Cell Mammosphere Research Articles

Abstract 4220: Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis

Abstract Introduction: Solid tumor cancers including breast cancer possess a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. These features render these cells as highly desirable albeit elusive therapeutic targets. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we establish the scientific rationale for targeting DCLK1 to inhibit breast cancer proliferation, tumor stem cell growth, self-renewal, EMT, circulating tumor cells (CTCa) and metastasis. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into breast cancer cell lines (MCF7 and MDAMB231 (triple negative breast cancer cell line)). For analyzing circulating tumor cells MDAMB231 cells are stably transfected with RFP (red fluorescence protein. Cell proliferation, colony formation, mammosphere formation assay (self-renewal), cell migration and invasion assays were assessed to evaluate the effect of DCLK1 knockdown on breast cancer cells self-renewal and metastatic potential. Isograft model was used to identify the role of DCLK1 knockdown on breast cancer growth and circulating tumor cells in vivo. The assays were analyzed utilizing experimental protocols for cell culture, clonogenic culture, circulating tumor cells by gradient centrifugation, immunohistochemistry, real-time PCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal tissue. DCLK1 siRNA inhibited the expression of DCLK1 mRNA and protein in breast cancer cells. Silencing DCLK1 decreased the proliferation (∼40%), colony formation (∼70%), and self-renewal (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to significantly less (40-80%) breast cancer cells migrated and invaded. Further, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were significantly (p<0.05) reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in isograft models resulted in reduced tumor growth (∼81%) and CTCs (∼98%) in vivo. Conclusions: DCLK1 expression is associated with breast cancer cell survival, self-renewal, metastasis and tumorigenesis. Silencing DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis, tumor growth and survival. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Vivian Taylor, William Berry, Randal May, Dongfeng Qu, Meghna Singh, Sripathi Sureban, Naushad Ali, Michael Bronze, Courtney Houchen. Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis. [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 4220. doi:10.1158/1538-7445.AM2015-4220

Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) “on-top” Matrigel, 3D “cell-embedded” Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

A novel molecular marker of breast cancer stem cells identified by cell-SELEX method.

Breast cancer stem cells (CSCs) are thought to initiate mammary tumors and render them resistant to anti-cancer therapies. However, there are currently no ideal biomarkers to identify this minority population in breast cancer. To find out the oligonucleotides with high specificity and affinity for mammosphere cells using a high capacity ssDNA library. We used the cell-SELEX (systematic evolution of ligands by exponential enrichment process) method. MCF-7 cells were cultured in serum-free media to form mammosphere cells as enriched stem cells, and were used as the positive target cells. The normal breast epithelial MCF-10A and MCF-7sal cells, which are MCF-7 cells treated with Salinomycin, were used as the negative target cells. We collected the ssDNA pools that were bound to positive target cells, and could not bind negative target cells. After 13 rounds of selection, we isolated the MS03 aptamer with high specificity and affinity for mammosphere cells. When compared with CD44(+)/CD24(-/low) cells, MS03(+) cells did not show any significant difference in sphere formation ability in vitro. In addition, 63.3% of MS03 aptamer-selected cells exhibited the CD44(+)/CD24(-/low) phenotype. Because the MS03 aptamer is synthesized easily and non-immunogenic, it is much more flexible than CD44/CD24 as a breast CSC biomarker. The MS03 aptamer may become a promising molecular probe during diagnostic and therapeutic applications in breast cancer.

Vitamin D compounds reduce mammosphere formation and decrease expression of putative stem cell markers in breast cancer

Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44+/CD24−/low and CD49f+/CD24−/low subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell-like population, potentially contributing to their inhibition of breast cancer.This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’.

Culture and characterization of mammary cancer stem cells in mammospheres.

Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation.

