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Association of Breast Cancer Progression with a Vitamin D Receptor Gene Polymorphism¹

Divisions of Cell Biology [A-C. L., P. S., B. E.] and Oncology [M. B-J., S. W.], Department of Biomedicine and Surgery, Faculty of Health Sciences, S 581 85 Linköping, Sweden

	ABSTRACT

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The vitamin D₃ receptor gene (VDR) contains a TaqI RFLP that is associated with increased VDR mRNA stability, increased serum levels of 1,25-dihydroxyvitamin D₃ (1,25-D₃), and decreased risk for prostate cancer. Determination of the TaqI genotype, in a group of young women with breast cancer (n = 111; age, <37 years) and a control population (n = 130), revealed no overall association to risk for breast cancer. However, patients without TaqI site (TT genotype) showed a significantly increased risk for lymph node metastasis (relative risk, 1.8, 95% confidence interval, 1.3–2.6). Furthermore, a tendency toward an increased survival was found among estrogen receptor-positive, tamoxifen-treated patients who were homozygous for the TaqI site (P = 0.075). We conclude that polymorphism in the VDR gene may influence tumor progression and tamoxifen treatment response in early-onset breast carcinomas.

	Introduction

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The physiologically active form of vitamin D, 1,25-D₃,⁴ possesses a regulatory function in bone metabolism and calcium homeostasis (1) . 1,25-D₃ exerts its growth-regulatory effects through binding to the VDR, a member of the steroid/thyroid/retinoic acid receptor family, which functions as a ligand-dependent transcription factor (1) . VDR in complex with its ligand forms heterodimers with the retinoid X receptor and associates with specific DNA sequences, i.e., vitamin D response elements. However, several reports have also demonstrated that 1,25-D₃ is involved in the regulation of growth and differentiation in breast and prostate cancer cells (1, 2, 3, 4, 5, 6, 7, 8) . The growth-inhibitory effects, found in breast cancer cell lines, are potentiated by simultaneous administration of 1,25-D₃ and antiestrogens (9, 10, 11, 12, 13) . Adjuvant treatment with the antiestrogen tamoxifen, which interacts with the ER, has been shown to improve the survival in breast cancer patients with tumors containing ERs (14) .

Several polymorphisms in the 3' untranslated region of the VDR gene are in strong linkage equilibrium with RFLPs both in intron 8 (Bsm1 and ApaI) and exon 9 (TaqI). The presence of the TaqI site (t) correlates with increased transcriptional activity, mRNA stability, and high serum levels of 1,25-D₃ (15) . Furthermore, individuals of tt genotype were found to have decreased risk of prostate cancer (16) . Polymorphism in genes involved in metabolism of and/or response to hormones, drugs, and other factors may affect the onset and/or prognosis of diseases and thus act as modifiers. We hypothesize that allelic variants of VDR influences onset and outcome of breast cancer, considering the T allele as a risk factor and the t allele as a protective factor, as well as the response to adjuvant antiestrogen treatment. To test this hypothesis, in a case control study, we compared the VDR genotypes in a group of young premenopausal breast cancer patients, with retained estrogen production, and in a healthy female control population.

	Materials and Methods

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Breast Cancer Patients.
The study included 111 young breast cancer patients diagnosed in the South-East Health Care Region between 1980 and 1993. At the time of diagnosis, the patients were between 24 and 36 years of age, with a median age of 34. Survival data from the Cause of Death Register, with a median follow-up time of 67 months, were provided by the National Board of Health and Welfare by December 31, 1996. The South-East Sweden Breast Cancer Group contributed data on lymph node status, staging, and treatment. Stages were reported from 81 patients. Twenty-six (32%), 51 (63%), and 4 (5%) were grouped in stages I, II, and III-IV, respectively. Available data of adjuvant treatment showed that 21 patients were treated with chemotherapy, and 27 were treated with tamoxifen. ER levels were determined by isoelectric focusing in polyacrylamide gel, and after January 1, 1988, we used an enzyme immunoassay. Tissue samples from archival material were obtained from the pathology departments of hospitals in Linköping, Norrköping, Jönköping, and Kalmar.

