR 95% CI Controls (n) Cases (n) OR 95% CI
Men 477 417 1.00 Reference 454 393 1.00 0.83, 1.21 123 75 0.71 0.52, 0.97
Combined rs6588147 and rs7799039
LEP rs 7799039 GG 363 287 1.00 Reference 166 146 1.00 Reference 156 118 0.89 0.64, 1.24 37 22 0.70 0.39, 1.25
GA 487 444 1.19 0.97, 1.46 216 204 1.12 0.83, 1.51 211 200 1.13 1.84, 1.53 58 37 0.76 0.48, 1.23
AA 210 157 0.98 0.75, 1.27 95 67 0.84 0.57, 1.25 87 74 1.02 0.69, 1.50 28 16 0.68 0.35, 1.32
Combined rs6588147 and rs2167270
LEP rs2167270 GG 438 349 1.00 Reference 207 160 1.00 Reference 177 162 1.20 0.89, 1.62 54 27 0.66 0.40, 1.10
GA 452 424 1.18 0.97, 1.43 190 210 1.44 1.08, 1.91 212 174 1.08 0.81, 1.44 48 39 1.07 0.67, 1.71
AA 165 111 0.84 0.64, 1.11 79 47 0.76 0.50, 1.16 65 54 1.09 0.72, 1.65 21 9 0.56 0.25, 1.26
Women 411 349 1.00 Reference 396 262 0.80 0.64, 0.99 108 80 0.90 0.65, 1.25
Combined rs6588147 and rs7799039
LEP rs2167270 GG 275 219 1.00 Reference 136 117 1.00 Reference 109 77 0.86 0.59, 1.27 28 24 1.09 0.59, 1.99
GA 459 345 1.01 0.80, 1.27 183 167 1.10 0.79, 1.53 204 134 0.84 0.60, 1.18 57 42 0.95 0.59, 1.52
AA 188 129 0.92 0.69, 1.24 81 64 1.01 0.66, 1.53 83 51 0.79 0.51, 1.22 23 14 0.78 0.38, 1.59
Combined rs6588147 and rs2167270
LEP rs2167270 GG 362 264 1.00 Reference 159 139 1.00 Reference 158 98 0.73 0.52, 1.02 44 27 0.73 0.43, 1.25
GA 417 345 1.14 0.92, 1.42 185 169 1.06 0.78, 1.44 178 134 0.89 0.65, 1.23 51 40 0.93 0.58, 1.50
AA 139 84 0.82 0.60, 1.13 66 39 0.67 0.42, 1.06 59 30 0.59 0.36, 0.97 13 13 1.20 0.54, 2.67
Everyone 881 759 1.00 Reference 848 650 0.91 0.79, 1.05 230 155 0.81 0.64, 1.02
Combined rs6588147 and rs7799039
LEP rs7799039 GG 634 499 1.00 Reference 301 260 1.00 Reference 265 193 0.88 0.69, 1.14 65 46 0.88 0.58, 1.34
GA 938 782 1.13 0.97, 1.33 408 368 1.13 0.90, 1.42 414 332 1.03 0.82, 1.29 114 79 0.89 0.63, 1.25
AA 393 284 0.98 0.80, 1.19 172 130 0.95 0.71, 1.28 169 124 0.92 0.68, 1.23 51 30 0.75 0.46, 1.22
Combined rs6588147 and rs2167270
LEP rs2167270 GG 794 611 1.00 Reference 361 297 1.00 Reference 335 260 0.96 0.76, 1.20 97 54 0.70 0.49, 1.02
GA 867 766 1.17 1.01, 1.35 375 378 1.24 1.00, 1.53 369 307 1.00 0.80, 1.24 99 79 1.02 0.72, 1.42
AA 304 190 0.79 0.64, 0.98 145 84 0.69 0.50, 0.94 124 83 0.81 0.59, 1.12 34 22 0.79 0.45, 1.39
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Adjusted for 5-year age group and race; everyone also adjusted for sex.

Because of previous associations with BMI observed only for men and for women who were recently exposed to estrogen because they were premenopausal or taking hormone replacement therapy,15 we present analyses for that group only. We did not observe a statistically significant interaction between BMI level and any of the LEP and LEPR polymorphisms (Table II). However, the GG genotype of the LEPR gene appeared to halve the risk of colon cancer for those with normal BMIs and those with a BMI of 30 or more. Among those with normal weight, the AA genotype of the LEP rs2167270 polymorphisms was associated with the greatest reduction in the risk of colon cancer (OR 0.63, 95% CI 0.39, 1.03) with a BMI of <25. There were no statistically significant interactions between these polymorphisms and other components of energy balance such as physical activity and energy intake (data not shown).

