Mammosphere Formation Assay Research Articles

Quercetin‑3‑methyl ether suppresses human breast cancer stem cell formation by inhibiting the Notch1 and PI3K/Akt signaling pathways.

Breast cancer is a leading cause of mortality among women with cancer worldwide. Quercetin‑3‑methyl ether, a natural compound occurring in various plants, has been indicated to have potent anticancer activity. Breast cancer cell growth and survival were examined by CCK‑8 and colony formation assay, whilst cell cycle and apoptosis were determined by flow cytometry. Cell invasion and migration were assessed by wound‑healing assay and Transwell assay. Cancer stem cell formation was analyzed by mammosphere formation assay and related signaling pathways were detected by western blotting. In the present study, it was observed that treatment with quercetin‑3‑methyl ether significantly inhibited cell growth, induced apoptosis and cell cycle arrest at the G2‑Mphase, and suppressed invasion and migration in human breast cancer cells, including the triple negative MDAMB‑231 cell line, and the estrogen receptor‑positive/progesterone receptor‑positive/human epidermal growth factor receptor2‑negative MCF‑7 and T47D cell lines. This compound also markedly suppressed the epithelial‑mesenchymal transition process as evidenced by the upregulated expression of E‑cadherin, and the concomitant downregulated expression of vimentin and MMP‑2. Furthermore, it was demonstrated that quercetin‑3‑methyl ether treatment inhibited mammosphere formation and the expression of the stemness‑related genes, SRY‑box2 and Nanog. Mechanistically, this compound decreased the expression of Notch1, and induced the phosphorylation of phosphoinositide3‑kinase (PI3K) and Akt. It also attenuated the human insulin growth factor1‑induced phosphorylation of PI3K, Akt and glycogen synthase kinaseβ. Additionally, the combination of quercetin‑3‑methyl ether and a secretase inhibitor (DAPT) exhibited additive suppression of the expression of Notch1, PI3K, Akt and mammalian target of rapamycin and a more marked inhibitory effect on cell proliferation and colony formation compared with either drug alone. Treatment with quercetin‑3‑methyl ether alone markedly suppressed the levels of tri‑methyl histoneH3 (Lys27), but had no effect on the expression of enhancer of zeste homolog2. Overall, these findings indicated that quercein‑3‑methyl ether may be a potential therapeutic compound for the treatment of triple negative and hormone‑sensitive breast cancer.

Diallyl Disulfide Inhibits Breast Cancer Stem Cell Progression and Glucose Metabolism by Targeting CD44/PKM2/AMPK Signaling.

It has been reported that diallyl disulfide (DADS) has anti-proliferative activity in many cancers. The purpose of this study was to investigate the functions of DADS and the underlying mechanisms of its effect in breast cancer stem cells (BCSCs). Mammosphere formation assay, glucose consumption assay, lactate production assay and mouse xenograft experiments were performed to explore the functions of DADS in BCSCs. ATPase activity assay, western blotting and immunohistochemistry (IHC) assay were conduct to explore the mechanisms underlying the effects of DADS in BCSCs. The results showed that DADS suppressed cell stemness and glucose metabolism in BCSCs. In vivo mouse xenograft experiments showed that DADS inhibited the proliferation and metastasis of BCSCs. Then, we continued to explore the mechanisms underlying the effects of DADS in BCSCs and found that DADS acts by targeting CD44, Pyruvate kinase M2 (PKM2) and AMP-activated protein kinase (AMPK) signaling pathways. IHC analysis of 125 breast cancer patients' tissues demonstrated that CD44, PKM2 and AMPK expression levels were positively correlated. In addition, positive CD44, PKM2 and AMPK expression was associated with poor patient overall survival (OS) and disease-free survival (DFS). In summary, DADS suppresses cell stemness, proliferation, metastasis and glucose metabolism in BCSCs partly through the inhibition of CD44/PKM2/AMPK. DADS may be used as a potential therapy for breast cancer treatment.

