Breast Cancer Stem Cell-like Cells Research Articles

Estrogen-related receptor alpha (ERRα) promotes the migration, invasion and angiogenesis of breast cancer stem cell-like cells.

Breast cancer progression, metastasis and recurrence are largely driven by breast cancer stem cells (BCSCs), which constitute a subset of tumor cells exhibiting stem cell characteristics. In this study, we evaluated the role of estrogen-related receptor alpha (ERRα) in the migration, invasion and angiogenesis of BCSCs. The inhibition of ERRα using XCT790 or knockdown of ERRα using shRNA inhibited the mammosphere formation efficiency, as well as the migration and invasion of BCSCs derived from the mammospheres of MCF7 and MDA-MB-231 (MB231) cells. Conversely, the overexpression of ERRα significantly increased the migration and invasion of BCSCs derived from the mammosphere. In addition, the XCT790 treatment or shERRα significantly downregulated the epithelial-mesenchymal transition (EMT), as evidenced by the downregulation in the expression of vimentin, Snail, Slug and N-cadherin in the mammospheres of MCF7 and MB231 cells. The chorioallantoic membrane assay showed that the conditioned media from XCT790-treated and shERRα cells significantly inhibited blood vessel formation and vessel length. Furthermore, XCT790 treatment or shERRα also downregulated the expression of molecular markers of angiogenesis, such as VEGF-A and Ang-2 in the mammospheres. Conversely, the overexpression of ERRα in MCF7 cells significantly increased both EMT and angiogenesis. These findings suggest that ERRα inhibits the migration, invasion and angiogenesis of BCSCs, suggesting as a potential target for breast cancer therapy.

Multi-Targeted and On-Demand Non-Coding RNA Regulation Nanoplatform against Metastasis and Recurrence of Triple-Negative Breast Cancer.

Dysregulation of microRNAs (miRs) is the hallmark of triple-negative breast cancer (TNBC), which is closely involved with itsgrowth, metastasis, and recurrence. Dysregulated miRs are promising targets for TNBC therapy, however, targeted and accurate regulation of multiple disordered miRs in tumors is still a great challenge. Here, a multi-targeting and on-demand non-coding RNA regulation nanoplatform (MTOR) is reported to precisely regulate disordered miRs, leading to dramatical suppression of TNBC growth, metastasis, and recurrence. With the assistance of long blood circulation, ligands of urokinase-type plasminogen activator peptide and hyaluronan located in multi-functional shells enable MTOR to actively target TNBC cells and breast cancer stem cell-like cells (BrCSCs). After entering TNBC cells and BrCSCs, MTOR is subjected to lysosomal hyaluronidase-induced shell detachment, leading to an explosion of the TAT-enriched core, thereby enhancing nuclear targeting. Subsequently, MTOR could precisely and simultaneously downregulate microRNA-21 expression and upregulate microRNA-205 expression in TNBC. In subcutaneous xenograft, orthotopic xenograft, pulmonary metastasis, and recurrence TNBC mouse models, MTOR shows remarkably synergetic effects on the inhibition of tumor growth, metastasis, and recurrence due to its on-demand regulation of disordered miRs. This MTOR system opens a new avenue for on-demand regulation of disordered miRs against growth, metastasis, and recurrence of TNBC.

Degradome-focused RNA interference screens to identify proteases important for breast cancer cell growth.

