Abstract
Background/Aim: Previous studies have associated certain variations in genes encoding factors of renin-angiotensin system (RAS), indirectly leading to higher angiotensin II (AngII) levels, with greater risk for basal cell carcinoma (BCC) development. Chymase (CMA1) is the main regulator of the RAS-independent AngII generation pathway and numerous studies have shown its oncogenic potential in several cancer types including BCC. In this study, we investigated the possible association between BCC pathogenesis and the functional DNA polymorphism A1903G (rs1800875) that affects expression of the CMA1 gene. Patients and Methods: We genotyped 199 DNA samples, isolated from 100 BCC patients and 99 age, sex, and ethnicity-matched healthy controls for the CMA1 A1903G polymorphism. Genotyping was performed with PCR amplification, followed by MboI enzyme digestion and agarose gel electrophoresis of the resulted DNA fragments. Results: The variant G allele that possibly increases CMA1 gene expression was not detected at a significantly different frequency between the groups of BCC patients and healthy controls. However, the AG heterozygous genotype was significantly increased in BCC patients compared with controls (p<0.001). Conclusion: The high expression CMA1 G allele carriers have an increased risk for BCC and elevated levels of chymase in the skin may have a carcinogenic effect.
Basal cell carcinoma (BCC) is the most common skin malignancy worldwide, which associates high morbidity but low mortality (1). BCC accounts for about 75% of all skin cancers in light-skinned populations and it is the most frequent skin cancer of the head and neck region with the majority of diagnosed cases presenting tumors on the face (2-5). It descends from undifferentiated cells of the basal layer of the epidermis that have undergone accumulative DNA damage, resulting in uncontrollable malignant proliferation (6). Although it rarely metastasizes, BCC can present increased local invasiveness that may be destructive to nearby soft tissues and bone structures.
BCC pathogenesis is a result of the interaction between environmental, phenotypic, and genetic factors (7). In particular, clear skin phenotypes, freckles in childhood, light eyes, and hair, alongside a BCC-positive family history are considered the most important predisposing factors. The most prevalent environmental risk factor is exposure to ultraviolet radiation (UVR), particularly in skin-sensitive life stages such as childhood and adolescence (8, 9). Other contributing factors include exposure to radioactivity or arsenic, persistent wounds that do not heal, chronic inflammatory skin conditions and complications from burns or infections (10). In terms of genetics, BCC patients often present with de novo or inherited mutations in the highly studied patched 1 (PTCH1) and tumor protein 53 (TP53) genes, as well as polymorphic variations in genes coding for key compounds of the renin-angiotensin system (RAS) (11-14).
RAS is intricately involved in the overall homeostasis, through the regulation of arterial pressure, peripheral vascular resistance as well as electrolyte balance and volume of extracellular fluids and exists both in a circulating form and several tissue equivalents in almost every organ system (15). RAS involves the conversion of angiotensinogen (AGT) by renin into angiotensin I (AngI), which is then hydrolyzed by the angiotensin converting enzyme (ACE) into active AngII, which after binding to its main receptor, promotes vasoconstriction, stimulates the sympathetic nervous system, and controls renal electrolyte flow (16, 17). In addition, AT1R bound AngII exerts hypoxic oxidative stress and inflammation effects, but it also promotes growth, cell proliferation and migration. The latter capabilities connect RAS with cancer development (15, 18-20).
Our group has previously shown that the involvement of RAS in BCC development is associated with certain functional polymorphisms in the genes of AGT and ACE, which indirectly increase the levels of AngII (11, 21). Our findings are in accordance with previous studies that have shown a strong association between the pharmacological treatment with either ACE inhibitors or angiotensin receptor blockers and reduced BCC risk (21, 22).
A RAS-independent pathway of AngII tissue generation includes the proteolytic enzyme chymase (CMA1), a serine protease present in mast cell secretory granules that plays an important role in inflammation and matrix remodeling (23). CMA1 is highly active in the epidermal tissue due to its involvement in the formation of collagen fibrils through the cleavage of type I procollagen, as well as its ability to penetrate and destroy the basal membrane and induce dermal–epidermal separation (24). In addition, CMA1 cleaves the decapeptide AngI and hydrolyzes it into AngII both in tissue and circulation, alternatively to ACE (25-29). In fact, CMA1 is responsible for 80% of total production of circulating AngII, while ACE generates the rest (29).
Interestingly, numerous studies have shown the oncogenic potential of chymase, as well as the association between increased CMA1 tissue levels and the development of several cancer types including BCC (24, 30-32). These observations raise the possibility that a genetically determined constitutive increase of chymase production and/or function may be a risk factor for carcinogenesis.
