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Association of polymorphisms of PTEN, AKT1, PI3K, AR, and AMACR genes in patients with prostate cancer

Abstract

Polymorphic variants in the PTEN (rs2735343), PI3K (rs2699887), AKT1 (rs2494750), AR (rs17302090), and AMACR (rs3195676) genes were evaluated as possible molecular markers of susceptibility, prognosis, and progression of prostate cancer (PCa), in a case-control study. Samples consisted of 277 patients with PCa and 277 controls from Londrina, PR, Brazil. SNPs were analyzed by real-time PCR. A family history of cancer, including PCa, as well as level of schooling were risk factors for PCa. The data were obtained via logistic regression, using odds ratios with a CI 95%. The genotypes of AKT1 and AKT1+AR demonstrated an association with protection for the disease. The combination of SNPs with the histopathological tumor data between allele variants of AMACR, AKT1+AR, and AKT1+AMACR indicated an association with protection against seminal vesicle invasion. The polymorphisms AKT1+AR and PI3K+AR were associated with protection against tumor bilaterality. The genotype combinations PTEN+AMACR and PTEN+AR were associated with the risk of extracapsular extension. Of the five genes studied, two were associated with protection for PCa, four were associated with protection for some prognostic variables, and only one was associated with risk. Thus, these SNPs are candidates for markers to discriminate men with better or worse prognosis for PCa.

Keywords:
SNP; prognosis; rs2494750; rs2735343; rs3195676

Introduction

Prostate cancer (PCa) is a heterogeneous and multifactorial pathology that presents itself in the form of indolent or very aggressive tumors and is a significant source of morbidity in the male population. In 2018, 1.3 million new cases were estimated and 359,000 associated deaths worldwide, however, the mortality rates do not follow the incidence rates. In certain countries, PCa still presents an elevated degree of mortality (Saini, 2016Saini S (2016) PSA and beyond: Alternative prostate cancer biomarkers. Cell Oncol 39:97-106.; Bray et al., 2018Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A (2018) Global Cancer Statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394-424.).

The most commonly utilized methods for the prognosis of this type of tumor are the TNM classification for staging, used in combination with the Gleason/tumor grade and prostate-specific antigen (PSA) level (Buyyounouski et al., 2017Buyyounouski MK, Choyke PL, McKenney JK, Sartor O, Sandler HM, Amin MB, Kattan MW and Lin DW (2017) Prostate cancer - major changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin 67:245-253.). However, the clinical applications of these indicators are limited. The American Urological Association encourages the identification of novel markers that can identify men at a greater risk of developing and progressing in the disease (Carter et al., 2013Carter HB, Albertsen PC, Barry MJ, Etzioni R, Freedland SJ, Greene KL, Holmberg L, Kantoff P, Konety BR, Murad MH et al. (2013) Early detection of prostate cancer: AUA Guideline. J Urol 190:419-426.). Studies have increasingly associated single-nucleotide polymorphisms (SNPs) with various diseases, resulting in SNPs being progressively utilized as markers associated with cancer, including PCa. Consequently, SNPs have received considerable attention for use as prognostic indicators in pre and post-treatment PCa patients (Qi et al., 2017Qi X, Wang Y, Hou J and Huang Y (2017) A single nucleotide polymorphism in HPGD gene is associated with prostate cancer risk. J Cancer 8:4083-4086.).

In recent decades, different SNPs have been associated with important cellular pathways, including the PI3K/PTEN/AKT signaling pathway (Jang et al., 2013Jang Y, Lu SA, Chen ZP, Ma J, Xu CQ, Zhang CZ and Wang JJ (2013) Genetic polymorphisms of CCND1 and PTEN in progression of esophageal squamous carcinoma. Genet Mol Res 12:6685-6691.), androgen action pathway (Prencipe et al., 2015Prencipe M, O’Neill A, O’Hurley G, Nguyen LK, Fabre A, Bjartell A, Gallagher WM, Morrissey C, Kay EW and Watson RW (2015) Relationship between serum response factor and androgen receptor in prostate cancer. Prostate 5:1704-1717.), and fatty acid metabolization pathway (Jiang et al., 2013Jiang N, Zhu S, Chen J, Niu Y and Zhou L (2013) A-methylacyl-coaracemase (AMACR) and prostate-cancer risk: A meta-analysis of 4,385 participants. PLoS One 8:e74386.). In this manner, they are related to the development of prostate adenocarcinoma.

