The multiple forms of exon 1 and the tissues in which each is expressed.
Estrogens play an important role in the development and progression of several types of cancers. The synthesis of estrogens occurs in almost all tissues of the body in addition to the gonads. The enzyme aromatase (CYP19A1) encoded by CYP19A1 gene is involved in the synthesis of estrogens. Genetic variations in CYP19A1 gene influence both the structure-function relationship of the enzyme and the rate of its synthesis. Extensive studies have reported different types of polymorphisms in the CYP19A1 gene and have shown that the polymorphisms, depending on their location in the gene, have different effects on the function and activity of the gene product. Association studies have been conducted and have led to the realization that interpopulation differences are widespread. Not only do polymorphic forms exert different effects on the development of different cancers, due possibly to the influence of other genetic variations, environmental, metabolic, and epigenetic factors, but also are important as they lead to the interindividual differences seen during treatment of the cancer state. This chapter covers important aspects of the aromatase function, the CYP19A1 gene structure, polymorphisms identified in the gene, different cancers and associated polymorphisms, and the role of the polymorphic forms in affecting the treatment strategies.
- aromatase inhibitors
By the late 1990s, several epidemiological and clinical studies had shown that estrogens play an important role in the development and progression of several types of cancers, in particular breast, endometrial, prostrate, and colorectal cancer (CRC). A strong connection was shown to exist between initiation/promotion of cancer and excess of estrogens, as the latter increased mitotic activity. Initially, extensive studies linked the administration of exogenous hormones to the development of these cancers , and later it was shown that several estrogen-sensitive tissues act as intracrine organs, producing estrogens and hence elevating local hormone levels, which accelerated proliferation and growth of cancer cell .
There are four major naturally occurring estrogens, estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4), which are produced only during pregnancy. Estradiol (E2) is an important estrogen, has the highest affinity to estrogen receptors, and is required for different physiological functions during all stages of life in both males and females . Aromatase, due to its critical role in the synthesis of the different forms of estrogens from androgens , specifically estradiol from testosterone, estrone from androstenedione, and estriol from 16α-hydroxylated dehydroepiandrosterone, is incriminated as a major player in cancer biogenesis.
2. The enzyme “aromatase”: a key player in estrogen synthesis
Aromatase (EC 184.108.40.206), also known as estrogen synthase, is the gene product of
Several studies suggest that many tumors are dependent upon estrogens for their development and continued growth . Blockage of any conversion in the pathway potentially leads to decreased estrogen production, but more specific suppression results from inhibition of the final step that is unique to estrogen biosynthesis, i.e., inhibition of aromatase. The key role of aromatase in estrogen biosynthesis has generated enormous interest in putative inhibitors of the enzyme and their use as therapy against endocrine-responsive tumors.
Initially, it was believed that the ovaries and placenta are the only site for the production of estrogens, which are involved in female reproductive functions. However, later studies conducted using many sophisticated and sensitive tests and equipment revealed that estrogens are also synthesized in the male gonadal tissues, i.e., the testis and epididymis, and in extra-gonadal tissues including liver, colon, prostate, brain, adrenal gland, skin, bone, hair follicles, adipose, and vascular tissues. This is due to the presence of aromatase, which is active in various tissues in both females and males, and hence estrogens are produced in gonads and in the extra-gonadal tissues [2, 7–9].
