Abstract
Background/Aim: Basal cell carcinoma (BCC) has been genetically associated with an increased expression of angiotensin-converting enzyme (ACE), an important factor of the renin–angiotensin system which produces vasoconstrictor angiotensin II. Other factors of this system include angiotensinogen (AGT) and angiotensin receptors AGTR1, AGTR2. We investigated the possible association of BCC with genetic variability in the AGT, AGTR1 and AGTR2 genes. Materials and Methods: DNA samples of 190 Greeks were studied, including 91 patients with BCC and 99 matched healthy controls. Molecular genotyping of patients and controls was performed for the polymorphisms AGT M235T, AGTR1 A1166C and AGTR2 G1675A. Results: The mutant T allele that increases AGT gene expression was detected in two-fold increased frequency in BCC patients in comparison to healthy controls (p <0.001). On the contrary, no significant difference was observed in AGTR1 and AGTR2 variants between patients and controls. Conclusion: Increased expression of AGT may be associated with BCC.
Basal cell carcinoma (BCC) is the most common type of skin cancer, representing about 75% of all cases (1-7). Basal cell carcinogenesis is a multifactorial process. Environmental factors, including life-style preferences such as prolonged exposure to sun ultraviolet light especially of fair skin phenotypes, and inherited risk predisposition seem to additively contribute in the appearance and evolution of this cancer type (2, 8-15).
The pathogenesis of BCC is still quite unclear with many genes involved (16). Nevertheless, there seem to be some gene variants that increase or decrease the risk for basal cell carcinogenesis. The frequency of BCC seems to be significantly reduced in individuals treated with either inhibitors of angiotensin-converting enzyme (ACE) or angiotensin receptor blockers (17-19). Based on these observations, we previously detected an association between low expression of an allele of ACE gene and a decreased risk for BCC (20).
Based on that previous study, we investigated the possible influence on BCC risk of other factors of the renin–angiotensin system. The AGT gene encodes angiotensinogen (AGT) which is hydrolysed by rennin for the production of angiotensin I (21). AGT hydrolysis into angiotensin I is rate-limiting step in the rennin-angiotensin system, which results in the production of the vasoactive peptides angiotensin II and III by ACE (21). Angiotensin II exerts its biological effects via specific receptors, namely angiotensin receptors 1 and 2 (AGTR1 and AGTR2) (22-28). AGTR1 mediates most of the known functions of angiotensin II such as vasoconstriction, antinatriuresis, cell proliferation, and the liberation of catecholamines from sympathetic nerve endings and the adrenal medulla (29). AGTR2 probably counterbalances the vasoconstrictor and anti-natriuretic effects produced by angiotensin II via AGTR1 (29, 30).
Following our previous results indicating that a functional polymorphism in ACE gene affecting its transcription is associated with decreased risk for BCC (20), we investigated the possibility that genetic variability in the AGT, AGTR1 and AGTR2 genes might have an impact on basal cell carcinogenesis.
Results of Hardy–Weinberg equilibrium comparison of genotypes in controls.
Materials and Methods
The protocol used was approved (27022019) by the Ethics Committee of the University Department of Oral and Maxillofacial Surgery in accordance with the standards of the 1964 Declaration of Helsinki. The individuals under study gave their informed consent to the study.
Study groups. A cohort of 190 Greeks that included 91 patients with basal cell carcinoma (BCC) and 99 healthy blood donors of equivalent age and gender was studied. The patients were diagnosed with BCC within the last three years, based on clinical signs and a biopsy compatible with BCC pathological findings. The ages of the 91 patients ranged between 28 and 96 years, while the patients' mean age was 71.5±12.2 years. Male patients were 41 (45.1%) and their mean age ranged between 44 and 96 years (70.1±12.5 years), while female patients were 50 (54.9%) and their age ranged between 28 and 88 years (72.2±11.2 years). The mean age for the whole group of 99 controls was 70.1±12.1 years. Male controls were 45 (45.5%) and their age varied between 42 and 90 years (68.2±11.6 years). The female controls were 54 (54.5%) and their age varied between 31 and 90 years (71.0±12.2 years).
Genotyping. DNA isolation was performed from stored biopsies of patients or blood samples of healthy subjects using NucleoSpin® Tissue or Blood kits (Macherey–Nagel GmbH, Düren, Germany). Molecular genotyping for each DNA polymorphism was realized with a combination of PCR amplification and restriction enzyme incubation followed by agarose gel electrophoresis of the resulted DNA fragments (Figure 1). For the functional M235T polymorphism in the AGT gene (chromosome 1q42-43) the primers used were: Forward: 5’-CAGGGTGCTGTCCACACTGGACCCC-3’ and Reverse: 5’-CCGTTTGTGCAGGGCCTGGCTCTCT-3’. The PCR conditions consisted of an initial denaturation step at 95°C, followed by 30 cycles of 95°C for 50 sec, 60°C for 60 sec, and 72°C for 50 sec, as well as a final elongation step at 72°C for 5 min. After treatment with restriction enzyme Tth111, in the presence of the M allele the PCR product of 165 bp remained intact, while in the presence of the T allele it was cleaved into two fragments of 141 bp and 24 bp. The A1166C functional polymorphism in the AGTR1 gene (chromosome 3q24) was detected by restriction fragment length polymorphism typing of PCR products. The primers used were: Forward: 5’-AGAAGCCTGCACCATGTTTTGAG-3’ and Reverse: 5’-CCTGTTGCTCCTCTAACGATTTA-3’. The PCR conditions consisted of an initial denaturation step at 95°C, followed by 30 cycles of 95°C for 50 sec, 60°C for 60 sec, and 72°C for 50 sec, as well as a final elongation step at 72°C for 5 min. After treatment with restriction enzyme DdeI, the PCR product of 404 bp remains intact in the presence of the mutant C allele, while it is cleaved into two fragments of 286 bp and 118 bp in the presence of the normal A allele. To detect the G1675A functional polymorphism in the AGTR2 gene (chromosome Xq23) the following primers were used: Forward: 5’-ATTACGTCCCAGCGTCTGAG-3’ and Reverse: 5’-GGCACTAAGCAAGCTGATTTAT-3’. The PCR conditions consisted of an initial denaturation step at 94°C, followed by 34 cycles of 95°C for 50 sec, 55°C for 60 sec, and 72°C for 50 sec, as well as a final elongation step at 72°C for 5 min. After treatment with restriction enzyme Hpy188III the PCR product of 255 bp remains intact in the presence of the mutant A allele, while it is cleaved into two fragments of 194 bp and 61 bp in the presence of the normal G allele.