The PTEN gene is a tumor suppressor with dual specificity. The principal substrate is phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The protein PTEN acts as an antagonist on the PI3K/AKT pathway by maintaining low PIP3 levels. The greater the concentration of PIP3, the greater the signaling of the PI3K/AKT pathway, which promotes inhibition of the cellular cycle, migration, metastasis, and apoptosis. The occurrence of loss of PTEN gene function is greater in aggressive metastatic disease, meaning that it can be used as a biomarker for prognosis in distinguishing indolent tumors from those that are aggressive (Di Cristofano and Pandolfi, 2000Di Cristofano A and Pandolfi PP (2000) The multiple roles of PTEN in tumor suppression. Cell 100:387-390.; Jang et al., 2013Jang Y, Lu SA, Chen ZP, Ma J, Xu CQ, Zhang CZ and Wang JJ (2013) Genetic polymorphisms of CCND1 and PTEN in progression of esophageal squamous carcinoma. Genet Mol Res 12:6685-6691.; Wise et al., 2017Wise HM, Hermida MA and Leslie NR (2017) Prostate cancer, PI3K, PTEN and prognosis. Clin Sci 131:197-210.).

Meanwhile, the PI3K gene is a proto-oncogene; the corresponding protein phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2), converting it into PIP3. Given the complexity of this intracellular signaling cascade and its association with the development of PCa, it is important to delineate the role of polymorphism in this gene with respect to prognosis and susceptibility (Toren and Zoubeidi, 2014Toren P and Zoubeidi A (2014) Targeting the PI3K/Akt in prostate cancer: Challenges and opportunities. Int J Oncol 45:1793-1801.).

In addition, the AKT1 gene is a proto-oncogene whose corresponding protein regulates events downstream in the PI3K signal transduction pathway. This protein plays an important role in inhibition of cellular viability, proliferation, differentiation, and apoptosis (Vivanco and Sawyers, 2002Vivanco I and Sawyers CL (2002) The phosphatidylinositol 3-kinase–AKT pathway in human cancer. Nat Rev Cancer 2:489-501.). The overexpression of this gene is related to different types of cancer, including the progression and biochemical recurrence of prostate tumors (Feldman and Feldman, 2001Feldman BJ and Feldman D (2001) The development of androgen-independent prostate cancer. Nat Rev Cancer 1:34-45.; Karyadi et al., 2015Karyadi DM, Zhao S, He Q, McIntosh L, Wright JL, Ostrander EA, Feng Z and Stanford JL (2015) Confirmation of genetic variants associated with lethal prostate cancer in a cohort of men from hereditary prostate cancer families. Int J Cancer136:2166-2171.).

Finally, the AR gene encodes the androgen receptor, a nuclear transcription factor that is involved in the development, maintenance, and regulation of the male reproductive system (Feldman and Feldman, 2001Feldman BJ and Feldman D (2001) The development of androgen-independent prostate cancer. Nat Rev Cancer 1:34-45.). Mutations in AR are associated with a series of diseases, ranging from androgen insensitivity syndrome to prostate cancer (Heemers and Tindall, 2007Heemers HV and Tindall DJ (2007) Androgen receptor (AR) coregulators: A diversity of functions converging on and regulating the AR transcriptional complex. Endocr Rev 28:778-808.). Among the mutations in this gene, SNPs are the most frequent type of alteration. Even a simple change in amino acids can result in either an increase in expression or a loss in gene function (Sun et al., 2010Sun T, Lee GSM, Werner L, Pomerantz M, Oh WK, Kantoff PW and Freedman ML (2010) Inherited variations in AR, ESR1, and ESR2 genes are not associated with prostate cancer aggressiveness or with efficacy of androgen deprivation therapy. Cancer Epidemiol Biomarkers Prev 19:1871-1878.; Wright et al., 2011Wright JL, Neuhouser ML, Lin DW, Kwon EM, Feng Z, Ostrander EA and Stanford JL (2011) AMACR polymorphisms, dietary intake of red meat and dairy and prostate cancer risk. Prostate 71:498-506.).

The peroxisomal and mitochondrial enzyme Alpha-methylacyl-CoA racemase (AMACR) is coded by the gene P504S (AMACR) and is involved in the biosynthesis and beta-oxidation of dietary fatty acids (Li et al., 2014Li CF, Fang FM, Lan J, Wang JW, Kung HJ, Chen LT, Chen TJ, Li SH, Wang YH, Tai HC et al. (2014) AMACR amplification in myxofibrosarcomas: A mechanism of overexpression that promotes cell proliferation with therapeutic relevance. Clin Cancer Res20:6141-6152.). Fatty acid metabolism represents a key process that influences different pathways and cellular characteristics, including cellular signaling, energetic processing, and membrane fluidity, among others (Liu et al., 2006Liu Y (2006) Fatty acid oxidation is a dominant bioenergetic pathway in prostate cancer. Prostate Cancer Prostatic Dis 9:230-234.).