TABLE II.

Association between BMI and Leptin (LEP) and Leptin Receptor (LEPR) Gene Polymorphisms and Colon Cancer Risk

BMI
Normal (<25 kg/m2)
 Overweight (25 to <30 kg/m2)
 Obese (≥30 kg/m2)
Controls (n) Cases (n) OR1 95%CI Controls (n) Cases (n) OR 95%CI Controls (n) Cases (n) OR 95%CI
Men and women recently exposed to estrogen2
LEPR (rs6588147)
   AA 241 161 1.00 277 215 1.21 0.92, 1.59 105 148 2.04 1.47, 2.83
   AG 220 143 1.01 0.75, 1.35 282 210 1.17 0.89, 1.53 115 134 1.78 1.29, 2.45
   GG 71 24 0.51 0.31, 0.85 65 47 1.16 0.76, 1.79 39 25 0.95 0.55, 1.65
p interaction 0.25
LEP (rs 7799039)
   GG 155 97 1.00 223 149 1.06 0.76, 1.48 85 103 1.80 1.22, 2.67
   GA 279 167 0.95 0.69, 1.31 289 233 1.33 0.98, 1.82 109 153 2.22 1.55, 3.17
   AA 99 65 1.04 0.69, 1.56 118 93 1.32 0.91, 1.93 66 51 1.19 0.76, 1.87
p interaction 0.11
LEP (rs 2167270)
   GG 217 139 1.00 250 186 1.23 0.92, 1.64 111 113 1.59 1.13, 2.23
   GA 243 159 1.07 0.79, 1.43 274 228 1.38 1.04, 1.83 108 144 2.07 1.48, 2.88
   AA 73 29 0.63 0.39, 1.03 101 59 0.93 0.63, 1.37 40 49 1.87 1.16, 3.02
p interaction 0.08
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Odds ratios and 95% confidence intervals adjusted for 5-year. age category, sex and race.

2

Women recently exposed to estrogen are defined as those women who were premenopausal or who had taken HRT within the past 2 years.

We observed a statistically significant interaction between use of aspirin/NSAIDs during the past 2 years and both LEP polymorphisms and risk of colon cancer (Table III). For both the LEPR and LEP rs2167270 markers, those with the lowest colon cancer risk were individuals who currently used aspirin/NSAIDs and had the GG (LEPR) and AA (LEP) genotypes. For the rs7799039 LEP polymorphism, those who used aspirin and had the GG genotype had the lowest colon cancer risk. The association with leptin genotypes and nonusers of NSAID is null.

TABLE III.

Asssociation between Aspirin/NSAIDS use, Leptin (LEP) and Leptin Receptor (LEPR) and Colon Cancer Risk

Aspirin/NSAID Use
Aspirin/NSAIDS Controls (n) Cases (n) OR1 No current use 95% CI Controls (n) Cases(n) OR Current use 95% CI
LEPR (rs6588147) AA 531 528 1.00 357 238 0.67 0.55, 0.83
AG 496 449 0.92 0.78, 1.10 354 206 0.60 0.48, 0.74
GG 129 109 0.88 0.78, 1.16 102 46 0.46 0.32, 0.67
p interaction 0.38
LEP (rs7799039) GG 358 358 1.00 280 150 0.55 0.43, 0.70
GA 555 540 1.03 0.85, 1.25 391 249 0.67 0.54, 0.84
AA 250 191 0.82 0.64, 1.04 148 95 0.68 0.50, 0.91
p interaction 0.04
LEP (rs2167270) GG 484 407 1.00 316 206 0.77 0.62, 0.96
GA 502 544 1.29 1.08, 1.55 367 225 0.74 0.59, 0.91
AA 171 135 0.93 0.71, 1.20 133 60 0.54 0.39, 0.75
p interaction 0.04
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Odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, race and center.

Evaluation of insulin-related genes and risk of colon cancer showed significant interactions between both IGF1 and IRS2 polymorphisms and the LEP rs2167270 polymorphism (Table IV). People with the AA genotype of the LEP rs2167270 polymorphism and did not have a 19 CA repeat of the IGF1 gene were at reduced risk of colon cancer (OR 0.63, 95% CI 0.38, 1.06) compared to people with the 19/19 CA repeat genotype and the AA rs2167270 LEP polymorphism (OR 1.02 95% CI 0.72, 1.44). Similar interactions were observed with the IRS2 G1057D polymorphism, although the greatest reduction in risk was observed for the combination of the GG IRS2 genotype and the AA LEP rs2167270 genotype.

