Research ArticleProceedings of the 4th International Symposium on Vitamin D and Analogs in Cancer Prevention and Therapy, May 20-21, 2011, Homburg/Saar, Germany

1Alpha,25-dihydroxyvitamin D3 Induces de novo E-cadherin Expression in Triple-negative Breast Cancer Cells by CDH1-promoter Demethylation

NAIR LOPES, JOANA CARVALHO, CECÍLIA DURÃES, BÁRBARA SOUSA, MADALENA GOMES, JOSÉ LUIS COSTA, CARLA OLIVEIRA, JOANA PAREDES and FERNANDO SCHMITT
Anticancer Research January 2012, 32 (1) 249-257;

Abstract

Background: The triple-negative subgroup of breast cancer includes a cluster of tumors exhibiting low E-cadherin expression (metaplastic carcinomas). In several cancer models, 1alpha,25-dihydroxyvitamin D3 (1α,25(OH)2D3) induces differentiation by increasing E-cadherin expression. The Vitamin D receptor (VDR) was evaluated as a possible therapeutic target for metaplastic carcinomas and 1α,25(OH)2D3 effects as a differentiating agent in triple-negative breast cancer cells were assessed. Materials and Methods: Metaplastic carcinomas were assessed for VDR expression by immunohistochemistry; differences in E-cadherin expression in triple-negative breast cancer cells were evaluated by real-time PCR, western blotting and Cadherin 1 (CDH1) methylation status. Results: Most of the metaplastic carcinomas were positive for VDR expression. Furthermore, 1α,25(OH)2D3 promoted differentiation of MDA-MB-231 cells by inducing de novo E-cadherin expression, an effect that was time- and dose-dependent. Also, E-cadherin expression was due to promoter demethylation. Conclusion: Metaplastic carcinomas may respond to 1α,25(OH)2D3, since they express VDR and 1α,25(OH)2D3 induces de novo E-cadherin expression in breast cancer cells by promoter demethylation.

Breast cancer is a heterogeneous disease, comprised of diverse molecular subtypes associated with different biological behaviours and clinical outcomes (1, 2). Among all breast cancer subgroups, the triple-negative basal-like type is the most aggressive, presents poor patient outcome (2) and comprises a rare cluster of carcinomas entitled metaplastic tumors (3-5). Our group and others have demonstrated that metaplastic carcinomas are distinguished by high levels of expression of classical basal-like markers, such as cytokeratin (CK) 5/6, CK14, epidermal growth factor receptor (EGFR), vimentin and P-cadherin, as well as E-cadherin down-regulation (5-7). Furthermore, patients harbouring metaplastic tumors display a worse prognosis, exhibiting lower rates of disease-free survival than those with invasive ductal carcinomas (8, 9). Due to their triple-negative phenotype, metaplastic carcinomas do not have a directed therapy. Since radiation and chemotherapy remain the only options to treat these carcinomas, intensive research on alternative therapeutic strategies is mandatory.

1Alpha,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active form of vitamin D, is a steroid hormone that exerts most of its biological activities by binding to a specific high-affinity receptor, the vitamin D receptor (VDR) (10). We previously reported that 56% of invasive breast carcinomas express the VDR and, among these, 56% of the cases classified as triple-negative basal-like tumors are positive for VDR expression (11), suggesting that they may be responsive to the anti-carcinogenic properties of 1α,25(OH)2D3. In several cancer models, 1α,25(OH)2D3 participates in cell growth regulation and cell differentiation (12). In breast cancer cells, it was demonstrated that 1α,25(OH)2D3 is able to induce cells to be more adhesive to each other, as well as to some substrates, through an increase in the expression of endogenous E-cadherin and other adhesion molecules (13). Additionally, 1α,25(OH)2D3 promotes the differentiation of colon cancer cells by inducing the expression of E-cadherin in VDR-expressing cells (14) and a similar result was obtained in prostate cancer with a 1α,25(OH)2D3 analogue (15).

These data provide good evidence for the ability of 1α,25(OH)2D3 to act as an epithelial differentiation-inducing agent. Therefore, the purpose of the current work was to study if the VDR could be a potential therapeutic target for metaplastic triple-negative breast carcinomas. Additionally, the in vitro effects of 1α,25(OH)2D3 as a differentiating agent in triple-negative breast cancer cell lines were evaluated.