Considering that the majority of cases of PCa are indolent, markers are needed that aid in classifying the degree of tumor aggressiveness and in providing individual treatment, in line with the genetic characteristics of each patient. In this sense, a case-control study was performed that evaluated the possible relationship between polymorphic variants of the genes PTEN, PI3K, AKT1, P504S (AMACR), and AR and prostate cancer susceptibility. In addition, data from the association study were correlated with the clinical parameters of the patients and the histopathological parameters of the tumors, with the aim of establishing prognostic molecular markers.

Subjects and Methods

Study cohorts

In total, 554 male individuals were studied, with 277 serving as a control, free of PCa with a prostate-specific antigen (PSA) value lower than 2 ng/mL, and with 277 patients diagnosed with PCa, as confirmed by histopathological analysis. Individuals were excluded if they had been submitted to radiotherapy and/or chemotherapy. The patients were paired with controls on a 1:1 basis, maintaining age (± 5 years), ancestry (European and African), and alcohol and tobacco use consistent between pairs. Peripheral blood samples of the patients and controls were collected from 2006 to 2015 at CISMEPAR (Consórcio Intermunicipal de Saúde do Médio Paranapanema), the Irmandade Santa Casa de Londrina, the State University of Londrina, and the Cancer Hospital of Londrina.

Disease stage was determined by pathology findings, pelvic computed tomography, or magnetic resonance image. Tumor staging was determined according to criteria established by the Union for International Cancer Control (2009) tumor-node-metastasis (TNM) classification system. Pathologic grading was recorded as the Gleason score and was further classified into 2 groups: Gleason score 3–7 (3+4) and Gleason 7(4+3)–10.

The patients and controls were volunteers and written informed consent was obtained from each participant that responded to the questionnaire, which was based on Carrano and Natarajam, (1988)Carrano AV and Natarajan AT (1988) International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC publication no. 14. Considerations for population monitoring using cytogenetic techniques. Mutat Res 204:379-406.. The study was approved by the Ethics Committee Involving Research on Human Beings of the State University of Londrina (CEPE/UEL 176/2013).

Extraction of genomic DNA

Peripheral blood was collected in tubes of EDTA (ethylenediaminetetraacetic acid), from which DNA was extracted using the High Pure PCR Template Preparation (Cat. 11796828001; Roche; IN, USA) commercial kit, following the manufacturer's instructions. DNA quantification was carried out via spectrophotometry, using a Nanodrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Absorption was measured at 260/280 nm in order to determine the purity of the nucleic acid, with samples with a ratio between 1.8 and 2.0 being considered pure.

Genotyping

Each gene selected for this study had one defined SNP chosen from the public Database of Single Nucleotide Polymorphisms (NCBI, 2016): PTEN (rs2735343), AKT (rs2494750), PI3K (rs2699887), AR (rs17302090), and P504S (AMACR) (rs3195676).

The genotypes were determined via real-time PCR molecular analysis using a hydrolysis probe (TaqMan System, Applied Biosystems, Foster City, CA, USA). The reactions contained the genotyping master mix (Applied Biosystems), 5 ng/mL of DNA, and the specific probe for each gene. Reagent quantities and cycling were carried out according to the manufacturer's specifications.

Statistical analysis

The χ2 test was used to determine if the genotype distributions were in a Hardy-Weinberg equilibrium. Allele frequency was calculated as [1(h + 2H)]/2 N, where h represents the heterozygous genotype, H is the homozygous genotype, and N is the sample size for each population.

First, the association between the following parameters: family history of cancer, family history of PCa, vasectomy, occupational exposure to agrochemicals, level of schooling, and consumption of red meat was determined through univariate logistic regression analysis, aiming to assess the impact of these variables on cancer risk. The association between the genotypes and cancer risk was also run by univariate logistic regression analysis.

Subsequently, multivariate logistic regression analyses were performed aiming to assess the impact of the studied SNPs on PCa risk. Variables that had a p-value ≤ 0.05 were included in the model: family history of cancer, family history of PCa, and level of schooling, avoiding possible bias in the analysis.

The different genotypes were evaluated with respect to histopathological parameters (Gleason score, bilaterality, seminal vesicle invasion, perineural invasion, lymphatic invasion, and extracapsular extension) and a clinical parameter (PSA).

Results are expressed as Odds Ratio (OR) with a confidence interval of 95% (CI95%) and assumed statistically significant for a p ≤ 0.05. Analyses were performed using SPSS 20.0 (IBM, Armonk, NY, USA).