Proteases are known to promote or impair breast cancer progression and metastasis. However, while a small number of the 588 human and 672 murine protease genes have been extensively studied, others were neglected. For an unbiased functional analysis of all genome-encoded proteases, i.e., the degradome, in breast cancer cell growth, we applied an inducible RNA interference library for protease-focused genetic screens. Importantly, these functional screens were performed in two phenotypically different murine breast cancer cell lines, including one stem cell-like cell line that showed phenotypic plasticity under changed nutrient and oxygen availability. Our unbiased genetic screens identified 252 protease genes involved in breast cancer cell growth that were further restricted to 100 hits by a selection process. Many of those hits were supported by literature, but some proteases were novel in their functional link to breast cancer. Interestingly, we discovered that the environmental conditions influence the degree of breast cancer cell dependency on certain proteases. For example, breast cancer stem cell-like cells were less susceptible to depletion of several mitochondrial proteases in hypoxic conditions. From the 100 hits, nine proteases were functionally validated in murine breast cancer cell lines using individual knockdown constructs, highlighting the high reliability of our screens. Specifically, we focused on mitochondrial processing peptidase (MPP) subunits alpha (Pmpca) and beta (Pmpcb) and discovered that MPP depletion led to a disadvantage in cell growth, which was linked to mitochondrial dysfunction.

Antagonizing RARγ Drives Necroptosis of Cancer Stem Cells

There is a need for agents that eliminate cancer stem cells, which sustain cancer and are also largely responsible for disease relapse and metastasis. Conventional chemotherapeutics and radiotherapy are often highly effective against the bulk of cancer cells, which are proliferating, but spare cancer stem cells. Therapeutics that target cancer stem cells may also provide a bona fide cure for cancer. There are two rationales for targeting the retinoic acid receptor (RAR)γ. First, RARγ is expressed selectively within primitive cells. Second, RARγ is a putative oncogene for a number of human cancers, including cases of acute myeloid leukemia, cholangiocarcinoma, and colorectal, renal and hepatocellular carcinomas. Prostate cancer cells depend on active RARγ for their survival. Antagonizing all RARs caused necroptosis of prostate and breast cancer stem cell-like cells, and the cancer stem cells that gave rise to neurospheres from pediatric patients’ primitive neuroectodermal tumors and an astrocytoma. As tested for prostate cancer, antagonizing RARγ was sufficient to drive necroptosis. Achieving cancer-selectively is a longstanding paradigm for developing new treatments. The normal prostate epithelium was less sensitive to the RARγ antagonist and pan-RAR antagonist than prostate cancer cells, and fibroblasts and blood mononuclear cells were insensitive. The RARγ antagonist and pan-RAR antagonist are promising new cancer therapeutics.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

Abstract 323: Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer

Abstract The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25-35% of the patients do not respond initially, and almost all patients eventually acquire resistance. Hence, understanding the mechanisms of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies. To identify mechanisms of resistance to CDK4/6 inhibition we developed MCF-7 and T47D palbociclib resistant cells in a step-wise manner by gradually increasing concentrations of palbociclib. These cells are not only resistant to palbociclib, but exhibited resistance to the other approved CDK4/6 inhibitors; ribociclib and abemaciclib. Additionally, we assessed if these resistant cells have an altered response to endocrine therapy and observed that these cells are also resistant to treatment with tamoxifen or fulvestrant by about 16-fold. Multi-omics analyses revealed enrichment of pathways known to regulate EMT and promote stem-like properties, as well as, downregulation of estrogen response and DNA repair pathways. Palbociclib resistant cells exhibited mammosphere formation and CD44high/CD24low population indicating the presence of increased breast cancer stem cell-like cells (B-CSC-L). Given the recently elucidated role of IL-6/STAT-3 mediated B-CSC-L phenotypes in drug resistance, we examined IL-6 mRNA levels, which increased by >12-fold in the resistant cells. Treatment with STAT-3 inhibitors, napabucasin and C188-9, significantly decreased the B-CSC-L population and mammosphere formation, indicating a crucial role for the IL-6/STAT-3 pathway in driving B-CSC-L phenotype and palbociclib resistance. Since DNA repair pathways were collectively downregulated in the palbociclib resistant cells, we examined their sensitivity to DNA damaging agents. Results showed that resistant cells were more sensitive to olaparib (PARP inhibition), with no effect on B-CSC-L population. Next, we examined if combined treatment with agents targeting STAT-3 and PARP would be synergistic in palbociclib resistant cells. Results show that combined treatment with olaparib and napabucasin or C-1889 significantly decreased B-CSC-L population, colony formation and increased cell death via apoptosis, when compared to no-treatment or single treatment controls of the palbociclib resistant cells. Lastly, we interrogated matched tumor samples from breast cancer patients who progressed on palbociclib for deregulation of estrogen receptor, DNA repair, and IL-6/STAT3 signaling and found that these pathways are altered as compared to the pre-treatment samples. Taken together, the results show that targeting IL-6/STAT-3 mediated cancer stem cells and DNA repair deficiency by PARP inhibitors in combination can effectively treat acquired resistance to palbociclib. Citation Format: Nicole M. Kettner, Smruthi Vijayaraghavan, Merih Guray Durak, Tuyen Bui, Mehrnoosh Kohansal, Min Jin Ha, Bin Liu, Xiayu Rao, Jing Yang, Min Yi, Jason P. Carey, Xian Chen, T. Kris Eckols, Akshara S. Raghavendra, Nuhad K. Ibrahim, Meghan Karuturi, Stephanie S. Watowich, Aysegul A. Sahin, David J. Tweardy, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 323.

