Rajeshwari R. Mehta

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.

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 PPARγs hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγs 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 PPARγs suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.

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

The present experiments were performed to determine the roles of estrogen receptors α and β (ERα and ERβ) 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 ERβ gene has been deleted (βERKO 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 βERKO 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 βERKO 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 βERKO 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 βERKO 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 ERβ 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.

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.

Abstract 3829: Inhibition of breast cancer metastasis by deguelin in an experimental model

Breast cancer is a second leading cause of cancer related deaths in women. Cancer related deaths in breast cancer patients are due to metastasis of disease. Thus new drugs which could successfully inhibit metastatic disease spread are highly desired. However till date there are only a few experimental models available to study metastatic progression of breast cancer. Murine 4T1 mammary breast cancer cells when transplanted s.c./or into mammary gland metastasizes to lungs, liver and bone similar to that observed in women with stage IV metastatic disease. Thus this model is highly appropriate for studies related to breast cancer metastasis. Previously we have shown that Deguelin, originally isolated from an African plant Mundulea sericea significantly inhibits the growth of triple negative breast cancer and that the Deguelin effect is mediated through inhibition of WNT signaling pathway. However their therapeutic effect in vivo is still unexplored. We evaluated the effects of Deguelin on growth (in vitro and in vivo) and lung metastasis of 4T1 cells. In vitro, Deguelin inhibited growth of 4T1 cells in time and concentration depended manner; optimum growth inhibition was obtained at 250nM Deguelin concentration after 72 hr. treatment. Toxicity study performed in 4-6 weeks old female BALB/c mice suggested that Deguelin administered as a suspension (saline/gum Arabic/Deguelin) daily for two weeks by intraperitoneal (i.p.) injection was well tolerated up to 16mg/kg body weight, and no toxicity (loss of appetite, body weight loss, mobility, morbidity, death) was observed in Deguelin treated animals. In vivo, as compared to vehicle treated animals, Deguelin (2 or 6mg/kg body weight) administered daily for 21 days i.p significantly (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3829. doi:1538-7445.AM2012-3829

Abstract 573: Deguelin inhibits the expression of estrogen receptor alpha in human breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The estrogen receptor alpha (ERα), a nuclear receptor and transcription factor governs the action of estrogen. This hormone is the major contributor to normal breast development and breast carcinogenesis. Thus, antiestrogens are widely utilized to treat and circumvent the progression of hormone receptor positive breast cancer. However, resistance to these agents develops after sustained use and major risk factors such as enhanced susceptibility to endometrial cancer have been associated with this type of treatment. In an effort to develop natural therapeutic compounds against ERα positive breast cancer, the effect of deguelin, a rotenoid isolated from an African plant; Mundulea sericea on ERα positive human breast cancer cells was investigated. Here we report that deguelin treatment downregulated the expression of ERα protein in a dose dependent manner in MCF-7 and T47D human breast cancer cell lines as determined by immunoblotting. Quantitative RT-PCR analysis revealed that the mRNA expression of ERα target genes Cathepsin D (CTSD), Trefoil factor 1 (TFF1) and the Progesterone receptor (PGR) was also downregulated by 50% in the presence of deguelin in MCF-7 cells. Furthermore, deguelin treatment significantly inhibited the proliferation of these two cell lines. Importantly, estrogen mediated stimulation of cell growth was reduced by 30% in MCF-7 cells after deguelin treatment. Together these findings suggest that deguelin suppresses ERα signaling, paving the way for the development of a novel natural therapeutic agent against endocrine receptor positive breast cancer. Future studies are aimed at deciphering the molecular mechanisms by which deguelin adversely affects ERα function. (This work was supported by NCI CA140321). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 573. doi:1538-7445.AM2012-573

Abstract 2580: Induction of estrogen receptor alpha by GW9662 sensitizes mammary glands to tamoxifen treatment in organ culture