Aromatase is a dimer, a complex of two polypeptides; one is a specific cytochrome P450, which is the product of the
2.1. Aromatase gene structure
This first exon (exon 1) occurs in multiple forms, which encode the 5′ untranslated region (UTR) of the
|Exon 1 type||Expressed in (tissue)|
|I.1 (1a) and I.2 (1e)||—Placenta|
|I.3 (1c) and PII (1d)||—Ovary|
|I.4 (1b)||—Adipose tissue|
—Vascular endothelial tissues
|II. I.3, I.7, and I.4||—Breast|
Investigations have shown the expression of aromatase gene in estrogen-dependent breast cancer (BC) tissues, endometrial carcinoma, and colorectal, gastric, liver, lung, ovarian, pancreatic, and prostatic cancers [2, 12]. The cell-specific expression of aromatase determines the presence or absence of aromatase activity in the tissue and hence the amount of available estrogens. The transcriptional regulation of aromatase has been extensively investigated since the 1980s. Many mechanisms have been proposed to explain the underlying control of
2.2. Genetic polymorphisms of the
Frequencies at which the alleles occur in different populations differ considerably, and it is also shown that the differences in the plasma levels of several sex hormones may be due to the presence of different alleles, particularly in postmenopausal women. It is hypothesized that the genetic polymorphisms provide a probable explanation for differences in cancer risk among different ethnic groups. Hence, the presence of the different
This section presents the polymorphisms identified in the
2.2.1. Short tandem repeat (STR) polymorphism
Short tandem repeats (STRs) were identified in the aromatase gene during the late 1990s, and some studies showed a higher incidence of cancers in individuals carrying different alleles of the
Several studies conducted in different populations reported association between the tetranucleotide repeat sequence and cancer, while others failed to do so. Kristensen et al.  reported that a rare polymorphic A1 allele of
Several studies were published from Russia, and it was shown by Artamonov et al.  that the allele (TTTA)8 was associated with BC (11.8 vs. 6.3%; p = 0.04). Risk of BC elevated if this allele was present with genotype A2/A2 of the tetranucleotide deletion (7.3 vs. 0%; p < 0.01). In Norwegian women , the association of breast cancer with the long allele (TTTA)12 was shown.
Kim et al.  reported that though there is no difference in the (TTTA)n genotype distribution between patients and controls, but there was a positive association between >(TTTA)10 and ER-negative tumors and between lower repeat polymorphism and ER-positive tumors (p = 0.019). A study on different ethnic groups (African-Americans, Japanese, Latinas, and non-Latina Whites) by Probst-Hensch et al.  reported contradictory results and showed no consistent association of (TTTA)n repeat polymorphism with breast cancer risk. This was in line with other studies from the USA  and Greece . These studies show that the tetranucleotide polymorphism occurs in different populations, and the repeat variants show association with breast cancer in some and not others. Often, contradictory results are reported from the same population.
The (TTTA)n polymorphism has been reported to modify susceptibility to prostate cancer development in several studies in different populations. Most of the studies have shown that the longer alleles (TTTA)7 or more are associated with a higher risk of prostate cancer, and in some studies, it is shown that the association is also with cancer-specific survival [32, 33]. A study on the Japanese men reported that (TTTA)7 and (TTTA)8 alleles show association with the risk of prostate cancer . It was also shown that when the patients were stratified according to the pathological grade or the clinical stage, there was no significant difference in the different genotypes. Tsuchiya et al.  regarded this polymorphism as a novel predictor of prostate cancer with bone metastasis. They showed that alleles longer than seven repeats (TTTA)7) were associated with worse cancer-specific survival. Tang et al.  further suggested that though these repeat polymorphism influence disease susceptibility, but the effect is modified by factors that alter hormone metabolism.
The (TTTA)n has also been investigated in endometrial cancer, and some studies have shown that the long alleles A6 and A7 occur at a higher frequency in the patients than the controls [37–39]. It was shown that the longer A6 and A7 alleles have higher intratumoral aromatase activity, thus predisposing to increased synthesis of estrogens and hence increasing the local estrogen concentration, which supports proliferation .
2.2.2. TCT insertion/deletion in intron 4
A TCT insertion/deletion (ins/del) polymorphism occurs in the intron 4 of the
2.2.3. Single nucleotide polymorphisms
The presence of single nucleotide variations in the DNA sequence of
Single nucleotide polymorphism (SNP) association studies have been reported in several cancers and provide several interesting cues about the role of these variations. This section presents different types of cancer and the SNPs identified and studied.
