Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) belong to a superfamily of nuclear receptors called steroid hormone receptors, which, upon binding ligand, dimerize and translocate to the nucleus where they activate or repress the transcription of a large number of genes, thus modulating critical physiologic processes. ERβ has multiple isoforms that show differing association with prognosis. Expression levels of the full length ERβ1 isoform are often lower in aggressive cancers as compared to normal tissue. High ERβ1 expression is associated with improved overall survival in women with breast cancer. The promise of ERβ activation, as a potential targeted therapy, is based on concurrent activation of multiple tumor suppressor pathways with few side effects compared to chemotherapy. Thus, ERβ is a nuclear receptor with broad-spectrum tumor suppressor activity, which could serve as a potential treatment target in a variety of human cancers including breast cancer. Further development of highly selective agonists that lack ERα agonist activity, will be necessary to fully harness the potential of ERβ.
Breast cancer is the most commonly diagnosed cancer in women. There is a continued interest for the development of more efficacious treatment regimens for breast carcinoma. Recombinant human tumor necrosis factor–related apoptosis-inducing ligand (rhTRAIL) shows potential as a potent anticancer therapeutic for the treatment of breast cancer, whereas displaying minimal toxicity to normal cells. However, the promise of rhTRAIL for the treatment of breast cancer is dismissed by the resistance to rhTRAIL-induced apoptosis exhibited by many breast cancers. Thus, a cotreatment strategy was examined by applying the natural compound quercetin (Q) as a sensitizing agent for rhTRAIL-resistant breast cancer BT-20 and MCF-7 cells. Quercetin was able to sensitize rhTRAIL-resistant breast cancers to rhTRAIL-induced apoptosis as detected by Western blotting through the proteasome-mediated degradation of c-FLIPL and through the upregulation of DR5 expression transcriptionally. Overall, these in vitro findings establish that Q is an effective sensitizing agent for rhTRAIL-resistant breast cancers.
IntroductionBreast cancer affects two million patients worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP is degraded by eATPases. Given that eATP is toxic to cancer cells, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy.MethodsTNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPases or specific antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively.ResultsIn the present study, we observed dose-dependent declines of cell viability and increases in eATP of paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases, but not of the MCF-10A cell line. These effects were reversed by specific antagonists of P2RXs. Similar results, as those observed with eATPase inhibitors, were seen with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels.ConclusionThese results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines, which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.
BackgroundAmong women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERβ plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERβ agonists without ERα activity has limited the exploration of ERβ activation as a strategy for ERα+ breast cancer.MethodsWe measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERβ agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERβ agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERβ agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes.ResultsIn this study, we demonstrate the efficacy of highly selective ERβ agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERβ agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERβ agonists and ERα antagonists suggested that the efficacy of ERβ agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERβ gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples.ConclusionOur results demonstrate that highly selective ERβ agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.
Background/Aim: Triple-negative breast cancer (TNBC) can be characterized as the deadliest breast cancer type considering the lack of efficacious therapeutics. Recombinant human tumor necrosis factor-related apoptosisinducing ligand (rhTRAIL) is an encouraging anti-cancer therapeutic with the capacity to induce apoptosis in cancer cells but there are TNBCs less susceptible to rhTRAIL. The aim of this study was to assess the potential of the natural product ursolic acid (UA) to sensitize of rhTRAIL-resistant TNBCs. Materials and Methods: In order to evaluate apoptosis induction in rhTRAIL and UA-treated TNBC BT-20 and HCC1937 cells that are resistant to rhTRAIL, western blot analysis and Annexin V/PI assays were executed. Results: UA increased the expression of death receptors 4 and 5 and decreased the expression of c-FLIP L transcriptionally sensitizing rhTRAIL-resistant TNBC cells to apoptosis induced by rhTRAIL. Conclusion: UA is a possible potent sensitizer of rhTRAIL-resistant TNBCs to rhTRAIL-induced apoptosis.
Malignant melanoma is the most commonly diagnosed skin cancer associated with a high rate of metastasis. Low-stage melanoma is easily treated, but metastatic malignant melanoma is an extremely treatment-resistant malignancy with low survival rates. The application of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) for the treatment of metastatic malignant melanoma holds considerable promise because of its selective proapoptotic activity towards cancer cells and not nontransformed cells. Unfortunately, the clinical utilization of rhTRAIL has been terminated due to the resistance of many cancer cells to undergo apoptosis in response to rhTRAIL. However, rhTRAIL-resistance can be abrogated through the cotreatment with compounds derived from ‘Mother Nature’ such as quercetin that can modulate cellular components responsible for rhTRAIL-resistance. Here, we show that rhTRAIL-resistant malignant melanomas are sensitized by quercetin. Quercetin action is manifested by the upregulation of rhTRAIL-binding receptors DR4 and DR5 on the surface of cancer cells and by increased rate of the proteasome-mediated degradation of the antiapoptotic protein FLIP. Our data provide for a new efficient and nontoxic treatment of malignant melanoma.
