Joseph Wahler

Potential therapeutic implications of IL-6/IL-6R/gp130-targeting agents in breast cancer

Interleukin-6 (IL-6) is a pleiotropic cytokine with known multiple functions in immune regulation, inflammation, and oncogenesis. Binding of IL-6 to the IL-6 receptor (IL-6R) induces homodimerization and recruitment of glycoprotein 130 (gp130), which leads to activation of downstream signaling. Emerging evidence suggests that high levels of IL-6 are correlated with poor prognosis in breast cancer patients. IL-6 appears to play a critical role in the growth and metastasis of breast cancer cells, renewal of breast cancer stem cells (BCSCs), and drug resistance of BCSCs, making anti–IL-6/IL-6R/gp130 therapies promising options for the treatment and prevention of breast cancers. However, preclinical and clinical studies of the applications of anti–IL-6/IL-6R/gp130 therapy in breast cancers are limited. In this review, we summarize the structures, preclinical and clinical studies, mechanisms of action of chemical and biological blockers that directly bind to IL-6, IL-6R, or gp130, and the potential clinical applications of these pharmacological agents as breast cancer therapies.

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.

Role of dietary bioactive natural products in estrogen receptor-positive breast cancer

Estrogen receptor (ER)-positive breast cancer, including luminal-A and -B, is the most common type of breast cancer. Extended exposure to estrogen is associated with an increased risk of breast cancer. Both ER-dependent and ER-independent mechanisms have been implicated in estrogen-mediated carcinogenesis. The ER-dependent pathway involves cell growth and proliferation triggered by the binding of estrogen to the ER. The ER-independent mechanisms depend on the metabolism of estrogen to generate genotoxic metabolites, free radicals and reactive oxygen species to induce breast cancer. A better understanding of the mechanisms that drive ER-positive breast cancer will help optimize targeted approaches to prevent or treat breast cancer. A growing emphasis is being placed on alternative medicine and dietary approaches toward the prevention and treatment of breast cancer. Many natural products and bioactive compounds found in foods have been shown to inhibit breast carcinogenesis via inhibition of estrogen induced oxidative stress as well as ER signaling. This review summarizes the role of bioactive natural products that are involved in the prevention and treatment of estrogen-related and ER-positive 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.

Vitamin D compounds inhibit cancer stem-like cells and induce differentiation in triple negative breast cancer

Triple-negative breast cancer is one of the least responsive breast cancer subtypes to available targeted therapies due to the absence of hormonal receptors, aggressive phenotypes, and the high rate of relapse. Early breast cancer prevention may therefore play an important role in delaying the progression of triple-negative breast cancer. Cancer stem cells are a subset of cancer cells that are thought to be responsible for tumor progression, treatment resistance, and metastasis. We have previously shown that vitamin D compounds, including a Gemini vitamin D analog BXL0124, suppress progression of ductal carcinoma in situ in vivo and inhibit cancer stem-like cells in MCF10DCIS mammosphere cultures. In the present study, the effects of vitamin D compounds in regulating breast cancer stem-like cells and differentiation in triple-negative breast cancer were assessed. Mammosphere cultures, which enriches for breast cancer cells with stem-like properties, were used to assess the effects of 1α,25(OH)2D3 and BXL0124 on cancer stem cell markers in the triple-negative breast cancer cell line, SUM159. Vitamin D compounds significantly reduced the mammosphere forming efficiency in primary, secondary and tertiary passages of mammospheres compared to control groups. Key markers of cancer stem-like phenotype and pluripotency were analyzed in mammospheres treated with 1α,25(OH)2D3 and BXL0124. As a result, OCT4, CD44 and LAMA5 levels were decreased. The vitamin D compounds also down-regulated the Notch signaling molecules, Notch1, Notch2, Notch3, JAG1, JAG2, HES1 and NFκB, which are involved in breast cancer stem cell maintenance. In addition, the vitamin D compounds up-regulated myoepithelial differentiating markers, cytokeratin 14 and smooth muscle actin, and down-regulated the luminal marker, cytokeratin 18. Cytokeratin 5, a biomarker associated with basal-like breast cancer, was found to be significantly down-regulated by the vitamin D compounds. These results suggest that vitamin D compounds may serve as potential preventive agents to inhibit triple negative breast cancer by regulating cancer stem cells and differentiation.

