PPARGC1A and ADIPOQ polymorphisms are associated with aggressive prostate cancer in Mexican-Mestizo men with overweight or obesity

Abstract

BACKGROUND:

Obesity constitutes a risk factor for the development of aggressive forms of prostate cancer. It has been proposed, that prostate cancer has a genetic predisposition and that PPARGC1A and ADIPOQ polymorphisms play a role in the development of this condition.

OBJECTIVE:

To analyse the association of two PPARGC1A and ADIPOQ polymorphisms as well as their haplotypes, with the development of aggressive prostate cancer in Mexican-Mestizo men with overweight or obesity.

SUBJECTS AND METHODS:

Two hundred fifty seven men with prostate cancer of Mexican-Mestizo origin were included. Body mass index (BMI) was determined and the degree of prostate cancer aggressiveness by the D’Amico classification. DNA was obtained. Rs7665116 and rs2970870 of PPARGC1A, and rs266729 and rs1501299 of ADIPOQ were studied by real-time PCR allelic discrimination. Pairwise linkage disequilibrium, between single nucleotide polymorphisms was calculated and haplotype analysis was performed.

RESULTS:

A higher-risk (D’Amico classification) was observed in 21.8% of patients. An association of cancer aggressiveness with rs2970870 of PPARGC1A, and rs501299 of ADIPOQ, as well as with one haplotype of ADIPOQ was documented.

CONCLUSIONS:

This is the first study regarding the relationship of PPARGC1A and ADIPOQ polymorphisms, and the aggressiveness of prostate cancer in men with overweight or obesity.

Keywords

Aggressive prostate cancer overweight obesity polymorphisms PPARGC1A ADIPOQ

1. Introduction

Prostate cancer is a global public health problem, being the second most common cancer in men [1]. In Mexico, mortality rate during 2013 was 10.3 deaths per 100,000 men [2].

It has been determined that prostate cancer is a multifactorial disease, where genetics and environmental factors play a key role in its development [3, 4]. It has been previously suggested that a higher body mass index (BMI), could be associated with the possibility of developing a more aggressive form of prostate cancer, thus increasing its mortality rate [5, 6, 7, 8]. Likewise, it has been stated that obesity could increase the risk of biochemical recurrence after primary treatment [9]. The after mentioned data is relevant, due to the fact that obesity has reached epidemic proportions worldwide, being México the second nation with the biggest obesity prevalence worldwide (32.4% in 2012); overweight prevalence in men aged 60–69 years, is at 49.8% in our country [10].

The Peroxisome proliferator-activated receptor $\gamma$ (PPAR $\gamma$ ), is part of the nuclear hormone receptor superfamily of ligand-dependent transcription factors and plays an important role in adipocyte differentiation, cellular proliferation, survival, and inflammatory response [11, 12, 13]. Likewise, it has also been implicated in tumorigenesis as a tumor suppressor gene [14], and it also seems to participate in the development of several neoplasias including prostate cancer [15, 16, 17, 18]. In addition, it has been described that the gene encoded by PPAR $\gamma$ , which is known as peroxisome proliferators-activated receptor $\gamma$ coactivator1 $\alpha$ (PPARGC1A), presents some polymorphisms and/or mutations that have been associated with different cancers and neurodegenerative disorders [19, 20, 21].

Low serum adiponectin concentrations, have also been described as a risk factor for the development of more aggressive forms of prostate cancer in subjects with obesity [22]. Different lines support the role of adiponectin as an anti-cancer adipokine, due to its anti- inflammatory effect [23, 24], to its inhibition of cell proliferation and to the generation of apoptosis [23, 25, 26]. Besides, polymorphisms of adiponectin (ADIPOQ) (rs266729 and rs1501299) associated with obesity [27, 28, 29] and with prostate cancer [27, 30, 31], have been described.

Obesity is considered an independent risk factor for the development of aggressive forms of prostate cancer and both diseases have a high genetic predisposition. Considering that polymorphisms of PPARGC1A and ADIPOQ may influence the risk of both entities, the aim of this study was to analyse the possible association of polymorphisms in these genes, as well as their haplotypes, with the presentation of aggressive forms of prostate cancer in Mexican-Mestizo men with overweight or obesity.

