Vitamin D mediates anti-proliferative and pro-differentiation signalling in various epithelial tissues, including the mammary gland . Therefore, it is reasonable to assume that disruption of the Vitamin D signalling and metabolic pathways may occur during tumour development. To explore this hypothesis, we have evaluated a cohort of 947 samples of human breast tissues for the presence of VDR, CYP27B1 and CYP24A1. Specifically, our series consisted of normal breast tissue (29 cases), preneoplastic benign mammary lesions (379 cases), carcinomas in situ (189 cases) and invasive breast carcinomas (350 cases). To the best of our knowledge, this is the first time that the expression of the VDR, CYP27B1 and CYP24A1 has been evaluated in histological sections of mammary lesions.
The three proteins have been found to be expressed in all breast tissues, although at different levels. VDR presented a nuclear localisation, as it would be expected for a nuclear receptor, while CYP27B1 and CYP24A1 enzymes displayed cytoplasmic staining with a granular pattern, which is consistent with their mitochondrial localisation. The immunohistochemical results were further validated and confirmed using quantitative real-time PCR and Western blotting.
Some studies have demonstrated that the VDR protein is expressed in samples from normal breast tissues and also in breast cancer biopsy specimens [14, 15, 24, 25]. Our results have shown that the VDR is expressed in carcinomas. However, the percentage of positive cases that we have obtained (47.3% in carcinomas in situ and 56.2% in invasive carcinomas) is lower than the 80% to 90% that had been previously described in the literature [26, 27]. This discrepancy can be explained by the development of new detection techniques and the use of different scoring methods. In this study, we have used the H-Score, the current method employed for other nuclear receptors, like ER , whereas in previous studies the presence of any staining was marked as positive. As far as we know, our study is the first to investigate the immunohistochemical expression of the VDR in a range of benign lesions and carcinomas in situ of the mammary gland. The percentage of positive cases for the VDR is higher in benign lesions than in invasive tumours (93.5% and 56.2%, respectively), while the carcinomas in situ display the lowest value of all (47.3%). There are some studies showing higher levels of VDR in tumour tissues [18, 28], but this discrepancy can be attributed to the use of different evaluation techniques.
An interesting finding is the correlation between the expression of the VDR and the ER in both in situ and invasive carcinomas. In fact, the VDR is expressed in most ER-positive cases (54.7% in in situ carcinomas and 65.5% in invasive tumours). It is thought that one of the VDR functions is to counteract oestrogen-mediated proliferation and maintain differentiation . Indeed, data support the concept that the anti-tumour effects of Vitamin D and its analogues on ER-positive human breast cancer cells are mediated through the down regulation of the ER itself and the attenuation of oestrogen responses, such as breast cancer cell growth [29, 30]. Thus, being the VDR mostly expressed in ER-positive carcinomas, Vitamin D or its analogues may become an alternative therapy for these tumours in cases of resistance to ER-targeted therapy.
The levels of protein expression of CYP27B1 and CYP24A1 have not been previously studied in breast cancer. In colon cancer, a study using immunohistochemistry has demonstrated that CYP27B1 is present at equally high levels in normal colonic epithelium and colorectal cancer . For CYP24A1 it has been shown that increasing amounts of this enzyme are present in normal colon tissue and pre-malignant lesions. In cancer, the expression of CYP24A1 decreases as a function of tumour cell dedifferentiation . In breast tissues, McCarthy et al. have demonstrated that CYP27B1 mRNA expression was significantly down regulated in adjacent non-cancerous tissue from women with breast cancer in comparison with individuals without cancer. Additionally, it has been shown that the expression of mRNA for CYP27B1 and the VDR was higher in carcinomas versus non-neoplastic tissue . Considering differences in expression in benign and malignant breast tissues, we have observed an increased expression of CYP24A1 and a decreased expression of CYP27B1 with malignant progression. In fact, CYP27B1 was expressed in 55.8% of the preneoplastic lesions and this percentage is decreased in invasive tumours (44.6%), while carcinomas in situ display the highest value (66.4%) and these differences are statistically significant. In contrast, CYP24A1 is augmented more than 2.5 fold in invasive tumours (53.7%), compared with benign breast lesions (19.0%) and this difference is also significant (p < 0.0001). The in situ carcinomas exhibit the highest percentage of positive cases (56.0%). These observations are consistent with the results of Townsend and colleagues , which have demonstrated that there was an up regulation of CYP24A1 mRNA in breast tumour tissue, in comparison with normal breast. It has also been described that the CYP24A1 gene is amplified in breast cancer . In contrast, another study has found no differences in the expression of the VDR, CYP27B1 and CYP24A1 mRNA in breast cancer and non-neoplastic mammary tissue . These contradictory results may be explained by recent reports where it is described that VDR and CYP24A1 are under the post-transcriptional control of miRNAs [35, 36].
Breast cancer is a process that evolves through the accumulation of (epi)genetic events that drive uncontrolled proliferation and resistance to apoptosis. The active form of Vitamin D is known for its capacity to modulate proliferation and induce apoptosis . Consequently, malignant cells would need to develop mechanisms to deregulate Vitamin D metabolic and signalling pathways in order to allow tumour development . Furthermore, it has been suggested that the Vitamin D produced in non-renal tissues is not released into the blood stream, but instead acts locally . Therefore, the amount of Vitamin D available in the tissue depends on the relative amounts of CYP27B1 (synthesis) and CYP24A1 (catabolism). Accordingly, our results show a deregulation of these two enzymes in the different stages of breast carcinogenesis. The crucial step of transformation introduces a clear unbalance in the Vitamin D signalling and metabolic pathways. A reduction in the expression of the VDR in carcinomas indicates lower sensitivity of the tissue to Vitamin D control. Furthermore, a strong increase in CYP24A1 positive cases points to an enhanced ability of the cells to degrade this hormone. In contrast, the stable levels of CYP27B1 throughout the transformation process, with only a small decrease in invasive carcinomas, may reflect a lower capacity to metabolize Vitamin D into its active form.