Materials and Methods

Immunohistochemistry. A series of 12 formalin-fixed paraffin-embedded metaplastic breast carcinomas were retrieved from the archives of the Federal University of São Paulo, Brazil and from the Federal University of Santa Catarina, Brazil. The cases were collected between 1994 and 2009. Immunohistochemical staining for the VDR was performed as described elsewhere (11).

Cell culture and treatments. All the breast cancer cells (MDA-MB-231, Hs578T and BT-549, commercially available from ATCC), representative of mesenchymal triple-negative breast cancer (16, 17) were grown in complete GIBCO, Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen, Carlsbad, CA, USA) in the presence of 10% foetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Treatments with 1α,25(OH)2D3 100 nM (Cayman Chemical, Denver, CO, USA), 5-aza-2-deoxycytidine 5 μM (5-aza-dC, Sigma, Munich, Germany), DMSO (dimethyl sulfoxide, vehicle for 5-aza-dC and Trichostatin A [TSA]) and ethanol (vehicle for 1α,25(OH)2D3) were performed for 72 hours, while the treatment with TSA 100 nM (Sigma) was performed only for 16 hours. Every 24 hours, the culture medium was changed and a fresh new treatment agent was added.

Western blotting. Total protein lysates were prepared from the cultured cells and the protein concentration was determined using the Bradford assay (Bio-Rad protein quantification system, Berkeley, CA, USA). Equal protein samples were separated in an 8% SDS-PAGE and the proteins were transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Chalfont St Giles, UK). For immunostaining, the membranes were blocked for non-specific binding with 5% (w/v) non-fat dry milk, in PBS containing 0.5% (v/v) Tween-20. The membranes were incubated with the primary antibodies (α-tubulin, clone DM1A, Sigma, 1:10000 for 1 hour; β-actin, clone I19, Santa Cruz [Santa Cruz, CA, USA], 1:1000 for 1 hour; E-cadherin, clone 24E10, Cell Signaling [Beverly, MA, USA], 1:1000 for 1 hour; and VDR, clone 9A7γE10.4, Calbiochem [Darmstadt, Germany], 1:400 overnight), followed by four 5 min washes in PBS/Tween-20; then they were incubated with horseradish peroxidase-conjugated secondary antibodies (all 1:1000, Santa Cruz) for 60 min. The membranes were then washed six times more for 5 min and the proteins detected using the ECL detection system (GE Healthcare Life Sciences).

RNA extraction, cDNA synthesis and quantitative real-time PCR. The RNA was extracted from the breast cancer cells using TRIzol® reagent (Invitrogen) and cDNA was synthesised from 1 μg of RNA, using an Omniscript Reverse Transcription kit (Qiagen, Düsseldorf, Germany), following the manufacturer's instructions. Real-time PCR was performed using TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA, USA), using 1 μL of cDNA and in accordance with the manufacturer's protocol. The TaqMan Gene Expression Assays used were Hs01023895_m1 (for CDH1 [Cadherin 1], Applied Biosystems) and TaqMan PreDeveloped Assay Reagents Human GAPDH (for GAPDH [Glyceraldehyde 3-phosphate dehydrogenase], Applied Biosystems). The reactions were performed using standard cycle parameters and relative transcript levels were determined using human GAPDH as an internal reference. Differences between samples were determined using the Quantitation–Relative Standard Curve method.

DNA extraction and CDH1 promoter methylation analysis. The DNA was extracted from the breast cancer cell lines using an ULTRAPrep Genomic DNA Blood and Cell Culture Kit (AHN Biotechnologie, Nordhausen, Germany), according to the manufacturer's instructions. Bisulfite treatment was performed on 300 ng of DNA, using an EpiTect Bisulfite kit (Qiagen) following the manufacturer's guidelines. Unmethylated cytosines were converted to uracil, whereas methylated ones remained unmodified. The 12 CpG sites (cytosine-phosphate-guanine) within the 90 base pairs upstream of the CDH1 translation start site (ATG) were analysed, as described elsewhere (18).