Inhibitory effects of γ- and δ-tocopherols on estrogen-stimulated breast cancer in vitro and in vivo.

Estrogens have been implicated as complete carcinogens for breast and other tissues through mechanisms involving increased cell proliferation, oxidative stress, and DNA damage. Because of their potent antioxidant activity and other effects, tocopherols have been shown to exert antitumor activities in various cancers. However, limited information is available on the effect of different forms of tocopherols in estrogen-mediated breast cancer. To address this, we examined the effects of α-, γ-, and δ-tocopherols as well as a natural γ-tocopherol–rich mixture of tocopherols, γ-TmT, on estrogen-stimulated MCF-7 cells in vitro and in vivo. For the in vivo studies, MCF-7 cells were injected into the mammary fat pad of immunodeficient mice previously implanted with estrogen pellets. Mice were then administered diets containing 0.2% α-, γ-, δ-tocopherol, or γ-TmT for 5 weeks. Treatment with α-, γ-, δ-tocopherols, and γ-TmT reduced tumor volumes by 29% (P < 0.05), 45% (P < 0.05), 41% (P < 0.05), and 58% (P < 0.01), as well as tumor weights by 20%, 37% (P < 0.05), 39% (P < 0.05), and 52% (P < 0.05), respectively. γ- and δ-tocopherols and γ-TmT inhibited the expression of cell proliferation–related genes such as cyclin D1 and c-Myc, and estrogen-related genes such as TFF/pS2, cathepsin D, and progesterone receptor in estrogen-stimulated MCF-7 cells in vitro. Further, γ- and δ-tocopherols decreased the levels of estrogen-induced oxidative stress and nitrosative stress markers, 8-hydroxy-2′-deoxyguanosine and nitrotyrosine, as well as the DNA damage marker, γ-H2AX. Our results suggest that γ- and δ-tocopherols and the γ-tocopherol–rich mixture are effective natural agents for the prevention and treatment of estrogen-mediated breast cancer. Cancer Prev Res; 10(3); 188–97. ©2017 AACR.

Differential Gene Regulation and Tumor-Inhibitory Activities of Alpha-, Delta-, and Gamma-Tocopherols in Estrogen-Mediated Mammary Carcinogenesis

Despite experimental evidence elucidating the antitumor activities of tocopherols, clinical trials with α-tocopherol (α-T) have failed to demonstrate its beneficial effects in cancer prevention. This study compared the chemopreventive efficacy of individual tocopherols (α-, δ-, and γ-T) and a γ-T–rich tocopherol mixture (γ-TmT) in the August-Copenhagen Irish (ACI) rat model of estrogen-mediated mammary cancer. Female ACI rats receiving 17β-estradiol (E2) implants were administered with 0.2% α-T, δ-T, γ-T, or γ-TmT for 30 weeks. Although α-T had no significant effects on mammary tumor growth in ACI rats, δ-T, γ-T, and γ-TmT reduced mammary tumor volume by 51% (P < 0.05), 60% (P < 0.01), and 59% (P < 0.01), respectively. Immunohistochemical analysis revealed that δ-T, γ-T, and γ-TmT reduced levels of the cell proliferation marker, proliferating cell nuclear antigen, in the rat mammary tumors. To gain further insight into the biological functions of different forms of tocopherols, RNA-seq analysis of the tumors was performed. Treatment with γ-T induced robust gene expression changes in the mammary tumors of ACI rats. Ingenuity Pathway Analysis identified “Cancer” as a top disease pathway and “Tumor growth” and “Metastasis” as the top signaling pathways modulated by γ-T. Although the results need further functional validation, this study presents an unbiased attempt to understand the differences between biological activities of individual forms of tocopherols at the whole transcriptome level. In conclusion, δ-T and γ-T have superior cancer preventive properties compared to α-T in the prevention of estrogen-mediated mammary carcinogenesis. Cancer Prev Res; 10(12); 694–703. ©2017 AACR.