The standardized Korean Red Ginseng extract and its ingredient ginsenoside Rg3 inhibit manifestation of breast cancer stem cell–like properties through modulation of self-renewal signaling

BackgroundThe ginsenoside Rg3, one of active components of red ginseng, has chemopreventive and anticancer potential. Cancer stem cells retain self-renewal properties which account for cancer recurrence and resistance to anticancer therapy. In our present study, we investigated whether the standardized Korean Red Ginseng extract (RGE) and Rg3 could modulate the manifestation of breast cancer stem cell–like features through regulation of self-renewal activity. MethodsThe effects of RGE and Rg3 on the proportion of CD44high/CD24low cells, as representative characteristics of stem-like breast cancer cells, were determined by flow cytometry. The mammosphere formation assay was performed to assess self-renewal capacities of breast cancer cells. Aldehyde dehydrogenase activity of MCF-7 mammospheres was measured by the ALDEFLUOR assay. The expression levels of Sox-2, Bmi-1, and P-Akt and the nuclear localization of hypoxia inducible factor-1α in MCF-7 mammospheres were verified by immunoblot analysis. ResultsBoth RGE and Rg3 decreased the viability of breast cancer cells and significantly reduced the populations of CD44high/CD24low in MDA-MB-231 cells. RGE and Rg3 treatment attenuated the expression of Sox-2 and Bmi-1 by inhibiting the nuclear localization of hypoxia inducible factor-1α in MCF-7 mammospheres. Suppression of the manifestation of breast cancer stem cell–like properties by Rg3 was mediated through the blockade of Akt-mediated self-renewal signaling. ConclusionThis study suggests that Rg3 has a therapeutic potential targeting breast cancer stem cells.

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study.

BackgroundBreast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial–mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes.MethodsA total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence.ResultsThe isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24− BCSCs from MCF-7 cells.DiscussionThis study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

Stat3/Oct-4/c-Myc signal circuit for regulating stemness-mediated doxorubicin resistance of triple-negative breast cancer cells and inhibitory effects of WP1066.

Doxorubicin (Dox) is widely used in the treatment of triple-negative breast cancer cells (TNBCs), however resistance limits its effectiveness. Cancer stem cells (CSCs) are associated with Dox resistance in MCF-7 estrogen receptor positive breast cancer cells. Signal transducer and activator of transcription3 (Stat3) may functionally shift non-CSCs towards CSCs. However, whether Stat3 drives the formation of CSCs during the development of resistance in TNBC, and whether a Stat3 inhibitor reverses CSC-mediated Dox resistance, remains to be elucidated. In the present study, human MDA-MB-468 and murine4T1 mammary carcinoma cell lines with the typical characteristics of TNBCs, were compared with estrogen receptor-positive MCF-7 cells as a model system. The MTT assay was used to detect cytotoxicity of Dox. In addition, the expression levels of CSC-specific markers and transcriptional factors were measured by western blotting, immunofluorescence staining and flow cytometry. The mammosphere formation assay wasusedto detect stem cell activity. Under long-term continuous treatment with Dox at a low concentration, TNBC cultures not only exhibited a drug-resistant phenotype, but also showed CSC properties. These Dox-resistant TNBC cells showed activation of Stat3 and high expression levels of pluripotency transcription factors octamer-binding transcription factor-4 (Oct-4) and c-Myc, which was different from the high expression of superoxide dismutase2(Sox2) in Dox-resistant MCF-7 cells. WP1066 inhibited the phosphorylation of Stat3, and decreased the expression of Oct-4 and c-Myc, leading to a reduction in the CD44-positive cell population, and restoring the sensitivity of the cells to Dox. Taken together, a novel signal circuit of Stat3/Oct-4/c-Myc was identified for regulating stemness-mediated Dox resistance in TNBC. The Stat3 inhibitor WP1066 was able to overcome the resistance to Dox through decreasing the enrichment of CSCs, highlighting the therapeutic potential of WP1066 as a novel sensitizer of Dox-resistant TNBC.

Citral induced apoptosis in MDA-MB-231 spheroid cells

BackgroundBreast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study.MethodsThe cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis.ResultsCitral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/β-catenin pathway.ConclusionThe results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis.