Results

The total sample was composed of 554 men, of which 85.6% were of European and 14.4% of African descent. The average age of the patients was 65.38 ± 7.18 years, while that of the control was 63.65 ± 8.21 years. In both groups, 68 individuals (24.5%) were tobacco users, while 141 individuals (50.9%) consumed alcohol.

Genotype frequencies were in Hardy-Weinberg equilibrium within a significance level of 5%. The univariate statistical analysis revealed significant differences between patients and controls with respect to a family history of cancer (58.8% of patients had at least one first-degree relative who was affected), a family history of PCa, and level of schooling. Other analyzed parameters, such as vasectomy, occupational exposure to agrochemicals, and red meat consumption, did not present statistically significant differences between patients and controls (Table 1).

Table 1
Demographic and clinical characteristics of prostate cancer patients and cancer-free controls.

The comparison of the genotypic variants and allele frequencies of each of the genes studied showed no statistically significant differences between patients with PCa and controls. A multivariate statistical analysis adjusted for the determined risk factors indicated a protecting effect for genotypes CG and CG + GG for the rs2494750 AKT1gene (Table 2). A multivariate analysis of the paired genes (Table S1) showed that the genotypes AKT1 (CG) + AR (A) provided protection against PCa (Table 3).

Table 2
Univariate and multivariate regression analysis of genotypes and allele of PTEN, AKT1, PI3K, AR, and AMACR genes in controls and patients with prostate cancer.
Table 3
Univariate and multivariate logistic regression analysis of the association between AKT1 and AR genes in prostate cancer patients and controls.

Table 4 presents the clinical and histopathological parameters obtained from the patient histories. In total, 147 patients presented a PSA level of 4.1-10.0 ng/mL, 115 patients presented a level greater than 10.0 ng/mL, and only 10 had a level below 4.0 ng/mL. Meanwhile, 133 patients had Gleason scores of 3-6, 117 patients had a score of 7, and only 25 had scores of 8-10. Tumor bilaterality was observed in 137 patients. According to the TNM system, 154 cases had tumors in stage T2, while 101were in stages T3 and T4. The other parameters were absent for the majority of the patients.

Table 4
Analysis of PSA levels of prostate cancer patients and histopathological characteristics of the tumors.

The genotypes of the five SNPs were evaluated for their relationship with the various histopathological and clinical parameters (Table S2). A comparison of the heterozygous and/or polymorphic genotypes with the prevalent genotypes indicated that only the isolated GA and GA+AA genotypes of the AMACR gene protected against seminal vesicle invasion (Table 5).

Table 5
Univariate regression analysis between genotypes of PTEN, AKT, PI3K, AR, and AMACR genes with clinicopathological features in prostate cancer patients.

When the genotypes of different SNPs were compared, several statistically significant results were obtained. The following associations indicated protection for seminal vesicle invasion: AKT1 (CC) + AR (A) and AKT1 (CC) + AMACR (GA). The associations that indicated protection for tumor bilaterality were: AKT1 (CC) + AR (A) and PI3K (GG) + AR (A) (Table 5).

Finally, the following combinations of genotypes indicated a greater risk of extracapsular extension: PTEN (CG) + AMACR (GG), PTEN (CG) + AR (G), and PTEN (CG) + AR (A) (Table 5).

Discussion

PCa is a heterogeneous pathology that currently has a lifetime possibility of detection of 19%, while the risk of death is just 8%. This discrepancy between incidence and mortality is attributed not just to treatment offered, but also to the increase in initial tumor detection and slow growth (Siegel et al., 2017Siegel RL, Miller KD and Jemal A (2017) Cancer Statistics, 2017. CA Cancer J Clin 67:7-30.). Patients with slow-growing tumors could be exposed to unnecessary risks as a consequence of therapy. As such, currently, the greatest challenge with PCa is the identification of markers that can be used to classify tumors according to their potential for aggressive behavior and the recognition of patients that need specific therapy (Vanacore et al., 2017Vanacore D, Boccellino M, Rossetti S, Cavaliere C, D'Aniello C, Di Franco R, Romano FJ, Montanari M, La Mantia E, Piscitelli R et al. (2017) MicroRNAs in prostate cancer: An overview. Oncotarget 8:50240–50251.).