A highly studied functional polymorphism in the CMA1 gene (locus14q12) is the A1903G (rs1800875) (29, 33). The G allele of the polymorphism is associated with increased CMA1 enzymatic activity and AngII accumulation as well the induction of vasoconstriction and hypoxic effects (32). Moreover, A1903G has been previously associated with cardiovascular diseases that implicate high AngII levels such as hypertension (29).
In the present study we investigated the possible association between the functional A1903G polymorphism of the CMA1 gene and the risk for BCC development, in order to determine whether another factor that takes place in AngII generation is involved in the pathogenesis of this particular carcinoma.
Patients and Methods
Ethics statement. The protocol used was approved by the Ethics Committee of the Department of Oral and Maxillofacial. Surgery (27022019) of the National and Kapodistrian University of Athens in accordance with the standards of the 1964 Declaration of Helsinki. The individuals under study gave their informed consent to be included in the study.
Subjects. A total of 199 individuals of Greek origin were enrolled in this study, including 100 BCC patients aged between 28 and 96 years, and 99 controls of the same age range. The patients included 41 males (45.1%) with a mean age of 70.1 (±12.5) years and 59 females (54.9%) with a mean age of 72.2 (±11.2) years. The healthy control group consisted of 45 males (45.5%) with a mean age of 68.2 (±11.6) years and 54 females (54.5%) with a mean age of 71.0 (±12.2) years. The diagnosis for all patients was confirmed by clinical examination and biopsy findings.
Genotyping. Genomic DNA was extracted from peripheral blood leukocytes with the standard phenol-chloroform method. The genotyping of CMA1 A1903G polymorphism was carried out by PCR amplification using the following primers: Forward: 5′-TCC ACC AAG ACT TAA GTT TTG AT 3′ and Reverse: 5′-GGA AAT GTG AGC AGA TAG CG-3′ based on the protocol developed by Wu et. al. (35). The initial denaturation step at 95 C for 4 min was followed by 31 cycles of 95°C for 1 min, 56°C for 1 min, 72°C for 1 min, and finally an elongation step at 72°C for 8 min, resulting in an amplified DNA product of 120 bp. Incubation with restriction endonuclease MboI in the presence of the G allele resulted in a 100 bp and a 20 bp fragment visualized after agarose gel electrophoresis (Figure 1).
Agarose gel electrophoresis results after incubation with restriction endonuclease MboI illustrating the A1903G polymorphism genotypes AG, AA, and GG. The normal allele A has an intact fragment size of 120 bp, while the variant allele G has a fragment of 100 bp after a restriction cleavage.
Statistical analysis. Statistical analysis was performed using SPSS v.21.0 (IBM Corp., Armonk, NY, USA). The frequencies of the alleles and genotypes of the whole group of patients were compared to the respective frequencies of the control group using the Fisher’s exact test. All the observed genotype and allele frequencies were prior tested for compliance with Hardy-Weinberg equilibrium. The Maentel-Haenzel method was used for the calculation of all odds ratios with a 95% confidence interval (CI). A p-value of less than 0.05 was considered statistically significant.
Results
In the group of healthy controls, the observed and expected frequencies of CMA1 A1903G genotypes were not significantly different (Table I). Therefore, the control population under study was in Hardy-Weinberg equilibrium for this polymorphic trait and further analysis was valid.
Hardy–Weinberg equilibrium comparison results for genotypes observed in the group of controls.
The detected genotypes of the CMA1 A1903G polymorphism in the groups of controls and BCC patients are shown in Table II. The frequency of the AG genotype differed significantly between patients and controls (p<0.001), while the frequencies of the AA and GG genotypes showed no statistical difference (p>0.1). Moreover, the A and G allelic frequencies did not differ between patients and healthy controls (p>0.1).
Comparison of genotype and allele frequencies concerning the CMA1 A1903G between controls and patients.
Within the control group, the percentage of AG carriers was 5 times higher than the percentage of GG homozygotes, while AA homozygotes were present in 39.4% of the control population. Within the group of BCC patients, the vast majority (86%) carried the AG genotype, while only 14% carried the AA genotype and none was found with GG genotype.
Discussion
Basal cell carcinoma (BCC) is the most common skin malignancy, which presents low mortality but extremely high morbidity worldwide (1). The indirect implication of the RAS in BCC pathogenesis was initially documented in the early 2010s when two studies demonstrated the chemopreventive properties of the pharmacological treatment with ACE inhibitors and angiotensin II (AngII) blockers (21, 22). In light of the already proven, well-documented oncogenic properties of AngII (17), and in accordance with the above findings, our group has shown that RAS is indeed involved in BCC development through certain functional polymorphisms in the genes of angiotensinogen (AGT) and angiotensin-converting enzyme (ACE), which indirectly increase the body’s AngII levels (11-13).