MiR-4319 Suppress the Malignancy of Triple-Negative Breast Cancer by Regulating Self-Renewal and Tumorigenesis of Stem Cells

Background/Aims: High levels of cancer stem cells (CSCs) in patients with triple-negative breast cancer (TNBC) correlate with risk of poor clinical outcome and possibly contribute to chemoresistance and metastasis in patients with highly malignant TNBC. Aberrant microRNA expression is associated with the dysfunction of self-renewal and proliferation in cancer stem cells, while there is little information about the TNBC-specific microRNAs in regulating CSC ability. Methods: Solexa deep sequencing was performed to detect the expression levels of TNBC or non-TNBC stem cells (CSCs) microRNAs. Mammosphere formation assay, qRT-PCR and the xenograft model in nude mice were performed. Bioinformatic analysis and microarray were used to select the target gene, and luciferase reporter assays were used to confirm the binding sites. Results: Solexa sequencing data exhibited differential expression of 193 microRNAs between TNBC and non-TNBC stem cells. The gene ontology analysis and pathways analyses showed that genes were involved in the maintenance of stemness. MiR-4319 could suppress the self-renewal and formation of tumorspheres in TNBC CSCs through E2F2, and also inhibited tumor initiation and metastasis in vivo. Moreover, increased E2F2 could reverse the effect of miR-4319 on the self-renewal in TNBC CSCs. Conclusions: MiR-4319 suppresses the malignancy of TNBC by regulating self-renewal and tumorigenesis of stem cells and might be a remarkable prognostic factor or therapeutic target for patients with TNBC.

A2B adenosine receptor agonist induces cell cycle arrest and apoptosis in breast cancer stem cells via ERK1/2 phosphorylation.

It has been reported that cancer stem cells (CSCs) may play a crucial role in the development, recurrence and metastasis of breast cancer. Targeting signaling pathways in CSCs is considered to be a promising strategy for the treatment of cancer. Here, we investigated the role of the A2B adenosine receptor (A2BAR) and its associated signaling pathways in governing the proliferation and viability of breast cancer cell line derived CSCs. CSCs were isolated fromthe breast cancercell lines MCF-7 and MDA-MB-231 using a mammosphere assay. The effect of the A2BAR agonist BAY606583 on cell proliferation was evaluated using XTT and mammosphere formation assays, respectively. Apoptosis was assessed using Annexin-V staining and cell cycle analyses were performed using flow cytometry. The expression levels of Bax, Bcl-2, cyclin-D1, CDK-4 and (phosphorylated) ERK1/2 were assessed using Western blotting. Our data revealed that the breast cancer cell line derived mammospheres were enriched for CSCs. We also found that A2BAR stimulation with its agonist BAY606583 inhibited mammosphere formation and CSC viability. In addition, we found that the application of BAY606583 led to CSC cell cycle arrest and apoptosis through the cyclin-D1/Cdk-4 and Bax/Bcl-2 pathways, respectively. Notably, we found that BAY606583 significantly down-regulated ERK1/2 phosphorylation inthe breast cancer cell line derivedCSCs. From our results we conclude that A2BAR induces breast CSC cell cycle arrest and apoptosis through downregulation of the ERK1/2 cascade. As such, A2BAR may be considered as a novel target for the treatment of breast cancer.

Abstract 938: Estrogen induced NRF1 signaling is a molecular mechanism underlying the generation of different breast cancer stem cell subpopulations leading to intratumoral heterogeneity