The rate of development of PCa varies substantially according to age (Rebbeck, 2017Rebbeck TR (2017) Prostate cancer genetics: variation by race, ethnicity, and geography. Semin Radiat Oncol 27:3-10.), ancestry (Bell et al., 2015Bell KJ, Del Mar C, Wright G, Dickinson J and Glasziou P (2015) Prevalence of incidental prostate cancer: A systematic review of autopsy studies. Int J Cancer 137:1749-1757.), and family history (Siegel et al., 2017Siegel RL, Miller KD and Jemal A (2017) Cancer Statistics, 2017. CA Cancer J Clin 67:7-30.). In this study, a family history of cancer was a risk factor (p = 0.001). Patients with PCa in their family also presented greater susceptibility to developing prostatic neoplasia (p = 0.031). These findings demonstrate the high contribution of heredity in the development of this carcinoma. Cremers et al. (2016)Cremers RG, Aben KK, van Oort IM, Sedelaar JP, Vasen HF, Vermeulen SH and Kiemeney LA (2016) The clinical phenotype of hereditary versus sporadic prostate cancer: HPC definition revisited. Prostate 76:897-904. demonstrated that, while there are inheritable sites that determine susceptibility to prostate cancer, there is not only one gene that alone determines susceptibility; the pathology of the disease involves a large number of genes, meaning that this illness is treated as being multifactorial.

Education is an indicator of socioeconomic status, and is inversely associated with the incidence of cancer in various organs; in general, the greater the level of schooling, the lower the risk of aggressive cancer (Mouw et al., 2008Mouw T, Koster A, Wright ME, Blank MM, Moore SC, Hollenbeck A and Schatzkin A (2008) Education and risk of cancer in a large cohort of men and women in the United States. PLoS One3:e3639.). In the studied sample, the individuals without formal schooling presented twice the risk of PCa when compared to their respective controls. These data are consistent with Wolf et al. (2006)Wolf MS, Knight SJ, Lyons EA, Durazo-Arvizu R, Pickard SA, Arseven A, Arozullah A, Colella K, Ray P and Bennett CL (2006) Literacy, race, and PSA level among low-income men newly diagnosed with prostate cancer. Urology 68:89-93., who stated that a higher degree of schooling offers greater comprehension of prostate health and, consequently, leads to individuals seeking out medical care. In this case, low patient schooling could lead to tumor diagnosis in more advanced stages and result in a higher mortality index.

The average patient age in this study was 65.38 ± 7.15 years, which corroborates the literature data that show that PCa is a disease of old age; the majority of cases of sporadic PCa are observed in men over the age of 65. This fact has been related to the accumulation of DNA damage (O'Connor, 2015O’Connor MJ (2015) Targeting the DNA damage response in cancer. Mol Cell 60:547-560.).

PSA is a glycoprotein produced by prostate epithelial cells that does not constitute a specific cancer marker, given that it can be changed in other diseases of the prostate (Hamdy et al., 2016Hamdy FC, Donovan JL, Lane JA, Mason M, Metcalfe C, Holding P, Davis M, Peters TJ, Turner EL and Martin RM (2016) 10-year outcomes after monitoring, surgery, or radiotherapy for localized prostate cancer. N Engl J Med 375:1415-1424.; Hendriks et al., 2017Hendriks RJ, van Oort IM and Schalken JA (2017) Blood-based and urinary prostate cancer biomarkers: A review and comparison of novel biomarkers for detection and treatment decisions. Prostate Cancer Prostatic Dis 20:12-19.); furthermore, no exact value exists that can determine the presence of a malignant disease. The PSA values at the time of diagnosis determined in this study ranged from 1.5 to 933.9 ng/mL, confirming that the specificity of this marker for PCa is low. Given this characteristic, the U.S. Preventive Services Task Force, as well as various other health entities around the world, recommend not tracking PCa using PSA, given that despite appearing in many PCa cases, its correlation with mortality is minimal (Jemal et al., 2015Jemal A, Fedewa SA, Ma J, Siegel R, Lin CC, Brawley O and Ward EM (2015) Prostate cancer incidence and PSA testing patterns in relation to USPSTF screening recommendations. JAMA 314:2054-2061.).

Considering the important role that SNPs play as biomarkers in various types of diseases, genes and SNPs that are candidates for PCa susceptibility and prognosis were chosen (Wang et al., 2016Wang C, Nie H, Li Y, Liu G, Wang X, Xing S, Zhang L, Chen X, Chen Y and Li Y (2016) The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC. Sci Rep 6:26526.). These include PTEN/PI3K/AKT signaling pathway components, which are important regulators for growth, metabolism, cell cycle, DNA repair, and apoptosis inhibition (Zhu et al., 2016Zhu J, Wang M, He J, Zhu M, Wang JC, Jin L, Wang XF, Yang YJ, Xiang JQ and Wei Q (2016) Polymorphisms in the AKT1 and AKT2 genes and oesophageal squamous cell carcinoma risk in an Eastern Chinese population. J Cell Mol Med 20:666-677.).