3. CYP19A1 polymorphisms in different cancers
3.1. Variations in CYP19A1 gene and breast cancer
Breast cancer remains as the most frequently occurring cancer in all races and all ethnic groups (https://nccd.cdc.gov/uscs/toptencancers.aspx). Estrogens affect proliferation and growth of the cells in the breast. Thus, polymorphisms of the genes, which are involved in the estrogen biosynthesis and metabolism, have been regarded as factors affecting the risk of breast cancer. Research conducted since the early 1970s confirmed that the major risk and predisposing factor for breast cancer is increased exposure to estrogens and progesterone . Obesity was also shown to be one of the factors since adipose tissues are an important source of endogenous estrogens . Furthermore, local production of estrogens in the breast tissue was shown to play a major role in elevating hormone levels in the breast tissue, which in turn accelerates proliferation and growth of breast cells and subsequent progression to malignant transformation . Genetic factors increase susceptibility to develop breast cancer, and in the 1990s, the identification of two breast cancer susceptibility genes, BRCA1 and BRCA2 , turned the attention of breast cancer research to the identification of possible genetic markers of breast cancer susceptibility. Extensive research has led to accumulation of knowledge about genetic variation in different genes, including the
|Population/study location||Clinical implication||Reference|
|rs10046||Inuit women||Nonsignificant association with BC|||
|rs10046||Iranian||Significant association with BC|||
|Swedish||Predictive marker for early breast cancer events in|
AI-treated patients with ER-positive tumors
|rs6493497||USA||Associated with decreased bone density with AI treatment|||
|rs4646||China||AA is significantly associated with improved clinical outcome of hormone therapy; prolonged DFS|||
|rs4646, rs10046, rs700518, rs749292 rs2289106, rs3759811, rs4775936||American||Significantly associated with decreases in triglycerides on letrozole treated|||
|rs10046||Xinjiang Uigur women||TC genotype and an abortion can reduce the risk of the breast cancer|||
|rs934635, rs60271534, rs700518||A meta-analysis||Treatment response|||
|rs4646, rs10046, rs936308||American||Were associated with bone AEs|||
|rs4646||Chinese||Related to DFS in early breast cancer and that the prognosis index of the homozygous for the minor allele (AA) may depend on menopause status|||
|American ancestry, Hispanic Americans, and Mexico Whites||Significantly associated with increased breast cancer risk|||
|rs10046||Japanese||Significant risk predictor|||
|rs700519||India||Could be used as molecular markers to assess breast cancer|||
|rs10046||Austrian population||Association with clinical characteristics|||
|rs10046||Spain||Related to levels of circulating estradiol and to the estradiol/testosterone ratio|||
|Taiwan||Associated with poor survival of premenopausal women but does not affect survival of postmenopausal women|||
|rs4646||American||May serve as a prognostic marker of the response to anastrozole in patients with MBC|||
|rs10046, rs4646, rs2830, rs9926298, and rs9939609||China||Nonsignificant association with BC|||
|rs700518, rs10459592, and rs4775936||Korea||Significantly associated with clinical efficacy|||
|rs10046, rs4646, rs74929,|
|Italian||The aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients|||
|Swedish||Involved in both breast cancer risk and prognosis|||
|Arg264Cys||Korean||Increased breast cancer risk|||
|rs1008805||USA||G allele associates with breast cancer|||
|rs2236722||Japanese||Trp more frequent in ER+|||
|rs1870049, rs1004982, rs28566535, rs936306, rs11636639, rs767199, rs4775936, rs11575899, rs10046, rs4646||Chinese||No association|||
The rs10046 polymorphism is a C/T transition in the 3′ UTR of
The rs4646 polymorphism is an A/C transversion, located in the 3′ UTR of the
Santa-Maria et al.  have recently reported that several SNPs influence the plasma lipid levels in patients treated with letrozole, where rs4646, rs10046, rs700518, rs749292, rs2289106, rs3759811, and rs4775936 decreased triglyceride levels and had a variable effect on the level of HDL-C.
A number of other SNPs have been investigated in breast cancer; some are associated with the risk of breast cancer, while others are not (Table 2). Some influence the clinical presentation of the disease, the prognosis, and the disease-free intervals, while still others modulate the effect of treatment and the associated complications.
