Amanda K. Smolarek

Mixed tocopherols prevent mammary tumorigenesis by inhibiting estrogen action and activating PPAR-γ

Purpose: Tocopherols are lipophilic antioxidants present in vegetable oils. Although the antioxidant and anticancer activities of α-tocopherol (vitamin E) have been studied for decades, recent intervention studies with α-tocopherol have been negative for protection from cancer in humans. The tocopherols consist of four isoforms, which are the α, β, γ, and δ variants, and recent attention is being given to other isoforms. In the present study, we investigated the inhibitory effect of a tocopherol mixture rich in γ- and δ-tocopherols against mammary tumorigenesis. Experimental Design: Female Sprague Dawley rats were treated with N-methyl-N-nitrosourea (NMU), and then fed diets containing 0.1%, 0.3%, or 0.5% mixed tocopherols rich in γ- and δ-tocopherols for 9 weeks. Tumor burden and multiplicity were determined, and the levels of markers of inflammation, proliferation, and apoptosis were evaluated in the serum and in mammary tumors. The regulation of nuclear receptor signaling by tocopherols was studied in mammary tumors and in breast cancer cells. Results: Dietary administration of 0.1%, 0.3%, or 0.5% mixed tocopherols suppressed mammary tumor growth by 38%, 50%, or 80%, respectively. Tumor multiplicity was also significantly reduced in all three mixed tocopherol groups. Mixed tocopherols increased the expression of p21, p27, caspase-3, and peroxisome proliferator activated receptor-γ, and inhibited AKT and estrogen signaling in mammary tumors. Our mechanistic study found that γ- and δ-tocopherols, but not α-tocopherol, activated peroxisome proliferator activated receptor-γ and antagonized estrogen action in breast cancer. Conclusion: The results suggest that γ- and δ-tocopherols may be effective agents for the prevention of breast cancer.

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.

Chemopreventive Activity of Vitamin E in Breast Cancer: A Focus on γ- and δ-Tocopherol

Vitamin E consists of eight different variants: α-, β-, γ-, and δ-tocopherols (saturated phytyl tail) and α-, β-, γ-, and δ-tocotrienols (unsaturated phytyl tail). Cancer prevention studies with vitamin E have primarily utilized the variant α-tocopherol. To no avail, a majority of these studies focused on variant α-tocopherol with inconsistent results. However, γ-tocopherol, and more recently δ-tocopherol, have shown greater ability to reduce inflammation, cell proliferation, and tumor burden. Recent results have shown that γ-enriched mixed tocopherols inhibit the development of mammary hyperplasia and tumorigenesis in animal models. In this review, we discuss the possible differences between the variant forms, molecular targets, and cancer-preventive effects of tocopherols. We recommend that a γ-enriched mixture, γ- and δ-tocopherol, but not α-tocopherol, are promising agents for breast cancer prevention and warrant further investigation.

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.

Gemini Vitamin D Analog Suppresses ErbB2-Positive Mammary Tumor Growth via Inhibition of ErbB2/AKT/ERK Signaling

Numerous synthetic vitamin D analogs have been studied for their effects on the prevention and treatment of breast cancer. However, the inhibitory effects of naturally occurring 1alpha,25-dihydroxyvitamin D3 or its synthetic analogs on ErbB2 overexpressing mammary tumorigenesis have not been reported. Gemini vitamin D analogs are novel synthetic vitamin D derivatives with a unique structure of two six-carbon chains at C-20. We have previously shown that Gemini vitamin D analogs significantly inhibited carcinogen-induced estrogen receptor (ER)-positive mammary tumorigenesis and reduced ER-negative MCF10DCIS.com xenograft tumor growth without hypercalcemic toxicity. In the present study, we have determined the inhibitory effect of a potent Gemini vitamin D analog BXL0124 (1alpha,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol) on the ErbB2/Her-2/neu overexpressing mammary tumorigenesis. The Gemini BXL0124 inhibits ErbB2-positive mammary tumor growth and down-regulates the phosphorylation of ErbB2, ERK and AKT in tumors of MMTV-ErbB2/neu transgenic mice. These effects of Gemini BXL0124 in vivo were confirmed by using the ErbB2 overexpressing tumor cells derived from the mammary tumors of MMTV-ErbB2/neu mice. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the growth of ErbB2 overexpressing mammary tumors through regulating the ErbB2/AKT/ERK signaling pathways, suggesting that Gemini vitamin D analog may be considered for translational studies.

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.

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.

Abstract 780: Knockdown of p53 increases tumorigenicity of human breast cancer cells.

The tumor suppressor p53 is mutated or functionally inactivated in ∼ 70% cancer. p53 causes cell cycle arrest, cellular senescence, or induces apoptosis in response to stress. Although knockdown of p53 has been shown to increase tumorigenicity of human colon and lung cancer cells, it is not known whether knockdown of p53 in human breast cancer cells can indeed lead to increased tumorigenicity. We show that knockdown of p53 in human breast cancer cells significantly increases tumorigenicity in vivo and colony formation on soft agar in vitro. Our ongoing studies are elucidating the underlying mechanisms. To the best of our knowledge, this is the first study demonstrating that knockdown of p53 increases tumorigenicity of human breast cancer cells. Our results highlight the importance of the tumor suppressor p53 in human breast cancer. Citation Format: Fang Liu, Tanima Roy, Jae Young So, Amanda Smolarek, You-Rong Lu, Nanjoo Suh. Knockdown of p53 increases tumorigenicity of human breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 780. doi:10.1158/1538-7445.AM2013-780 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.