Abstract LB-49: Defining hierarchical organization of breast cancer cells and cancer activated fibroblasts using single-cell gene expression profiling: switch-like and transient transitions between novel stem cell and progenitor pools

Abstract The cancer stem cell (CSC) model holds that cancers are organized in a hierarchical structure and derived from cellular populations with stem cell properties. CSC characteristics include self-renewal, tumorigenicity, multi-lineage differentiation as well as increased resistance to radio- and chemotherapy-induced cell death. In order to better understand the complex hierarchical structure of breast cancer cells in vivo as well as phenotypes of stromal cells as cancer associated fibroblasts, tools need to be developed, enabling analyses of key regulators and biomarkers on a single-cell level. We therefore established single-cell reverse transcription quantitative real-time PCR and gene expression profiling, including more than 100 key regulators associated with the cell cycle, differentiation, EMT, stemness and fibroblast activation and performed extensive single-cell PCR analyses, using cell lines and primary breast cancer samples. Three strategies were used to enrich for cancer stem cells and progenitor populations corresponding to 1) cells surviving anchorage-independent growth conditions, 2) cells in mammospheres showing few cell divisions, defined by a cell division tracker and 3) cells grown under hypoxic conditions. All methods have earlier been shown to enrich for cells that have an increased cancer initiating ability in mouse model systems. Interestingly, all strategies increased two separate tumor subpopulations defined by lower proliferative features and various EPCAM, ERBB2, CDH1, ID1, ABCG2, POU5F1, NANOG and SOX2 levels. Besides, a clear switch-like transition between the various populations, a transition state linking the cancer stem cell and progenitor populations with the high proliferative main cluster was observed. When specifically analyzing the few cells that had only undergone one asymmetrical cell division in 5 days mammosphere cultures, it was clear that this potential stem cell pool had a distinctive expression profile based on the included key regulators. We next analyzed immortalized primary fibroblasts and tumor activated counterparts using single-cell PCR analyses. The two cell types clearly separated from each other and showed various activation states defined by key regulators as COL1A1, ALDH1A3, TGFB, PDGFA, ACTA2, CXCL12 and cell cycle regulators. The data support a continuing transition towards myofibroblast differentiation with gradual loss of proliferation, whereas the activation markers peak in an intermediate state before entering a final low proliferative condition. When analyzing primary tumors we observed distinct clusters of epithelial cells and fibroblasts supporting the validity of the defined assay. A detailed description of primary breast cancer samples and important microenvironmental components combined with functional assays on a single cell level are now ongoing. Within this comprehensive program we have identified diverse previously unknown CSC/progenitor pools or distinct cellular states and we are now able to model how cells actually transfer between various differentiation and stem cells states, thereby better understanding the hierarchical composition of breast cancer on a single cell level. Citation Format: Anders Ståhlberg, Nina Akrap, Daniel Andersson, Pernilla Gregersson, Susann Busch, Göran P. Landberg. Defining hierarchical organization of breast cancer cells and cancer activated fibroblasts using single-cell gene expression profiling: switch-like and transient transitions between novel stem cell and progenitor pools. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-49. doi:10.1158/1538-7445.AM2014-LB-49

Abstract 1236: A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells

Abstract It has been suggested that cancer stem cells (CSCs), which represent a subset of tumor cells, are responsible for the possible origination and maintenance of tumors. Moreover, CSCs are believed to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). The involvement of CSCs in the progression of DCIS to IDC has yet to be elucidated. In order to investigate the role of CSCs in DCIS progression to IDC, we utilized the MCF10DCIS.com cell line in the mammosphere cell culture system, which enriches for mammary progenitor cells and mammary CSCs. 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), inhibits the progression of DCIS to IDC. Therefore, we have determined whether BXL0124 plays a role in regulating the CSC or progenitor cell populations, resulting in reduced tumorigenicity. MCF10DCIS.com cells were plated in ultra-low attachment plates and treated with BXL0124 or 1α25(OH)2D3 over the course of 1, 3 and 5 days. Starting at day 2, MCF10DCIS.com cells began to form mammospheres of 100 µm in size. MCF10DCIS.com mammospheres showed enrichment in the CD44+/CD24- and the CD49f+/CD24- populations compared to monolayer cell culture. The phenotype of MCF10DCIS.com mammospheres showed a disorganized, undefined and irregular shape. However, when the MCF10DCIS.com mammospheres were treated with BXL0124 or 1α25(OH)2D3, mammospheres form a more organized, symmetrical and circular shape, which is similar to the phenotype of the spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. In addition, the mammosphere forming efficiency (MFE) was decreased upon BXL0124 and 1α25(OH)2D3 treatment, showing that there is an inhibitory effect on mammosphere development. Treatment with BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, Notch1 and JAG1. Furthermore, BXL0124 demonstrated the reduced expression of MMPs, such as MMP2, 9, 14, 15, and 16, in mammospheres, suggesting that BXL0124 reduces the tumorigenicity and invasive potential of MCF10DCIS.com cells by targeting the stem cell-like population. In conclusion, the Gemini vitamin D analog BXL0124 represses the CSC population, potentially contributing to the inhibition of the progression of DCIS to IDC. (This work was supported in part by the National Institutes of Health National Cancer Institute R01-CA127645 and the National Institute of Environmental Health Sciences Grant ES005022). Citation Format: Joseph Wahler, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. A gemini vitamin D analogue, BXL0124, represses mammosphere formation and decreases expression of stem cell markers in MCF10DCIS breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2014-1236

