Abstract
Background: International mortality and frequency rates for breast cancer have been associated with the wet type of human earwax. It was recently found that earwax type is determined by a single nucleotide polymorphism (SNP), 538G>A (Gly180Arg), in ABCC11. The G allele determines the wet type of earwax as a Mendelian trait with a dominant phenotype. The present study examined the association between the frequency rate of breast cancer and the frequency of the G allele of ABCC11. Patients and Methods: Using blood samples from patients with invasive breast cancer (n=270) and control volunteers (n=273), the 538G>A SNP in ABCC11 was genotyped using the SmartAmp method. Results: The frequency of the G allele in breast cancer patients was higher than that in healthy controls. The odds ratio for the genotypes (G/G+G/A) to develop breast cancer was estimated to be 1.63 (p-value=0.026), suggesting that the G allele in ABCC11 is associated with breast cancer risk. Conclusion: This study showed that Japanese women with wet earwax have a higher relative risk of developing breast cancer than those with dry earwax. The ABCC11 SNPs that determine these phenotypes should be further investigated in order to obtain insights into the mechanisms by which breast cancer develops and progresses.
Breast cancer is the most common cancer among women in the industrialised world, where it accounts for 22% of all cancers in women. There has been increased interest in the genetic predisposition for many common cancer types, including breast cancer (1). In 1971, Nicholas L. Petrakis first reported that international mortality and frequency rates for breast cancer appeared to be associated with the frequency of the allele for wet-type earwax (2). Caucasian and African-American women in the USA as well as German women exhibited approximately four-fold higher rates of breast cancer mortality than Japanese and Taiwanese women (2). Nevertheless, the phenotypic association of the wet type of earwax with breast cancer has remained controversial (2, 3).
Recent studies (4, 5) have provided evidence that the type of earwax is determined by one single nucleotide polymorphism (SNP), 538G>A (Gly180Arg), in the ATP-binding cassette (ABC) transporter ABCC11 located on human chromosome 16q12.1 (6). The G/G and G/A genotypes correspond to the wet type of earwax, whereas A/A corresponds to the dry type (5). Wide ethnic differences have been observed in the frequencies of those alleles (4, 7). Among worldwide populations, the ‘G’ (wild-type) allelic frequency shows striking downward geographical gradient distributions from Africa to Far East Asia, supporting previous phenotypic observations (8). Interestingly, there are strong associations among wild-type ABCC11, earwax type (4), axillary osmidrosis (5, 9, 10), and apocrine colostrum secretion from the mammary gland (11). Furthermore, ABCC11 mRNA is highly expressed in breast tumours (6, 12, 13).
At the present time, it is not well understood whether wild-type ABCC11 actually influences breast cancer risk. The present study therefore genotyped the 538G>A SNP in 543 Japanese women to examine the association between the frequency rate of breast cancer and the allele frequency of the wild-type. For this purpose, the SmartAmp method was used to measure 538G>A SNP frequency in ABCC11 (14, 15). The results suggest that the wild-type allele in ABCC11 is associated with breast cancer risk.
Patients and Methods
Collection of blood samples from breast cancer patients and control volunteers. Blood samples from 270 Japanese female patients with invasive breast cancer who had been diagnosed at Yokohama City University Medical Centre from 1991 to 2008. In addition, blood samples were also collected from 273 Japanese female volunteers as controls. All blood samples were collected in 2Na-EDTA-coated blood collection tubes. All study participants provided written informed consent and protocols for the present study were approved by the Institutional Review Boards at both Yokohama City University Medical Centre and Mitsui Memorial Hospital. This clinical research study was conducted according to the Declaration of Helsinki Principles. Genotyping of ABCC11 in the blood samples by the SmartAmp method was approved by the Research Ethical Committee at RIKEN Yokohama Institute.
Clinicopathological data. For breast cancer patients, clinicopathological data was acquired such as age, body mass index (BMI), tumour size, lymph node metastasis (N), the status of the oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), triple negative (ER− PR− HER2−) tumour phenotype, nuclear grade, and the St. Gallen risk assignment criteria. ER, PR, and HER2 status in breast cancer was determined by immunohistochemistry.