TABLE IV.

Associations between Insulin-Related Genes, Leptin (LEP), Leptin Receptor (LEPR) and Colon Cancer Risk

IGF1
19/19
 19/non 19
 non 19/non 19
Controls (n) Cases (n) OR 95%CI Controls (n) Cases (n) OR 95%CI Controls (n) Cases (n) OR 95%CI
LEPR (rs6588147)
   AA 341 296 1.00 421 354 0.97 0.78, 1.20 114 109 1.00 0.73, 1.37
   AG 352 264 0.87 0.70, 1.09 385 292 0.89 0.71, 1.10 102 89 1.02 0.73, 1.42
   GG 101 62 0.72 0.50, 1.03 94 74 0.91 0.65, 1.29 28 18 0.78 0.42, 1.46
   p interaction 0.55
LEP (rs7799039)
   GG 232 192 1.00 294 229 0.93 0.71, 1.21 104 83 0.85 0.59, 1.23
   GA 403 322 1.01 0.79, 1.29 436 359 1.03 0.81, 1.31 93 102 1.37 0.97, 1.93
   AA 163 112 0.85 0.62, 1.16 176 134 0.97 0.72, 1.31 49 31 0.80 0.49, 1.31
   p interaction 0.13
LEP (rs2167270)
   GG 331 224 1.00 368 284 1.16 0.92, 1.46 87 91 1.48 1.04, 2.09
   GA 358 321 1.37 1.09, 1.72 393 343 1.31 1.05, 1.64 103 101 1.43 1.03, 1.99
   AA 108 76 1.02 0.72, 1.44 140 94 0.98 0.71, 1.34 53 24 0.63 0.38, 1.06
   p interaction <0.01
IRS2
LEPR (rs6588147) GG GD/DD
   AA 380 357 1.00 495 406 0.90 0.74, 1.10
   AG 343 287 0.92 0.74, 1.13 499 356 0.79 0.64, 0.96
   GG 97 68 0.78 0.55, 1.10 126 85 0.75 0.55, 1.02
   p interaction 0.88
LEP (rs7799039)
   GG 274 226 1.00 357 273 0.99 0.78, 1.26
   GA 384 363 1.24 0.98, 1.57 550 421 1.01 0.81, 1.27
   AA 167 126 1.00 0.74, 1.34 220 155 0.93 0.70, 1.23
   P interaction 0.44
LEP (rS2 167270)
   GG 358 282 1.00 427 323 0.98 0.79, 1.21
   GA 334 356 1.36 1.10, 1.69 523 408 1.02 0.83, 1.25
   AA 131 75 0.72 0.52, 0.99 171 116 0.88 0.66, 1.17
   p interaction 0.04
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Odds rations (OR) and 95% confidence intervals (CI) adjusted for age category, sex and race.

The rs65888147 LEPR and the rs7799039 LEP polymorphisms significantly interacted with the Bsm1 VDR gene (Table V). For the rs67888147 marker, those with a B allele had the greatest reduction in risk if the also had the GG genotype (OR 0.75 95% CI 0.55, 1.02). Those with the VDR bb genotype and the AA rs7799039 LEP genotype had statistically significantly lower risk than those with the bb and GG genotypes. Both of the LEP markers interacted the Fok1 VDR polymorphisms. Among those with the f allele, those with the GG genotype of rs7799039 LEP and the AA genotype of the rs2167270 marker had the greatest reduced risk of colon cancer.

TABLE V.

Association between, VDR, Leptin (LEP) and Leptin Receptor (LEPR) and Colon Cancer Risk