The rs2735343 gene PTEN, studied herein is located in an intronic region. According to Jang et al. (2013)Jang Y, Lu SA, Chen ZP, Ma J, Xu CQ, Zhang CZ and Wang JJ (2013) Genetic polymorphisms of CCND1 and PTEN in progression of esophageal squamous carcinoma. Genet Mol Res 12:6685-6691., it can influence splicing, protein expression, and, consequently, the regulation of the cell cycle and the risk of cancer. The aforementioned authors showed that carriers of the homozygous GG genotype, the same SNP evaluated in this study, presented a greater risk for esophageal squamous cell carcinoma (ESCC). This SNP was also considered by Ma et al. (2012)Ma J, Zhang J, Ning T, Chen Z and Xu C (2012) Association of genetic polymorphisms in MDM2, PTEN and P53 with risk of esophageal squamous cell carcinoma. J Hum Genet 57:261-264. as a factor of susceptibility for ESCC in patients that presented CG and GG genotypes. Song et al. (2017)Song DD, Zhang Q, Li JH, Hao RM, Ma Y, Wang PY and Xie SY (2017) Single nucleotide polymorphisms rs701848 and rs2735343 in PTEN increases cancer risks in an Asian population. Oncotarget 8:96290-96300. performed a meta-analysis with different types of cancer, including PCa and the associated risk of cancer for carriers of the G allele in an Asian population. In this study, we did not find any association between the variants of this gene and susceptibility to PCa. Nonetheless, in all genotypic combinations in which we detected a greater risk of extracapsular extension, the G allele of the (rs2735343) PTEN gene was present (Table 4), which seems to confirm the above-cited hypotheses.

The heterozygous genotype (CG) in these cases can create a non-functional protein, given that, according to Jang et al. (2013)Jang Y, Lu SA, Chen ZP, Ma J, Xu CQ, Zhang CZ and Wang JJ (2013) Genetic polymorphisms of CCND1 and PTEN in progression of esophageal squamous carcinoma. Genet Mol Res 12:6685-6691., this tumor suppressor gene presents very unusual characteristics. As opposed to the majority of tumor suppressor genes, the loss of just a single allele results in an abnormal phenotype, i.e., it is considered a haploinsufficient suppressor and, as a result, does not follow the classical genetic model of biallelic inactivation. The loss of activity of this protein seems to be common in different types of cancer (Inoue and Fry, 2017Inoue K and Fry EA (2017) Haploinsufficient tumor suppressor genes. Adv Med Biol 118:83-122.) and explains our findings.

Several studies have demonstrated that tumors with extracapsular extension had a higher risk of progression than those confined to the prostate and this can be considered an indicator of poor prognosis (Troyer et al., 2015Troyer DA, Jamaspishvili T, Wei W, Feng Z, Good J, Hawley S, Fazli L, McKenney JK, Simko J, Hurtado-Coll A et al. (2015) A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer. Prostate 75:1206-1215.; Epstein et al., 2016Epstein JI, Egevad L, Amin MB, Delahunt B, Srigley JR, Humphrey PA, Al-Hussain T, Algaba F, Aron M, Berman D et al. (2016) The 2014 International Society of Urological Pathology (ISUP) Consensus Conference on Gleason Grading of Prostatic Carcinoma: Definition of grading patterns and proposal for a new grading system. Am J Surg Pathol 40:244-52.). Troyer et al. (2015)Troyer DA, Jamaspishvili T, Wei W, Feng Z, Good J, Hawley S, Fazli L, McKenney JK, Simko J, Hurtado-Coll A et al. (2015) A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer. Prostate 75:1206-1215. reported that a PTEN deletion was significantly associated with higher extracapsular extension. Our results, added to these, suggest that alterations in this gene have the ability to predict aggressive disease.