Genotyping of ABCC11 by the SmartAmp method. Detection of the 538G>A SNP in ABCC11 was performed as described previously (5). Prior to the SmartAmp reaction, the blood samples were incubated at 98°C for 3 min to destroy RNA and to denature proteins and genomic DNA. After chilling on ice, each sample (1 μl) was added directly into the SmartAmp reaction mixture (final total volume of 25 μl). The reaction mixtures were then incubated at 60°C for 40 min under an isothermal condition in a real-time PCR model Mx3000P system (Agilent technologies, La Jolla, CA, USA), where the fluorescence intensity of SYBR® Green I dye indicating DNA amplification was monitored during the reaction.
Statistical rationale. The sample size required for this clinical study was calculated by assuming that 26% of Japanese breast cancer patients and 16% of control volunteers carry G/G homozygote or G/A heterozygote genotypes. The rationale for this assumption originated from Petrakis' pilot study (2), which showed that more Japanese women with breast cancer (9 out of 31; 29%) had the wet type of earwax than did women of the control group (9 out of 52; 17%). The minimum number of subjects needed for confirming significant differences between the cancer and control groups with 80% power was calculated to be 270 in total for a two-sided model at a 5.0% significance level. Thus, for this clinical study, blood samples were collected from a total of 270 Japanese breast cancer patients and 273 Japanese control volunteers.
Statistical analysis. To evaluate the statistical significance of observed data, χ2 tests were performed using the Dr. SPSS software (SPSS11.5J for Windows; SPSS Inc., Chicago, IL, USA) for univariate analysis and logistic regression for multivariate analyses.
Results
Detection of the 538G>A SNP in ABCC11 by the SmartAmp method. The SmartAmp method can genotype ABCC11 in blood samples within 40 min (5). Figure 1A depicts the time courses for the SmartAmp reaction that clearly discriminated the three different genotypes (538G/G homozygote, 538G/A heterozygote, and 538A/A homozygote) in human ABCC11, where blood samples were pre-treated and incubated as described in the legend of Figure 1A.
The mean age was 52.9±12.52 and 53.3±9.96 years old (mean±S.D.) for the breast cancer patient group and control group, respectively. The mean body mass index (BMI) value was 23.0±3.87 for the breast cancer patient group and 21.0±3.02 for the control group.
Table I compares the ABCC11 genotypes and earwax type in the study population. The odds ratio was calculated to be 1.63 (p-value=0.026) (Table I).
Figure 1B shows that the relative ratio of breast cancer patients carrying the homozygous 538G/G allele was 1.77-fold greater than that of the corresponding healthy volunteers. This relative ratio was even greater than that (1.41-fold) for breast cancer patients carrying the heterozygous 538G/A allele. The G allele appears to be positively related to breast cancer frequency in the groups of Japanese women studied.
Clinicopathological observations. The prognosis for breast cancer patients with wet earwax has been reported to be worse than for that for patients with dry earwax (2). In this context, it was anticipated in this study that the wet earwax genotypes might have some relation to specific clinicopathological features, such as the status of growth factor receptors or nuclear grade. Since histopathology data were available for all breast cancer patients involved in this clinical study, a possible relationship between ABCC11 genotypes and clinicopathological features including tumour size, lymph node metastasis, ER, PR, and HER2 status, triple negative tumour phenotype, nuclear grade, tumour stage, and St. Gallen risk was investigated (Table II). Nevertheless, neither chi-square tests nor logistic regression analysis revealed any statistically significant difference between the wet earwax genotypes (538G/G+538G/A) and the dry earwax genotype (538A/A) with respect to the clinicopathological features investigated in this study.
SmartAmp method-based genotyping of human ABCC11 in blood samples from breast cancer patients and control subjects. A: Blood was mixed with 50 mM NaOH (1:2 v/v) and incubated at 98°C for 3 min. After chilling on ice, 1 μl of the sample was mixed with the SmartAmp reaction mixture (a final total volume of 25 μl) containing 2.0 μM folding primer, 2.0 μM turn-back primer, 1.0 μM boost primer, 0.25 μM of each outer primer, 20 μM competitive probe, 1.4 mM dNTPs, 5% DMSO, 20 mM Tris-HCl (pH 8.0), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% (v/v) Tween®20, SYBR® Green I (Takara Bio Inc., Shiga, Japan) diluted 1/100,000, and 0.24 unit/μl Aac DNA polymerase (K.K. DNAFORM, Yokohama, Japan). SmartAmp reaction mixtures were incubated at 60°C for 40 min in a real-time PCR model Mx3000P system (Agilent Technologies, La Jolla, CA, USA). The fluorescence intensity of the SYBR® Green I dye was monitored. The time courses represent SmartAmp reactions with ABCC11 allele-specific primers: 538G (red) and 538A (blue). B: Relative ratios of breast cancer patients carrying 538G/G, 538G/A, and 538 A/A alleles. Data are normalised to the controls (female volunteers).