Abstract Life time exposure to elevated levels of estrogen (E2) is a major risk factor for breast cancer. Among women with ER/PR positive tumors, only 50 to 60% of women respond to endocrine therapy. Intra-tumoral heterogeneity of breast cancer cells may be one of the reason for unresponsiveness to endocrine therapy in more than 40% of ER-positive breast cancer cases. In this study we examined that heterogeneous breast cancer stem cells (BCSCs) are produced by exposure to E2 and/or ectopic expression of molecular risk factor - nuclear respiratory factor 1 (NRF1). To elucidate this, we used single cell confocal imaging, flow sorting, ChIP and qRT-PCR approaches. We also measured the key regulators of pluripotency, epithelial-mesenchymal transition (EMT), stemness, cell apoptosis and cell cycle regulation. The functional assay for cancer stem cells enrichment employed were selection of cells in B27 medium as spheroids, anchorage-independent growth and mammosphere formation assays. Carcinogenic treatments of E2 to MCF10 A CD24- cells induced formation of ~1.46% CD44+/CD24+ and 26.02% of CD44+/CD24- subpopulations, which are a typical phenotypes associated with human breast tumor-initiating cells (BTICs) or BCSCs. These subpopulations are capable of forming self-renewing mammospheres. In contrast, CD24+ or CD24-/CD44- cells did not form mammospheres. Stable NRF1 overexpression induced formation of 2.36% CD44+/CD24+ and of 64% CD44+/CD24- subpopulations. Carcinogenic treatments of E2 to stable NRF1 overexpressing MCF10 A cells, induced to acquire 21.54% CD44+/CD24+ and 44.54% CD44+/CD24- subpopulations. E2 treatment to NRF1 overexpressing BSCSs markers significantly increased mammosphere forming capability, compared to NRF1 alone. The dominant negative form of NRF1 diminished the effects of E2 and/or NRF1 induced acquiring of BCSCs antigen markers and their capability of forming mammospheres. E2 induced BTICs/BCSCs were heterogeneous. Each subpopulation was characterized by a different transcriptional and biomarker profiles of CD24, CD44, CD49f, CD133, ALDH1A1, CXCR4, and NRF1. The expression of different markers for pluripotency (OCT4, SOX2 and Nanog), EMT (E-cadherin, N-cadherin and Vimentin), stemness (CD24, CD44, CD39f, CD133, ALDH1A1), cell cycle (p16,CDC2,CDC25C, cyclinB1) and metastasis (CXCR4), were associated with BTICs produced by E2 and/or NRF1. In summary, we observed new roles of NRF1 in contributing to acquire breast tumor initiating stem-like cells and in regulating EMT and invasiveness of BCSCs, thus opens a new direction of NRF1’s role in breast cancer research. A better understanding of how E2 dependent breast neoplasm heterogeneity depends on NRF1 network may open new avenues for therapeutic strategy against breast cancer. This work was supported partly by VA MERIT Review (VA BX001463) grant to DR. Citation Format: Jayanta Kumar Das, Deodutta Roy. Estrogen induced NRF1 signaling is a molecular mechanism underlying the generation of different breast cancer stem cell subpopulations leading to intratumoral heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 938. doi:10.1158/1538-7445.AM2017-938

Abstract 4788: Abraxane eliminates cancer stem cells in triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) represents about 20 percent of all breast cancers, with a poorer survival rate and being associated with high-grade tumors and the least favorable prognosis. Due to the absence of hormone receptors and Her2 receptor, no targeted therapy is available and chemotherapy is the standard treatment for TNBC patient. Paclitaxel is a widely used chemotherapy drug for various cancers with severe side effects due to its solvent. Abraxane is an albumin-bound nanoparticle of paclitaxel with less severe side effects. Here we investigated whether cancer stem cells (CSCs) in TNBC response differentially to abraxane and paclitaxel both in cell culture and xenograft mouse models. Using a series of human TNBC cell lines, we showed that there is no difference between solvent-based paclitaxel and abraxane in vitro in term of efficacy and eliminating cancer stem cells using MTS assay, mammosphere formation assay, and flow analysis of cancer stem cells. Interestingly, we demonstrated that abraxane shows superior efficacy than solvent-based paclitaxel in human SUM149 xenograft NOD SCID mouse model in adjuvant treatment. More important, we demonstrated that abraxane eliminates cancer stem cells in xenograft NOD SCID mouse model in advanced treatment, while solvent-based paclitaxel increases cancer stem cells. Cell uptake assay indicated that TNBC cells take more abraxane than solvent-based paclitaxel, which partially attributes to its superior efficacy in xenograft mouse models. Taken together, our data confirmed that abraxane is superior to solvent-based paclitaxel and may be a better option for TNBC patients in adjuvant or metastatic treatment. Citation Format: Hebao Yuan, Hongwei Guo, Feng Li, Joseph Burnett, Miao He, Nathan Truchan, Ila Myers, Duxin Sun. Abraxane eliminates cancer stem cells in triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2017-4788

