Vitamin D Receptor Cdx-2 Polymorphism and Premenopausal Breast Cancer Risk in Southern Pakistani Patients

Background

Vitamin D3 is a secoster oid that exerts its effect by binding to its nuclear receptor called vitamin D receptor (VDR), inducing apoptosis and thereby inhibiting cell proliferation in cancer cells. The VDR receptor, located in the nucleus, is known to regulate the functions of over 200 genes. Various allelic forms of hVDR have been discovered that increase susceptibility to various cancers. The VDR-Cdx2 polymorphism, located in the promoter region of exon 1e in the VDR gene, influences the functional activity of the receptor, since the hVDR lacks consensus TATA and CAAT boxes. The current investigation examines the association between VDR-Cdx2 polymorphism and breast cancer in premenopausal females from Southern Pakistan.

Methods

We conducted a case control study on 264 subjects (103 cases and 161 controls) who were recruited from a tertiary hospital located in Karachi, Pakistan. Genomic DNA was extracted from peripheral blood using a commercial kit method, and the VDR-Cdx2 polymorphism was genotyped using tetraprimer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Pearson chi square test was used to assess the association between VDR-Cdx2 genotype and breast cancer while genotype distribution in controls was evaluated by Hardy-Weinberg equilibrium (HWE). Breast cancer risk was calculated using odds ratios and 95% confidence intervals.

Results

The genotype distribution in the control group was in HWE (p > 0.05) for the VDR-Cdx2 polymorphism. A non-significant association was observed between VDR cdx2 polymorphism and breast cancer, however the GG genotype was at risk (OR = 1.832, 95% CI = 0.695–4.828) of developing breast cancer.

Conclusion

The GG genotype of Cdx2-VDR gene polymorphism may increase the risk of developing breast cancer in young female patients in South Pakistan. Further investigations examining additional single nucleotide polymorphisms (SNPs) in VDR are required to assess their relationships with breast cancer.     

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

Background

The FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)₂D₃, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)₂D₃ downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.

Methods/Findings

To elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)₂D₃ treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.

Conclusions/Significance

These results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.     

Vitamin D Receptor Gene Polymorphisms and Prognosis of Breast Cancer among African-American and Hispanic Women

Background

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts.

Methods

Blood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method.

Results

An increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome.

Conclusions

VDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer.     

Anti-Proliferative Effects of Evodiamine on Human Breast Cancer Cells

Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and –independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10⁻⁶ and 1×10⁻⁵ M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Human Breast Cancer Cell Lines Co-Express Neuronal,Epithelial, and Melanocytic Differentiation Markers In Vitro and In Vivo

Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL) and glioblastoma cell lines (U87 and D54). These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.     

Association between the BsmI Polymorphism in the Vitamin D Receptor Gene and Breast Cancer Risk: Results from a Pakistani Case-Control Study

Background

Vitamin D is postulated to decrease the risk of breast cancer by inhibiting cell proliferation via the vitamin D receptor (VDR). Two common single nucleotide polymorphisms (SNPs) in the VDR gene, rs1544410 (BsmI) and rs2228570 (FokI), are inconsistently associated with breast cancer risk in Caucasian populations, while data for Asians are scarce. Here, we investigated the possible contribution of these SNPs to breast cancer risk in Pakistani breast cancer patients and in controls participating in a hospital-based breast cancer case-control study (PAK-BCCC).

Methods

Genotyping of the BsmI and FokI SNPs was performed by PCR-based restriction fragment length polymorphism (RFLP) analysis of 463 genetically enriched female breast cancer cases with known BRCA1/2 status and in 1,012 controls from Pakistan. The association between SNP genotypes and breast cancer risk was investigated by logistic regression adjusted for potential breast cancer risk factors and stratified by BRCA1/2 status and family history. Odds ratios (ORs) and 95% confidence intervals (CIs) were reported.

Results

The b allele of the BsmI was associated with an increased breast cancer risk (per b allele OR 1.28, 95% CI 1.09–1.49, P = 0.003). Subgroup analysis revealed that this effect was restricted to BRCA1/2 non-carriers (per b allele OR 1.33, 95% CI 1.11–1.59, P = 0.002) and was stronger in those who reported a positive family history of breast and/or ovarian cancer (per b allele OR 1.64, 95% CI 1.20–2.22, P = 0.002). No association with breast cancer risk was detected for the FokI SNP.

Conclusions

The BsmI polymorphism in the VDR gene may be associated with an increased breast cancer risk in Pakistani women negative for BRCA1/2 germline mutations.     