Immunofluorescence. The cells were seeded on coverslips and fixed with formaldehyde 4% (v/v) for 30 min The coverslips were washed three times with PBS for 5 min, followed by incubation with 50 mM NH4Cl in PBS for 10 min. Following another set of three 5 minute washes with PBS, the coverslips were incubated with Triton X-100 0.2% (v/v) for 5 min and washed with PBS three times for 5 min. Subsequently, they were blocked for non-specific binding with BSA 5% in PBS, containing 0.5% (v/v) Tween-20, for 30 min and incubated with the primary antibody for E-cadherin (Zymed, San Francisco, CA, USA, clone HECD1, 1:100) for 1 hour. After three 5 minute washes with PBS, the coverslips were incubated with a goat anti-mouse secondary antibody (Alexa Fluor 594, 1:500, Invitrogen), washed with PBS for 3 times 5 min and mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA).

Transfection with siRNA for VDR. MDA-MB-231 cells (2.5×105 cells) were cultured in 6-well plates for 24 hours. For each well, 150 nmol of siRNA against VDR (Hs_VDR_8 FlexiTube siRNA, Qiagen) or control siRNA (Qiagen) was used according to the manufacturer's instructions. After 5 hours of incubation, the cell medium was replaced and the cells were treated with 1α,25(OH)2D3 100 nM and ethanol. The evaluation of siRNA efficiency occurred 48 hours after transfection.

Statistical analysis. Differences between groups were assessed using Student's t-test. Differences with p-values <0.05 were considered statistically significant. All the presented results are representative of at least three independent experiments, unless stated otherwise.

Results

VDR expression in metaplastic breast carcinomas. Out of the 12 metaplastic breast carcinomas, 8 cases (66.7%) were positive for the expression of VDR (Figure 1).

VDR expression in triple-negative breast cancer cell lines. By western blotting, it was shown that all the cell lines studied were positive for VDR expression. The MDA-MB-231 and BT-549 cells seem to be more sensitive to 1α,25(OH)2D3, as in these cells there was a clear increase in VDR expression upon hormonal treatment (Figure 2).

Figure 1.
Figure 1.

H&E, magnification ×630 (A) and VDR, magnification ×400 (B) staining in a case of metaplastic breast carcinoma.

Figure 2.
Figure 2.

Western blot of VDR expression in MDA-MB-231, Hs578T and BT-549 breast cancer cell lines.

Effect of 1α,25(OH)2D3 on the expression of E-cadherin. A de novo expression of E-cadherin, by western blotting, was observed upon 1α,25(OH)2D3 treatment in the MDA-MB-231 cells (Figure 3A). As presented in Figure 3B, the expression of E-cadherin was dependent on the duration of treatment. The protein expression was first detected at 24 hours and increased with time. With 72 hours of treatment the E-cadherin expression level was dependent on the dose of 1α,25(OH)2D3 and was identified even with the very low dose of 1 nM (Figure 3C).

In the MDA-MB-231 cells, 1α,25(OH)2D3 was a potent inducer of CDH1 mRNA expression, displaying more than 10-fold induction, compared with the control (p<0.01) (Figure 4A). Furthermore, the level of expression induced by 1α,25(OH)2D3 was 2-fold higher than that produced by the demethylating agent 5-aza-dC alone and 3-fold higher than that induced by the histone deacetylation (HDAC) inhibitor agent TSA alone. However, both agents displayed an additive effect to 1α,25(OH)2D3, the highest levels of expression being induced when the three drugs were combined. These results were also confirmed by the protein expression (Figure 4A). In the BT-549 cells, 1α,25(OH)2D3 was unable to induce E-cadherin expression on its own. However, in the Hs578T cells CDH1 expression was significantly induced upon 1α,25(OH)2D3 treatment (Figure 4B). Furthermore, in both cell lines CDH1 mRNA expression was induced upon treatment with 5-aza-dC. Interestingly, 1α,25(OH)2D3 seemed to display an additive effect when administered with both 5-aza-dC and TSA. Again, the highest levels of CDH1 expression were achieved whenever all the agents were added together and, in this case, the BT-549 cells were more responsive than the Hs578T cells, which corroborated the VDR expression results. Furthermore, these results were confirmed by the protein expression (Figure 4B and 4C).

