Insulin Receptor H1085H C>T and Insulin Receptor Substrate 1 G972R Polymorphisms and Prostate Cancer Risk: A Pilot Study

Abstract

Background: In recent years, numerous studies have focused their attention on genes that are part of the insulin/insulin-like growth factor 1 signaling pathway, such as the insulin receptor (INSR) and the insulin receptor substrate 1 (IRS1) genes. Aim: We aimed to examine the association of INSR H1085H C>T and IRS1 G972R polymorphisms with prostate cancer (PC). We also aimed to examine possible association with cancer severity assessed by Gleason score. Materials and methods: We have studied 180 consecutive patients referred for PC screening. The genotyping of two polymorphisms (INSR H1085H C>T and IRS1 G972R) was performed by the polymerase chain reaction-restriction fragment length polymorphism method. Results: There was no difference in genotype (p = 0.794) or allelic (p = 0.621) frequency of the IRS1 G972R polymorphism between PC (n = 119) and control (n = 61) groups. However, a significant difference was found in INSR H1085H C>T polymorphism genotype and allelic distribution. Carriers of the polymorphic allele (C/T + T/T) were more frequent in control group patients than in the PC group (54.10% vs. 37.82%; p = 0.040; odds ratio [95% confidence interval] = 0.52 [0.28-0.96]). The IRS1 and INSR polymorphism distribution did not differ in subgroups according to Gleason score. Conclusion: INSR H1085H C>T polymorphism seems to be associated with PC risk, whereas IRS1 G972R is not. However, because of the limited power of this study, there is a possibility that some modest effects of the IRS1 G972R polymorphism on PC risk went undetected. Neither polymorphism is associated with the degree of PC malignancy.

Introduction

Prostate cancer (PC) is a major health issue in men worldwide. The latest reports show that PC in men accounts for the highest cancer incidence and is the second most common cause of cancer mortality (Jemal et al., 2009).

Because of the well-established fact that genetic factors play an important role in cancer development, many efforts are being made to identify specific genes that contribute to cancer susceptibility. Numerous studies have focused their attention on genes that play a crucial role in cell signaling and survival, apoptosis, proliferation, differentiation, tissue development, and growth (Jakubowska et al., 2007; Reljic et al., 2007; Chen et al., 2009). Two such possible gene candidates are the insulin receptor (INSR) and the insulin receptor substrate 1 (IRS1) genes, both part of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. IRS1 is activated by both IGF-1 receptor and INSR and therefore has an important function in signal conduction.

While many reports confirm the role of insulin/IGF-1 signaling pathway in tumorigenesis in general as well as in PC development (Pollak et al., 2004; Renehan et al., 2004), results regarding the association of IRS1 gene polymorphisms with PC are inconsistent (Li et al., 2005; Neuhausen et al., 2005; Fall et al., 2008). In addition, only one study has so far investigated the association of INSR gene polymorphisms and PC susceptibility (Moore et al., 2007).

For these reasons, the aim of this exploratory study was to examine the association of INSR H1085H C>T and IRS1 G972R polymorphisms with PC susceptibility. We also aimed to explore possible association with cancer severity assessed by Gleason score.

Materials and Methods

Subjects

A consecutive series of 180 Caucasian patients of Croatian origin who were referred to the Department of Urology of the Sestre Milosrdnice University Hospital in the period from July 2003 till March 2006 for PC screening were included in this prospective unmatched case-control study. All patients were informed about the study and have consented to participate.

All patients underwent digital rectal exam (DRE) and had their prostate-specific antigen (PSA) values determined. If the PSA value was greater than 4 μg/L and/or DRE was positive, patients underwent transrectal prostate biopsy using a biopsy gun with 18-gauge needle (Bard Biopsy Systems, Tempe, AZ). Between 8 and 12 tissue samples were obtained from each patient. Samples were stained according to the standard hemalaun-eosin staining method.

Gleason score was assessed in 116 patients and they were divided into subgroups of higher (Gleason score 7-9) and lower (Gleason score 4-6) grades of malignancies according to the Gleason grading system (Gleason, 1966; Bostwick and Foster, 1999).

PC-free patients (control group) were those with (a) negative biopsy or (b) PSA <4 and negative DRE.