Curcumin Targets Breast Cancer Stem–like Cells with Microtentacles That Persist in Mammospheres and Promote Reattachment

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3.MethodsThe tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed.ResultsThe AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil.ConclusionsOur work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.

Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.

Downregulation of Ezh2 methyltransferase by FOXP3: New insight of FOXP3 into chromatin remodeling?

Transcription factor FOXP3 (forkhead box P3) is found initially as a key regulator in regulatory T cells. Recently its expression has been demonstrated in some non-lymphoid normal and cancerous cells. Now FOXP3 has been proven to regulate cancer-related genes, especially suppressor genes in breast cancer. But the mechanisms by which FOXP3 regulates suppressor genes are not fully determined. In this study, we found the inverse correlation between FOXP3 and Ezh2, an enzyme for histone H3K27 trimethylation (H3K27me3) and a central epigenetic regulator in cancer. The overexpression of FOXP3 weakened Ezh2's enhancement on the mammosphere formation, cell proliferation, directional migration, and colony forming ability of T47D cells. We demonstrated that FOXP3 could downregulate Ezh2 protein level and this depended on not only the FOXP3 expression amount, but also the nuclear localization of FOXP3. More importantly, we demonstrated FOXP3 accelerated Ezh2 protein degradation through the polyubiquitination–proteasome pathway by enhancing the transcription of E3 ligase Praja1 directly. These results provided a new mechanism for FOXP3 in histone modifications as an Ezh2 suppressor and supported new evidence for FOXP3 as a tumor suppressor in breast cancer.

Differences in expression of Notch1, E-cadherin, vascular endothelial growth factor A and C, and chemokine receptor 4 gene in breast suspension mammosphere cells and normal adherent cells

Notch1是参与调控细胞分化的重要分子,其表达异常与许多肿瘤的转移密切相关.在乳腺癌发展过程中,Notch1分子可能通过增强乳腺癌干细胞迁徙活性促进其转移,如果证实这一机制,对于研制靶向于乳腺癌干细胞Notch1分子的药物,阻断其转移有重要意义。

Abstract LB-252: Synergy of epithelial and mesenchymal cells under anoikis-inducing conditions.