VDR Fok1
FF
 Ff/ff
Controls (n) Cases (n) OR 95% CI Controls (n) Cases (n) OR 95% CI
LEPR (rs6588147)
   AA 312 315 1.00 510 405 0.81 0.66, 0.99
   AG 289 246 0.86 0.68, 1.08 515 375 0.74 0.60, 0.92
   GG 84 56 0.68 0.46, 0.98 130 82 0.65 0.47, 0.90
   p interaction 0.45
LEP (rs7799039)
   GG 223 232 1.00 373 234 0.63 0.49, 0.81
   GA 319 285 0.91 0.71, 1.16 568 457 0.82 0.66, 1.04
   AA 145 103 0.73 0.53, 1.00 221 174 0.80 0.61, 1.06
   p interaction <0.01
LEP (rs2167270)
   GG 292 228 1.00 444 353 1.04 0.83, 1.30
   GA 297 293 1.27 1.00, 1.61 524 428 1.08 0.87, 1.34
   AA 96 95 1.26 0.90, 1.76 189 83 0.57 0.42, 0.78
   p interaction <0.01
VDR Bsm1
bb
 bB/BB
Controls (n) Cases (n) OR1 95% CI Controls (n) Cases (n) OR 95% CI
LEPR (rs6588147)
   AA 320 261 1.00 546 492 1.12 0.91, 1.38
   AG 297 238 1.01 0.79, 1.28 539 400 0.93 0.76, 1.15
   GG 68 63 1.17 0.80, 1.72 153 90 0.75 055, 1.02
   p interaction 0.02
LEP (rs7799039)
   GG 221 197 1.00 400 303 0.87 0.68, 1.11
   GA 318 278 1.05 0.81, 1.35 610 497 0.97 0.77, 1.22
   AA 152 89 0.70 0.50, 0.97 233 186 0.96 0.73, 1.26
   p interaction 0.04
LEP (rs2167270)
   GG 277 211 1.00 504 384 1.01 0.81, 1.27
   GA 298 279 1.24 0.98, 1.59 551 478 1.16 0.94, 1.45
   AA 113 70 0.82 0.58, 1.16 184 123 0.88 0.66, 1.18
   p interaction 0.76
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Odds ratios (OR) and 95% confidence intervals (CI) adjusted for age, sex, race and center.

Discussion

In this study, we evaluated the associations between the rs6588147 LEPR rs7799039 LEP, and the rs2167270 LEP markers and risk of colon cancer. The two leptin markers were chosen because they represent polymorphic loci that have previously been associated with variability in secreted leptin levels and obesity. 19,20,23 Marker rs6588147 located in intron 2 of the leptin receptor gene was chosen for its proximity to the 5′ end of the gene and its relatively common minor allele frequency of ~0.39 in the Caucasian population. The LEP markers studied also are in linkage disequilibrium with two other LEP markers, rs2167270 and rs3828942. We observed that various combinations of the LEPR and LEP genotypes may alter colon cancer risk, providing support for a link between leptin and its receptor and colon cancer. Because of the diverse biological activities of leptin, we evaluated several factors for which we believe there was a physiological basis for interaction with LEP and LEPR and could influence colon cancer risk. Our data provide little support for LEP and LEPR polymorphisms modulating energy balance, although they do support associations with factors related to insulin and inflammation as well as VDR

Leptin is derived from adipocytes and circulating levels of leptin are associated with adipose tissue mass.33 Leptin influences regulation of food intake and energy expenditure in conjunction with its receptors.34,35 Studies of leptin, the protein coded by the ob gene in mice, showed that mice with a mutation in the gene developed obesity and subsequent treatment with leptin caused weight loss.36,37 Therefore, much of the early work on leptin has focused on its associations with obesity. Because of the associations between energy balance as a contributor to the development of several types of cancer, including colon cancer,14 assessment of leptin as a modulator of colon cancer is reasonable. Data from a cohort study in Norway detected an almost threefold increased risk of colon cancer among people with high leptin levels,11 the association was independent of BMI, suggesting that mechanisms other than energy balance were involved. Similar associations, although slightly weaker, were observed in a nested case-control study conducted in Sweden.9 We observed a weak protective effect with LEP and LEPR variant alleles of the polymorphisms assessed. However, our data suggest that the LEP and LEPR polymorphisms assessed do not interact with BMI or energy-balance related factors such as physical activity and energy intake. It is possible that other LEP and LEPR polymorphisms may have different associations with BMI, although it also is possible that LEP and LEPR associations with colon cancer risk include biological mechanisms other than energy balance. It is also recognized that other composition of body composition, such as total adipose tissue mass and distribution of adipose tissue may be important to clearly define the association between leptin and body size.

