A novel and cost-effective ex vivo orthotopic model for the study of human breast cancer in mouse mammary gland organ culture.

Mouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to in vivo carcinogenesis models. Here, we developed a new ex vivo MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics in vivo orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured in vitro with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected ex vivo and in vivo Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15 days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive ex vivo model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.

Differential roles of ERα and ERß in normal and neoplastic development in the mouse mammary gland.

The present experiments were performed to determine the roles of estrogen receptors α and ß (ERα and ERß) in normal and neoplastic development in the mouse mammary gland. In wild-type mice, in vivo administration of estradiol (E) + progesterone (P) stimulated mammary ductal growth and alveolar differentiation. Mammary glands from mice in which the ERß gene has been deleted (ßERKO mice) demonstrated normal ductal growth and differentiation in response to E + P. By contrast, mammary glands from mice in which the ERα gene has been deleted (αERKO mice) demonstrated only rudimentary ductal structures that did not differentiate in response to E + P. EGF demonstrates estrogen-like activity in the mammary glands of αERKO mice: treatment of αERKO mice with EGF + P (without E) supported normal mammary gland development, induced expression of progesterone receptor (PR), and increased levels of G-protein-coupled receptor (GPR30) protein. Mammary gland development in ßERKO mice treated with EGF + P was comparable to that of wild-type mice receiving EGF + P; EGF had no statistically significant effects on the induction of PR or expression of GPR30 in mammary glands harvested from either wild-type mice or ßERKO mice. In vitro exposure of mammary glands to 7,12-dimethylbenz[a]anthracene (DMBA) induced preneoplastic mammary alveolar lesions (MAL) in glands from wild-type mice and ßERKO mice, but failed to induce MAL in mammary glands from αERKO mice. Microarray analysis of DMBA-treated mammary glands identified 28 functional pathways whose expression was significantly different in αERKO mice versus both ßERKO and wild-type mice; key functions that were differentially expressed in αERKO mice included cell division, cell proliferation, and apoptosis. The data demonstrate distinct roles for ERα and ERß in normal and neoplastic development in the mouse mammary gland, and suggest that EGF can mimic the ERα-mediated effects of E in this organ.

Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells.

BACKGROUND: Treatment of breast cancer patients with antiestrogens and aromatase inhibitor(s) or Herceptin have shown significant success in steroid receptor positive or Her-2+ breast cancers respectively. However, choice of treatments for breast cancer patients with negative status for estrogen, progesterone receptors and HER2/neu is limited. As a result, search for appropriate therapy regimen for these triple negative breast cancers (TNBC) has become a major focus of investigations for many laboratories. Recently, Deguelin, a natural product isolated from African plant Mundulea sericea (Leguminossae) has shown both antiproliferative actions in various cancers including breast as well as chemoprenventive activity against carcinogen induced experimental cancers. In this report we evaluated efficacy and mechanism of action of Deguelin in triple negative breast cancer cell lines. METHODS/FINDINGS: In vitro, Deguelin in a dose and time dependent manner inhibited the growth of MDA-MB-231, MDA-MB-468, BT-549 and BT-20 cells. Deguelin (2 or 4 mg/kg body weight), when injected intraperitoneally, reduced the in vivo tumor growth of MDA-MB-231 cells transplanted subcutaneously in athymic mice. Moreover it was nontoxic as evident from daily observations on mobility, food and water consumption and comparison of bodyweight and other visceral organ weights with those in control animals at the termination of the study. The western blot analyses and immunostaining studies indicated that the deguelin effects may be mediated through EGFR-PAKT/c-Met p-ERK and NF-κB by down regulating their downstream targets such as p-STAT3, c-Myc, Survivin. CONCLUSION/SIGNIFICANCE: These results suggest that Deguelin may have a significant therapeutic value for the treatment of TNBC patients.

Efficacy and mechanism of action of Deguelin in suppressing metastasis of 4T1 cells.

Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients.

PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687-694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER- patients.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 µM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.

Vitamin D and breast cancer: emerging concepts.