The gene AKT1 belongs to the AKT family and the protein that it codes plays an important role in cellular survival via apoptosis control; it is related to the development of various diseases, including cancer (Painter et al., 2016Painter JN, Kaufmann S, O’Mara TA, Hillman KM, Sivakumaran H, Darabi H, Cheng THT, Pearson J, Kazakoff S, Waddell N et al. (2016) A common variant at the 14q32 endometrial cancer risk locus activates AKT1 through YY1 binding. Am J Hum Genet 98:1159–1169.). The relationship between PCa and rs2494750SNP is controversial. Karyadi et al. (2015) and Fitzgerald et al. (2018)FitzGerald LM, Zhao S, Leonardson A, Geybels MS, Kolb S, Lin DW, Wright JL, Eeles R, Kote-Jarai Z and Govindasami K (2018) Germline variants in IL4, MGMT and AKT1 are associated with prostate cancer-specific mortality: An analysis of 12,082 prostate cancer cases. Prostate Cancer Prostatic Dis 21:228-237. associated this SNP with death specifically for PCa patients carrying allele G. Nonetheless, this SNP, in the present study, protected the carriers of the CG genotypes against PCa. The heterozygous genotype, when combined with the A allele of the (rs17302090) AR gene, also protects against PCa. Other research has failed to associate the (rs2494750) AKT1 gene with squamous cell carcinoma of the esophagus (Zhu et al., 2016Zhu J, Wang M, He J, Zhu M, Wang JC, Jin L, Wang XF, Yang YJ, Xiang JQ and Wei Q (2016) Polymorphisms in the AKT1 and AKT2 genes and oesophageal squamous cell carcinoma risk in an Eastern Chinese population. J Cell Mol Med 20:666-677.) or with cancer of the endometrium (Fallah et al., 2015Fallah S, Korani M, Hajimirza M and Seifi M (2015) Association between genetic variants of Akt1 and endometrial cancer. Biochem Genet 53:281-290.). Given these conflicting results, it would be interesting to investigate this question further with larger samples of PCa patients from other populations.

Studies, both in vitro and in vivo, have demonstrated that PI3K/AKT/mTOR signaling interacts with the AR gene in an antagonistic manner; when the pathway is inhibited, it increases expression of AR, and as such could serve as a therapeutic target for patients with androgen-dependent prostate cancer (Kaarbø et al., 2010Kaarbø M, Mikkelsen OL, Malerød L, Qu S, Lobert VH, Akgul G, Halvorsen T, Maelandsmo GM and Saatcioglu F (2010) PI3K-AKT-mTOR pathway is dominant over androgen receptor signaling in prostate cancer cells. Cell Oncol 32:11-27.). According to these authors, it appears that the AR gene signaling pathway, depending on downstream genes, could be involved in the malignant transformation of PCa.

Some of the SNPs studied in the AR gene have already demonstrated that the coded protein changes binding with cofactors. This change is associated with androgen insensitivity syndrome, which can affect the activity and level of circulating androgens (Naeyer et al., 2014Naeyer H, Bogaert V, Spaey A, Roef G, Vandewalle S, Derave W, Taes Y and Kaufman JM (2014) Genetic variations in the androgen receptor are associated with steroid concentrations and anthropometrics but not with muscle mass in healthy young men. PLoS One 9:e86235.). In our study, the rs17302090 SNP was not associated with PCa. Sun et al. (2010)Sun T, Lee GSM, Werner L, Pomerantz M, Oh WK, Kantoff PW and Freedman ML (2010) Inherited variations in AR, ESR1, and ESR2 genes are not associated with prostate cancer aggressiveness or with efficacy of androgen deprivation therapy. Cancer Epidemiol Biomarkers Prev 19:1871-1878. evaluated the rs1204038, which presents strong linkage disequilibrium with rs17302090, evaluated in this study, and did not identify any association between risk of aggressive PCa and androgen deprivation therapy. On the other hand, Lindström et al. (2007)Lindström S, Adami HO, Bälter KA, Xu J, Zheng SL, Stattin P, Grönberg H and Wiklund F (2007) Inherited variation in hormone-regulating genes and prostate cancer survival. Clin Cancer Res13:5156-5161. demonstrated that carriers of the rare allele A (rs17302090) who received primary hormonal therapy presented twice the risk of death from PCa compared to individuals that had the prevalent allele. The authors further suggested that this polymorphism would act as a modifier in the hormonal treatment of the disease leading to death by PCa.

Nonetheless, allele A of the AR gene, when combined with the gene AKT1 (CC), conferred protection against seminal vesicle invasion and tumor bilaterality; when combined with the gene PI3K (GG), it also conferred protection against tumor bilaterality. The finding of seminal vesicle invasion at the time of radical prostatectomy is an adverse pathologic finding that confers a decrease in long-term freedom from biochemical recurrence, exceeded in magnitude only by the finding of lymph node metastases (Potter et al., 2000Potter SR, Epstein JI and Partin AW (2000) Seminal vesicle invasion by prostate cancer: Prognostic significance and therapeutic implications. Rev Urol 2:190–195.).