Breast cancer is the most commonly diagnosed cancer in women in the United States. There is a continued need for the development of selective and specific treatment options for all types of breast cancer, including hormone-dependent and triple-negative subtypes. Recombinant human Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (rhTRAIL), the optimized form of the endogenous death ligand, shows potential as an effective anti-cancer therapeutic due to its ability to induce apoptosis in cancer independent of wild-type p53 function, while displaying minimal toxicity to normal cells. However, a majority of breast cancer cell lines exhibit resistance to TRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors 4 and 5. To overcome TRAIL resistance, a cotreatment option has been explored utilizing the natural compound Quercetin (Q). Q is a flavonol found in certain fruits, vegetables, and teas. As a single agent, Q has been shown to have antiproliferative and pro-apoptotic effects on a variety of cancer cell lines. The aim of this study is to examine the capacity of Q to enhance TRAIL's pro-apoptotic and antiproliferative effects on breast cancer cells. Sulphorhodamine B (SRB) assays were performed on hormone dependent (MCF-7) and triple negative (BT-20) breast cancer cell lines to determine if the cotreatment of Q and TRAIL hinders cell growth. Growth for both MCF-7 and BT-20 cells was substantially inhibited by single agent Q treatments (12.5 μM; ∼20%, 25 μM; ∼40%, 50 μM; ∼60%) but not by single agent TRAIL treatment (100 ng/mL; <20%). Moreover, BT-20 and MCF-7 cell growth was further inhibited by cotreatment with both agents; for example, the cotreatment of 50 μM Q and 100 ng/mL TRAIL inhibited BT-20 and MCF-7 cell growth by 80% and 90%, respectively. As a single agent, Q was able to induce apoptosis in a dose-dependent manner in both breast cancer cells; thereafter, Q's ability to sensitize breast cancer cells to TRAIL-induced apoptosis was examined by western blot analysis. Compared to single agent treatments, the combination of Q and TRAIL enhanced the induction of apoptosis as indicated by increased PARP cleavage (a hallmark of apoptosis) and the activation of the executioner caspases 3 and 7. Furthermore, the cotreatment of breast cancer cells with Q and TRAIL enhanced the activation of the extrinsic and intrinsic apoptotic pathways, as assessed by the activation of caspase 8 and the release of cytochrome c from the mitochondria, respectively. Overall, these findings suggest that the cotreatment of Q and rhTRAIL possesses the potential to be an anti-breast cancer therapeutic by enhancing pro-apoptotic and anti-proliferative effects in hormone dependent and triple-negative breast cancer cells. Citation Format: Jasmine M. Manouchehri, Michael Kalafatis, Daniel Lindner. Evaluation of the efficacy of TRAIL plus quercetin as a potential breast carcinoma therapeutic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1295.
Background:Triple negative breast cancer (TNBC) represents about 10-15% of all breast cancers. Given the lack of targeted therapies, chemotherapy and immunotherapy are the only treatment options. The five-year survival rate of TNBC patients is approximately 15% less than other forms of breast cancer. Therefore, there is an urgent need to develop novel effective therapeutics for TNBC.Material and Methods:RNA isolated from human immortalized mammary epithelial MCF10A, and various TNBC cell lines was DNase treated and quantitative RT-PCR (qRT-PCR) performed using gene-specific primers spanning exon-exon junctions that include introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. Immuno-blot analyses were performed on lysates prepared from cells in log phase of growth. Proteins were detected by Enhanced Chemi-Luminescence (ECL) method. Viability of cells treated with ERβ specific agonists was determined by using CellTiter-Glo® 2.0 assay. MB231 cells were treated with ERβ agonists following transfection with pcDNA3 (vector) or Flag-tagged ESR2 plasmids. Western blot analyses using specific antibodies and qRT-PCR were performed with the protein extracts and RNA isolated from the cells, respectively. Results:Our experiments demonstrate that ESR2 (ERβ) is differentially expressed and its level of expression is about 3 to 22-fold higher in TNBC cell lines tested than MCF10A. We have shown that viability of estrogen receptor (ER) positive breast cancer cell lines can be significantly inhibited by selective activation of ERβ. A highly selective ERβ agonist has been invented in the Drug Development Institute at the Ohio State University. Treatment of TNBC lines with these ERβ agonists exhibited a dramatic reduction of cell viability. While comparable IC50 values were observed with our in house and commercially available compounds, TNBC had 2-3-fold lesser IC50 values than ER-positive cell lines. Increased expression of FOXO3 and c-Myc1 and decreased expression of c-Myc2 isoform was noted in ERβ overexpressing MB231 cells upon treatment with ERβ agonists.Conclusion:Our results demonstrate that treatment with highly selective ERβ agonists could be an effective therapeutic strategy for TNBC patients. To improve and develop novel therapeutics for TNBC understanding the mechanism of action of these compounds will be crucial. Citation Format: Jharna Datta, Natalie Willingham, Jasmine Manouchehri, Joel David, Mirisha Sheth, Bhuvaneswari Ramaswamy, Ramesh Ganju, Mathew A Cherian. Estrogen receptor beta agonists for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-39.
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