Properties Of Breast Cancer Stem Cells Research Articles

Abstract CN06-02: Breast cancer stem cells.

Abstract CN06-02: Breast cancer stem cells.

Abstract 3724: Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4.

Abstract Salinomycin isolated from Streptomyces albus and a potassium ionophore. It has been used to eliminate bacteria, fungi and parasites. Also, Cancer stem cells (CSCs) are a subpopulation of cells within the tumor that has ability to self-renew and undergo differentiation to phenotypically diverse cell populations. So, CSCs are involved in metastasis, chemoresistance and even cancer relapse. Recently, there are many evidences to CSCs subpopulations enhance capability of DNA repair and reduce the apoptosis. Furthermore, salinomycin has been reported to act as selective breast cancer stem cell inhibitor based on its property of reducing the cancer stem cell and to induce the apoptosis in cancer cell. Therefore, we investigated the effect of salinomycin on breast cancer stem cells. First, Mammosphere formation assay was performed to observe stem cell-like property after salinomycin treat in anchorage-independent state. We confirmed salinomycin reduces number and size of mammosphere at 3, 6, 9 day. Also, we compared mRNA expressing level of stem cell marker such as SOX2 and OCT4 by RT-PCR. Consequently, salinomycin was the down-regulated stem cell marker mRNA level as well as protein level. To confirm the reduction of the side population level, we performed FACS analysis using CD44+ and CD24- markers. Results by FACS analysis revealed that salinomycin treated with breast cancer stem cell reduced stemness by downregulating CD44+/CD24- population. Consistently, when mammosphere implanted into the mammary fat pads of BALB/c female nude mice cleared of endogenous epithelium, the tumor size was decreased. In conclusion, we concluded that effect of salinomycin to reduce stemness is mediated by downregulation of SOX2 and OCT4 in human breast cancer cell. Citation Format: Hyun Sook An, Han Na Kang, Myoung Hee Kang, Jung Lim Kim, Jun Suk Kim, Jae Hong Seo. Salinomycin reduced breast cancer stem cell properties by reducing SOX2 and OCT4. [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 3724. doi:10.1158/1538-7445.AM2013-3724

Abstract 250: Growth characteristics of breast cancer stem cells in vitro and in vivo.

Abstract Breast cancer (BCa) is the most common malignancy in United States women, accounting for greater than 40,000 deaths each year. Studies have attributed the presence of cancer stem cells (CSC) with resistance to chemotherapy as a key component of refractories of treatments in BCa. Breast cancer stem cells (BCa-SC) are characterized by CD24lo and CD44hi phenotype with mesenchymal properties, which differ from the epithelial CD24hi and CD44hi phenotype expressed in the majority of BCa cells. Thus, further understanding properties of BCa-SC may lead to the development of novel treatment options towards treating refractory cancer cells. Fundamental to this, further characterization of BCa-SC will be needed to identify therapeutic targets based CSC biology. To this end, we sought to characterize the plasticity of BCa-SC based on cell cycle in vitro and growth kinetics in vivo. A murine BCa cell line, 4T1, was cultured in CSC medium and analyzed every 6 hrs for CD24 and CD44 expression by flow cytometry. Results of this analysis indicated that specific BCa-SC markers peaked every 24 hours. This data supports a possible reprogramming of BCa-SC based on cell cycle regulation, suggesting the use of cell cycle inhibitors to block cells in particular stages of the cell cycle, which may improve therapeutic approaches to induce cell death. The stemness of these cells was further confirmed by real-time PCR. BCa-SC showed increased expression of ALDH1, TWIST, and TGF-β3 with decreased E-Cadherin and CD24 levels correlating to the phenotypic changes observed with CD24 and CD44 expression. To further examine the role of BCa-SC in tumor growth in vivo, BALB/c mice were subcutaneously implanted with populations of 4T1 cells that constituted CSC and non- cancer stem cells (NCSC) in different combinations and tumor growth was analyzed. Results showed that the growth kinetics CSC and NCSC when used alone was significantly lower in vivo compared to mixed populations containing both CSC and NCSC, suggesting CSC may attribute for the repopulation of the disease but an aggressive growth requires differentiated non-stem cells in the tumor microenvironment. Citation Format: Carnella M. Lee, Anandi Sawant, Ha-Ram Cha, Selvarangan Ponnazhagan. Growth characteristics of breast cancer stem cells in vitro and in vivo. [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 250. doi:10.1158/1538-7445.AM2013-250

Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo

Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing nicastrin and HCC1806 stably depleted of nicastrin, in vivo, we demonstrate that nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of nicastrin-targeted therapies in breast cancer.