Peroxisome proliferator-activated receptor gamma (PPARδ) are ligand activated nuclear receptor and binds to natural and synthetic ligands. Earlier we had reported that PPARδ agonist troglitazone inhibited the development of precancerous lesions induced by DMBA in mouse mammary gland organ culture (MMOC). In the present study we evaluated the effects of PPARδ agonist GW7845 and antagonist GW9662 on the development of 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary alveolar lesions (MAL) in ER negative mammary glands in MMOC. The results showed that both GW7845 and GW9662 inhibited development of ER- MAL in MMOC. Since PPARδ and ER cross talk has been reported in the literature, we determined the effects of GW9662 on the induction of ER and PR in this model. Real-time PCR studies and immunohistochemical analyses showed that GW9662 induced ERα in the absence of estradiol, whereas GW7845 did not induce ERα under these conditions. Both GW7845 and GW9662 failed to suppress mRNA expression of PPARδ in the mammary glands that are otherwise ER negative. However GW9662 suppressed the expression of PPARδ response genes adipsin and aP2. Conversely, GW7845 an agonist of PPARδ enhanced the expression of aP2 and adipsin. These results suggested that the effects of these agents are mediated by PPARδ in MMOC. Finally, Tamoxifen does not inhibit MAL in MMOC, however sequential treatments with GW9662 followed by Tamoxifen suppressed development of DMBA-induced MAL in these glands. Since the ER negative breast cancer patients are generally not treated with SERM, inducing ERα in the mammary cells by GW9662 possibly could make them responsive to Tamoxifen or other SERM. [This work was supported by N01 CN433303] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2580. doi:1538-7445.AM2012-2580

Abstract 3275: Development of a new ex-vivo orthotopic model-Human breast cancer cells in mouse mammary gland organ culture

Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogen-induced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC)). Three to four week old female BALB/c mice were sensitized with estradiol (1μg) + progesterone (1mg) for 9 days. On the 10 th day animals were sacrificed and 2.5x10 4 T47Dparental or T47D aromatase overexpressing cells were injected into the fourth pair of thoracic mammary glands. The glands were excised then cultured at 37°C under 95% O 2 / 5% CO 2 in hMEM medium containing 10% charcoal stripped FBS/supplemented with Testosterone (1nM) and progesterone (1uM) and growth promoting hormones (5 µg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific Ck-18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex-vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3275. doi:1538-7445.AM2012-3275

Abstract 3812: Deguelin as a novel therapeutic agent for Herceptin resistant breast cancers

Human breast cancers overexpressing HER-2 are generally highly aggressive. Current therapeutic strategies to target HER-2 signaling pathway with antibodies or small molecule tyrosine kinase inhibitors have shown impressive success in the management of these tumor types, although most women exhibit short term response to these therapies. Acquired resistance to HER-2 target drugs, especially Herceptin is a major challenge to clinicians. Patients receiving Herceptin therapy invariably recur and most often with highly aggressive tumors. Thus identifying new drugs which target multiple molecular pathways is desired to treat Herceptin resistant breast cancer. Using paired HER-2 overexpressing parental Herceptin responsive (BT-474) and its Herceptin resistant variant cells (BT-474HR) we showed that acquired resistance to Herceptin is associated with enhanced expression of HER-2, EGFR, CyclinD1, P-AKT (Ser-473), PI-3K, JAB1 proteins and decreased expression of nuclear pten and p27 proteins. These results are similar to those observed in recurrent breast tumors in patients following Herceptin treatment. Deguelin, a natural product derivative of rotenoids and has been shown to inhibit growth of diverse cancer types. Deguelin is a known AKT inhibitor, however its efficacy is never tested in breast cancers resistant to Herceptin which express high levels of p-AKT. We examined effect of Deguelin on HER-2 overexpressing BT-474 BT-474-HRcells. Deguelin inhibited growth of both, BT-474 and BT-474HR cells in concentration (0-250nM) and time dependent (24-72h) manner. There was a 40-50%- inhibition of both BT474and BT-474HR cells at 25nM Deguelin treatment at 72h. Deguelin decreased cell surface expression of HER-2 in both these cell lines. In parental BT-474 cells both Herceptin 5-10ug/ml and Deguelin down regulated HER-2 expression, but only Deguelin reduced HER-2 (125-250nM) over expression in both BT-474 and BT-474HR cells. Down regulation of HER-2 following Deguelin treatment was associated with simultaneous down regulation of nuclear accumulation of cyclin D1, and JAB1, and p-AKT (Ser-473) proteins and up regulation of nuclear p27 and p53in both BT-474 and BT474 HR cells. These results suggest that Deguelin inhibits growth of both HER-2 overexpressing and those resistant to Herceptin therapy breast cancer cells. Effect of Deguelin appears to be mediated through down regulation of HER-2, PI3K AKT, JAB1, Cyclin D1, and p27 pathway. Our preliminary results warrant further investigation into Deguelin as a potential therapeutic agent for Herceptin resistant breast cancers. (This work was supported by NCI CA140321.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3812. doi:1538-7445.AM2012-3812