Abstract 1385: New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer

Abstract Background: Epigenetic modifiers have been emerging as new players in breast cancer development. FAD-dependent histone demethylases have been proved to play important roles in regulating breast tumor initiation and development. While the role of histone lysine-specific demethylase 1 (LSD1) in breast cancer progression has been well characterized, the activity of its homolog, lysine-specific demethylase 2 (LSD2), in breast cancer is still a riddle that needs to be uncovered. Methods: To explore the precise role of LSD2 in breast cancer, we overexpressed LSD2 protein in MDA-MB-231 cells (LSD2-OE). The phenotypic effect of LSD2-OE on MM231 cells was characterized by proliferation assay, soft agar assay (3D growth), trans-well cell migration and invasion assay and mammosphere formation assay. To extend stem cell-like features study of LSD2 overexpressed MM231 cells, we examined CD44+/CD24- subpopulation by flow cytometry and the expression of several stem cell markers by qPCR and western blot. In addition, we also investigated the potential altered phenotypes of LSD2 knockdown in MM231 cells via RNAi. Results: Results from Oncomine database show that LSD2 expression level is greatly increased in invasive ductal carcinoma (IDC) vs. non-invasive breast cancers. The cellular proliferation assay revealed that LSD2-OE promoted MM231 cell growth. Similarly, LSD2 overexpressed MM231 cells were capable of forming large colonies in soft agar. Trans-well cell migration and invasion assay results indicated LSD2-OE significantly decreased both migration and invasion in MM231 cells. Mammosphere formation assay results showed LSD2 overexpression increased the number of primary and tertiary mammospheres in MM231 cells. Consistent with increase of mammosphere forming efficiency, NANOG and SOX2 were increased by LSD2 overexpression at the protein level. However, LSD2-OE significantly decreased CD44+/CD24- subpopulation. Moreover, siRNA mediated LSD2 depletion significantly decreased cell growth in multiple breast cancer cell lines without altering migration and invasion in MM231 cells. Finally, no obvious phenotypic changes were observed in MM231 cells expressing stable LSD2 shRNA. Conclusion: The multifaceted effects of LSD2 on breast cancer phenotypes may reflect the profound influence of LSD2 activity on chromatin structure and gene expression in the breast cancer genome. Our studies suggest that overexpression of LSD2 facilitates growth and confers stem cell-like traits to breast cancer cells, and transient LSD2 inhibition by siRNA hinders breast cancer cell growth. These results suggest that LSD2 might be a valuable therapeutic target in breast cancer. Citation Format: Lin Chen, Shauna N. Vasilatos, Ye Qin, Steffi Oesterreich, Nancy E. Davidson, Yi Huang. New insights into the roles of histone lysine-specific demethylase 2 (LSD2) in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1385. doi:10.1158/1538-7445.AM2017-1385

Abstract 3183: Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone

Abstract Postmenopausal women with early-stage metastatic estrogen-dependent breast cancer are generally treated with aromatase inhibitors (AIs) (e.g., letrozole). However, acquired resistance remains a major clinical obstacle. Previously, our group revealed a global proteomic signature of a letrozole-resistant cell line (LTLT-Ca) associated with hormone independence, enhanced cell motility and epithelial to mesenchymal (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC), we chose to utilize a two-dimensional (2D) vs three-dimensional (3D) culture system to compare the proteome of LTLT-Ca cells, as 3D culture not only enriches for CSC, but more accurately recapitulates the tumor microenvironment, morphology, function and response to therapy compared to conventional 2D culture. We hypothesize utilizing a novel systems biology approach may reveal previously unconsidered molecular changes that could aid in understanding complex signaling networks and be exploited as therapeutic targets. To address this hypothesis ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with LTLT-Ca or letrozole sensitive cells (AC-1) and intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44high/CD24low while AC-1 tumors were CD44-/CD24low. Mammosphere formation assays were conducted and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index than AC-1 cells. A quantitative proteomic analysis of whole cell lysates from LTLT-Ca (2D adherent cells) versus LTLT-Ca (3D mammospheres) was conducted. Results identified significant protein expression changes within a panel of 1010 proteins; 173 were upregulated and 186 downregulated (p<0.05, fold change >1.20). Additionally, functional enrichment analyses were performed and 19 gene ontology (GO) terms and one KEGG pathway (hsa03010:Ribosome) were over-represented (BH adjusted p-value < 0.01) by the cognate genes. Notably, there was a 35.04-fold increase in midasin (MDN1), a nuclear chaperone protein required for maturation and nuclear export of pre-60S ribosome subunit. Increased MDN1 expression was strongly correlated with highly tumorigenic breast cancer spheres. Additionally, Kaplan-Meier survival plots demonstrate that increased MDN1 levels were positively correlated with decreased relapse free survival in estrogen receptor negative breast cancer, and is constitutively expressed as breast tumors progress from atypical ductal hyperplasia to ductal carcinoma in situ to invasive, metastatic breast cancer. The TGCA database was interrogated and MDN1 was frequently amplified or mutated in breast tumors. Taken together our study for the first time implicates a role for ribosomal assembly in AI resistant cells enriched for CSCs and underscores a potential for MDN1 in the progression and responsiveness to therapy. Citation Format: Syreeta L. Tilghman, Jamal Pratt, Shawn D. Llopis, A. Michael Davidson, Rashidra R. Walker, Patrick Carriere, Ian R. Davenport, Wensheng Zhang, Karen Zhang. Proteomic characterization of aromatase inhibitor resistant mammospheres reveal the presence of a novel nuclear chaperone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2017-3183