Determination of the target proteins in chemotherapy resistant breast cancer stem cell-like cells by protein array

Breast cancer comes second among the causes of cancer deaths of women. Although new generation hormone therapy is a promising strategy, re-occurrence or emergence of drug resistance limits the success. According to the theory of cancer stem cells (CSCs); CSCs are immortal, tumor inducing and self renewing pluripotent cells and multiply as chemotherapy proceeds, making the chemotherapy inefficient. Emerging scientific reports indicate that the mechanisms of drug resistance are the main features that CSCs gain actually. Due to this fact, cancer stem cell markers should be clarified to target CSCs and this will play important role to reverse drug resistance.In this study, MCF-7/Pac, a cell line resistant to microtubule inhibitor paclitaxel and multiple drugs permanently, was used as a reference cell line for drug resistant mammary cancer. It has some properties that breast cancer stem cells possess so it is considerable to isolate breast cancer stem cell-like cells from MCF-7/Pac population. The chemotherapy resistant breast cancer stem–like (BCSC-like) cells were sorted from MCF-7/Pac population by using markers CD44, CD24 and ALDH. At the next step the proteins that are up-regulated in BCSC-like cells were determined by protein array analysis. Additionally the effect of paclitaxel on BCSC-like cell proliferation was determined.The MCF-7/Pac population contains 12.4% BCSC-like cells. The cells bearing BCSC-like cell phenotype exhibited resistance to paclitaxel. The over-expressed growth factors, MMP proteins, Frizzled proteins and IL-23 were found to be related to the BCSC-like cell proliferation. These results will guide both basic science and medical science.

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

MiR-130a-3p inhibits migration and invasion by regulating RAB5B in human breast cancer stem cell-like cells

Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. MicroRNAs (miRNAs) are involved in tumorigenesis by regulating specific oncogenes and tumor suppressor genes, and their roles in BCSCs are becoming more apparent. We try to reveal the mechanism by which specific miRNA plays its function in BCSCs. Herein, we show that miR-130a-3p is down-regulated in human breast cancer tissues and exosomes from circulating blood. Overexpression of miR-130a-3p in BCSCs inhibited cellular proliferation, migration, and invasion, and silencing of miR-130a-3p had the opposite effects. We also confirmed that RAB5B is directly down-regulated by miR-130a-3p. Knockdown of RAB5B also inhibited cell proliferation, migration and invasion. Furthermore, we found that lower levels of exosome-derived miR-130a-3p are associated with lymph node metastasis and advanced TNM stage. Taken together, our results demonstrate that miR-130a-3p may act as a disease progression monitoring indicator and therapeutic target in breast cancer.