Figure 3.
Figure 3.

Effect of 1α,25(OH)2D3 on E-cadherin expression in MDA-MB-231 breast cancer cells treated for 72 hours (A) or for various times (B) or at various dose rates (C), and assessed by western blotting.

As shown by immunofluorescence in Figure 5, upon treatment with 1α,25(OH)2D3, the MDA-MB-231 cells exhibited expression of E-cadherin at the plasma membrane. In contrast, the expression of E-cadherin induced by 5-aza-dC alone was granular and dispersed throughout the cytoplasm. However, when these cells were treated with both agents, the E-cadherin expression was located at the membrane.

Mediation of 1α,25(OH)2D3-induced expression of E-cadherin. Since 1α,25(OH)2D3 alone induced E-cadherin expression at the protein level only in the MDA-MB-231 cells, the experiments using VDR knockdown with siRNA were only conducted in this cell line. Upon silencing of the VDR in the MDA-MB-231 cells, the E-cadherin expression after hormonal treatment was abrogated (Figure 6).

Mechanism of E-cadherin expression. Upon 1α,25(OH)2D3 treatment, partial demethylation of the CDH1 promoter in the MDA-MB-231 cells was observed (Figure 7). Demethylation was detected in 7 out of the 12 CpG sites analysed.

Discussion

The majority of the metaplastic breast carcinomas studied were positive for VDR expression, suggesting that they might be responsive to treatment with 1α,25(OH)2D3. In addition, 67% of the tumors had previously been characterised as negative for E-cadherin expression and 83.3% exhibited vimentin expression (unpublished results), showing that these tumors were indeed undifferentiated and could benefit from the differentiation-inducing properties of 1α,25(OH)2D3 treatment. In the in vitro model, 1α,25(OH)2D3 induced a de novo E-cadherin (epithelial differentiation marker) expression in the triple-negative MDA-MB-231 breast cancer cell line. This is an important finding, given the major role of E-cadherin as a tumor suppressor protein in lobular breast carcinomas and other cancer models (19, 20) and since down-regulation of E-cadherin is required to initiate breast cancer metastatic growth (21). Furthermore, this effect was dependent on the duration of treatment and the quantity of 1α,25(OH)2D3 supplied to the cells. As far as we know, this is the first study demonstrating the de novo induction of E-cadherin expression in breast cancer cells by 1α,25(OH)2D3 due to CDH1 promoter demethylation, although it has been reported that 1α,25(OH)2D3 can augment the expression of endogenous E-cadherin in mammary tumor cells (13). In addition, it has been demonstrated that a 1α,25(OH)2D3 analogue, increased the expression of E-cadherin in prostate cancer cells (15). In colon carcinoma cells, 1α,25(OH)2D3 is also known to promote differentiation by inducing E-cadherin expression and other adhesion proteins, an effect only observed in VDR positive cells (14). Likewise, in the MDA-MB-231 cells, E-cadherin expression was dependent on the presence of the VDR, suggesting it could mediate this effect.

Figure 4.
Figure 4.
Figure 4.

Effect of 1α,25(OH)2D3, 5-aza-dC and TSA on CDH1 mRNA expression and E-cadherin expression in MDA-MB-231 (A), Hs578T (B) and BT-549 (C) breast cancer cells (*p<0.05, **p<0.01).

Figure 5.
Figure 5.

Immunofluorescence of E-cadherin expression in MDA-MB-231 breast cancer cells (magnification ×400).

Figure 6.
Figure 6.

Effect on E-cadherin expression induced by 1α,25(OH)2D3 of VDR knockdown by siRNA in MDA-MB-231 cells.

Figure 7.
Figure 7.

Methylation analysis of CDH1 promoter in MDA-MB-231 breast cancer cells. ● – methylation, Embedded Image – hemimethylation, ○ – demethylation (A); Example of DNA sequences treated with ethanol or 1α,25(OH)2D3 (B).

