Targets Breast Cancer Stem-like Cells Research Articles

Ganglioside GD2: a novel therapeutic target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by lack of hormone receptor expression and is known for high rates of recurrence, distant metastases, and poor clinical outcomes. TNBC cells lack targetable receptors; hence, there is an urgent need for targetable markers for the disease. Breast cancer stem-like cells (BCSCs) are a fraction of cells in primary tumors that are associated with tumorigenesis, metastasis, and resistance to chemotherapy. Targeting BCSCs is thus an effective strategy for preventing cancer metastatic spread and sensitizing tumors to chemotherapy. The CD44hi CD24lo phenotype is a well-established phenotype for identification of BCSCs, but CD44 and CD24 are not targetable markers owing to their expression in normal tissues. The ganglioside GD2 has been shown to be upregulated in primary TNBC tumors compared with normal breast tissue and has been shown to identify BCSCs. In this review, we discuss GD2 as a BCSC- and tumor-specific marker in TNBC; epithelial-to-mesenchymal transition and the signaling pathways that are upstream and downstream of GD2 and the role of these pathways in tumorigenesis and metastasis in TNBC; direct and indirect approaches for targeting GD2; and ongoing clinical trials and treatments directed against GD2 as well as future directions for these strategies.

Anti-Breast Cancer Activities of Ketoprofen-RGD Conjugate by Targeting Breast Cancer Stem-Like Cells and Parental Cells.

Cancer Stem Cells (CSCs) play an important role in various stages of cancer development, advancement, and therapy resistance. Ketoprofen-RGD has been revealed to act as an anti-cancer agent against some tumors. We aimed to explore the effects of a novel Ketoprofen-RGD compound on the suppression of Breast Cancer Stem-like Cells (BCSCs) and their parental cells. Mammospheres were developed from MCF-7 cells and assessed by CSC surface markers through flowcytometry. The anti-proliferative and pro-apoptotic activities of Ketoprofen-RGD were measured by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative Real Time-PCR (qRT-PCR) and western blotting, respectively. Intracellular Reactive Oxygen Species (ROS) was measured using a cell permeable, oxidant-sensitive fluorescence probe (carboxy-H2DCFDA). Ketoprofen-RGD significantly reduced the mammosphere formation rate and the expression of three out of six stemness markers and remarkably decreased viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that Ketoprofen-RGD induced intrinsic pathway, suggesting a mechanism by which Ketoprofen-RGD triggers apoptosis. ROS production was also another way to induce apoptosis. Results of western blot analysis also revealed a marked diminish in the phosphorylation of JAK2 and STAT proteins. Our study, for the first time, elucidated an anti-BCSC activity for Ketoprofen-RGD via declining stemness markers, inducing toxicity, and apoptosis in these cells and parental cells. These findings may suggest this compound as a promising anti-breast cancer.

Targeting breast cancer stem-like cells using chloroquine encapsulated by a triphenylphosphonium-functionalized hyperbranched polymer.

Cancer stem cells (CSCs) have garnered increasing attention over the past decade, as they are believed to play a crucial role in tumor progression and drug resistance. Accumulating evidence provides insight into the function of autophagy in maintenance and survival of CSCs. Here, we studied the impact of a mitochondriotropic triphenylphosphonium-functionalized dendrimeric nanocarrier on cultured breast cancer cell lines, grown either as adherent cells or as mammospheres that mimic a stem-like phenotype. The nanocarrier manifested a substantial cytotoxicity both alone as well as after encapsulation of chloroquine, a well-known autophagy inhibitor. The cytotoxic effects of the nanocarrier could be ascribed to interference with mitochondrial function. Importantly, mammospheres were selectively sensitive to encapsulated chloroquine and this depends on the expression of the gene encoding ATM kinase. Ataxia-telangiectasia mutated (ATM) kinase is an enzyme that functions as an essential signaling mediator that enables growth of cancer stem cells through the regulation of autophagy. We noted that this ATM-dependent sensitivity of mammospheres to encapsulated chloroquine was independent of the status of the tumor suppressor gene p53. Our study suggests that breast cancer stem cells, as they are modeled by mammospheres, are sensitive to encapsulated chloroquine, depending on the expression of the ATM kinase, which is thereby characterized as a potential biomarker for sensitivity to this type of treatment.