An ACE-independent molecular mechanism for the production of angiotensin II (AngII), the effector hormone of the RAS, involves an alternative angiotensin II converting enzyme known as chymase (CMA1) that accounts for the majority of tissue and circulating AngII production (29). CMA1 is a serine chymotryptic protease, which is highly active in the epidermal tissue both in matrix remodeling and inflammation. Being a major AngII generator, CMA1 has demonstrated oncogenic properties in the skin and other tissues according to several studies (24, 30-32).
In the present study we investigated the possible association between the functional A1903G promoter polymorphism (rs1800875) of the CMA1 gene and the risk for BCC development, in order to determine whether the genetically determined increase of CMA1 gene expression that results in increased production of that AngII generating factor is implicated in BCC pathogenesis. Our hypothesis was based on the fact that A1903G has been previously associated with AngII accumulation, vasoconstriction and blood vessel hypoxia, as well as with the development of AngII-related pathologies such as hypertension (29, 34).
The results of our analysis showed no significant difference of allelic frequencies between the group of BCC patients and matched healthy controls. Nevertheless, there was a significant increased frequency of the AG genotype of the CMA1 A1903G polymorphism in patients versus controls (p<0.001). In fact, the great majority of BCC patients (86%) carried the AG genotype.
In light of the above, the A1903G polymorphism of the CMA1 gene is strongly associated with increased risk for BCC development. The findings of the present study indicate that the high expression CMA1 G allele carriers have an increased risk for BCC and provide a genetic confirmation that elevated levels of chymase in the skin may indeed have a carcinogenic effect. It is known that the G allele of the polymorphism is associated with increased CMA1 enzymatic activity and AngII accumulation as well the induction of vasoconstriction and hypoxic effects in blood vessels, in addition to hypertension implicating high AngII levels in cardiovascular diseases (29, 34-36). The observation of the present study that there was lack of GG genotypes in the group of BCC patients could possibly indicate that homozygosity for the high activity variant allele G might result in more aggressive phenotypes with higher mortality than BCC. According to the literature, increased levels of active CMA1 are more prevalent in aggressive malignancies (30-32), as well as in severe cardiovascular diseases (34-36). Perhaps the GG genotype might cause an increased risk either in aggressive malignancy or severe hypertension and people who carry this genotype would not have time to live long enough to develop BCC. Further research in various human populations could clarify this point.
On the other hand, the AG genotype was detected in a remarkably high frequency in our study’s BCC group, compared to healthy controls. Consequently, our results suggest that the AG genotype may be associated with benign or slow-growing tumors such as BCC. Due to their low mortality rates, mild neoplasms are rarely investigated as thoroughly and by using alternative approaches as in the present study. To validate or refute this assumption, however, it is necessary to conduct additional research on this particular polymorphic trait, including patients with other mild neoplasms such as adenomas and hemangioblastomas, and to compare the results to those of studies pertaining to aggressive carcinomas. This may also clarify whether the A allele itself exerts protective anticancer properties or is associated with a favorable prognosis.
In conclusion, the current study is the first to genetically associate high expression of CMA1 gene with the development of a skin cancer type. The main source of chymase is the mast cells of the skin and blood vessels, and chymase is responsible for 80% of the produced AngII levels in the circulation. Since chymase is not affected by ACE-inhibitors and angiotensin-receptor blockers, the CMA1 AG genotype may serve as a potential biomarker to evaluate an individual’s risk of developing BCC, alongside the ID and MT genotypes of ACE and AGT genes, respectively (11, 12). Further studies are needed to investigate the relative association of the previously mentioned and other functional polymorphisms leading to AngII production with basal cell carcinogenesis. Such genetic association studies may eventually lead to better strategies for skin cancer prevention in at-risk individuals in the general population.
Acknowledgements
The Authors wish to thank all individuals (BCC patients and healthy controls) who donated blood samples and made this study possible. This work was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH-CREATE-INNOVATE (Project code: T2EDK-Milksafe).
Footnotes
Authors’ Contributions
Sevastiana Charalampidou performed the main work of molecular analysis and prepared the initial draft of the manuscript; Iphigenia Gintoni assisted in initial laboratory work and in the initial draft of the manuscript; Dimitris Avgoustidis performed statistical analysis; Dimitris Vlachakis assisted in research logistics and corrected the manuscript; Veronica Papakosta 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 in relation to this study.
- Received July 25, 2022.
- Revision received September 12, 2022.
- Accepted September 13, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