Targeting 1alpha,25-dihydroxyvitamin D3 antiproliferative insensitivity in breast cancer cells by co-treatment with histone deacetylation inhibitors

Proliferation of the non-malignant breast epithelial cell line, MCF-12A, is sensitively and completely inhibited by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (ED90 = 70 nM), We used real time RT-PCR to demonstrate that the relative resistance to 1alpha,25(OH)(2)D(3) of MDA-MB-231 cells (ED50 > 100 nM) correlated with significantly reduced Vitamin D receptor (VDR) and increased NCoR1 nuclear receptor co-repressor mRNA (0.1-fold reduction in VDR and 1.7-fold increase in NCoR1 relative to MCF-12A (P < 0.05)). This molecular lesion can be targeted by co-treating cells with 1alpha,25(OH)(2)D(3) or potent analogs and the histone deacetylation inhibitor trichostatin A (TSA). For example, the co-treatment of 1,25-dihydroxy-16,23,Z-diene-26,27-hexafluoro-19-nor Vitamin D(3) (RO-26-2198) (100 nM) plus TSA results in strong additive antiproliferative effects in MDA-MB-231 cells. This may represent novel chemotherapeutic regime for hormone insensitive breast cancer.     

The immunomodulatory drug lenalidomide restores a vitamin D sensitive phenotype to the vitamin D resistant breast cancer cell line MDA-MB-231 through inhibition of BCL-2: potential for breast cancer therapeutics

1α,25-Dihydroxyvitamin D3, (1,25-D3) the biologically active form of vitamin-D, is well established as a cancer cell growth inhibitor in addition to maintaining bone mineralization. In breast cancer cells, inhibitory effects on angiogenesis, and metastasis have been observed together with enhancement of apoptosis and induction of cell cycle arrest. There is a correlation between vitamin-D receptor expression on breast cancer cells and patient survival. However vitamin-D resistance and hypercalcaemia are key limiting factors in clinical use. The IMiD^? immunomodulatory drug lenalidomide, (Revlimid^?, CC-5013) used in myeloma, can also modulate apoptotic and growth signalling. We studied whether lenalidomide treated breast cancer cells would acquire sensitivity to 1,25-D3 with resulting growth inhibition. The cell lines MCF-12A, MCF-7 and MDA-MB-231, representing non-tumorogenic, tumorogenic, and vitamin-D resistant lines respectively were treated with lenalidomide and/or 1,25-D3(at 100 nM). Whereas lenalidomide alone had no effect on cell growth, a 50% inhibition of cell growth by 1,25-D3 was achieved with additional 1 μM lenalidomide in resistant cells. This effect was through apoptosis measured by PARP cleavage and annexin-V expression. An apoptosis protein array showed that the 1,25-D3 and lenalidomide combination increased pro-apoptotic proteins (phosphorylated p53) and decreased BCL-2 expression. BCL-2 inhibition is proposed as a mechanism of action for the combined drugs in the MDA-MB-231 cell line. In vitamin D resistant cell lines MCF-7VDR and HBL-100 where the combination does not affect BCL-2—no inhibitory effect is observed. These results demonstrate the potential for the combinatorial use of lenalidomide and 1,25-D3 for vitamin D refractory tumours.     

Pattern of serum immunoreactivity against breast cancer cell lysates may predict severity of disease in breast cancer patients

Humoral tumor-specific immunity has been investigated as a potential tool to identify tumor-associated antigens and evaluate cancer diagnosis and prognosis. Using SDS-PAGE and western blotting techniques we investigated the humoral immune response against tumor cell antigens in 36 breast cancer patients, 17 node-positive (NP) and 19 node-negative (NN). As a source of antigens, we prepared protein lysates from four breast cancer cell lines (AU565, BT474, MCF-7 and MDA-MB-231) which in vitro exhibit different features of invasion, estrogen receptor/progesterone receptor status and HER2/neu expression thereby potentially representing mild to aggressive forms of clinical disease. A higher number of immunocomplexes Ag–Ab were formed when serum from NN patients was immunoreacted against lysates from AU565 and MCF-7 in comparison to serum from NP patients (P < 0.01). BT474 cells were not a good antigenic source. MDA-MB-231 cells could not significantly discriminate between NN and NP patients since both groups showed higher amounts of reactivity against the lysate. However, comparative analysis of protein preparations purified from MCF-7 and MDA-MB-231 cells and immunodetected concomitantly with the same serum samples showed that serum from patients with cancers with worse prognosis (stage, nodality, HER2/neu and hormonal status) reacted more intensely to proteins purified from the relatively more invasive cell line MDA-MB-231 compared to MCF-7. These findings suggest that the study of serum antibody reactivity to antigens purified from breast cancer cell lines with different invasive properties should be further investigated for its potential in providing beneficial prognostic information in breast cancer. Supported by the United States Military Cancer Institute and the Department of Clinical Investigation at Walter Reed Army Medical Center. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.     