3.2. Variations in CYP19A1 gene and endometrial cancer
Endometrial cancer (EC) is one of the most frequently encountered gynecologic malignancies, and a strong association is shown to exist between excess of estrogens and initiation and promotion of endometrial cancer . As early as 1975, it became evident that estrogen may act as a carcinogen when unopposed by an adequate amount of progesterone. Many studies demonstrated higher risk of endometrial cancer in females on hormone replacement therapy . Since exposure to endogenous estrogens was regarded as an important determinant of risk of endometrial cancer, several studies were initiated to identify genetic variants and the role they play as risk factor for the development of endometrial cancer. One of the earliest studies was reported from Russia and implicated genetic variants of
3.3. Variations in CYP19A1 gene and prostate cancer
Prostate cancer is among the most frequently encountered non-cutaneous malignancy in men. Extensive research has been carried out to identify the etiology and pathological mechanisms, but the mechanism of prostate cancer development is not fully clear. Several factors have been implicated in its etiology including environmental, dietary, hormonal, lifestyle, and genetic factors. Studies have confirmed that estrogens may be closely involved in predisposing to or even causing cancer . Aromatase is shown to be altered in patients with prostate cancer, and its expression is elevated almost 30 times in the cancer tissue compared to the normal tissue [91, 92]. The mechanisms by which estrogens induce carcinogenesis in prostate tissue have been hypothesized in several studies and involve genotoxicity, after chronic inflammation, epigenotoxicity, hyperprolactinemia, and prostatic ER-mediated changes. The genetic factors in the patient are gaining considerable interest, and genetic polymorphisms are being regarded as prognostic predictors of metastatic prostate cancer .
A few studies have reported the influence of
A study recently reported on two populations of African ancestry failed to show any association between rs60271534 and prostate cancer risk . Another SNP 1531 C > T was investigated in the Turkish prostate cancer patients, but no significant association was observed . Lévesque et al.  reported a study in which results obtained in Caucasians and Taiwanese were compared. It was shown that rs12900487, rs4441215, and rs2470152 in
The effect of two or more coexisting SNPs in influencing predisposition to prostate cancer is shown in several studies, thereby implying that the SNPs may behave synergistically or have antagonistic effect and thus bringing further heterogeneity to SNP action . Cussenot et al.  reported that the long allele (>175 bp) of the TTTA repeat of
Interestingly, it was shown that some of the SNPs in
The plasma level of estrogens may also be altered by the presence of different SNPs in the
The presence of some SNPs also influences the effect of different nutritional and therapeutic agents used for protection from cancer. Sonoda et al.  report from Japan that the protective effect shown by isoflavones against prostate cancer is modified by the (TTTA) long repeat alleles and coexisting minor alleles of rs10046 in
3.4. Variations in
CYP19A1gene and colorectal cancer
It is well documented that estrogens play a role in the development and progression of colorectal cancer (CRC) [107–109]. The beneficial role played by estrogens in preventing CRC is obvious since males have a higher prevalence of CRC than premenopausal females, but the prevalence increases in menopausal females. Furthermore, females on hormone replacement therapy have a lower susceptibility to CRC . However, it is shown that estrogens are locally produced in the colorectal tissue and result in a higher level of E2 and a lower level of E1. This imbalance in E2/E1 ratio may result in an increase in cell proliferation and concomitant decrease in apoptosis, thus increasing the risk of CRC [111–113]. Normally, in the colon, E2 is converted to E1 by 17β-HSD2 and 17β-HSD4. The E1 is antiproliferative, and the E2/E1 ratio keeps a check on the cell cycle. In colon cancer since this ratio is altered, proliferation is accelerated [111, 112]. A study on Chinese men showed that there were elevated E2 levels and the presence of CT/TT genotype of ESR2 receptor increased the risk of CRC to 2.3 (95% CI = 1.4–3.9), compared to those who had lower levels of E2 and the ESR2 genotype CC .
