Jae Young So

A novel Gemini vitamin D analog represses the expression of a stem cell marker CD44 in breast cancer

CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 μg/kg body weight) or orally (0.03 or 0.1 μg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells.

Combination of atorvastatin with sulindac or naproxen profoundly inhibits colonic adenocarcinomas by suppressing the p65/β-catenin/cyclin D1 signaling pathway in rats.

Evidence supports the protective role of nonsteroidal anti-inflammatory drugs (NSAID) and statins against colon cancer. Experiments were designed to evaluate the efficacies atorvastatin and NSAIDs administered individually and in combination against colon tumor formation. F344 rats were fed AIN-76A diet, and colon tumors were induced with azoxymethane. One week after the second azoxymethane treatment, groups of rats were fed diets containing atorvastatin (200 ppm), sulindac (100 ppm), naproxen (150 ppm), or their combinations with low-dose atorvastatin (100 ppm) for 45 weeks. Administration of atorvastatin at 200 ppm significantly suppressed both adenocarcinoma incidence (52% reduction, P = 0.005) and multiplicity (58% reduction, P = 0.008). Most importantly, colon tumor multiplicities were profoundly decreased (80%–85% reduction, P < 0.0001) when given low-dose atorvastatin with either sulindac or naproxen. Also, a significant inhibition of colon tumor incidence was observed when given a low-dose atorvastatin with either sulindac (P = 0.001) or naproxen (P = 0.0005). Proliferation markers, proliferating cell nuclear antigen, cyclin D1, and β-catenin in tumors of rats exposed to sulindac, naproxen, atorvastatin, and/or combinations showed a significant suppression. Importantly, colon adenocarcinomas from atorvastatin and NSAIDs fed animals showed reduced key inflammatory markers, inducible nitric oxide synthase and COX-2, phospho-p65, as well as inflammatory cytokines, TNF-α, interleukin (IL)-1β, and IL-4. Overall, this is the first report on the combination treatment using low-dose atorvastatin with either low-dose sulindac or naproxen, which greatly suppress the colon adenocarcinoma incidence and multiplicity. Our results suggest that low-dose atorvastatin with sulindac or naproxen might potentially be useful combinations for colon cancer prevention in humans. Cancer Prev Res; 4(11); 1895–902. ©2011 AACR.

Targeting CD44-STAT3 signaling by Gemini vitamin D analog leads to inhibition of invasion in basal-like breast cancer.

Background CD44, a transmembrane glycoprotein, is a major receptor for extracellular proteins involved in invasion and metastasis of human cancers. We have previously demonstrated that the novel Gemini vitamin D analog BXL0124 [1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluro-cholecalciferol] repressed CD44 expression in MCF10DCIS.com basal-like human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth. In the present study, we investigated potential factors downstream of CD44 and the biological role of CD44 repression by BXL0124 in MCF10DCIS cells. Methods and Findings The treatment with Gemini vitamin D BXL0124 decreased CD44 protein level, suppressed STAT3 signaling, and inhibited invasion and proliferation of MCF10DCIS cells. The interaction between CD44 and STAT3 was determined by co-immunoprecipitation. CD44 forms a complex with STAT3 and Janus kinase 2 (JAK2) to activate STAT3 signaling, which was inhibited by BXL0124 in MCF10DCIS cells. The role of CD44 in STAT3 signaling and invasion of MCF10DCIS cells was further determined by the knockdown of CD44 using small hairpin RNA in vitro and in vivo. MCF10DCIS cell invasion was markedly decreased by the knockdown of CD44 in vitro. The knockdown of CD44 also significantly decreased mRNA expression levels of invasion markers, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), in MCF10DCIS cells. In MCF10DCIS xenograft tumors, CD44 knockdown decreased tumor size and weight as well as invasion markers. Conclusions The present study identifies STAT3 as an important signaling molecule interacting with CD44 and demonstrates the essential role of CD44-STAT3 signaling in breast cancer invasion. It also suggests that repression of CD44-STAT3 signaling is a key molecular mechanism in the inhibition of breast cancer invasion by the Gemini vitamin D analog BXL0124.

Dietary tocopherols inhibit cell proliferation, regulate expression of ERα, PPARγ, and Nrf2, and decrease serum inflammatory markers during the development of mammary hyperplasia