Breast cancer stem cell-like cells generated during TGFβ-induced EMT are radioresistant.

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24−/low/CD44+ CSCs and their corresponding CD24+/CD44low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor β (TGFβ) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFβ-induced cell reprogramming. We showed that mesenchymal CD24−/low/CD44+ CSCs are more resistant to radiation compared with CD24+/CD44low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24−/low/CD44+ cells acquired during EMT.

High CD44 expression mediates p62-associated NFE2L2/NRF2 activation in breast cancer stem cell-like cells: Implications for cancer stem cell resistance

Cluster of differentiation 44 (CD44) is the most common cancer stem cell (CSC) marker and high CD44 expression has been associated with anticancer drug resistance, tumor recurrence, and metastasis. In this study, we aimed to investigate the molecular mechanism by which CD44 and nuclear factor erythroid 2-like 2 (NFE2L2; NRF2), a key regulator of antioxidant genes, are linked to CSC resistance using CD44high breast CSC-like cells. NRF2 expression was higher in CD44high cell populations isolated from doxorubicin-resistant MCF7 (ADR), as well as MCF7, MDA-MB231, and A549 cells, than in corresponding CD44low cells. High NRF2 expression in the CD44highCD24low CSC population (ADR44P) established from ADR cells depended on standard isoform of CD44. Silencing of CD44 or overexpression of CD44 resulted in the reduction or elevation of NRF2, respectively, and treatment with hyaluronic acid, a CD44 ligand, augmented NRF2 activation. As functional implications, NRF2 silencing rendered ADR44P cells to retain higher levels of reactive oxygen species and to be sensitive to anticancer drug toxicity. Moreover, NRF2-silenced ADR44P cells displayed tumor growth retardation and reduced colony/sphere formation and invasion capacity. In line with these, CD44 significantly colocalized with NRF2 in breast tumor clinical samples. The molecular mechanism of CD44-mediated NRF2 activation was found to involve high p62 expression. CD44 elevation led to an increase in p62, and inhibition of p62 resulted in NRF2 suppression in ADR44P. Collectively, our results showed that high CD44 led to p62-associated NRF2 activation in CD44high breast CSC-like cells. NRF2 activation contributed to the aggressive phenotype, tumor growth, and anticancer drug resistance of CD44high CSCs. Therefore, the CD44-NRF2 axis might be a promising therapeutic target for the control of stress resistance and survival of CD44high CSC population within breast tumors.

Ultrasound reverses chemoresistance in breast cancer stem cell like cells by altering ABCG2 expression.

Doxorubicin (DOX) resistance in breast cancer largely results from the breast cancer stem cell like cells (BCSCs) which could be targetted to improve the efficacy of chemotherapy. Cell permeabilization using microbubbles (MBs) and ultrasound (US) have the potential for delivering molecules into the cytoplasm. We aim to evaluate a new methodology of US on BCSCs. First, our findings indicated that ALDHA1+ spheres which were derived from fresh primary breast cancer samples displayed stem cell like features and were resistant to DOX. In patient cohort, we revealed the presence of a variable fraction of ALDHA1+cells in nine out of ten. We, for the first time, showed a new US-MB treatment condition which could be used on ALDHA1+ BCSCs by fluorescence measurement and calcein assay. Next, we demonstrated the efficacy of combined treatment on human BCSCs in vitro and in vivo using DOX and US-MB: the combined treatment with much reduced drug dosage significantly suppressed the stem cell like features of BCSCs and induced BCSCs apoptosis. Furthermore, we suggested that decreased ABCG2 level might be one of the mechanisms by which US-MB medicated DOX treatment. In conclusion, this new US-MB treatment condition has clinical potential in breast cancer therapy by targetting BCSCs; thereby holding benefits for breast cancer patients.

Enhanced SLC34A2 in breast cancer stem cell-like cells induces chemotherapeutic resistance to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling.