Association of Vitamin D Receptor Polymorphisms (FokI (Rs2228570), ApaI (Rs7975232), BsmI (Rs1544410), and TaqI (Rs731236)) with Gastric Cancer in a Kurdish Population from West of Iran

Background:The association of 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and its receptor, vitamin D receptor (VDR), with cancer types have been studied. However, there are controversial findings regarding the association of specific VDR polymorphisms with different kinds of cancers. In the current study, we investigated the association of VDR polymorphisms (Fok1 (rs2228570), ApaI (rs7975232), BsmI (rs1544410), and TaqI (rs731236)) with the risk of gastric cancer in a Kurdish population of Kermanshah in Iran for the first time. Methods:In this case-control study, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used in 99 gastric cancer patients and 100 healthy subjects as controls.Results:The frequencies of f (FokI), b (BsmI), t (TaqI), and a (ApaI) alleles were: 55.6%, 27.3%, 62.1%, and 44.95% in the patient group, respectively and 42%, 29.5%, 54.5%, and 46.0% in the control group, respectively. Analysis of the results indicated that there was a positive association between the frequency of FokI genotypes with gastric cancer risk (p= 0.021). However, no statistically significant association of BsmI, Taq1, and ApaI polymorphisms of VDR was detected in gastric patients when compared with healthy individuals.Conclusion:VDR-FokI polymorphism could increase the risk of GC development and predispose to the disease by mechanisms.Key Words: Gastric cancer, PCR-RFLP, Polymorphism, Vitamin D receptor     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Synthesis and anti breast cancer activity of biphenyl based chalcones

A series of (2E,2′E)-1,1′-(3-hydroxy-5-methylbiphenyl-2,6-diyl)-bis(3-pheylprop-2-ene-1-ones (5–33) were prepared by the reaction of 1,3-diacetyl biphenyls (1–4) with different aldehydes in presence of catalytic amount of solid KOH in ethanol in excellent yields. The compounds were evaluated for anticancer activity against human breast cancer MCF-7 (estrogen responsive proliferative breast cancer model) and MDA-MB-231 (estrogen independent aggressive breast cancer model) cell lines, HeLa (cervical cancer) cell line, and human embryonic kidney (HEK-293) cells. Most of the compounds preferentially inhibited the growth of the aggressive human breast cancer cell lines, MDA-MB-231 in the range of 4.4–30 μM. The two compounds 9 and 29 proved to be better anticancer agents than the standard drug tamoxifen against the MDA-MB-231 cell lines. Mode of action of these compounds was established to be apoptosis, cell cycle arrest and loss of mitochondrial membrane potential.     

Breast cancer cell regulation by high-dose Vitamin D compounds in the absence of nuclear vitamin D receptor

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) inhibits growth and induces apoptosis in breast cancer cells in vivo and in vitro. To examine the role of the Vitamin D receptor (VDR) in mediating the actions of 1,25D(3) at nanomolar and micromolar concentrations, mammary epithelial tumor cell lines generated in wild type (WT) and VDR knockout (VDRKO) mice were utilized. WT cells express VDR and are growth inhibited by 1,25D(3) and synthetic analogs EB1089 and CB1093 at 1nM concentrations, while VDRKO cells do not express VDR and are insensitive to Vitamin D compounds at concentrations up to 100nM. In the current studies, we have confirmed and extended these previous observations. At nanomolar concentrations of 1,25D(3) and all analogs tested, including EB1089, CB1093, MC1288, and KH1230, WT cells are growth inhibited and exhibit apoptotic morphology, while VDRKO cells show no growth inhibition or apoptosis. At concentrations of 1-10microM, however, 1,25D(3) and synthetic analogs induce growth inhibition and apoptotic morphology in both WT and VDRKO cell lines. These data indicate that nanomolar concentrations of 1,25D(3) and analogs mediate growth regulatory effects via mechanisms requiring the nuclear VDR, but that micromolar concentrations of Vitamin D compounds can exert non VDR-mediated effects.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.