Control Group.
Female blood donors (n = 130) at the Blood Donor Center, Linköping University Hospital, were used as a control population. Their ages ranged between 19 and 64 years, with a mean age of 37 years. We compared the VDR TaqI RFLP distribution in the oldest group (n = 40; 42–64 years) with the youngest population (n = 40; 19–30 years), and no age-related differences were found in VDR genotype.

Isolation of DNA.
DNA from the breast cancer patients was isolated from formalin-fixed, paraffin-embedded lymph nodes without cancer cells. The tissue sections were deparaffinized with xylene, and DNA was isolated by proteinase K digestion and phenol-chloroform extraction. DNA from the controls was isolated from blood samples and collected in EDTA tubes, using the Genomic DNA Purification kit (Promega, Inc.).

PCR Assay.
The TaqI VDR RFLP was determined by a PCR-based method. A 198-bp DNA fragment was generated using PCR primers (VDR99F, 5'-GTG GGA TTG AGC AGT GAG-3' and VDR99R, 5'-TGG ATC ATC TTG GCA TAG AG-3') located within intron 8 and exon 9. Exon 9 contained a silent polymorphism in codon 352, ATC or ATT, coding for isoleucine. The TaqI restriction enzyme recognizes and cleaves the ATC allele, which is designated t. DNA amplifications were performed in a PCR reaction of 20 µl containing 1x PCR buffer provided by the Taq polymerase supplier (Life Technologies, Inc.), 20 ng of DNA, 2 mM MgCl₂, 200 µM deoxynucleotide triphosphate, each primer (1 µM), 1.5 units of Taq polymerase (5 units/µl), and DMSO to a final concentration of 5%. The genomic DNA was initially denatured at 95°C for 5 min and thereafter subjected to 35 cycles of PCR amplification with denaturation for 0.5 min at 95°C, annealing for 0.5 min at 60°C, extension for 1 min at 72°C, and a final extension at 72°C for 5 min. For a few samples, a secondary PCR reaction was performed; 1 µl of the primary reaction was reamplified for 16 cycles in the same PCR mix as described above. Thereafter, 15 µl of the PCR products were incubated for 2 h at 65°C with 26 units of TaqI (MBI Fermenta) and then separated on a 3% agarose gel containing ethidium bromide (0.5 µg/ml). An intact PCR product indicates absence of TaqI site (T allele), whereas the presence of the TaqI site (t allele) displays a DNA fragment of 99 bp (Fig. 1) .

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Restriction fragments after incubating a 198-bp product from intron 8 and exon 9 in the VDR gene with TaqI restriction enzyme for 2 h in 65°C. Separation is made on 3% agarose gel loaded with ethidium bromide. Two 100-bp ladders are used. tt represents homozygous presence of a Taq restriction site, TT is the homozygous absence, and Tt is the heterozygous form.

	Results

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The determination of the TaqI genotype in women with breast cancer and in the control population displayed similar frequencies and revealed no association to risk of breast cancer (Table 1) . The distribution of clinicopathological factors in genotypes and calculated RRs are shown in Table 2 . A tendency toward a decreased mortality rate was found in patients homozygous for the t allele (22%) as compared with women homozygous (41%) or heterozygous (44%) for the T allele (Table 2) . Furthermore, women of the TT genotype expressed a significantly increased risk (RR, 1.8; 95% confidence interval, 1.3–2.6; P = 0.004) for lymph node metastasis (N⁺) as compared with patients who were carriers of the t allele (Table 2) .

	Discussion

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As shown in several reports from studies on malignant cell lines, including leukemia, colon, prostate, and breast cancer cells, 1,25-D₃ influences both cell proliferation and cell differentiation (1, 2, 3, 4, 5, 6, 7) . A decrease in malignant behavior, such as invasive growth and tumor progression, has been found after treatment with 1,25-D₃ (2, 3, 4, 5, 6, 7) . In addition, inhibition of tumor progression by 1,25-D₃ treatment has also been demonstrated in a nitrosomethylurea-induced rat mammary tumor model (2) . The present study indicates that lymph node metastasis is associated with the TT genotype, and this finding can be compared with an earlier report, where low protein levels of VDR is correlated with lymph node metastasis (17) . The significance of VDR in primary breast cancer is further supported by an earlier observation showing that the majority of tumors express the VDR receptor and the significantly longer disease-free survival, found in patients with VDR-positive tumors (3) . It has also been reported that individuals homozygous for the t allele are significantly underrepresented among prostate cancer patients (16) , illustrating a protective role for the t allele in prostate carcinogenesis. However, no difference in VDR genotype between the breast cancer patients and the control group was found in our study, indicating that VDR RFLP cannot predict early-onset breast cancer.