2. Subjects and methods

2.1 Subjects

The study was approved by the Human Research Committees of the participating institutions; informed written consent was obtained from all men before participation, and all procedures were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Two hundred fifty seven unrelated overweight or obese men, with histologically confirmed prostate cancer, of Mexican-Mestizo ethnic origin who visited our outpatient clinic were included. Only men born in Mexico, who had a Spanish-derived last name and Mexican ancestors back to the third generation, were considered Mexican-Mestizo. Men with a family history of prostate cancer or men, who had presented other previous neoplasias, were excluded from the study.

Body weight and height were measured at the first visit, with the subject in a standing position, wearing a hospital gown and without shoes. Weight and height were used to calculate body mass index (BMI) (kg/m ${}^{2}$ ). Individuals were categorized as being overweight if BMI was in the range of 25.0–29.9 kg/m ${}^{2}$ and being obese if BMI was $\geqslant$ 30.0 kg/m ${}^{2}$ .

Patients were categorized according to the classification proposed by D’Amico into low, intermediate and high risk, based on prostate specific antigen (PSA), Gleason grade and clinical stage [32].

2.2 Methods

Peripheral blood samples were obtained from all individuals and genomic DNA was purified using the salting out procedure described by Miller et al. [33]. The rs7665116 and rs2970870 of PPARGC1A and the rs266729 and rs1501299 of ADIPOQ were studied.

Real-time PCR allelic discrimination TaqMan assay (AB) was used for genotyping analysis. All PCR reactions contained 10 ng of DNA, 5.0 $\mu$ l TaqMan Universal Master Mix (AB) (2X), 0.25 $\mu$ l primers and probes (10X), as well as water, for a final volume of 10 $\mu$ l, including the appropriate negative controls in all assays. For all polymorphisms, the assay used probes and primers designed by assay-on-demand services from Applied Biosystem: for rs7665116, assay ID: C__31279675_10; for rs2970870, assay ID: C___1643241_10, both SNPs of PPARGC1A; for rs266729, assay ID: C___2416786_10; for rs1501299, assay ID: C___7497299_10, both SNPs of ADIPOQ.

Real-time PCR was performed on a LightCycler ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 480 Instrument (Roche Diagnostics Ltd, Switzerland). Conditions for all polymorphisms were 95 ${}^{\circ}$ C for 10 min and 40 cycles of amplification (95 ${}^{\circ}$ C for 15 sec and 60 ${}^{\circ}$ C for 1.30 min). For each cycle, the software determined the fluorescent signal from the VIC- or FAM-labeled probe. Allelic discrimination was performed using specific primers and probes for each allele. A 5% random sample from each genotype was independently verified twice for quality control.

2.3 Statistical analysis

This is a cross sectional and retrospective study. Data from the overall patient population was summarized as absolute and relative frequencies for qualitative variables. Allele frequency differences between groups were assessed by $\chi^{2}$ tests. $\chi^{2}$ was used to test for associations between genotypes, haplotypes and aggressiveness of prostate cancer (according to D’Amico’s classification). Statistical analyses were carried out using SPSS v.16.0 (SPSS Inc., Chicago, IL, USA); a $P$ value $<$ 0.05 was accepted as statistically significant. Deviations from Hardy-Weinberg equilibrium were tested using the $\chi^{2}$ test. Pairwise linkage disequilibrium ( $D$ ) estimations between polymorphisms, haplotype reconstruction and a permutation test to obtain a corrected $P$ -value, were performed with Haploview version 4:1. Haplotypes with a frequency $<$ 0.05 were excluded (Broad Institute, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, USA). Statistical power to detect an association of all polymorphisms analyzed with prostate cancer aggressiveness, at an alpha of 0.05, was calculated taking into account the frequencies of the polymorphisms.