375P - Targeting Breast Cancer Stem Cells for Treatment Failure Cases of Late Stage Breast Cancer

ABSTRACT Disclosure All authors have declared no conflicts of interest. Targeting breast cancer stem cells (BCSCs) present in bone marrow and breast tissues is an attractive alternative for ‘event-free survival’ in breast cancer as it is an extremely heterogeneous disease. The invasive and mesenchymal properties of BCSCs with CD44 + /CD24low/ALDH1+ phenotype has made them a promising target for eliminating the metastatic capacity of primary tumors. We hypothesize that the ability to therapeutically attack stem cells will be decisive for the development of specific target therapies. Ten chemo-failure late stage patients with biopsy-proven triple-negative metastatic breast cancer were selected randomly. Breast cancer cells were isolated from whole tumor and cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel and correlation was drawn between cell differentiation and drug response. Accordingly chemotherapy was designed for a particular patient. BCSCs were also isolated from the whole tumor, cultured and chemotherapy was designed. We detected chemo-failure in 65% cases for whole cell chemo-predictive assays, while BCSCs isolated from those non-responders responded to chemotherapy. Among positive samples, 89% of patients showed platinum sensitivity and rest were found to be anthracycline sensitive. No sensitivity towards docetaxel was observed. In lieu of this, cisplatin was applied ex vivo and percentage of BCSCs came down to 6.58% from an initial 11.16% (for a representative case). Thus the primary aim to target BCSCs to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival.

52P Breast Cancer Stem Cells: A Novel Therapy for Breast Cancer

ABSTRACT Simple BRCA screening is insufficient for ‘event-free survival’ as breast cancer is clinically and pathologically an extremely heterogeneous disease. Targeting breast cancer stem cells (BCSCs) present in bone marrow and breast tissues is an attractive alternative. Identification of BCSCs is the salient aspect of our research. The invasive and mesenchymal properties of BCSCs with CD44+/CD24low/ALDH1+ phenotype has made them a promising target for eliminating the metastatic capacity of primary tumors. We hypothesize that the ability to therapeutically attack stem cells hinges upon identifying unique targets such as cell surface markers which will be decisive for the development of specific target therapies. A total of 10 early chemo-naive patients with biopsy-proven triple-negative metastatic breast cancer in age group 18-36 years (mean age 28 years) were selected randomly and tested for CD44/CD24 cell surface markers following immunosorting using a magnetic cell sorter and immunophenotyping by flow cytometric analysis. Isolated BCSCs were cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel and correlation was drawn between cell differentiation, percentage of stem cells and drug response. Accordingly chemotherapy was designed for a particular patient. The percentage of BCSCs in pre- and post-chemotherapeutic conditions were further compared. We have detected BCSCs in 90% of the cases. Among positive samples, 89% of patients showed platinum sensitivity and the rest were found to be anthracycline sensitive. No sensitivity to docetaxel was observed. In lieu of this, cisplatin was applied in vivo and the percentage of BCSCs came down to 6.58% from an initial 11.16% (for a representative case). Thus the primary aim to target BCSCs at onset of tumors in breast cancer patients to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate the ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival. Finally, if no BCSCs were to prevail after chemotherapy, then patients would be kept under observation and if traces were found, we would proceed to stem cell replacement. Disclosure All authors have declared no conflicts of interest.

Abstract 5197: Quantitative phosphorproteomics identified GPER-initiated PKA signal transduction event in breast cancer stem cell

Abstract Human breast cancer stem cells (BCSC) identified recently have the ability to initiate neoplastic growth with differentiation and display higher resistance to chemotherapy and radiotherapy. The allure of targeting this specific group of cells to reduce mortality of disease has attracted research efforts toward BCSC. Although some unique properties of BCSC have been deciphered, the molecular mechanisms are still poorly understood. Here, we applied in-house developed techniques to quantitatively compare the phosphoproteomic profiles between BCSC and non-cancer stem cells (nonCSC) derived from a xenograft of human breast cancer. For quantitative analysis, we utilized a SEMI-strategy for multiplexed label-free quantitation combining pH/acid-controlled IMAC chromatography and LC-MS/MS for relative quantitation of site-specific phosphorylation. This strategy effectively increases the phosphopeptide quantitation coverage. The differentially expressed phosphoproteins were mapped to multiple signaling pathways including NOTCH, CDK/ERK and JAK-STAT pathways, which potentially orchestrate the self renewal and stemness of BCSC. We also demonstrated ∼2 fold greater expression of GPER in BCSC than non-BCSC population, and GPER signaling could induce PKA-mediated phosphorylation of BAD in BCSC. Silencing of GPER via RNAi or mutation of phosphorylation sites of BAD led to reduced proliferation to 40.8 and 53.2 % of control, respectively, and the RNAi silencing of GPER decreased the mammosphere formation of BCSC to 43.5 % of control. These findings implies the importance of GPER and its downstream PKA pathway to the maintenance of stemness characteristics of BCSC. 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 5197. doi:1538-7445.AM2012-5197