Exclusion criteria from our research were other malignancies and age ≥85 years.

Genotyping

Genomic DNA was extracted from peripheral blood leukocytes using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). Both of the polymorphisms were genotyped by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method.

The PCR amplification for IRS1 G972R polymorphism (rs1801278) was performed using forward (5′-GCTTTCCACAGCTCACCTTC-3′) and reverse (5′-GGTAGGCCTGCAAATGCTA-3′) primers (van Dam et al., 2004). The PCR product of 198 bp was digested with SmaI (New England Biolabs, Beverly, MA) at 37°C overnight. The wild-type allele (G allele) was cleaved, producing fragments of 171 and 27 bp, whereas polymorphic (A allele) remained undigested (198 bp).

The PCR amplification for INSR H1085H C>T polymorphism (rs1799817) was performed by the protocol described by Siegel et al. (2002) with several modifications. Briefly, primer sequences were forward 5′-TCAGGAAAGCCAGCCCATGTC-3′ and reverse 5′-CCAAGGATGCTGTGTAGATAAG-3′. The PCR product of 317 bp was digested with Pml1 (New England Biolabs, Beverly, MA) at 37°C overnight. The wild-type allele (C allele) remained uncleaved (317 bp), whereas the polymorphic (T allele) was digested, producing fragments of 274 and 43 bp.

All fragments were separated on 4% NuSieve 3:1 Agarose (Lonza, Rockland, ME) using the SEA 2000^® electrophoresis apparatus (Elchrom Scientific AG, Cham, Switzerland).

Ten percent of all samples were double genotyped. Four controls (one wild type, one heterozygous, one variant homozygous, and one negative control—H₂O instead of DNA) were used per 20 samples.

Statistical analysis

All categorical data were presented as direct counts and percentages. Quantitative data were presented as mean and standard deviation if normally distributed and as median and interquartile range in case of non-Gaussian distribution. The differences between observed and expected genotype frequencies for calculating Hardy-Weinberg equilibrium were tested using chi-square test. Distribution of IRS1 and INSR polymorphisms between the PC and control groups as well as their distributions according to Gleason score were tested using Fisher exact test. Assuming the dominant model of the inheritance and expected allele frequency for the IRS1 variant allele of 6%, we could have detected an odds ratio (OR) of 3.5 with 80% power in this sample.

The results were analyzed with the MedCalc^® 9.0.1.0. (F. Schoonjans, Belgium). Power analysis was done using freely available statistical software (QUANTO, version 1.2.4, Jim Gauderman, Ph.D. and John Morrison, M.S., University of Southern California). A p-value of <0.05 was considered significant.

Results

PC was diagnosed in 119 patients, and 61 remaining patients were PC free (control group of patients).

Genotype frequencies for the control group were in Hardy-Weinberg equilibrium. There were no differences between observed and expected genotype frequencies for the control group of subjects (p = 0.739 and 0.329 for the IRS1 and INSR polymorphisms, respectively).

The patients in the control group were significantly older than PC patients (71 ± 9 vs. 68 ± 7 years; p = 0.013); however, we did not consider this age difference to be clinically relevant.

The characteristics of patients with PC are presented in Table 1. In 3 of 119 patients, Gleason score could not be accessed because of inadequate biopsy material.

Table 1.

Characteristics of Patients with Prostate Cancer

Variable	PC (n = 119)
PSA (μg/L)	7.5 (3.7-13.2)
Gleason score^a (n, %)
4	1 (0.9%)
5	11 (9.5%)
6	37 (31.9%)
7	49 (42.2%)
8	14 (12.1%)
9	4 (3.4%)
Digital rectal examination^a
T0	36 (31.0%)
T1	20 (17.2%)
T2	30 (25.9%)
T3	24 (20.7%)
T4	6 (5.2%)

Values were missing for three patients.

PC, prostate cancer; PSA, prostate-specific antigen.

There were no differences in either genotype or allelic frequencies of the IRS1 G972R polymorphism between the control and PC groups (p = 0.794 and 0.621, respectively). However, a significant difference was observed for the INSR H1085H C>T polymorphism. Carriers of the polymorphic allele (C/T + T/T) were more frequent in controls than in the PC group (54.10% vs. 37.82%; p = 0.040; OR [95% confidence interval or CI] = 0.52 [0.28-0.96]). Accordingly, variant T allele was also overrepresented in the control group (30.3% vs. 19.8%; p = 0.035; OR [95% CI] = 0.57 [0.34-0.93]) (Table 2).