The growth-suppressing effect from 1,25-D₃, has been shown to be mediated by the VDR in a prostate cancer cell line. However, this cellular response did not correlate with the receptor content, suggesting that there are additional factors involved in vitamin D-mediated growth regulation (18) . Contradictory results are reported regarding the interaction between VDR and ER. Vink-van Wijngaarden et al. (12) observed no effect on the ER content by 1,25-D₃ treatment of MCF-7 cells, whereas James et al. (11) found a dose-dependent down-regulation of the ER in the same cell line. Love-Shimenti et al. (13) examined whether ER-positive (ER+) and ER-negative (ER-) cells were differentially affected by 1,25-D₃ and analogues and found ER- cells to be more responsive to 1,25-D₃ than the ER+ cell population. They also reported that in the presence of estradiol (E₂), low doses of 1,25-D₃ provided a proliferative response, whereas at high doses, the effect was inhibitory in ER+ cells. Withdrawal of E₂ from the ER+ cell culture or addition of antiestrogens abolished this biphasic response to one resembling ER- cells.

In the present study of premenopausal women, it was of interest to assess the survival of tamoxifen-treated, ER+ patients and the relation to VDR genotype. The analysis indicated a tendency of increased survival rate among patients homozygous for the t allele as compared with women with other genotypes. Although the number of cases in this analysis is low (n = 15), it is tempting to speculate that the elevated serum levels of 1,25-D₃, which is reported to correlate with this genotype (15 , 16) , potentiate the effects of tamoxifen. Another possibility may be that the tt genotype is associated with enhanced VDR mRNA stability or higher transcriptional activity (15) , which permits the 1,25-D₃ to act more efficiently than in Tt and TT genotypes. The advantage with combined treatment of tamoxifen and 1,25-D₃ in ER+ cells (13) might also result from the antiestrogenic effect of tamoxifen, converting a biphasic response to 1,25-D₃ to a monophasic response and abolish the growth-promoting effect, allowing growth-inhibitory effects from the physiological levels of 1,25-D₃.

Finally, we observed that among breast cancer patients homozygous for the t allele, significantly more individuals received tamoxifen treatment as compared with Tt and TT genotypes. This indicates that subjects homozygous for t display a phenotype and/or clinical characteristics indicative for tamoxifen treatment. However, the significance of the VDR TaqI RFLP in breast cancer may differ in pre- and postmenopausal women because of differences in steroid hormonal status or tumor properties, suggesting that additional and larger investigations are needed to elucidate the influence of VDR.

	ACKNOWLEDGMENTS

 
We thank all of the women, cases and controls, who have made this investigation possible.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Swedish Cancer Society.

² To whom requests for reprints should be addressed, at Division of Oncology, Department of Biomedicine and Surgery, Faculty of Health Sciences, S 581 85 Linköping, Sweden. Phone: 46 13 22 34 89; Fax: 46 13 22 28 46; E-mail: sten.wingren{at}onk.liu.se

³ Members of the South-East Sweden Breast Cancer Group: B. Nordenskjöld, L. G. Arnesson, A. Malmström (Linköping), H. Bång (Motala), E. Einarsson (Eksjö-Nässjö), A. C. Källström (Norrköping), B. Norberg (Jönköping), Å. Henning (Oskarshamn), M. Sundqvist (Kalmar), W. Adlouni (Värnamo), and G. Tejler (Västervik).

⁴ The abbreviations used are: 1,25-D₃, 1,25-dihydroxyvitamin D₃; VDR, vitamin D receptor; ER, estrogen receptor; RR, relative risk.

Received 1/19/99. Accepted 4/ 1/99.

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