Abstract 1919: Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancers, is highly refractory to chemotherapy and has a high incidence of metastasis and tumor relapse. Targeted therapies for TNBC do not exist because this subtype lacks druggable hormone receptors and HER2 amplification or overexpression. Although Taxane-based chemotherapy is a standard of care for TNBC treatment, paclitaxel (Taxol)-induced cancer stem cell (CSC) enrichment presents a significant challenge to the success of breast cancer treatment. Thus, there is a critical need to understand the mechanisms by which chemotherapy induces CSC enrichment in residual tumors following anticancer therapies. Here we report that Taxol-induced enrichment for CSCs correlates well with a marked upregulation of Twist1 expression in human TNBC cells. Knockdown of Twist1 inhibits the clonogenic capacity of TNBC cells in vitro and TNBC tumorigenesis in vivo. Mammosphere formation assays indicate that silencing of Twist1 greatly diminishes the tumor sphere-forming potential of breast CSCs. Furthermore, limited dilution assays and stem cell xenotransplantation reveal that knockdown of Twist1 expression decreases the frequency of CSCs by 72-fold in human TNBC cells. More importantly, we show that silencing of Twist1 blocks Taxol-induced CSC enrichment in residual tumor cells that have survived drug treatment. Together, these results demonstrate a previously unrecognized role for Twist1 in mediating Taxol-induced CSC enrichment in residual tumors of human TNBC and suggest that targeted inhibition of the Twist1 signaling pathway may represent a novel therapeutic strategy to tackle therapy-induced CSC enrichment in breast cancer treatment. Citation Format: Aimin Yang, Xiaoyuan He, Bradley A. Schulte, Steven L. Carroll, Stephen P. Ethier, Gavin Y. Wang. Twist1 modulates paclitaxel-induced cancer stem cell enrichment in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1919. doi:10.1158/1538-7445.AM2017-1919