Previous clinical and epidemiological studies of vitamin E have used primarily α‐tocopherol for the prevention of cancer. However, γ‐tocopherol has demonstrated greater anti‐inflammatory and anti‐tumor activity than α‐tocopherol in several animal models of cancer. This study assessed the potential chemopreventive activities of a tocopherol mixture containing 58% γ‐tocopherol (γ‐TmT) in an established rodent model of mammary carcinogenesis. Female ACI rats were utilized due to their sensitivity to 17β‐estradiol (E2) to induce mammary hyperplasia and neoplasia. The rats were implanted subcutaneously with sustained release E2 pellets and given dietary 0.3% or 0.5% γ‐TmT for 2 or 10 wk. Serum E2 levels were significantly reduced by the treatment with 0.5% γ‐TmT. Serum levels of inflammatory markers, prostaglandin E2 and 8‐isoprostane, were suppressed by γ‐TmT treatment. Histology of mammary glands showed evidence of epithelial hyperplasia in E2‐treated rats. Immunohistochemical analysis of the mammary glands revealed a decrease in proliferating cell nuclear antigen (PCNA), cyclooxygenase‐2 (COX‐2), and estrogen receptor α (ERα), while there was an increase in cleaved‐caspase 3, peroxisome proliferator‐activated receptor γ (PPARγ), and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) in γ‐TmT‐treated rats. In addition, treatment with γ‐TmT resulted in a decrease in the expression of ERα mRNA, whereas mRNA levels of ERβ and PPARγ were increased. In conclusion, γ‐TmT was shown to suppress inflammatory markers, inhibit E2‐induced cell proliferation, and upregulate PPARγ and Nrf2 expression in mammary hyperplasia, suggesting that γ‐TmT may be a promising agent for human breast cancer prevention.

Tocopherols Inhibit Oxidative and Nitrosative Stress in Estrogen-Induced Early Mammary Hyperplasia in ACI Rats

Oxidative stress is known to play a key role in estrogen‐induced breast cancer. This study assessed the chemopreventive activity of the naturally occurring γ‐tocopherol‐rich mixture of tocopherols (γ‐TmT) in early stages of estrogen‐induced mammary hyperplasia in ACI rats. ACI rats provide an established model of rodent mammary carcinogenesis due to their high sensitivity to estrogen. Female rats were implanted with 9 mg of 17β‐estradiol (E2) in silastic tubings and fed with control or 0.3% γ‐TmT diet for 1, 3, 7, and 14 d. γ‐TmT increased the levels of tocopherols and their metabolites in the serum and mammary glands of the rats. Histological analysis revealed mammary hyperplasia in the E2 treated rats fed with control or γ‐TmT diet. γ‐TmT decreased the levels of E2‐induced nitrosative and oxidative stress markers, nitrotyrosine, and 8‐oxo‐dG, respectively, in the hyperplastic mammary tissues. 8‐Isoprostane, a marker of oxidative stress in the serum, was also reduced by γ‐TmT. Noticeably, γ‐TmT stimulated Nrf2‐dependent antioxidant response in the mammary glands of E2 treated rats, evident from the induced mRNA levels of Nrf2 and its downstream antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. Therefore, inhibition of nitrosative/oxidative stress through induction of antioxidant response is the primary effect of γ‐TmT in early stages of E2‐induced mammary hyperplasia. Due to its cytoprotective activity, γ‐TmT could be a potential natural agent for the chemoprevention of estrogen‐induced breast cancer.

A synthetic triterpenoid CDDO-Im inhibits tumorsphere formation by regulating stem cell signaling pathways in triple-negative breast cancer.

Triple-negative breast cancer is associated with poor prognosis because of a high rate of tumor recurrence and metastasis. Previous studies demonstrated that the synthetic triterpenoid, CDDO-Imidazolide (CDDO-Im) induced cell cycle arrest and apoptosis in triple-negative breast cancer. Since a small subpopulation of cancer stem cells has been suggested to be responsible for drug resistance and metastasis of tumors, our present study determined whether the effects of CDDO-Im in triple-negative breast cancer are due to the inhibition of a cancer stem cell subpopulation. CDDO-Im treatment markedly induced cell cycle arrest at G2/M-phase and apoptosis in the triple-negative breast cancer cell lines, SUM159 and MDA-MB-231. Because SUM159 cells were more sensitive to CDDO-Im than MDA-MB-231 cells, the effects of CDDO-Im on the cancer stem cell subpopulation were further investigated in SUM159 cells. SUM159 cells formed tumorspheres in culture, and the cancer stem cell subpopulation, CD24−/EpCAM+ cells, was markedly enriched in SUM159 tumorspheres. The CD24−/EpCAM+ cells in SUM159 tumorspheres were significantly inhibited by CDDO-Im treatment. CDDO-Im also significantly decreased sphere forming efficiency and tumorsphere size in both primary and secondary sphere cultures. PCR array of stem cell signaling genes showed that expression levels of many key molecules in the stem cell signaling pathways, such as Notch, TGF-β/Smad, Hedgehog and Wnt, were significantly down-regulated by CDDO-Im in SUM159 tumorspheres. Protein levels of Notch receptors (c-Notch1, Notch1 and Notch3), TGF-β/Smad (pSmad2/3) and Hedgehog downstream effectors (GLI1) also were markedly reduced by CDDO-Im. In conclusion, the present study demonstrates that the synthetic triterpenoid, CDDO-Im, is a potent anti-cancer agent against triple-negative breast cancer cells by targeting the cancer stem cell subpopulation.