Anti-Breast Cancer Activities of 8-Hydroxydaidzein by Targeting Breast Cancer Stem-Like Cells.

Cancer stem cells (CSCs) play an important role in various stages of cancer development and therapy refractoriness. 8-Hydroxydaidzein (8-OHD) has revealed anti-cancer activity in different tumors. Accordingly, we aimed to assess the effects of 8-OHD on the suppression of breast cancer stem-like cells (BCSCs). The anti-proliferative and pro-apoptotic properties of 8-OHD were examined by MTS assay and flowcytometry. The expression levels of stemness markers and JAK2/STAT proteins were measured by quantitative real time-PCR (qRT-PCR) and western blotting, respectively. 8-OHD significantly decreased three out of six stemness markers and remarkably reduced viability and induced apoptosis of spheroidal and parental cells compared to controls. Further experiments using CD95L, as a death ligand, and ZB4 antibody, as an extrinsic apoptotic pathway blocker, showed that 8-OHD induced apoptosis through the intrinsic pathway, proposing a mechanism by which 8-OHD triggers apoptosis. Results of western blot analysis also revealed a marked decline in the phosphorylation of JAK2 and STAT proteins. Our study, for the first time, elucidated an anti-BCSC activity for 8-OHD via decreasing stemness markers, inducing toxicity and stimulating apoptosis in these cells and parental cells. Our results also suggested a novel mechanism by which 8-OHD induces apoptosis in BCSCs.

Abstract 155: KDM4 inhibition targets breast cancer stem-like cells

Abstract Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSC regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. Citation Format: Jochen Maurer. KDM4 inhibition targets breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 155.

376 - Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumours

376 - Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumours

Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells.

Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44+/CD24- stem-like population. Salinomycin is an antibiotic that has been shown to target cancer stem cells (CSC); however, the mechanisms of action involved have not been well characterized. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in MDA-MB-231 breast cancer cells. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44+/CD24- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. These findings support the notion that salinomycin may be potentially efficacious for targeting breast cancer stem-like cells through the inhibition of STAT3 activation.

Abstract 2319: Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors

Abstract Sulforadex (SFX) is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within α-cyclodextrin. SFN has been studied as an anti-cancer compound for many years however its development has hitherto been hampered due to its inherent instability. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are believed to be the drivers of breast cancer metastasis. We investigated SFX effects on breast CSC activity using mammosphere formation and aldehyde dehydrogenase (ALDH) activity in patient samples and patient-derived xenograft (PDX) tumours. Mammosphere formation efficiency (MFE) assessed in cells from primary (n = 12) and metastatic (n = 15) samples treated with SFX (5 μM) or vehicle control (water) showed a significant reduction in breast CSC activity following SFX treatment. A reduction greater than 50% in the MFE was observed in both primary (0.52%±0.06 vs 0.19±0.02, p<0.001) and metastatic (0.93%±0.07 vs 0.43±0.04, p<0.001) cells treated with SFX. Importantly, SFX also inhibits breast CSC activity of early (HBCx34) and metastatic (BB3RC31) patient-derived xenograft (PDX) tumours. Using a 14 day in vivo ‘window’ treatment, we observe that SFX (300mg/Kg/day) decreases ALDH+ cells using ALDEFLUOR assay (HBCx34 6.3%±0.4 vs 3%±0.6, p<0.01; BB3RC31 3%±0.6 vs 1±0.2, p<0.05) and MFE (HBCx34 0.64%±0.09 vs 0.35%±0.03, p<0.01; BB3RC31 0.89%±0.06 vs 0.78±0.04). The majority of breast cancers express the estrogen receptor (ER) but these hormone-responsive breast cancers frequently develop resistance to hormonal therapies (e.g. tamoxifen). We have established that CSCs in ER+ BC lack ER expression, representing a potential mechanism of therapeutic resistance. However, SFX targets such surviving CSCs identified by ALDH positivity and mammosphere-initiating capacity following tamoxifen treatment in vivo in ER+ PDX tumours. Combinatorial treatment of tamoxifen (10 mg/kg/day) and SFX (300mg/Kg/day) in a 14 day ‘window’ treatment reduced ALDH+ cells (HBCx34 10%±0.4 vs 4.2%±0.4, p<0.01; BB3RC31 11.3%±1.1 vs 6.9±0.6, p<0.05) and MFE (HBCx34 0.81%±0.07 vs 0.34%±0.02, p<0.01; BB3RC31 0.47%±0.06 vs 0.27±0.04, p<0.05) compared to tamoxifen treatment alone. Mechanistically SFX potently inhibited the canonical Wnt pathway in MCF-7 cells and their endocrine-resistant derivatives and future studies will explore SFX activity on other CSC regulatory pathways. Our data demonstrate that SFX might produce clinically meaningful improvements to endocrine therapy in ER+ BC by reversing CSC mediated resistance. Clinical trials of endocrine therapy in combination with SFX are currently being planned. Citation Format: Bruno M. Simões, Denis Alferez, Rachel Eyre, Kath Spence, Angélica Santiago-Gomez, Iris Tanaka, Bertram Kohler, David Howat, Sacha J. Howell, Robert B. Clarke. Sulforadex targets breast cancer stem-like cells in patient-derived cells and xenograft tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2015-2319