Agarose gel electrophoresis of restriction enzyme fragments. M: DNA weight marker-scale per 100 bp. A: Genotyping of BCC patients for the M235T mutation in the AGT gene; (1,2,5,7): Homozygous for the normal allele; (3,4,6,8,9,10,11): Heterozygous. B: Genotyping of BCC patients for the A1166C mutation in the AGTR1 gene; (1,2,3,8,11): Homozygous for the mutant allele; (7): Homozygous for the normal allele (4,5,6,9,10): Heterozygous. C: Genotyping of female controls for the G1675A mutation in the AGTR2 gene; (2,3,4,5,6,7,9,10,11): Homozygous for the normal allele (1): Homozygous for the mutant allele.
Statistical analysis. Data were analyzed using SPSS v.21.0 (IBM Corp., Armonk, NY, USA). All the observed genotype and allele frequencies were initially tested for compliance with Hardy-Weinberg equilibrium. The frequencies of alleles and genotypes of the patients' group were compared to the respective frequencies of the control group, using the Fisher's exact test. The calculation of all odds ratios with a 95% confidence interval (CI) was done using the Maentel-Haenzel method. A p-value of less than 0.05 was considered as statistically significant.
Results
In the group of controls, the observed and expected genotype frequencies did not differ significantly for the three genes (Table I). Therefore, the control population under study was in Hardy-Weinberg equilibrium for those variants and further analysis was legitimate.
The detected genotypes in controls and patients with BCC are shown in Table I. For the M235T functional polymorphism in the AGT gene, the frequencies of heterozygotes among controls and patients were significantly different (p<0.001). Furthermore, the patients had two-fold higher frequencies of the mutant allele (that increases gene expression rate) and mutant carrier status in comparison to the controls (p<0.001). On the contrary, there was no significant differences in the observed frequencies of genotypes, mutant alleles and carrier status of AGTR1 and AGTR2 gene variants between the cohort of patients and the group of controls (Table II).
Discussion
The molecular mechanism for the production of angiotensin II, the effector hormone of the rennin-angiotensin axis (21), involves both angiotensinogen (AGT) and angiotensin-converting enzyme (ACE). Angiotensin II exerts its biological effects by binding to its receptors AGTR1 and AGTR2 (21).
Comparison of AGT, AGTR1 and AGTR2 genotypes and allele frequencies between controls and patients.
We have previously observed that an ACE allele resulting in high gene expression is associated with increased risk for basal cell carcinogenesis (20). In accordance with our genetic findings, an association of angiotensin-converting-enzyme inhibitors (ACEIs) and angiotensin-receptor blockers (ARBs) with decreased risk for BCC has been reported (17, 18, 31).
In this study we have investigated the possibility that genetic variability in AGT, AGTR1 and AGTR2 genes might be associated with the risk for BCC development. The single base mutation resulting in threonine instead of methionine (M235T) in the 235 amino-acid position of AGT protein was detected in two-fold increased frequency in BCC patients in comparison to healthy controls. It is known that the mutant T allele increases the rate of AGT gene expression, and that it is associated with increased hypertension, as well as increased risk for thrombosis and malignancy (22, 32-34). Obviously, the increased levels of either AGT or ACE proteins result in increased production of the vasoactive peptide angiotensin II, which significantly attributes a higher risk for malignancy, including for basal cell carcinogenesis.
On the contrary, no difference in variant allele frequencies between BCC patients and controls was observed regarding an AGTR1 functional polymorphism previously detected to be significantly increased in hypertensive subjects or an AGTR2 functional polymorphism previously associated with vasoconstrictor and anti-natriuretic effects (24, 26). It seems that the AGTR1 and AGTR2 polymorphisms do not play a significant role in basal cell carcinogenesis because their function depends on the available amount of angiotensin II. In accordance to our findings, increased levels of AGTR1 were observed only in the tumor cells of basal cell carcinoma with follicular differentiation (35).
Further studies are needed to further decipher the association between basal cell carcinogenesis and factors of the renin–angiotensin system and their inhibitors or blockers. The findings of the present genetic association study and other relevant ones may increase our knowledge on BCC development and could ultimately result in establishing a preventive therapy safeguarding the health status of light-skinned at-risk individuals in the general population.
Acknowledgements
The Authors wish to thank all individuals (BCC patients and healthy subjects) who participated in this study.
Footnotes
Authors' Contributions
John Papaggelopoulos collected patients and did initial laboratory work; Antonia Angelopoulou performed the main work of molecular analysis and prepared the initial draft of the manuscript; Dimitris Avgoustidis performed statistical analysis; Nikolas Koronellos assisted in initial laboratory work; Spyridoula Derka and Stavros Vassiliou collected patients and made corrections in the manuscript; Christos Yapijakis conceived the study, supervised molecular analysis and authored the final draft of the manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received July 27, 2019.
- Revision received August 26, 2019.
- Accepted August 27, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