3. Results

Table 1
General characteristics of 257 Mexican-Mestizo men with prostate cancer

Variable $N$ (%)

Age (years)

$<$ 60 101 (39.3)

$\geqslant 60$ 156 (60.7)

PSA

$<$ 6 65 (25.3)

$\geqslant 6$ 192 (74.7)

Gleason

$<$ 7 110 (42.8)

$\geqslant 7$ 147 (57.2)

Tumor stage

T1 139 (54.1)

T2 102 (39.7)

T3 9 (3.5)

T4 7 (2.7)

D’Amico classification

Low-risk 73 (28.4%)

Intermediate-risk 128 (49.8%)

High-risk 56 (21.8%)

Variable	$N$	(%)
Age (years)
$<$ 60	101	(39.3)
$\geqslant 60$	156	(60.7)
PSA
$<$ 6	65	(25.3)
$\geqslant 6$	192	(74.7)
Gleason
$<$ 7	110	(42.8)
$\geqslant 7$	147	(57.2)
Tumor stage
T1	139	(54.1)
T2	102	(39.7)
T3	9	(3.5)
T4	7	(2.7)
D’Amico classification
Low-risk	73	(28.4%)
Intermediate-risk	128	(49.8%)
High-risk	56	(21.8%)

PSA $=$ prostate-specific antigen.

Two hundred fifty seven men presenting overweight or obesity and prostate adenocarcinoma were included. General characteristics of all individuals included are shown in Table 1. Age ranged from 55–78 years. According to the World Health Organization criteria, among the 257 men, 187 (72.8%) presented overweight and 70 (27.2%) obesity. One hundred and ninety two patients (74.7%) had PSA concentrations $\geqslant$ 6 ng/ml at the moment of prostate cancer diagnosis. Regarding cancer aggressiveness, 73 (28.4%) were at a low-risk, 128 (49.8%) at an intermediate-risk and 56 (21.8%) at a high-risk (Table 1).

A Hardy-Weinberg equilibrium test was performed for the polymorphisms, showing that the distribution of the observed genotypes did not differ from what was expected in men with prostate cancer and overweight or obesity ( $P$ $>$ 0.05). Genotype and allele frequencies of rs7665116 and rs2970870 of PPARGC1A, and of rs266729 and rs1501299 of ADIPOQ, according to the D’Amico classification, are shown in Table 2. Genotype distribution of rs2970870 of PPARGC1A and rs1501299 of ADIPOQ differed significantly between groups, whilst genotypes of rs7665116 of PPARGC1A and rs266729 of ADIPOQ showed no significant differences.

Table 2

Allele and Genotype frequencies of PPARGC1A and ADIPOQpolymorphisms in Mexican-Mestizo men with prostate cancer and overweight or obesity according to the D’Amico classification

dbSNP	Genotype	Low-risk	Intermediate-risk	High-risk	$P$ -value
Reference number		( $N=$ 73)	( $N=$ 128)	( $N=$ 56)
rs7665116 of PPARGC1A	T/T	49 (67.1%)	102 (79.7%)	36 (64.3%)	0.175
	C/T	23 (31.5%)	25 (19.5%)	19 (33.9%)
	C/C	1 (1.4%)	1 (0.8%)	1 (1.8%)
Allele frequencies	T	82.9%	89.5%	81.2%
	C	17.1%	10.5%	18.8%
rs2970870 of PPARGC1A	C/C	42 (57.5%)	79 (61.7%)	24 (42.9%)	0.035
	C/T	30 (41.1%)	39 (30.5%)	26 (46.4%)
	T/T	1 (1.4%)	10 (7.8%)	6 (10.7%)
Allele frequencies	C	78.1%	77.0%	66.1%
	T	21.9%	23.0%	33.9%
rs266729 ofADIPOQ	C/C	29 (39.7%)	48 (37.5%)	25 (44.6%)	0.671
	C/G	31 (42.5%)	64 (50.0%)	23 (41.1%)
	G/G	13 (17.8%)	16 (12.5%)	8 (14.3%)
Allele frequencies	C	61.0%	62.5%	65.2%
	G	39.0%	37.5%	34.8%
rs1501299 of ADIPOQ	C/C	40 (54.8%)	70 (54.7%)	34 (60.7%)	0.031
	C/A	24 (32.9%)	55 (43.0%)	20 (35.7%)
	A/A	9 (12.3%)	3 (2.3%)	2 (3.6%)
Allele frequencies	C	71.2%	76.2%	78.6%
	A	28.8%	23.8%	21.4%