Abstract 1600: Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells.

Abstract Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths although contributing to a small percentage (∼1%) of total breast cancer cases each year. The hallmarks of IBC are skin involvement and a high propensity to metastasize. It is thought that a small population of stem-like cancer cells is responsible for the metastatic potential and treatment resistance to therapies such as radiation therapy in IBC. In this study we explored the radiosensitization of several breast cancer cell lines in vitro with simvastatin and the mechanism for this. Methods: In order to determine cell proliferation, breast cancer cell lines SUM149, SUM159 and MCF-7 were grown in 96 well plates with different concentrations of simvastatin for one week. In order to determine the effect of simvastatin in stem-like cancer cells, cells were seeded in serum free, growth factor enriched, anchorage independent, treated with simvastatin for 24 hours and mammosphere formation was quantified a week later. In addition, SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and in stem cell enriching anchorage independent cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated using Sigmaplot 8.0 and t-test was used to compare surviving fraction (SF) of groups. Finally, cell lysates were obtained from cultures untreated and treated with simvastatin and radiation and analyzed through western blot. Results: Simvastatin significantly decreased cancer cell proliferation in vitro in all cell lines (p<0.002). Cell lines SUM 159 and MCF 7 grown in mammosphere enriched media showed significant decreased mammosphere formation when grown in the presence of simvastatin (p<0.05). Moreover, simvastatin radiosensitized all cell lines in both monolayer and anchorage independent clonogenic assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p<0.0001) and in stem cell enriching cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p<0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p<0.001) than in stem cell enriching cultures. Combination of simvastatin and radiation therapy decreased protein expression of phospho-rho A, VEGF-A, and phospho-ras. Conclusions: IBC remains a difficult disease to treat with a high rate of local recurrence after radiation therapy. Statins have been shown to increase tumor free survival in breast cancer. Herein we show simvastatin targets breast cancer stem-like cells resulting in radiosensitizing of IBC in vitro. Citation Format: Adam R. Wolfe, Lara Lacerda, Richard Larson, Bisrat G. Debeb, Wei Xu, Naoto T. Ueno, Wendy A. Woodward. Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory 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 1600. doi:10.1158/1538-7445.AM2013-1600

112 Inhibition of Heat Shock Protein (Hsp) 27 Potentiates the Suppressive Effect of Hsp90 Inhibitors in Targeting Breast Cancer Stem-like Cells

112 Inhibition of Heat Shock Protein (Hsp) 27 Potentiates the Suppressive Effect of Hsp90 Inhibitors in Targeting Breast Cancer Stem-like Cells

1018 Disulfiram, an Antialcoholism Drug, Targets Breast Cancer and Glioblastoma Stem-like Cells

1018 Disulfiram, an Antialcoholism Drug, Targets Breast Cancer and Glioblastoma Stem-like Cells

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.