The benefit of vitamin D in cancer prevention and to certain extent therapy has been well recognized. The active form of vitamin D, 1,25-dihydroxycholecalciferol (1,25(OH)2 D3) is a natural ligand for vitamin D receptor (VDR). Since 1,25(OH)2D3 exerts toxic effects at a concentration that is beneficial, nearly 1500 analogs of vitamin D have been synthesized and evaluated for their efficacy in a variety of carcinogenesis and human cancer models both in vitro and in vivo. Among these only a handful of them have been approved for evaluation in clinical trials for leukemia, breast, prostate and colon cancers. The mechanism of vitamin D action is mediated by the nuclear VDR and the signaling cascade for its action is extensively reported. In this review we focus on the newer concepts for vitamin D action. These include (1) differential effects of vitamin D in maintaining cell proliferation when the cells are under stress but suppressing cell growth when the cells are transformed; (2) functional significance of VDR polymorphism in potential vitamin D responsiveness; (3) regulation of constitutive splicing of vitamin D target gene, CYP24a, by the hormone and its significance; and (4) regulation of microRNA by vitamin D in breast cancer. It is anticipated that the new work in these selective areas would expand the understanding of vitamin D in breast cancer prevention and therapy.

Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation.

Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D(3) (1,25D(3)) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D(3) transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.

Regulation of CYP24 splicing by 1,25-dihydroxyvitamin D3 in human colon cancer cells.

CYP24 is a well-established vitamin D receptor (VDR) target gene. The active VDR ligand 1,25(OH)2D3 regulates its own catabolism by increasing CYP24 expression. It is well known that in the presence of 1,25(OH)2D3, VDR binds to VDREs in the promoter region of CYP24 and initiates CYP24 transcription. However, little is known about the role of 1,25(OH)2D3 in the posttranscriptional modulation of CYP24. In this study, we investigated the functional significance of 1,25(OH)2D3 in CYP24 RNA splicing in colon cancer cells. Using RT-PCR, we found that 1,25(OH)2D3 actively induces CYP24 splicing in a time-dependent manner and CYP24 splicing pattern could be cell type or tissue specific. The induction of RNA splicing by 1,25(OH)2D3 was mainly CYP24 selective. Treatment of cells with parathyroid hormone inhibited basal CYP24 splicing, but failed to inhibit 1,25(OH)2D3-induced CYP24 splicing. Further experiments demonstrated that new RNA synthesis was required for the induction of CYP24 splicing by vitamin D. In addition, alteration of multiple signaling pathways also affected CYP24 splicing and cellular sensitivity in response to vitamin D appeared to correlate with the induction of CYP24 splicing. These results suggest that 1,25(OH)2D3 not only regulates CYP24 transcription, but also plays an important role in posttranscriptional modulation of CYP24 by inducing its splicing. Our findings reveal an additional regulatory step that makes the vitamin D mediated action more prompt and efficient.

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt.

Amino acids 50-77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin's preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

A 28-amino-acid peptide fragment of the cupredoxin azurin prevents carcinogen-induced mouse mammary lesions.

Azurin, a member of the cupredoxin family of redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and apoptotic effects. Azurin and amino acids 50-77 (p28) of azurin also produce a dose-dependent reduction in the proliferation of human mammary cancer by increasing the level of the tumor suppressor protein p53 in the cancer cell nucleus. We show that the development of 7,12-dimethylbenz[a]anthracene-induced hormone-dependent premalignant mammary ductal lesions and hormone-independent mammary alveolar lesions in mouse mammary gland organ culture is also significantly reduced by azurin and p28. The dose-dependent reduction in carcinogen-induced mammary cell proliferation by p28 was associated with an increase in the expression of p53. p28 also enhanced the inhibitory effect of a low dose of the antiestrogen tamoxifen on the development of hormone-dependent mammary ductal lesions, but did not enhance the inhibitory activity of fenretinide (N-4-hydroxyphenyl retinamide) on hormone-independent mammary alveolar lesions. These observations suggest that cupredoxins and fragments derived from them can exert a chemopreventive effect on carcinogen-induced mammary gland transformation, irrespective of hormonal environment, and enhance the inhibitory effects of tamoxifen in this model of preneoplastic mammary development.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells.

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G(2)-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents.

Noncationic peptides obtained from azurin preferentially enter cancer cells.

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Prohibitin is a novel target gene of vitamin D involved in its antiproliferative action in breast cancer cells.