Abstract Tumor metastasis is the ultimate cause for death of cancer patients. In epithelial tumors such as breast cancer the early steps of the metastatic cascade require tumor cells to migrate, invade and survive detachment from the extracellular matrix in suspension which in epithelial (E) cells results in cell death (anoikis). Therefore, all of these earlier steps appear to be accomplished by mesenchymal (M) cells. In a process called epithelial to mesenchymal transition (EMT) E tumor cells can become M. However, a central paradox of this model is that in most patients the metastatic tumor has an E morphology just as the primary tumor. Analogous to primary tumors and their corresponding metastases, in transformed and immortalized mammary epithelial HMLER cells we frequently found E cells in cultures of replated derivatives of HMLER cells grown in suspension, as well as in suspended mammospheres. In order to disentangle the role of E and M cells under suspension culture conditions in the heterogeneously E and M HMLER cells, we generated mammospheres of HMLER cells, and of isolated E or M HMLER clones. As expected, only the heterogeneous HMLER cells and the M clones were able to form mammospheres, and in gene expression arrays we detected a significant upregulation of genes encoding the pluripotency factor OCT4 and its targets. Importantly, the M cell clone significantly upregulated E genes during suspension conditions. Intriguingly, amongst those were many secreted growth factors and inflammatory cytokines such as SLPI, IL1B, MMP7, S100 proteins and CXCL1, several of which have previously been described to enhance aggressive cancer growth and suppress apoptosis at the metastatic site. In order to directly test the effect of E cells on M cells in suspended mammospheres we generated suspension cultures of isolated E and M cells or of E and M co-cultures. We found that i) E cells can survive in suspension only in conjunction with M cells, and ii) co-culture of E and M cells in suspension resulted in significantly larger and higher number of mammospheres than culture of isolated M cells. Moreover, treatment of M cells with supernatant of E cells could mimic the coculture effect of increased and enlarged mammosphere formation, suggesting that soluble E factors are responsible for enhanced proliferation of M HMLER cells in suspension. In summary, in stem cell-enriched mammospheres of M cells we found high induction of secreted growth and survival factors which are constitutively high expressed in adherent as well as in suspended E cells. Indeed, co-culture of suspended E and M cells resulted in a synergistic effect of enhanced proliferation of M cells in mammospheres and survival of E cells. Synergy of E and M cells in suspension can explain how epithelial cells could be retained under anoikis-inducing conditions in the vasculature and why metastatic tumors are E like their primary counterparts. Citation Format: Anne Grosse-Wilde, Gokhan Ertaylan, Robert West, Kathie Walters, Adrian Ozinsky. Synergy of epithelial and mesenchymal cells under anoikis-inducing conditions. [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 LB-252. doi:10.1158/1538-7445.AM2013-LB-252

Abstract 5575: Strigolactones: a novel class of plant hormones inhibit cancer cell and cancer stem-like cell growth and survival.

Abstract Strigolactones (SLs) are a novel class of plant hormones produced in roots and regulate new above ground shoot branching, by inhibiting self-renewal of undifferentiated meristem cells. Here we show that Strigolactone analogues are able to inhibit proliferation and induce apoptosis of several different types of cancer cells including breast, prostate, lung, colon and osteosarcoma cells but not ‘non-cancer’ lines. Given the therapeutic problem of cancer recurrence which is hypothesized to be due to drug resistant cancer stem cells, we also tested the ability of Strigolactone analogues to inhibit cancer stem cell growth. We show that Strigolactones are potent inhibitors of self-renewal and survival of breast cancer cell lines grown as cancer stem cell enriched mammospheres and even a short exposure leads to irreversible effects on mammosphere dissociation and cell death. Cell cycle analysis showed that all five strigolactone analogues induced G2/M arrest, coupled with increased percentages of cells in the sub-G1 fraction. AnnexinV staining confirmed that Strigolactone treatment induced apoptosis. Strigolactone treatment was hallmarked by increased expression of genes involved in stress signaling, as well as apoptosis, metabolic function and cellular adhesion. Immunoblot analysis of strigolactone treated cells also revealed increased phosphorylation of the stress activated MAPKs: p38 and JNK1/2 and decreased phospho-ERK1/2 and phospho-AKT. In summary, Strigolactones are promising candidates for anticancer therapy by their ability to induce cell cycle arrest and apoptosis in cell lines derived from a wide array of different cancers. Citation Format: Ronit I. Yarden, Claire B. Pollock. Strigolactones: a novel class of plant hormones inhibit cancer cell and cancer stem-like cell growth and survival. [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 5575. doi:10.1158/1538-7445.AM2013-5575 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract P4-05-01: Oncolytic Herpes Simplex Virus Vector G47Δ Effectively Targets Breast Cancer Stem Cells