Other possible effects of leptin on colon cancer risk might involve inflammation-related pathways. It is now recognized that adipose tissue secretes adipocytokines such as adiponectin and leptin. Leptin and its receptor are considered pro-inflammatory cytokines and play a major role in modulating inflammation and immune response.33,38 Studies also have shown that prostaglandin E2 stimulates leptin secretion from cultured human adipose-tissue cells and that COX2 inhibitors prevented the increased leptin production. 39 Our results showed a statistically significant interaction between recent use of aspirin/NSAIDs and LEP polymorphisms suggest that leptin may influence colon cancer through mechanisms involving COX2 inhibitors and that greater pro-inflammatory response possibly associated with genotype may be modified by aspirin/NSAID.

Insulin and leptin interact at multiple levels within a complex network of adipose tissue signaling pathways,1 providing another mechanism that could link leptin to colon cancer. It is thought that insulin is involved in the secretion of leptin from the adipocyte, and thus is involved in the leptin signal-transduction pathway. The pathway that connects IRS-1 to the insulin signaling cascade can be modulated by leptin and the leptin receptor.33 Several studies have examined the associations between leptin and IGF-1 and IGFBP-3.40–43 We observed a statistically significant interaction between LEP polymorphisms and both IGF1 and IRS2 genotypes. Those without a 19 CA repeat of the IGF1 gene had the greatest reduction in risk of colon cancer if they also had the AA genotype of the rs2167270 LEP gene. Studies have shown that the 19/19 CA repeat of the IGF1 gene is associated with lower serum levels of IGF-1, which may imply lower colon cancer risk.44 We observed that the non-19/19 IGF1 genotype was associated with the greatest risk, as expected, but only in the presence of the LEP AA genotype, and thus, possibly lower leptin levels.18 For the same rs216720 LEP polymorphism, we observed a statistically significant interaction with IRS2, with the greatest reduced risk for those with the AA genotype who also had the GG IRS2 genotype. These IRS2 polymorphisms (DD genotype) have been associated with obesity and insulin resistance45 and therefore, the association with LEP may indicate interaction with obesity or insulin.

Of interest is our observation of a statistically significant interaction between the LEP and LEPR polymorphisms and VDR genotypes with colon cancer risk. Given previous work has shown that vitamin D3 can regulate leptin levels and that leptin can influence expression of renal 25-hydroxyvitamin D3-1α-hydroxylase in mice there appears to be support for our observed association between VDR and LEP and LEPR.7,8 Vitamin D, like leptin, is thought to have multiple biological roles that could relate to cancer, including both inflammation and insulin46 and has been shown to be associated with colon cancer.47

There are limitations to our work, including the limited number of polymorphisms examined. The LEP polymorphisms we examined were thought to have functional role based on the literature 19,20,23; however, other polymorphisms may help further define the relationship with colon cancer and the pathways being examined. Even with the limited number of polymorphisms, we have made many comparisons and it is possible that findings are from chance. Thus, it is important that others replicate these observations as a means of validation of our results. Additionally, exposure information was based on participant recall which is subject to bias in any epidemiological study. From previous analyses we know that nonparticipants were more likely to be older and have more advanced disease stage48; however, we do not believe that these factors would contribute to associations since we did not observe an association between these genotypes and survival, and associations did not differ by age at diagnosis.

Our results add to the limited available data on the association between leptin and the leptin receptor as they relate to risk of colon cancer. Although we did not observe meaningful interaction between these markers of LEP and LEPR and indicators of energy balance, we did observe important interactions with indicators of insulin and inflammation-related pathways and with VDR. One of the challenges in defining an association between colon cancer, obesity, insulin, inflammation, and leptin is to disentangle their roles and influences upon each other, especially within the context of epidemiologic studies. It remains to be determined whether leptin plays a causal role in the risk of developing CRC, or whether it is merely a marker for other risk-contributing processes. Research to understand the associations and the functionality between leptin and CRC are therefore needed. It is possible that leptin may be at an important junction of pathways that are central to the development of CRC.

Acknowledgements

This study was funded by NCI grants CA48998, CA85846, and CA61757 to Dr. Slattery. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official view of the National Cancer Institute. We would like to acknowledge the contributions of Mr. Michael Hoffman, Ms. Erica Wolff and Ms. Thao Tran for genotyping and Ms. Sandra Edwards, Mr. Roger Edwards, Ms. Leslie Palmer, Ms. Donna Schaffer and Ms. Judy Morse for data management and collection.

Grant sponsor: NCI grants; Grant numbers: CA48998, CA85846, CA61757; Grant sponsor: National Cancer Institute; Grant number: N01-PC-67000; Grant sponsors: State of Utah Department of Health, the Northern California Cancer Registry, the Sacramento Tumor Registry.

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