Oral Administration of a Gemini Vitamin D Analog, a Synthetic Triterpenoid and the Combination Prevents Mammary Tumorigenesis Driven by ErbB2 Overexpression

HER2 (or ErbB2), a member of ErbB receptor tyrosine kinases, is overexpressed in approximately 20% of human breast cancer, and the ErbB2 signaling pathway is a critical therapeutic target for ErbB2-overexpressing breast cancer. We investigated the inhibitory effects of the Gemini vitamin D analog BXL0124, the synthetic triterpenoid CDDO-Im and the combination on the tumorigenesis of ErbB2-overexpressing breast cancer. MMTV-ErbB2/neu transgenic mice were treated with BXL0124, CDDO-Im, or the combination from three months of age until the end of the experiment. Formation and growth of MMTV-ErbB2/neu mammary tumors were monitored every week, and all three treatments delayed the development of mammary tumors without significant toxicity. Decreased activation of ErbB2 as well as other ErbB receptors, ErbB1 and ErbB3, in MMTV-ErbB2/neu mammary tumors was shown by all treatments. Protein levels of downstream targets of the ErbB2 signaling pathway, including activated-Erk1/2, activated-Akt, c-Myc, CycD1, and Bcl2, were repressed by all three treatments, with the combination treatment exhibiting the strongest effects. To investigate therapeutic efficacy, the combination of BXL0124 and CDDO-Im was given to MMTV-ErbB2/neu mice after mammary tumors were established between 23 and 30 weeks of age. Short-term treatment with the combination did not show effects on tumor growth nor the ErbB2 signaling pathway. The present study shows BXL0124, CDDO-Im, and the combination as potential agents for prevention, but not treatment, against the tumorigenesis of ErbB2-overexpressing breast cancer. Cancer Prev Res; 6(9); 959–70. ©2013 AACR.

Dietary γ-Tocopherol–Rich Mixture Inhibits Estrogen-Induced Mammary Tumorigenesis by Modulating Estrogen Metabolism, Antioxidant Response, and PPARγ

This study evaluated the anticancer activity and mechanism of action of a γ-tocopherol–rich tocopherol mixture, γ-TmT, in two different animal models of estrogen-induced breast cancer. The chemopreventive effect of γ-TmT at early (6 weeks), intermediate (18 weeks), and late (31 weeks) stages of mammary tumorigenesis was determined using the August-Copenhagen Irish rat model. Female rats receiving 17β-estradiol (E2) implants were administered with different doses (0%, 0.05%, 0.1%, 0.3%, and 0.5%) of γ-TmT diet. Treatment with 0.3% and 0.5% γ-TmT decreased tumor volume and multiplicity. At 31 weeks, serum concentrations of E2 were significantly decreased by γ-TmT. γ-TmT preferentially induced expression of the E2-metabolizing enzyme CYP1A1, over CYP1B1 in the rat mammary tissues. Nrf2-dependent antioxidant response was stimulated by γ-TmT, as evident from enhanced expression of its downstream targets, NQO1, GCLM, and HMOX1. Serum concentrations of the oxidative stress marker, 8-isoprostane, were also decreased in the γ-TmT–treated groups. Treatment with γ-TmT increased expression of PPARγ and its downstream genes, PTEN and p27, whereas the cell proliferation marker, PCNA, was significantly reduced in γ-TmT–treated mammary tumors. In an orthotopic model in which human MCF-7 breast cancer cells were injected into the mammary fat pad of immunodeficient mice, γ-TmT inhibited E2-dependent tumor growth at all the doses tested. In conclusion, γ-TmT reduced mammary tumor development, in part through decreased E2 availability and reduced oxidative stress in mammary tissues; γ-TmT could thus be an effective agent for the prevention and treatment of E2-induced breast cancer. Cancer Prev Res; 8(9); 807–16. ©2015 AACR.

Inhibition of the Transition of Ductal Carcinoma In Situ to Invasive Ductal Carcinoma by a Gemini Vitamin D Analog

Ductal carcinoma in situ (DCIS) is a nonmalignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model, which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In this study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions. Cancer Prev Res; 7(6); 617–26. ©2014 AACR.

Structural analysis and biological activities of BXL0124, a gemini analog of vitamin D

Gemini analogs of calcitriol, characterized by the extension of the C21-methyl group of calcitriol with a second chain, act as agonists of the vitamin D receptor (VDR). This second side chain of gemini is accommodated in a new cavity inside the VDR created by the structural rearrangement of the protein core. The resulting conformational change preserves the active state of the receptor and bestows gemini compounds with biological activities that exceed those of calcitriol. Of particular interest are geminis anti-cancer properties, and in this study we demonstrate anti-proliferative and tumor-reducing abilities of BXL0124 and BXL0097, differing only by the presence or absence, respectively, of the methylene group on the A ring. BXL0124 acts as a more potent VDR agonist than its 19-nor counterpart by activating VDR-mediated transcription at lower concentrations. In a similar manner, BXL0124 is more active than BXL0097 in growth inhibition of breast cancer cells and reduction of tumor volume. Structural comparisons of BXL0097 and BXL0124, as their VDR complexes, explain the elevated activity of the latter.