Comparisons of ABCC11 genotypes and earwax type between Japanese breast cancer patients and control subjects.
Discussion
Potential role of ABCC11 in breast cancer. The present study provides evidence that the wild type allele of the ABCC11 gene is associated with breast cancer risk, at least in the Japanese population. About 40 years ago, Petrakis (2) assumed that genetically determined variation in the apocrine system might influence susceptibility to breast cancer, although the genetic determinant (538G>A SNP in ABCC11) was not known at that time. It is only recently that more than 10 nonsynonymous SNPs have been found in the human ABCC11 gene (5, 7). Among those SNPs, one SNP (rs17822931; 538G>A, Gly180Arg) is thought to be a clinically important polymorphism that may related with breast cancer risk.
Genetic polymorphisms of ABCC11 gene. The wild-type (538G or Gly180) human ABC transporter ABCC11 expressed in apocrine glands plays a pivotal role in earwax secretion (4, 5), axillary osmidrosis (5, 9, 10, 16), and apocrine colostrum secretion from the mammary gland (11). Human ABCC11 reportedly functions as an ATP-dependent efflux pump for amphipathic anions, including oestrone 3-sulfate, dehydroepiandrosterone 3-sulfate (DHEAS), and oestradiol 17-β-D-glucuronide (16-18), suggesting a potential role of ABCC11 in the secretion of steroid metabolites from secretory cells within apocrine glands (16). Indeed, the transport activity of ABCC11 appears to be related to the size of the apocrine glands (5). The genetic polymorphism, on the other hand, has an impact on the N-linked glycosylation of ABCC11, intracellular sorting, and proteasomal degradation of the variant protein (5). The SNP variant (538A; Arg180), which lacks N-linked glycosylation, is recognised in the endoplasmic reticulum as a misfolded protein that is readily ubiquitinated and proteasomally degraded. Thus, the dry type of earwax was determined to be a Mendelian trait with a recessive phenotype (5). As a consequence, the SNP variant (538A or Arg180) fails to perform its transport function (4, 5), and the apocrine glands that are formed are notably small in size (5). Therefore, it is hypothesised that the function of ABCC11 per se or a metabolite transported by ABCC11 may stimulate the proliferation of apocrine gland cells. As far as the cell cycle machinery is operating normally, proliferation of apocrine gland cells should stop at certain levels. However, once somatic mutation has occurred in BRCA1, BRCA2, p21, or p53 gene, deleterious and unregulated proliferation of those cells may start.
Comparison of clinicopathological features between sub-populations with wet-type (538G/G + 538G/A in ABCCC11) and dry-type (538A/A in ABCC11) earwax in Japanese breast cancer patients.
It has also been reported that ABCC11 is potentially involved in drug resistance in breast cancer. ABCC11 mRNA is highly expressed in breast tumours (6, 12, 13), in particular, in invasive ductal adenocarcinomas (https://www.oncomine.org/resource/logn.html). Its expression is reportedly regulated by ER-α (19) and induced by 5-fluorouracil (5-FU) (20). In addition, it has been reported that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) (20-22). It remains to be elucidated, however, whether the expression of wild-type ABCC11 (538G) is related to drug resistance in breast cancer and high rates of mortality. In conclusion, the present study shows that Japanese women with wet earwax have a higher relative risk of developing breast cancer than those with dry earwax. Further examination of the ABCC11 SNPs that determine these phenotypes may provide useful insights into the mechanisms by which breast cancer develops and progresses, including drug resistance and chemosensitivity.
Acknowledgements
The Authors thank Drs. Susan E. Bates and Stephen J. Chanock (National Cancer Institute, NIH) for their helpful discussions. This study was supported by a Research Grant for RIKEN Omics Science Centre from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and the Japan Science and Technology Agency (JST) research project named, ‘Development of the World's Fastest SNP Detection System.’ Yu Toyoda is a research fellow of Japanese Society for the Promotion of Science (JSPS).
- Received September 20, 2010.
- Revision received October 24, 2010.
- Accepted October 27, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