Previously, we showed that N-methyl-N-nitrosourea-transformed MCF12F breast epithelial cells exhibited differential expression of several genes, including up-regulation of prohibitin and elevated sensitivity to a relatively noncalcemic vitamin D analogue, 1alpha-hydroxyvitamin D5 [1alpha(OH)D5]. In this report, we evaluated the functional significance of prohibitin in relation to the cellular response to vitamin D. The in silico screening for putative transcription factor binding sites identified two vitamin D receptor (VDR)/retinoid X receptor binding sites in the 1-kb promoter region of prohibitin. Prohibitin up-regulation by 1alpha(OH)D5 treatment at both transcriptional and translational levels was confirmed by real-time reverse transcription-PCR and Western blot analysis in breast cancer cells, identifying prohibitin as a vitamin D target gene. Confocal microscopic analysis showed that prohibitin was localized in the nuclei of MCF-7 cells and a portion of prohibitin was colocalized with VDR, but direct physical interaction between VDR and prohibitin in cell lysates was not detectable. In MCF-7 cells expressing tetracycline-inducible prohibitin (Tet-On model), the overexpression of prohibitin inhibited cell proliferation and enhanced vitamin D-induced antiproliferative activity. Knockdown of prohibitin was accompanied by increased number of cells incorporating bromodeoxyuridine in the whole population and increased cell distribution in the S phase of cell cycle. In addition, prohibitin level had no significant effect on the vitamin D-induced transactivation of CYP24, a VDR target gene. This is the first report to suggest that prohibitin serves as a novel vitamin D target gene, which is involved in the antiproliferative action of vitamin D without affecting CYP24 transactivation in breast cancer cells.

Clinical trials using chemopreventive vitamin D analogs in breast cancer.

This article comprehensively reviews the clinical trials and considers the future directions of the use of vitamin D and its analogs in the treatment or chemoprevention of breast cancer. Chemopreventive treatment strategies strive to delay the onset of certain cancers, prevent the progression of malignant disease after diagnosis, or delay the advent of recurrence after curative treatment. We first summarize the epidemiological evidence that led to the hypothesis that vitamin D may have an anti-cancer activity. Vitamin D shows great potential as a therapy for breast cancer; however, its use in clinical trials has been hindered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. This has led to the development of less calcemic analogs of vitamin D. We review the clinical trials with breast cancer patients using vitamin D analogs.

Growth inhibition of carcinogen-transformed MCF-12F breast epithelial cells and hormone-sensitive BT-474 breast cancer cells by 1alpha-hydroxyvitamin D5.

Several studies have established the active form of vitamin D(3) as an effective tumor-suppressing agent; however, its antitumor activity is achieved at doses that are hypercalcemic in vivo. Therefore, less calcemic vitamin D(3) analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), was evaluated for its potential use in breast cancer chemoprevention. Previously, 1alpha(OH)D5 showed anticarcinogenic activity in several in vivo and in vitro models. However, its effects on growth of normal tissue were not known. The present study was conducted to determine the effects of 1alpha(OH)D5 on the growth of normal mouse mammary gland and normal-like human breast epithelial MCF-12F cells and to compare these effects with carcinogen-transformed MCF-12F and breast cancer cells. No significant difference was observed in the growth or morphology of cultured mouse mammary gland and MCF-12F cells in the presence of 1alpha(OH)D5. However, the transformed MCF-12F cells underwent growth inhibition (40-60%, P < 0.05) upon 1alpha(OH)D5 treatment as determined by cell viability assays. Cell cycle analysis showed marked increase (50%) in G-1 phase for cells treated with 1alpha(OH)D5 compared with the controls. Moreover, the percentage of cells in the synthesis (S) phase of cell cycle was decreased by 70% in transformed MCF-12F, BT-474 and MCF-7 cells. The growth arrest was preceded by an increase in expression of cell cycle regulatory proteins p21(Waf-1) and p27(Kip-1). In addition, differential expression studies of parent and transformed MCF-12F cell lines using microarrays showed that prohibitin mRNA was increased 4-fold in the transformed cells. These results indicate that the growth inhibitory effect of 1alpha(OH)D5 was achieved in both carcinogen-transformed MCF-12F and breast cancer cells at a dose that was non-inhibitory in normal-like breast epithelial cells.

Clinical trials involving vitamin D analogs in prostate cancer.

Vitamin D shows significant potential as a therapy for prostate cancer. However, its use in clinical trials has been hampered by its induction of hypercalcemia at serum concentrations required to suppress cancer cell proliferation. This has spurred the development of less calcemic analogs of vitamin D. In this article, we review the clinical trials and consider the future directions of the use of vitamin D and its analogs in the treatment or chemoprevention of prostate cancer. First, we summarize the epidemiological evidence leading to the hypothesis that vitamin D has anticancer activity. We then review the clinical trials using vitamin D analogs that involve patients with prostate cancer and conclude with a brief overview of our planned study with vitamin D5, [1alpha(OH)D5], which will begin shortly. Data for this review were identified by searches of PubMed, the Cochrane Library, Biosis, and references from relevant articles, using the search terms "vitamin D," "prostate cancer," "chemoprevention" and "vitamin D analog." Abstracts from recent international meetings were also reviewed but were only included when they were the only known reference to the clinical trial or the research mentioned. Only papers published in English were included.

A novel RARbeta isoform directed by a distinct promoter P3 and mediated by retinoic acid in breast cancer cells.