Table 3

Frequencies of different haplotypes of the ADIPOQ gene in Mexican-Mestizo men with prostate cancer presenting overweight or obesity grouped by the D’Amico classification

Haplotypes	Low-risk	Intermediate/	$P$ -value
of ADIPOQ		Higher risk
$C$ - $C$	41.8%	40.5%	0.31
$G$ - $C$	35.1%	34.8%	0.85
$C$ - $A$	21.5%	22.2%	0.54
$G$ - $A$	1.6%	2.5%	0.03

The first allele of the haplotype corresponds to rs266729, and the second to rs1501299.

When we analyzed linkage disequilibrium (LD) among the two SNPs of PPARGC1A we observed a value of $D^{\prime}=$ 0.22, whilst for the two SNPs of ADIPOQ we found a value of $D^{\prime}=$ 0.72. With these results, we proceeded to perform the haplotype analysis only for the ADIPOQ polymorphisms, observing that the most frequent haplotype in our population was the one formed by the two ancestral alleles: $C$ - $C$ (41.1%). Besides, we detected a significant association between the $G$ - $A$ haplotype (formed by the polymorphic alleles) and the aggressiveness of prostate cancer, when comparing patients that had lower-risk (1.6%) vs those with intermediate/higher-risk (2.5%) ( $P=$ 0.03) (Table 3).

4. Discussion

Taking into consideration that obesity constitutes a risk factor for the development of aggressive forms of prostate cancer, thus increasing its mortality rate and also knowing that both pathological entities have a genetic predisposition, we considered it was important to study the association of some genetic variants in patients presenting prostate cancer and overweight or obesity. Therefore, in the present study we analyzed the association among two PPARGC1A (rs7665116 and rs2970870), and two of ADIPOQ (rs266729 and rs1501299) polymorphisms, as well as their haplotypes, with the aggressiveness of prostate cancer in Mexican-Mestizo men presenting overweight or obesity.

PPARGC1A is a master regulator gene of adipose tissue differentiation [11], whilst PPARg is expressed in several prostate cancer cell lines and also in human prostate epithelium, in normal as well as in tumoral tissue [15]. Different polymorphisms of PPARGC1A (like, rs7665116 and rs2970870), have been studied in neurodegenerative diseases and left ventricular diastolic function [20, 21, 34, 35, 36]. Che et al. [21] found that both polymorphisms were associated with age-at-onset of Huntington Disease; meanwhile, Soyal et al. [36] only observed that rs2970870 was associated with this condition. Further, Taherzadeh-Fard et al. [20] and Ramos et al. [35] found that rs7665116 presented a marginal association with the age-at-onset of the disease in both additive and dominant models. Juang et al. [34] carried out an association study of rs7665116 and rs2970870 with left ventricular diastolic function in Caucasian individuals, observing a marginal association of rs2970870 with latent left ventricular diastolic dysfunction. However, to our knowledge, there are no reports of genetic studies that analyze these polymorphisms in the context of men with prostate cancer regarding its aggressiveness. Due to the fact that, rs2970870 is located in the promoter region, which could influence gene expression, and rs7665116 is located in intron 2 a highly conserved region, which is suggested as a potential recombination hot spot, we analyzed both SNPs in our population with prostate cancer.

We found that only rs2970870 of PPARGC1A was associated with the presence of aggressive prostate cancer in men with overweight or obesity. When we compared the genotypes among the different groups, according with the D’Amico’s classification, we observed in additive and in recessive models that CC genotypes were protective for the development of aggressive prostate cancer (data not shown). In contrast, Che et al. [21] observed, that the CC genotype was associated with an earlier onset of symptoms in Huntington disease when comparing these patients with those exhibiting other genotype. The relative distribution of the $C$ allele of rs2970870 varies remarkably among populations, as it is evident from the data described in HapMap: the frequency of this allele is 46.7% in Europeans, 51.3% in Japanese and 67.4% in Han-Chinese individuals (http://www.ncbi. nlm.nih.gov/snp/?term $=$ rs2970870); our patients exhibit the highest frequency documented for this allele (74.9%).