The last gene evaluated in this study codes the enzyme AMACR, which is overexpressed in prostate tumor tissue. This overexpression seems to be involved in carcinoma initiation, as this type of alteration has been described in prostatic intraepithelial neoplasia (Jarvis et al., 2015Jarvis TR, Chughtai B and Kaplan SA (2015) Testosterone and benign prostatic hyperplasia. Asian J Androl 17:212–216.; Jemal et al., 2015Jemal A, Fedewa SA, Ma J, Siegel R, Lin CC, Brawley O and Ward EM (2015) Prostate cancer incidence and PSA testing patterns in relation to USPSTF screening recommendations. JAMA 314:2054-2061.). The enzyme AMACR participates in the metabolism of fatty acids. The main dietary source of branched-chain fatty acids in the human diet includes red meat, which is considered a PCa risk factor. The consumption of these fatty acids is associated with the overexpression of AMACR in cancerous prostate cells, as it consistently increases oxidation in these cells, resulting in the production of reactive oxygen species that can damage DNA (Wright et al., 2011Wright JL, Neuhouser ML, Lin DW, Kwon EM, Feng Z, Ostrander EA and Stanford JL (2011) AMACR polymorphisms, dietary intake of red meat and dairy and prostate cancer risk. Prostate 71:498-506.).

The rs3195676 SNP, located on exon 1 in a coding region, results in the substitution of valine for methionine. The use of bioinformatics tools to predict the influence of this exchange of amino acids on protein integrity has demonstrated that this substitution is not tolerated in carriers of recessive alleles of this SNP, suggesting that this results in protein damage and lower expression (Levin et al., 2007Levin AM, Zuhlke KA, Ray AM, Cooney KA and Douglas JÁ (2007) Sequence variation in α-methylacyl-CoA racemase and risk of early-onset and familial prostate cancer. Prostate 67:1507-1513.).

Lee et al. (2013)Lee SJ, Joung JY, Yoon H, Kim JE, Park WS, Seo HK, Chung J, Hwang JA, Hong SH and Nam S (2013) Genetic variations of α-methylacyl-CoA racemase are associated with sporadic prostate cancer risk in ethnically homogenous underwent radical prostatectomy in Koreans. Korean Biomed Res Int 2013:394285. studied this polymorphism and demonstrated that male carriers of allele G with a Gleason score ≥ 7 and ≥ pT2c present a greater risk of developing PCa. An in vitro experiment also demonstrated that a decrease in the level of AMACR protein results in a decrease in the proliferation of prostate adenocarcinoma cells (Zha et al., 2003Zha S, Ferdinandusse S, Denis S, Wanders RJ, Ewing CM, Luo J, De Marzo AM and Isaacs WB (2003) Alpha-methylacyl-CoA racemase as an androgen-independent growth modifier in prostate cancer. Cancer Res 63:7365-7376.). This explains the results obtained herein, given that whenever the A allele of the SNP was present, it provided protection against more aggressive PCa - the AMACR protein was damaged and likely less expressed, improving the prognosis for the patient carriers of the rare allelei n relation to seminal vesicle invasion. Genotypes of the AMACR gene (GA/GA+AA) alone or in combination with the AKT1 gene (CC) significantly protected the occurrence of seminal vesicle invasion.

The AKT1 and AR genes, alone or combined, showed protection for PCa, while AKT1, PI3K, AR, and AMACR genes protected against PCa aggressiveness. Only the PTEN gene associated with AMACR or AR indicated a risk for extracapsular extension. These results, showing the influence of the SNPs assessed on the clinical and pathological outcomes of PCa, are summarized in Figure 1.

Figure 1
An individual's genetic profile can: (A) Identify men with genotypes that confer protection for prostate cancer. (B) Discriminate men with a better or worse prognosis for prostate cancer.

The identification and implementation of novel molecular markers to improve the prediction of tumor behavior in patients with PCa will reduce the variability among observers regarding the distinction of many histopathological parameters. This is important for treatment decisions that depend heavily on the opinions of an individual pathologist (Hoogland et al., 2014Hoogland AM, Kweldam CF and Leenders GJLH (2014) Prognostic histopathological and molecular markers on prostate cancer needle-biopsies: A review. Biomed Res Int 2014:341324). The results of our study indicate SNPs as potential molecular markers to be used in prognostic panels for PCa, however, these findings still need additional confirmation before use in clinical practice.

Acknowledgments

The authors thank the Cancer Hospital of Londrina, the Consórcio Intermunicipal de Saúde do Médio Paranapanema, and Irmandade Santa Casa de Londrina, Paraná, Brazil, for their support and partnership, as well as the volunteers, both patients and control, that participated in this study. This research was supported by the Araucaria Foundation of Support for the Scientific and Technological of Parana (185/2014);

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  • Associate Editor: Mara H. Hutz

Publication Dates

  • Publication in this collection
    01 June 2020
  • Date of issue
    2020

History

  • Received
    07 Nov 2018
  • Accepted
    31 July 2019
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