In MDA-MB-231 cells, CDH1 trancription is silenced due to promoter methylation (22). Interestingly, the levels of CDH1 expression upon 1α,25(OH)2D3 treatment in the MDA-MB-231 cells were 2- and 3-fold higher than those induced by the demethylating agent 5-aza-dC and by the HDAC inhibitor TSA, respectively, while the combination of 1α,25(OH)2D3 with either of these molecules promoted an additive effect, which was further confirmed by the protein expression. In gastric cancer cells, 1α,25(OH)2D3 has been shown to work in synergy with 5-aza-dC and TSA (23), thus supporting the effect obtained in the present study. Additionally, in colon cancer cells with silenced HDAC3, E-cadherin expression increased upon treatment with 1α,25(OH)2D3 (24), a result that mimics that observed in the MDA-MB-231 cells upon treatment with TSA and 1α,25(OH)2D3. In the other cells tested (Hs578T and BT-549) the results were not so encouraging when 1α,25(OH)2D3 was used alone; however, CDH1/E-cadherin expression was detectable when the cells were treated with 1α,25(OH)2D3 together with 5-aza-dC or TSA.

Also remarkably, the 1α,25(OH)2D3 treatment promoted the correct localisation of E-cadherin at the cell membrane in the MDA-MB-231 cells, suggesting a functional adhesion molecule, unlike the granular and dispersed pattern of expression induced by treatment with 5-aza-dC, which is suggestive of a non-functional protein. Similarly, in colon carcinoma, upon 1α,25(OH)2D3 treatment, E-cadherin expression was observed at the cell membrane (14). However, this 1α,25(OH)2D3 effect on E-cadherin induction is not exclusive of disease settings, as in normal keratinocytes, the treatment with 1α,25(OH)2D3 stimulates the assembly of adherens junctions, assessed by translocation of E-cadherin to the cell membrane (25). Surprisingly, when the MDA-MB-231 cells were treated with both 1α,25(OH)2D3 and 5-aza-dC, the effect induced by 1α,25(OH)2D3 prevailed over the 5-aza-dC-induced effect and there was a rescue of E-cadherin expression back to the membrane, hinting that 1,25(OH)2D3 is indeed inducing not only the expression of E-cadherin, but, apparently, it is also important for the correct membrane localisation of the protein as a cell-cell adhesion molecule. Unlike the current results, 5-aza-dC was found to be necessary to sensitise leukaemia cells to differentiate in response to 1α,25(OH)2D3 treatment (26).

For the first time, 1α,25(OH)2D3 was found to promote partial CDH1 promoter demethylation, suggesting that 1α,25(OH)2D3 can work as a demethylating agent in MDA-MB-231 breast cancer cells. To the best of our knowledge, only one study has correlated 1α,25(OH)2D3 with methylation and reported that it induced methylation of CYP27B1 (the enzyme responsible for its synthesis) and, thus, silenced its expression (27). In colon cancer cells, where 1α,25(OH)2D3 induces E-cadherin expression, a new mechanism involving phosphoinositide signalling was recently proposed (28). Also in colonic cancer cells, a novel mechanism involving 1α,25(OH)2D3 in epigenetic events was reported, where the knockdown of KDM6B/JMJD3, a histone demethylase induced by 1α,25(OH)2D3, down-regulated E-cadherin expression (29). Studies addressing the importance of these mediators in breast cancer are still lacking.

In summary, the majority of metaplastic carcinomas examined were positive for VDR expression, hinting that this rare type of aggressive cancer may be responsive to the antitumor effects of 1α,25(OH)2D3. Furthermore, 1α,25(OH)2D3 induced the de novo expression of the epithelial differentiation marker E-cadherin in the highly metastatic, triple-negative MDA-MB-231 breast cancer cell line. To the best of our knowledge, this is the first report of the de novo induction of E-cadherin in breast cancer cells by 1α,25(OH)2D3 due to CDH1 promoter demethylation, therefore, revealing a novel mechanism for the action of 1α,25(OH)2D3 in breast cancer cells. The induction of differentiation promoted by 1α,25(OH)2D3 in triple-negative metaplastic breast cancer may decrease the aggressiveness of this subtype of mammary carcinomas and improve patient outcome, but further studies are necessary to confirm this hypothesis.