Abstract Breast tumors can be classified according to their gene-expression profiles into different molecular subtypes with distinct biological and clinical features. Moreover, different subtypes show distinct responses to different therapeutic regimens, thereby resulting in markedly different outcomes. Recently, accumulating evidence has suggested that breast cancer originates from cancer stem cells (CSCs), which comprise a small percentage of the overall tumor but are highly tumorigenic and pluripotent with unlimited proliferation potential. Furthermore, cancer stem cells are highly resistant to conventional treatment, which potentially explains certain difficulties in treating cancer with current therapy options. The use of oncolytic viruses is a relatively new strategy in cancer therapy, oncolytic herpes simplex virus (oHSV) has been shown to be a safe and effective therapeutic approach for a variety of different cancers. In this study, different types of breast cancer cell lines were cultured in anchorage-independent conditions, the cells typically form mammospheres within 10 days of anchorage-independent culture, which showed properties of CSCs. The mammospheres are capable of being passaged indefinitely and single dissociated mammosphere cells were able to regenerate spheres. Moreover, compared to the parental cell lines, mammoshpere cells were enriched for CD44+CD24− cells and highly expression of the alternative breast cancer stem cell marker ALDH-1. The third generation oHSV vector G47Δ effectively killed different subtypes of breast cancer cells in vitro, with more than 98% of the tumor cells killed by day 5, even at multiplicities of infection(MOI) 0.01. Moreover, G47Δ equally targeted non-cancer stem cells (NCSCs) and CSCs which showed resistance to paclitaxel. We also demonstrated that G47Δ effectively replicated and spread among CSCs. Moreover, G47Δ impaired the self-renewal ability of CSCs, as the viable cells unable to form secondary tumor spheres. We also showed that G47Δ was able to induce the regression of tumor xenografts in BALB/c nude mice and demonstrated the ability of G47Δ to synergize with paclitaxel for killing both NCSCs and CSCs. This is the first report that oHSV effectively targets different breast cancer NCSCs and CSCs, thereby suggesting that oHSV may be an effective treatment modality for patients with breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-05-01.

Abstract B27: Osteoprotegerin's key role in inflammatory breast cancer: Bane or Boon.

Abstract Inflammatory breast cancer (IBC), the most aggressive form of breast cancer is presented with a rapidly enlarging erythematous breast, often with no discernible breast mass. IBC is lethal, highly metastatic and accounts for less than 5% of all diagnosed breast cancers; although in young African-American women recurrence rates are as high as 50%. Despite multimodality treatments such as systemic chemotherapy, adjuvant therapy, surgery and radiation, there is dismal prognosis with a 3-year survival rate of only about 40% compared to 85% among patients with non-IBC. Our long-term goal is to understand IBC biology, to evaluate its unique tumor microenvironment (TME), and to use that information to improve diagnosis, management, and survival rate in IBC patients. A proactive, complex and dynamic TME adds to the grim scenario of the disease by accumulating inflammatory and angiogenic growth factors and creating a niche for tumor cells. These cells rely on their TME which can drive a latent tumor to grow into different from the gross metastasis. Here, we performed thorough analysis and comparison of cytokine levels in the conditioned media obtained from inflammatory breast cancer (SUM149PT, SUM1315MO2; cells obtained from primary inflammatory ductal carcinoma of the breast) and normal cell lines (Primary human mammary epithelial cells; HMEC, 184B5; cells of normal mammary tissue obtained from a normal reduction mammoplasty). Inflammatory breast cancer cell lines secrete higher levels of cytokines such as osteoprotegerin (OPG), GRO, GRO-α, MIF, and MIP-1α. To fully comprehend the role of inflammatory cytokines in breast cancer tumorigenesis, we used “mammospheres” also known as tumorspheres or multicellular spheroids. Mammospheres represents an in vitro cultivation system which allows undifferentiated human mammary epithelial cells in suspension to propagate and form non-adherent colonies which recapitulate the in vivo spatial orientation. We compared the composition of conditioned media obtained from inflammatory breast cancer cell line and primary human mammary epithelial cell mammospheres. We observed striking differences in the level of osteoprotegerin (OPG) in the inflammatory breast cancer mammosphere conditioned medium as compared the one obtained from primary human mammary epithelial cells. We focused our study on OPG, a secreted member of the TNF superfamily that acts as a decoy receptor for the receptor activator of nuclear factor kappa B ligand (RANKL). Our work highlights the potential involvement of OPG in IBC tumor cell survival, metastases and reprogramming of mammosphere characteristics (size, number and, proliferation index) thus adding to the severity of disease. Collectively, our study significantly contributes to the understanding the role of OPG in cellular and molecular events involved in IBC progression and opens new avenues for strategic targeting of OPG and the development of novel therapeutic modalities for IBC. Citation Format: Sudeshna Goswami, Neelam Sharma-Walia. Osteoprotegerin's key role in inflammatory breast cancer: Bane or boon. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B27.