Abstract 3341: TGFβ/ TNFα-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2011-3341

Stem Cells: Their Role in Breast Cancer Development and Resistance to Treatment

About 20% of the total cells from primary breast tumors could generate palpable tumors in non-obese diabetic severe combined immunodeficient (NOD/SCID) immunocompromised mice. All the tumorigenic cells originate from a normal mammary stem cell. Human mammary stem cells are sensitive to oncogenic mutations and in mouse models they share similarities with breast cancer stem cells (BrCSCs). Tumorigenicity, invasion, progression and metastasization are further BrCSCs properties likely depending on their CD44+/CD24- phenotype. Local invasion and tumor metastasization seem to be facilitated by the epithelial to mesenchymal transition (EMT) program. This program may be reactivated from stable genetic alterations or through exposure of cancer cells to factors present in the surrounding micro-environment, or by an up-regulation of EMT-inducing transcription factors. One main explanation for resistance to treatment by cancer cells is that a rare subpopulation of cells in residual tumors with tumorigenic potential is intrinsically resistant to therapy. Consistent with this hypothesis, in human breast tumors, the subpopulation of tumor-initiating cancer cells with CD44(high)/CD24(low) cell surface-marker profile was found more resistant to cancer therapies (chemo, hormone and radiotherapy) than is the major population of more differentiated breast cancer cells. The reasons for CSC resistance to chemotherapy, hormone therapy and radiotherapy also have been examined and they opened new scenarios for cancer therapy.

MiR-495 is upregulated by E12/E47 in breast cancer stem cells, and promotes oncogenesis and hypoxia resistance via downregulation of E-cadherin and REDD1

MicroRNAs (miRNAs) are involved in tumorigenecity by regulating specific oncogenes and tumor suppressor genes, and their roles in breast cancer stem cells (BCSCs) are becoming apparent. Distinct from the CD44(+)/CD24(-/low) sub-population, we have isolated a novel PROCR(+)/ESA(+) BCSC sub-population. To explore miRNA-regulatory mechanisms in this sub-population, we performed miRNA expression profiling and found miR-495 as the most highly upegulated miRNA in PROCR(+)/ESA(+) cells. Coincidently, high upregulation of miR-495 was also found in CD44(+)/CD24(-/low) BCSCs, reflecting its potential importance in maintaining common BCSC properties. Ectopic expression of miR-495 in breast cancer cells promoted their colony formation in vitro and tumorigenesis in mice. miR-495 directly suppressed E-cadherin expression to promote cell invasion and inhibited REDD1 expression to enhance cell proliferation in hypoxia through post-transcriptional mechanism. miR-495 expression was directly modulated by transcription factor E12/E47, which itself is highly expressed in BCSCs. These findings reveal a novel regulatory pathway centered on miR-495 that contributes to BCSC properties and hypoxia resistance.

Breast cancer stem cells

Since the initial discovery of leukemia stem cells nearly a decade ago, a great deal of cancer research has focused on the identification of cancer stem cells (CSCs) in many types of solid tumors, including breast cancer. Through analysis of cell surface markers and xenotransplant models, a subpopulation of putative human breast cancer stem cells (BCSCs) that is CD24-negative/CD44-positive (CD24(-)/CD44+) and bears high aldehyde dehydrogenase 1 activity has been isolated in clinical samples of breast cancer tissues. Human BCSCs are considered to be derived from basal cells that reside in the basal membranes of alveolar units in human adult mammary glands. Furthermore, BCSCs have been shown to express higher levels of oxidative stress-responsive genes, which could confer part of their ability to resist anti-cancer therapy, than non-CSCs. The emerging picture of the biological properties of BCSCs would contribute for devising innovative therapies for breast cancer, targeting the intrinsic and extrinsic factors that maintain the BCSCs.

Suspension culture combined with anticancer regimens for screening breast cancer stem cells

Breast cancer stem cells (BrCSCs)have been first identified in solid tumors. To be more exact, they are defined as tumorigenic cancer cells which exhibit the capacity to form tumors distinct from the majority non-tumorigenic cancer cells. However it is only a minor subset that cancer stem cells are highly enriched in. It is still unknown how to find the definitive research subject - "a single breast cancer stem cell" - that can cause a new tumor by itself. Since recent studies suggest that BrCSCs have a higher proportion in suspension culture and have a greater resistance to chemotherapy regimens, we propose a novel strategy to isolate and identify BrCSCs:suspension culture combined with anticancer regimens. This double sorting method by means of the unique properties of BrCSCs may be helpful to screen genuine cancer stem cells(CSCs).