Moreover, adiponectin a cytokine mainly secreted in adipose tissue could be the link between obesity and prostate cancer [22, 23, 24, 25, 26]; furthermore, polymorphisms of ADIPOQ (i.e. rs266729 and rs1501299) with a functional impact have been associated with adiponectin levels, obesity or prostate cancer [27, 28, 29, 30, 31]. Beebe-Dimmer et al. [27] studied rs266729 and rs1501299 not finding any association with prostate cancer risk. On the other hand, Kaklamani et al. [31] found an association of both polymorphisms with prostate cancer in Caucasians. Dhillon et al. [30] also studying Caucasian men, found an association of rs266729 with an overall risk of prostate cancer, as well as with lower adiponectin levels in plasma.

Regarding our results, of ADIPOQ polymorphisms, we found that only rs1501299 was associated with prostate cancer aggressiveness in men with overweight or obesity. Furthermore, according with the D’Amico’s classification we observed, in all genetics models tested, that the CC genotype was associated with aggressive prostate cancer (data not shown). The latter is important, since the frequency of the $C$ allele (75.3%) of this polymorphism in our patients is the highest ever reported, when comparing it to those described in HAPMAP (http://www.ncbi.nlm.nih.gov/snp) for Caucasians (72%), for two populations of Asian origin (Han Chinese and Japanese, 67.4% and 69.2%, respectively), and one with an African ethnic origin (61.9%).

Additionally, due to the fact that haplotypes could provide a better tool than a single marker variation and because of its strong LD, we analyzed the relationship of haplotypes formed by rs266729 and rs1501299 of ADIPOQ, with aggressive prostate cancer in men with overweight or obesity, observing that these SNPs are inherited as a block. We found that the $G$ - $A$ haplotype, formed by the two minor alleles, was significantly associated with an intermediate/higher-risk of prostate cancer.

This study has some limitations. The first one is that the design does not allow us to determine a causal relationship between the polymorphisms analyzed and prostate cancer aggressiveness in individuals with overweight or obesity. Another limitation was the limited statistical power to detect association of rs7665116 of PPARGC1A and rs266729 of ADIPOQ, with prostate cancer aggressiveness. Despite these limitations, the strengths of the study are the analysis of this cancer aggressiveness according to D’Amico classification and the statistical power, according to the frequency, of the minor allele of rs2970870 of PPARGC1A and rs1501299 of ADIPOQ, as well as for the haplotypes ( $>$ 80%, $P<$ 0.05).

In conclusion, to our knowledge, this is the first study regarding the relationship of rs2970870 of PPARGC1A and rs1501299 of ADIPOQ, as well as the haplotype formed by both SNPs of ADIPOQ, with the aggressiveness of prostate cancer in men presenting overweight or obesity, due to the importance of trying to find an obesity biomarker, which could be related to prostate cancer aggressiveness. Regarding the other two SNPs studied, we did not find a significant association relating to the aggressiveness of prostate cancer. This could be due to the fact that there was not enough power to detect differences; therefore, further studies in a larger number of individuals should explore this possibility. In addition, in our patients, the allelic and genotypic frequencies of the SNPs analyzed were different when compared to other populations. For this reason, it is important to carry out association studies in different populations worldwide, in order to determine whether genetic markers associated with prostate cancer aggressiveness in individuals with overweight or obesity, are the same or vary depending on the populations studied.

Footnotes

Acknowledgments

This work was supported by the División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, México, and by the Consejo Nacional de Ciencia y Tecnología (Apoyo al Fortalecimiento y Desarrollo de la Infraestructura Científica y Tecnológica), México; Grant: 250786). Jesús Benítez Granados was supported by a Consejo Nacional de Ciencia y Tecnología (CONACyT), México, fellowship award. María Elena Tejeda and André Tapia were supported by a Sistema Nacional de Investigadores (CONACyT), México, fellowship award.

Conflict of interest

All authors declare that there are no conflicts of interest.

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