Table 2.

Distribution of Insulin Receptor Substrate 1 G972R and Insulin Receptor H1085H C>T Polymorphisms Between Prostate Cancer and Controls

	Controls (n = 61)	PC (n = 119)	OR (95% CI)	p^a
IRS1 G972R
G/G	56 (91.80%)	106 (89.08%)	1.37 (0.47-4.05)	0.794
G/A + A/A	5 (8.20%)	13 (10.92%)
G	117 (95.90%)	224 (94.12%)	1.47 (0.51-4.16)	0.621
A	5 (4.10%)	14 (5.88%)
INSR H1085H C>T
C/C	28 (45.90%)	74 (62.18%)	0.52 (0.28-0.96)	0.040
C/T + T/T	33 (54.10%)	45 (37.82%)
C	85 (69.67%)	191 (80.25%)	0.57 (0.34-0.93)	0.035
T	37 (30.33%)	47 (19.75%)

Fisher exact test.

CI, confidence interval; IRS1, insulin receptor substrate; INSR, insulin receptor; OR, odds ratio.

The polymorphism frequencies according to Gleason score are presented in Table 3.

Table 3.

Insulin Receptor Substrate 1 G972R and Insulin Receptor H1085H C>T Polymorphism Distribution According to Gleason Score in Patients with Prostate Cancer

	Gleason score 4-6 (n = 49)	Gleason score 7-9 (n = 67)	OR (95% CI)	p^a
IRS1 G972R
G/G	46 (93.88%)	58 (86.57%)	2.38 (0.61-9.30)	0.234
G/A + A/A	3 (6.12%)	9 (13.43%)
G	94 (95.92%)	125 (93.28%)	1.69 (0.51-5.67)	0.565
A	4 (4.08%)	9 (6.72%)
INSR H1085H C>T
C/C	30 (61.22%)	41 (61.19%)	1.00 (0.47-2.13)	1.000
C/T + T/T	19 (38.78%)	26 (38.81%)
C	78 (79.59%)	107 (79.85%)	0.98 (0.52-1.89)	1.000
T	20 (20.41%)	27 (20.15%)

Fisher exact test.

The IRS1 and INSR polymorphism genotype and allelic distribution did not differ in subgroups according to Gleason score.

Discussion

The goal of our pilot study was to investigate the association between INSR H1085H C>T and IRS1 G972R polymorphisms with PC susceptibility as well as to explore potential association with cancer severity. The key finding of our study was a significant difference observed in INSR H1085H C>T polymorphism genotype and allelic distribution between the PC and control patient groups. Carriers of the polymorphic T allele were more frequent in controls than in the PC group, suggesting its possible protective role.

Although a complete pathophysiological mechanism of PC is not yet fully elucidated, it has been hypothesized that impaired insulin sensitivity, together with hyperinsulinemia, may be associated with an increased risk of cancer development (Giovannucci, 2003; Godsland, 2009), most probably because of impaired cell signaling and stimulation of cell proliferation (Sentinelli et al., 2006). In addition, previous studies have shown that the IRS1 G972R polymorphism impairs insulin signaling in different cell types (Almind et al., 1996; Hribal et al., 2000; Federici et al., 2001; Sentinelli et al., 2006). Moreover, Sentinelli et al. (2006) have suggested that because of insulin signaling impairment, cell proliferation instead of differentiation is stimulated. All these results indicate that G972R polymorphism is an eligible candidate for cancer research studies. Unfortunately, INSR has not been as intensively studied as IRS1. Nevertheless, it is an interesting candidate for cancer research because of its major role in the insulin signaling pathway.