Retinoids regulate gene transcription through activating retinoic acid receptors (RARs)/retinoic X receptors (RXRs). Of the three RAR receptors (alpha, beta, and gamma), RARbeta has been considered a tumor suppressor gene. Here, we identified a novel RARbeta isoform-RARbeta5 in breast epithelial cells, which could play a negative role in RARbeta signaling. Similar to RARbeta2, the first exon (59 bp) of RARbeta5 is RARbeta5 isoform specific, whereas the other exons are common to all of the RARbeta isoforms. The first exon of RARbeta5 does not contain any translation start codon, and therefore its protein translation begins at an internal methionine codon of RARbeta2, lacking the A, B, and part of C domain of RARbeta2. RARbeta5 protein was preferentially expressed in estrogen receptor-negative breast cancer cells and normal breast epithelial cells that are relatively resistant to retinoids, whereas estrogen receptor-positive cells that did not express detectable RARbeta5 protein were sensitive to retinoid treatment, suggesting that this isoform may affect the cellular response to retinoids. RARbeta5 isoform is unique among all of the RARs, because a corresponding isoform was not detectable for either RARalpha or RARgamma. RARbeta5 mRNA was variably expressed in normal and cancerous breast epithelial cells. Its transcription was under the control of a distinct promoter P3, which can be activated by all-trans-retinoic acid (atRA) and other RAR/RXR selective retinoids in MCF-7 and T47D breast cancer cells. We mapped the RARbeta5 promoter and found a region -302/-99 to be the target region of atRA. In conclusion, we identified and initially characterized RARbeta5 in normal, premalignant, and malignant breast epithelial cells. RARbeta5 may serve as a potential target of retinoids in prevention and therapy studies.

Effect of vitamin D analog (1alpha hydroxy D5) immunoconjugated to Her-2 antibody on breast cancer.

We previously showed that a new vitamin D analog, 1alpha(OH)D5 (D5), induced differentiation and inhibited the growth of breast cancer cells. In this report, we examined whether D5 specifically delivered to breast cancer cells could have any therapeutic effect. D5 was linked to Her-2 antibody using sulfosuccinimidyl 6-4 azido nitrophenylamido hexanode (SANPAH) as a linker. The Her-2 antibody selected in our study had no significant effect on the in vitro or in vivo growth of breast cancer cells; however, it had cell-differentiating action. In vitro, D5-Her-2 antibody conjugate (IMC) showed the ability to specifically bind to Her-2-expressing cells, to compete with Her-2 antibody for surface receptor and to cause internalization. IMC (equivalent to 5 microg Her-2 antibody given intraperitoneally once weekly for 6 weeks) significantly inhibited the growth of BT-474 cells transplanted into athymic mice. The in vivo growth-inhibitory effect of IMC treatment was similar to that observed in animals receiving D5 continuously as a dietary supplement. These results show that the targeted delivery of D5 by immunoconjugation to cell surface receptor antibodies may be of potential therapeutic value for the treatment of Her-2 positive breast cancer.

Designing a randomized phase I/II prostate cancer chemoprevention trial using 1alpha-hydroxy-24-ethyl-cholecalciferol, an analogue of vitamin D3.

Prostate cancer continues to be a significant source of morbidity and mortality among older men. One possible means of reducing its impact on overall health and vitality is via cancer chemoprevention, both in the population that is unaffected but at some risk and in those who have undergone some form of curative therapy after the onset of the disease. Chemoprevention holds significant promise, but large phase III clinical trials evaluating chemopreventive agents in prostate cancer can require vast numbers of enrollees and require the commitment of significant financial resources and time before any therapeutic benefit may become apparent. One technique to shorten the time required for chemoprevention clinical trials is to use surrogate endpoint biomarkers in place of the currently used actual endpoints of cancer incidence or survival. The validation of such surrogate endpoint biomarkers will require small, well-designed phase I and/or II trials to accumulate data on the modulation of the surrogate biomarkers and the endpoints of cancer incidence or survival by the chemopreventive agent. Careful statistical correlation and clinical validation of the data will then allow us to justify the use such surrogates in place of the actual endpoint in large, randomized trials, potentially shortening trial duration, improving financial efficiency, and accelerating approval of the chemopreventive agent. To that end, we first review the theoretical construct of cancer chemoprevention trials with particular reference to prostate cancer. We thereafter describe the design of a small, randomized, double-blinded, placebo-controlled phase I/II clinical trial of an analogue of vitamin D, vitamin D5, which we believe could serve as a model for data accumulation on surrogate biomarkers and correlation with other clinical endpoints.