MicroRNA expression analysis of mammospheres cultured from human breast cancers

There is accumulating evidence suggests that tumors are initiated and maintained by a small fraction of tumor-initiating cells (TICs). TICs can be enriched by mammospheres culturing without surface markers. MicroRNAs participated in many important processes of life including regulating tumorigenicity of TICs. However, roles of miRNAs in TICs of breast cancer are still unknown. We compared mammospheres formation of four breast cancer cell lines, cultured mammospheres from breast cancers specimens of three patients and compared microRNAs profiling of mammospheres cultured from breast cancer specimens with differentiated progenies. Three of the four breast cancer cell lines showed the ability of mammospheres formation. The proportions of CD24(-)cells in mammospheres were increased significantly in the three cell lines. The expression of genes associated with stem cells and chemoresistance increased significantly after mammospheres formation. Breast cancer cells isolated from patients' specimens survived in nonadherent culture conditions generated mammospheres with ability of self-renewal and bilineage potential. MicroRNA expression profiling of mammospheres compared with differentiated progenies isolated and propagated from the three patients identified 17 microRNAs. And the target genes of these miRNAs are involved in several key signaling pathways. The results suggested that mammospheres were enriched in TICs and proved a valuable model for studies of breast cancer TICs in vitro, microRNAs played critical roles in maintenance of stemness properties of mammospheres and provided novel insights into breast cancer therapy.

The research of the transforming growth factor β1 and p21 effect on breast cancer stem cells

Objective To detect the enrichment of breast cancer stem cells induced by transforming growth factor β1 (TGF-β1),and the difference of p21 expression between breast cancer stem cells and MCF7/ADM cells.Methods Flow cytometry detect the proportion of aldehyde dehydrogenase Ⅰ (ALDH1) positive cells in MCF7/ADM,breast cancer stem cells enriched by mammosphere and breast cancer stem cells induced by TGF-β1.To detect the mammoshpore formation ability in above three teams by mammoshpere formation test.To detect the p21 expression in three teams above by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Results The proportion of ALDH1 positive cells in the three teams were 8.49％,14.94％,23.44％ respectively.The mammosphere formation rate were 5.29％,11.67％,23.13％ respectively.The expression of p21 increased in the three teams.Conclusion TGF-β1 can induce the enrichment of breast cancer stem cells,and enhance it' s self-renewal ability.The expression of p21 is higher in breast cancer stem cells than common breast cancer cells. Key words: Breast cancer stem cell; Tranforming growth factor-β1 ; Aldehyde dehydrogenase 1 ; p21

Strigolactones: a novel class of phytohormones that inhibit the growth and survival of breast cancer cells and breast cancer stem-like enriched mammosphere cells

Several naturally occurring phytohormones have shown enormous potential in the prevention and treatment of variety of different type of cancers. Strigolactones (SLs) are a novel class of plant hormones produced in roots and regulate new above ground shoot branching, by inhibiting self-renewal of undifferentiated meristem cells. Here, we study the effects of six synthetic SL analogs on breast cancer cell lines growth and survival. We show that SL analogs are able to inhibit proliferation and induce apoptosis of breast cancer cells but to a much lesser extent "non-cancer" lines. Given the therapeutic problem of cancer recurrence which is hypothesized to be due to drug resistant cancer stem cells, we also tested the ability of SL analogs to inhibit the growth of mammosphere cultures that are typically enriched with cancer stem-like cells. We show that SLs are potent inhibitors of self-renewal and survival of breast cancer cell lines grown as mammospheres and even a short exposure leads to irreversible effects on mammosphere dissociation and cell death. Immunoblot analysis revealed that SLs analogs induce activation of the stress response mediated by both P38 and JNK1/2 MAPK modules and inhibits PI3K/AKT activation. Taken together this study indicates that SLs may be promising anticancer agents whose activities may be achieved through modulation of stress and survival signaling pathways.

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.