Acknowledgements

This study was supported by five research grants (Nair Lopes: SFRH/BD/39208/2007; Joana Carvalho: SFRH/BD/44074/2008; Cecília Durães: SFRH/BPD/62974/2009; Bárbara Sousa: SFRH/BD/69353/2010; Madalena Gomes: PIC/IC/83264/2007; José Luis Costa: SFRH/BPD/20370/2004) and by a scientific project (PIC/IC/83264/2007), all financed by Fundação para a Ciência e Tecnologia (Portugal). Salary support was provided for Joana Paredes and Carla Oliveira by the POPH – QREN/Type 4.2, European Social Fund and Portuguese Ministry of Science and Technology. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by Fundação para a Ciência e Tecnologia.

  • Received September 28, 2011.
  • Revision received November 10, 2011.
  • Accepted November 11, 2011.

References

  1. ↵
    1. Perou CM,
    2. Sorlie T,
    3. Eisen MB,
    4. van de Rijn M,
    5. Jeffrey SS,
    6. Rees CA,
    7. Pollack JR,
    8. Ross DT,
    9. Johnsen H,
    10. Akslen LA,
    11. Fluge O,
    12. Pergamenschikov A,
    13. Williams C,
    14. Zhu SX,
    15. Lonning PE,
    16. Borresen-Dale AL,
    17. Brown PO,
    18. Botstein D
    : Molecular portraits of human breast tumours. Nature 406(6797): 747-752, 2000.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Sorlie T,
    2. Perou CM,
    3. Tibshirani R,
    4. Aas T,
    5. Geisler S,
    6. Johnsen H,
    7. Hastie T,
    8. Eisen MB,
    9. van de Rijn M,
    10. Jeffrey SS,
    11. Thorsen T,
    12. Quist H,
    13. Matese JC,
    14. Brown PO,
    15. Botstein D,
    16. Eystein Lonning P,
    17. Borresen-Dale AL
    : Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98(19): 10869-10874, 2001.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Korsching E,
    2. Jeffrey SS,
    3. Meinerz W,
    4. Decker T,
    5. Boecker W,
    6. Buerger H
    : Basal carcinoma of the breast revisited: an old entity with new interpretations. J Clin Pathol 61(5): 553-560, 2008.
    OpenUrlAbstract/FREE Full Text
    1. Kuroda N,
    2. Fujishima N,
    3. Inoue K,
    4. Ohara M,
    5. Hirouchi T,
    6. Mizuno K,
    7. Hayashi Y,
    8. Lee GH
    : Basal-like carcinoma of the breast: further evidence of the possibility that most metaplastic carcinomas may be actually basal-like carcinomas. Med Mol Morphol 41(2): 117-120, 2008.
    OpenUrlPubMed
  5. ↵
    1. Reis-Filho JS,
    2. Milanezi F,
    3. Steele D,
    4. Savage K,
    5. Simpson PT,
    6. Nesland JM,
    7. Pereira EM,
    8. Lakhani SR,
    9. Schmitt FC
    : Metaplastic breast carcinomas are basal-like tumours. Histopathology 49(1): 10-21, 2006.
    OpenUrlCrossRefPubMed
    1. Gwin K,
    2. Buell-Gutbrod R,
    3. Tretiakova M,
    4. Montag A
    : Epithelial-to-mesenchymal transition in metaplastic breast carcinomas with chondroid differentiation: expression of the E-cadherin repressor Snail. Appl Immunohistochem Mol Morphol 18(6): 526-531, 2010.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Reis-Filho JS,
    2. Milanezi F,
    3. Paredes J,
    4. Silva P,
    5. Pereira EM,
    6. Maeda SA,
    7. de Carvalho LV,
    8. Schmitt FC
    : Novel and classic myoepithelial/stem cell markers in metaplastic carcinomas of the breast. Appl Immunohistochem Mol Morphol 11(1): 1-8, 2003.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Bae SY,
    2. Lee SK,
    3. Koo MY,
    4. Hur SM,
    5. Choi MY,
    6. Cho DH,
    7. Kim S,
    8. Choe JH,
    9. Lee JE,
    10. Kim JH,
    11. Kim JS,
    12. Nam SJ,
    13. Yang JH
    : The prognoses of metaplastic breast cancer patients compared to those of triple-negative breast cancer patients. Breast Cancer Res Treat 126(2): 471-478, 2011.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Okada N,