Other researchers have also investigated the association of INSR H1085H C>T and IRS1 G972R polymorphisms with PC. As mentioned previously, Moore and his associates (2007) were the first to evaluate the association between PC risk and INSR polymorphisms, and by the time of writing this article, this is the second study that has examined this association. Moore and his colleagues conducted a large case-control study (1053 case/control pairs) and found no association for any of the studied genes (insulin, INSR, IRS1, and IRS2) with PC susceptibility. Further, an interesting finding of their study was a reduced risk of advanced PC for the carriers of the C allele at the INSR IVS7-126 C>T locus. Additionally, Wang and coworkers (2007) have conducted a nested case-control study on 488 subjects to examine the associations between single-nucleotide polymorphisms in energy regulation candidate genes and postmenopausal breast cancer risk. They found significantly lower breast cancer risk among women homozygous for the T allele of INSR H1085H C>T, being consistent with our finding in PC patients. These findings indicating a possible protective role of some INSR polymorphisms in cancer risk might motivate researchers to further investigate the role of INSR gene in cancer development.

Our results concerning the association of IRS1 G972R polymorphism with PC confirm some earlier findings (Li et al., 2005; Moore et al., 2007). Considering that both these studies were case-control studies with a large sample size, it is very likely that they had adequate power to detect substantial association. On the contrary, Neuhausen Neuhausen et al. (2005) found a 2.8-fold (95% CI = 1.5-5.1) higher risk of PC risk for A allele (GA/AA) carriers. However, compared with the two previously mentioned studies, this study comprised a rather small sample size (199 newly diagnosed PC cases). This could be the reason for the inconsistency of their results with those previously published. In addition, Neuhausen and associates also found a 6.3-fold (95% CI = 2.3-17.6) higher risk of Gleason score 8-10 for IRS1 GR/RR genotypes, whereas our results show no association of any of the polymorphisms with the degree of PC malignancy.

Because of the design of our study, there is a substantial probability of misclassifying a certain proportion of PC patients as controls, as a result of the (a) lack of repeated biopsies in all patients with negative first biopsy and (b) low sensitivity of applied diagnostic criteria for biopsy (PSA > 4 μg/L and/or positive DRE). It is well established that proper definition of the control group is a major drawback of PC studies, because there are many different ways to define controls. A large body of evidence for this inconsistency exists in the literature. Although some authors use quite rigorous inclusion criteria for controls (healthy individuals with negative DRE, negative PC family history, and PSA <1 ng/mL) (Karatzas et al., 2010), there are also studies (like ours) using less-strict criteria (defining controls as patients having negative DRE and PSA <4 ng/mL) (Ma et al., 2008), or even using only random-digit telephone dialing and self-declared health status without undergoing any diagnostic procedure (Holt et al., 2009).

Lack of data on some well-established PC risk factors (smoking, diet, obesity, and family history) might also be considered as a limitation to our study. Although this surely could have provided a deeper insight into the complex multifactorial origin of PC, because of some organizational issues we were, unfortunately, not able to collect those data.

Another possible source of bias in this study might be the small sample size and insufficient power. Unfortunately, this study was powered to detect an OR of 3.5 for the IRS1 variant allele. Therefore, a more modest effect could go undetected because of the low statistical power.

However, because of a rather scarce number of conducted studies about the impact of INSR H1085H C>T and IRS1 G972R polymorphisms on PC, we utterly believe that the results of this pilot study are a valuable contribution to possible future larger studies and meta-analyses.

Within the last year the role of the IGF-1 system has been intensively studied not only in PC (Rowlands et al., 2009) but also in colorectal (Takahari et al., 2009), renal (Dong et al., 2009), breast (Eng-Wong et al., 2009), and other types of cancer. Many reports point out the significant roles of INSR and IRS1 in PC (Reiss et al., 2001; Cox et al., 2009), some even proposing IRS1 as a potential biomarker for tumors (Baserga, 2009). Still, well-designed studies with large sample sizes and matching controls are needed to broaden our knowledge about the role of genetic variations in cancer development and behavior as well as for finding new and better therapeutic options.

To conclude, out of the two investigated polymorphisms, only INSR H1085H C>T polymorphism seems to be associated with PC risk and neither polymorphism is associated with the degree of PC malignancy. However, because of the limited power of this study, there is a possibility that some modest effects of the IRS1 G972R polymorphism on PC risk went undetected.

Footnotes

Acknowledgment

This work was supported by the Ministry of Science, Education, and Sports, Republic of Croatia (project number: 134-1340227-0200).

Disclosure statement

No competing financial interests exist.

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