Polymorphisms are reported in
It has also been reported that aromatase also participates in metabolizing various compounds produced endogenously, including sex hormones, lipids, and other lipid derivatives. The rate of metabolism of these compounds depends on the amount and activity of the enzyme, which in turn may be altered by the alleles of the different SNPs. Metabolic end products produced may increase or decrease the risk of CRC and hence the interindividual differences in inherited metabolic susceptibility to CRC. Inflammatory response to different exogenous and endogenous factors may also have a role in CRC .
3.5. Variations in
CYP19A1gene and ovarian cancer
Several clinical trials have provided evidence implicating hormone replacement therapy as a risk factor for development of ovarian cancer. However, the role played by estrogen in the etiology of ovarian cancer has yet to be unveiled. Polymorphisms in
3.6. Variations in CYP19A1 gene and hepatocellular carcinoma
Worldwide, the prevalence of hepatocellular carcinoma (HCC) is high, classifying it as one of the most common malignancies. Studies have suggested that sex hormones, including androgen and estrogen, may be involved in HCC development and progression , pointing toward aromatase variants in HCC development. However, studies on
3.7. Variations in
CYP19A1gene and esophageal adenocarcinoma
Esophageal adenocarcinoma (EA) prevalence is on the rise in the young Western population. A strong gender bias is shown in epidemiological studies, with a sex ratio of 8:9.1. It is suggested that the estrogens may be a protecting factor in females, since estrogens have been shown to stimulate apoptosis and decrease the growth of the esophageal squamous cells . It also decreases the expression of Ki-67 while increasing E-cadherin expression . However, not many studies have explored the role of SNPs in
Recently, a study by Lagergren et al.  pooled 14 studies from three continents (Australia, Europe, and North America) and investigated the effect of 60 SNPs in
3.8. Variations in
CYP19A1gene and gastric cancer
Gastric cancer is the fourth most common cause of cancer-related death in the world [http://www.who.int/mediacentre/factsheets/fs297/en/]. Studies have suggested that long exposure to estrogens, of ovarian or exogenous origin, may provide a protection against development of cancer [127, 128]. This finding has led to the implication of estrogen receptor defects in the development of gastric cancer . There are very few reports on the association between CYP19A1 gene polymorphism and gastric cancer risk. Freedman et al.  investigated 58 SNPs in six genes (
3.9. Variations in
CYP19A1gene and testicular germ cell tumor
In young men, testicular germ cell tumor (TGCT) is reported to be the most common cancer. It is hypothesized that an imbalance in the in utero level of androgens and estrogens may be the major predisposing factor in influencing TGCT risk. Kristiansen et al.  conducted an investigation on Norwegian-Swedish case-parent trios and genotyped 105 SNPs in 20 genes whose gene products were involved in the sex hormone pathways. Three SNPs (rs2414099, rs8025374, and rs3751592) showed a statistically significant association with TGCT risk in the case-parent analysis. For each of the studied SNP, the T alleles were associated with an elevated risk of TGCT (OR = 1.30, 1.30, and 1.21, respectively). No differences were identified in allelic effect estimates when the parental inherited genetic variation was correlated with the TGCT risk in the offspring. Furthermore, no differences were observed between the Norwegian and the Swedish populations for each of the studied SNP. It was concluded that aromatase may be a factor playing a role in the etiology of TGCT. However, this statement needs confirmation from further population-based studies.
4. Effect of SNPs on prognosis and survival of breast cancer patients
Some of the SNPs in CYP19A1 gene have been linked to disease prognosis and survival. It was shown that rs28566535, rs730154, and rs936306 are significantly associated with plasma levels of estrone as well as with breast cancer survival [133, 134]. Long et al.  showed an association between genetic polymorphisms of the CYP19A1 gene and breast cancer survival. Udler et al.  presented preliminary evidence suggesting that germline variation in genes involved in steroid hormone metabolism may alter breast cancer prognosis.
5. Effect of SNPs on hormonal parameters
Variations related to the effect of SNPs on biochemical and hormonal parameters are also reported in a few studies. Huhtaniemi et al.  did not find any associations between
Estrogen levels are influenced by the presence of different genotypes of a SNP, as reported in some studies but not in others. Thompson et al.  showed in a comprehensive study that the SNP rs727479 was associated most strongly with circulating E2 concentrations in postmenopausal healthy controls and its effect was stronger in obese females. Cai et al.  showed that rs1902584 in block 1 was associated with estradiol only in overweight postmenopausal women.