    2. Hasebe T,
    3. Iwasaki M,
    4. Tamura N,
    5. Akashi-Tanaka S,
    6. Hojo T,
    7. Shibata T,
    8. Sasajima Y,
    9. Kanai Y,
    10. Kinoshita T
    : Metaplastic carcinoma of the breast. Hum Pathol 41(7): 960-970, 2010.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Eisman JA,
    2. Martin TJ,
    3. MacIntyre I,
    4. Moseley JM
    : 1,25-Dihydroxyvitamin-D-receptor in breast cancer cells. Lancet 2(8156-8157): 1335-1336, 1979.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Lopes N,
    2. Sousa B,
    3. Martins D,
    4. Gomes M,
    5. Vieira D,
    6. Veronese LA,
    7. Milanezi F,
    8. Paredes J,
    9. Costa JL,
    10. Schmitt F
    : Alterations in Vitamin D signalling and metabolic pathways in breast cancer progression: a study of VDR, CYP27B1 and CYP24A1 expression in benign and malignant breast lesions. BMC Cancer 10: 483-492, 2010.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Deeb KK,
    2. Trump DL,
    3. Johnson CS
    : Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 7(9): 684-700, 2007.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Pendas-Franco N,
    2. Gonzalez-Sancho JM,
    3. Suarez Y,
    4. Aguilera O,
    5. Steinmeyer A,
    6. Gamallo C,
    7. Berciano MT,
    8. Lafarga M,
    9. Munoz A
    : Vitamin D regulates the phenotype of human breast cancer cells. Differentiation 75(3): 193-207, 2007.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Palmer HG,
    2. Gonzalez-Sancho JM,
    3. Espada J,
    4. Berciano MT,
    5. Puig I,
    6. Baulida J,
    7. Quintanilla M,
    8. Cano A,
    9. de Herreros AG,
    10. Lafarga M,
    11. Munoz A
    : Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. J Cell Biol 154(2): 369-387, 2001.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Campbell MJ,
    2. Elstner E,
    3. Holden S,
    4. Uskokovic M,
    5. Koeffler HP
    : Inhibition of proliferation of prostate cancer cells by a 19-nor-hexafluoride vitamin D3 analogue involves the induction of p21waf1, p27kip1 and E-cadherin. J Mol Endocrinol 19(1): 15-27, 1997.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Charafe-Jauffret E,
    2. Ginestier C,
    3. Monville F,
    4. Finetti P,
    5. Adelaide J,
    6. Cervera N,
    7. Fekairi S,
    8. Xerri L,
    9. Jacquemier J,
    10. Birnbaum D,
    11. Bertucci F
    : Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene 25(15): 2273-2284, 2006.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Neve RM,
    2. Chin K,
    3. Fridlyand J,
    4. Yeh J,
    5. Baehner FL,
    6. Fevr T,
    7. Clark L,
    8. Bayani N,
    9. Coppe JP,
    10. Tong F,
    11. Speed T,
    12. Spellman PT,
    13. DeVries S,
    14. Lapuk A,
    15. Wang NJ,
    16. Kuo WL,
    17. Stilwell JL,
    18. Pinkel D,
    19. Albertson DG,
    20. Waldman FM,
    21. McCormick F,
    22. Dickson RB,
    23. Johnson MD,
    24. Lippman M,
    25. Ethier S,
    26. Gazdar A,
    27. Gray JW
    : A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6): 515-527, 2006.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Oliveira C,
    2. Sousa S,
    3. Pinheiro H,
    4. Karam R,
    5. Bordeira-Carrico R,
    6. Senz J,
    7. Kaurah P,
    8. Carvalho J,
    9. Pereira R,
    10. Gusmao L,
    11. Wen X,
    12. Cipriano MA,
    13. Yokota J,
    14. Carneiro F,
    15. Huntsman D,
    16. Seruca R
    : Quantification of epigenetic and genetic 2nd hits in CDH1 during hereditary diffuse gastric cancer syndrome progression. Gastroenterology 136(7): 2137-2148, 2009.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Becker KF,