Even though early detection methods and treatment options are greatly improved, chemoresistance is still a tremendous challenge for breast cancer therapy. Breast cancer stem cells (BCSCs) represent a subpopulation that is central to chemoresistance. We aim to investigate the relationship between SLC34A2 and chemoresistance in BCSCs and identify the underlying mechanisms by which SLC34A2 regulates chemoresistance in BCSCs. Fluorescence Activated Cell Sorting (FACS) analysis showed the presence of a variable fraction of CD44(+)CD24(-) cells in 25 out of 25 breast cancer samples. We cultured primary breast cancer sample cells and breast cancer cell line cells to induce sphere formation in serum-free medium. Following sorting of CD44(+)CD24(-) cells from spheres, we showed that CD44(+)CD24(-) cells displayed stem cell-like features and were resistant to chemotherapy drug doxorubicin. Significantly, enhanced SLC34A2 expression correlated with chemoresponse and survival of breast cancer patients. We subsequently indicated that increased SLC34A2 expression in BCSCs directly contributed to their chemoresistance by a series of in vitro and in vivo experiments. Furthermore, we demonstrated that SLC34A2 induced chemoresistance in BCSCs via SLC34A2-Bmi1-ABCC5 signaling. Finally, we showed that ABCC5 was a direct transcriptional target of Bmi1 by chromatin immunoprecipitation (ChIP). In conclusion, our work indicated that decreased SLC34A2 expression sensitized BCSCs to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling and shed new light on understanding the mechanism of chemoresistance in BCSCs. This study not only bridges the missing link between stem cell-related transcription factor (Bmi1) and ABC transporter (ABCC5) but also contributes to development of potential therapeutics against breast cancer.

Breast Cancer Stem Cell-Like Cells Are More Sensitive to Ionizing Radiation than Non-Stem Cells: Role of ATM

There are contradictory observations about the different radiosensitivities of cancer stem cells and cancer non-stem cells. To resolve these contradictory observations, we studied radiosensitivities by employing breast cancer stem cell (CSC)-like MDA-MB231 and MDA-MB453 cells as well as their corresponding non-stem cells. CSC-like cells proliferate without differentiating and have characteristics of tumor-initiating cells [1]. These cells were exposed to γ-rays (1.25–8.75 Gy) and survival curves were determined by colony formation. A final slope, D0, of the survival curve for each cell line was determined to measure radiosensitivity. The D0 of CSC-like and non-stem MDA-MB-453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB-231 cells (0.94 Gy vs. 1.56 Gy). After determination of radiosensitivity, we investigated intrinsic cellular determinants which influence radiosensitivity including cell cycle distribution, free-radical scavengers and DNA repair. We observed that even though cell cycle status and antioxidant content may contribute to differential radiosensitivity, differential DNA repair capacity may be a greater determinant of radiosensitivity. Unlike non-stem cells, CSC-like cells have little/no sublethal damage repair, a low intracellular level of ataxia telangiectasia mutated (ATM) and delay of γ-H2AX foci removal (DNA strand break repair). These results suggest that low DNA repair capacity is responsible for the high radiosensitivity of these CSC-like cells.

MiR‐21 regulates epithelial‐mesenchymal transition phenotype and hypoxia‐inducible factor‐1α expression in third‐sphere forming breast cancer stem cell‐like cells

Cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to their "stemness", but the molecular mechanism of CSCs in regulating metastasis remains to be elucidated. Epithelial-mesenchymal transition (EMT), hypoxia-inducible factor (HIF)-1α, and miR-21, all of which contribute to cell migration for metastasis, are interrelated with CSCs. In the present study, third-sphere forming (3-S) CSC-like cells, which showed elevated CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and sphereforming capacity as well as migration and invasion capacities, were cultured and isolated from breast cancer MCF-7 parental cells, to evaluate the role of miR-21 in regulating the CSC-like cell biological features, especially EMT. EMT, which was assessed by overexpression of mesenchymal cell markers (N-cadherin, Vimentin, alpha-smooth muscle actin [α-SMA]) and suppression of epithelial cell marker (E-cadherin), was induced in 3-S CSC-like cells. Moreover, both of HIF-1α and miR-21 were upregulated in the CSC-like cells. Interestingly, antagonism of miR-21 by antagomir led to reversal of EMT, downexpression of HIF-1α, as well as suppression of invasion and migration, which indicates a key role of miR-21 involved in regulate CSC-associated features. In conclusion, we demonstrated that the formation of CSC-like cells undergoing process of EMT-like associated with overexpression of HIF-1α, both of which are regulated by miR-21.

Abstract 3367: Breast cancer stem cell-like cells are sensitive to ionizing radiation: Role of ATM

Abstract The aim of this study was to determine the radiosensitivity of cancer stem (tumor-initiating) cells (CSC), which are known to be responsible for tumorogenicity and metastasis. We used breast cancer stem cell (CSC)-like MDA-MB231 and MDA-MB453 cell lines which were established by Dr. Edward V. Prochownik (Sajithlal et al., Stem Cells, 28:1008, 2010). Interestingly, these cells can proliferate without differentiation and have characteristics of tumor-initiating cells. CSC-like cells and their alternate subset non-CSC cells were exposed to α-rays (1.25-8.75Gy) and survival curves were determined by colony formation. A final slope, D0, of the survival curve for each cell line was determined to measure radiosensitivity. D0 of CSC-like MDA-MB453 cells and that of non-CSC MDA-MB453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB231 cells. We further examined whether CSC-like cells are relatively radiosensitive owing to different intrinsic factors including cell cycle distribution, free-radical scavengers and DNA repair. We observed that, unlike non-CSC cells, CSC-like cells have little/no sublethal damage repair and a low intracellular level of ATM. These results suggest that low DNA repair capability is responsible for the high radiosensitivity of these CSC-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3367. doi:1538-7445.AM2012-3367

Preclinical Studies of Salinomycin In Multiple Myeloma (MM) Models: Targeting of Side Population (SP) Cells In the Context of Tumor – Microenvironment Interactions.

AbstractAbstract 1574Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be >100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values <1 uM in 10/15 cell lines tested, including >80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 >>10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses >20 times lower than those required for in vitro effect against the bulk <<main population>> of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures:McMillin:Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

Mutant TNF-α negatively regulates human breast cancer stem cells from MCF7 in vitro

Understanding the biology of breast cancer stem cells and trying new ways to obliterate these cells would be a key step in developing cures for breast cancer. The objective of this study was to investigate the effect of mutant TNF-α on human breast cancer stem cells derived from MCF7 cell line under the characterization of biologic features of these cells in vitro. By FACS analysis and sorting, we got MCF7 side population (SP) cells and showed that MCF7 SP cells possess cancer stem cell characteristics using the accepted breast cancer stem cell markers, but do not express multiple drug resistance transporters. Furthermore, by RT-PCR, these stem cells were found to constitutively express TNFR-p55 and TNFR-p75. After being treated with Mt rh471 TNF-a, SP cells displayed a decreased self-renewal ability and an increased apoptosis by three different methods. When monocolony antibody against TNFR-p55 was added into the culture medium, the inhibitory effect of Mt rh471 TNF-α on self-renewal was blocked completely, but this was not the case for that of Wt rhTNF-α. The possible reasons might be that the increased binding of Mt rh471 TNF-α mainly to TNFR-p55 results in induction of apoptosis of SP cells, while Wt rhTNF-α could bind to both TNFR-p55 and TNFR-p75 which would lead to NF-κB activity, resulting in a discounted apoptotic effect. These data suggest that Mt rh471 TNF-a might be a negative regulator of the breast cancer stem cell-like cells and have the potential to treat breast cancer in clinic.