6. Influence of SNPs in CYP19A1 gene on treatment with aromatase inhibitors
The treatment strategies have been extensively investigated in breast cancer, due to the high prevalence and associated morbidity and motility. There are a number of options for the treatment of the different types of cancer, and generally a multidisciplinary approach is preferred. The options are dependent on the type of cancer, patients’ history, and the characteristics of the tumor. Some of the more common treatment strategies are surgery, radiotherapy, chemotherapy, and hormonal therapy. The two common antiestrogen therapies are tamoxifen and aromatase inhibitors. The former is used generally for the treatment of ER+ breast cancer in premenopausal women, while the latter is under investigation for treatment of premenopausal breast cancer patients. In the postmenopausal women, the aromatase inhibitors (AIs) are reported to have a higher efficacy compared to tamoxifen in the postmenopausal group in relation to metastasis and prognosis in the presence of adjuvant treatment. However, ethnic differences and interindividual differences are frequently reported and are related to genetic variations.
As stated in the earlier part of this chapter, there are a significantly large number of SNPs in the CYP19A gene. Some of these have an influence on aromatase activity and hence influence the level of estrogens. Such mutations play a role in the effectiveness of the clinical efficacy related to treatment strategies.
Several studies have evaluated the effect of the genotype on the efficacy of the AI used for treatment of cancer. There are several contradictory reports, and the SNP may or may not associate with AI treatment complications. Those SNPs, which influence aromatase activity and are associated with elevated levels of estrogens, such as rs6493497 and rs7176005, seem to alter the effectiveness of AI . On the other hand, it was shown that rs700518, rs10459592, and rs4775936 were significantly associated with higher clinical benefit rate with letrozole treatment . Ferraldeschi et al.  investigated the effect of 56 SNPs on AI treatment and concluded that none of the variants independently were associated with improved AI efficacy and emphasized the significance of further studies on genetic biomarkers as prognostic factors in pharmacogenetic studies.
Table 3 lists a few SNPs and their influence on the outcome of AI treatment.
|SNP||Effect on AI treatment||Reference|
|rs6493497||Almost 13-fold increase estradiol concentration in post-anastrozole. Alters effectiveness of AI treatment|
associated with decreased bone density in the exemestane-treated patients
|rs7176005||Almost 11-fold increase estradiol concentration in post-anastrozole. Alters effectiveness of AI treatment|||
|rs4646||Associated with poor response to the AI, letrozole (p = 0.03)||[134, 142]|
|rs700518||—GG genotype of rs700518 increases risk for significant loss of fat-free mass and increase in truncal fat with AI therapy|
—AA genotype of rs700518 is at risk for AI-associated bone loss
|rs700518||Significantly associated with higher clinical benefit rate with letrozole treatment|||
|rs1062033||CC genotype in postmenopausal patients had lower spine and hip bone mineral density compared with the GG genotype. No correlation with AIs was established|||
|rs60271534||Lower incidence of AI-associated arthralgia. But the effect was not definite as two different AIs were used|||
|rs10046||Five-year disease-free survival was enhanced in premenopausal women on AI therapy|||
|rs700519||Associated with lower aromatase activity, but no studies showed a significant correlation with AIs|||
Aromatase is an essential enzyme required for the synthesis of estrogens. The polymorphic forms of aromatase gene seem to contribute to the development of different forms of cancer, and several avenues await exploration. Population differences in the frequencies of different SNPs and the association with the different disease states need further detailed study. Association studies are required to confirm if there is a risk or protective effect of the SNP genotype. Studies on disease prognosis, in relation to the different genotypes of a SNP, are required. Finally, the influences of the SNP on treatment strategies are warranted. Individualized medicine is the dream of present-day clinicians. The role played by SNPs may contribute to achieve this dream.
We thankfully appreciate the support provided by the Central Laboratory during the preparation of this chapter.