    2. Atkinson MJ,
    3. Reich U,
    4. Becker I,
    5. Nekarda H,
    6. Siewert JR,
    7. Hofler H
    : E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res 54(14): 3845-3852, 1994.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Berx G,
    2. Cleton-Jansen AM,
    3. Nollet F,
    4. de Leeuw WJ,
    5. van de Vijver M,
    6. Cornelisse C,
    7. van Roy F
    : E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. EMBO J 14(24): 6107-6115, 1995.
    OpenUrlPubMed
  21. ↵
    1. Wendt MK,
    2. Taylor MA,
    3. Schiemann BJ,
    4. Schiemann WP
    : Downregulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol Biol Cell 22(14): 2423-2435, 2011.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Gagnon J,
    2. Shaker S,
    3. Primeau M,
    4. Hurtubise A,
    5. Momparler R L
    : Interaction of 5-aza-2’-deoxycytidine and depsipeptide on antineoplastic activity and activation of 14-3-3sigma, E-cadherin and tissue inhibitor of metalloproteinase 3 expression in human breast carcinoma cells. Anticancer Drugs 14(3): 193-202, 2003.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Pan L,
    2. Matloob AF,
    3. Du J,
    4. Pan H,
    5. Dong Z,
    6. Zhao J,
    7. Feng Y,
    8. Zhong Y,
    9. Huang B,
    10. Lu J
    : Vitamin D stimulates apoptosis in gastric cancer cells in synergy with trichostatin A /sodium butyrate-induced and 5-aza-2’-deoxycytidine-induced PTEN upregulation. FEBS J 277(4): 989-999, 2010.
    OpenUrlPubMed
  24. ↵
    1. Godman CA,
    2. Joshi R,
    3. Tierney BR,
    4. Greenspan E,
    5. Rasmussen TP,
    6. Wang HW,
    7. Shin DG,
    8. Rosenberg DW,
    9. Giardina C
    : HDAC3 impacts multiple oncogenic pathways in colon cancer cells with effects on Wnt and vitamin D signaling. Cancer Biol Ther 7(10): 1570-1580, 2008.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Gniadecki R,
    2. Gajkowska B,
    3. Hansen M
    : 1,25-Dihydroxyvitamin D3 stimulates the assembly of adherens junctions in keratinocytes: involvement of protein kinase C. Endocrinology 138(6): 2241-2248,1997.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Niitsu N,
    2. Hayashi Y,
    3. Sugita K,
    4. Honma Y
    : Sensitization by 5-aza-2’-deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3. Br J Haematol 112(2): 315-326, 2001.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Kim MS,
    2. Fujiki R,
    3. Kitagawa H,
    4. Kato S
    : 1Alpha,25(OH)2D3-induced DNA methylation suppresses the human CYP27B1 gene. Mol Cell Endocrinol 265-266: 168-173, 2007.
    OpenUrlPubMed
  28. ↵
    1. Kouchi Z,
    2. Fujiwara Y,
    3. Yamaguchi H,
    4. Nakamura Y,
    5. Fukami K
    : Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells. Biochem Biophys Res Commun 408(4): 523-529, 2011.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Pereira F,
    2. Barbachano A,
    3. Silva J,
    4. Bonilla F,
    5. Campbell M J,
    6. Munoz A,
    7. Larriba MJ
    : KDM6B/JMJD3 histone demethylase is induced by vitamin D and modulates its effects in colon cancer cells. Hum Mol Genet, 2011 (to be published).
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
1Alpha,25-dihydroxyvitamin D3 Induces de novo E-cadherin Expression in Triple-negative Breast Cancer Cells by CDH1-promoter Demethylation
NAIR LOPES, JOANA CARVALHO, CECÍLIA DURÃES, BÁRBARA SOUSA, MADALENA GOMES, JOSÉ LUIS COSTA, CARLA OLIVEIRA, JOANA PAREDES, FERNANDO SCHMITT
Anticancer Research Jan 2012, 32 (1) 249-257;
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section