Abstract 2875: Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest

Abstract We found that ganglioside GD2 is a biomarker for breast cancer stem cell (BCSCs) in triple negative breast cancer (TNBC) and that GD2 biosynthesis is tightly regulated by enzyme ST8SIA1 (GD3 synthase) in these cells. We have reported that ST8SIA1 is highly expressed in TNBC and its expression is highly correlated with TP53 mutations in primary tumors (based on TCGA data set). Here we hypothesized that ST8SIA1 has a functional role in BCSC mediated tumorigenesis in TNBC. To test this hypothesis, we deleted ST8SIA1 in SUM159 cells using CRISPR-Cas9 technology. As expected, deletion of ST8SIA1 in SUM159 cells reduced the absolute number of GD2+ cells from 17 ± 1.5% to 0.3 ± 0.1%. Although there was no significant difference in 2D cell growth, anchorage-independent growth by soft-agar and the mammosphere formation assays revealed a complete loss of colony formation in ST8SIA1-KO cells. Moreover, transplantation of ST8SIA1-KO- or Cas9 control- SUM159 cells in mammary fat pad of NSG mice revealed that ST8SIA1-KO cells failed to produce tumor formation even after a 15-wk after implantation. In addition, most of the cas9 control cell injected mice died within 4 wk after cell implantation whereas no deaths were observed in the ST8SIA1-KO cells as long as 100 d after tumor implantation. To investigate the mechanism involved in tumor growth inhibition associated with the loss of ST8SIA1 expression, we analyzed mRNA expression in ST8SIA1-KO- and Cas9 control- SUM159 cells by RNA sequencing. At p < 0.05 and fold change >2, we found 1502 genes down-regulated and 842 genes up-regulated in the ST8SIA1-KO-cells compared to controls. Ingenuity pathway analysis revealed that several stem cell-associated signaling pathways, including wnt, stat3, NFκB, nanog, and IL8, were down-regulated. Conversely, the potent tumor suppressor PTEN was induced in the ST8SIA1-KO-cells compared to controls. In addition, gene ontology by gene set enrichment analysis (GSEA) revealed inhibition of oncogenic signaling pathways including KRAS, p53, AKT, and RB. We have also identified a significant down-regulation of breast cancer-associated genes including BCL11A, PDGFRb, VCAM1, CXCR4, and wnt5a. Interestingly DKK1, which acts as an antagonist for wnt-β-catenin signaling, was up-regulated in the ST8SIA1-KO cells. These findings were validated by qRT-PCR, flow cytometry and western blot analysis. Ganglioside GM1 has been reported to have major in anti-tumor effect by inhibiting ERK signaling. Flow cytometry analysis revealed that ST8SIA-KO cells expressed ~10-fold higher GM1 compared to Cas9 control cells. In addition, pERK was down-regulated in ST8SIA-KO cells, suggesting a loss of tumorigenic potential of these cells. In conclusion, our data suggests that inhibition of ST8SIA1 in TNBC cells depletes BCSCs and inhibits tumorigenesis by down-regulating oncogenic pathways and up-regulation of PTEN and GM1 mediated signaling. Citation Format: Khoa Nguyen, Yuanqing Yan, Chandra Bartholomeusz, Naoto Ueno, Kim-Anh Do, Michael Andreeff, V. Lokesh Battula. Knockout of ST8SIA1 inhibits tumorigenesis in triple negative breast cancer by inducing PTEN and ganglioside GM1 mediated tumor growth arrest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2875. doi:10.1158/1538-7445.AM2017-2875

HN1 contributes to migration, invasion, and tumorigenesis of breast cancer by enhancing MYC activity

BackgroundHematological and neurological expressed 1 (HN1) is upregulated in many tumors, but the role of HN1 in breast cancer progression and its regulatory mechanism have not been well understood.MethodsTo study the role of HN1 in the initiation and progression of breast cancer, we examined HN1 levels in breast cancer cells and tissues and analyzed the relationship between HN1 levels and patient survival. We used mammosphere formation assay, side population analysis, wound healing assay, transwell assay, soft agar formation assay, and xenografted tumor model to determine the effect of HN1 on the expansion of breast cancer stem cells, and the migration, invasion and tumorigenesis of breast cancer. To determine whether HN1 regulates MYC, we used quantitative real-time PCR and Western blot analysis to assess the expression of MYC and their targeted genes to determine the phenotype caused by knockdown of MYC in breast cancer cell with HN1 overexpression.ResultsIn this study, we found that HN1 was upregulated in breast cancer tissues. Patients with high levels of HN1 expression had significantly shorter survival than those with low HN1 expression. In breast cancer cell line, ectopic overexpression of HN1 not only promoted the expansion of breast cancer stem cells, but also promoted cell migration, invasion, and tumorigenesis, while knockdown of HN1 reduced these effects. Furthermore, there was a positive correlation between MYC (also known as c-MYC) level and HN1 level, mechanism analysis suggested HN1 promoted the expression of MYC and its targeted genes like CDK4, CCND1, p21, CAV1, and SFRP1. Downregulation of MYC abrogated the effect of HN1 overexpression in breast cancer cell lines.ConclusionTaken together, these data reveal that HN1 promotes the progression of breast cancer by upregulating MYC expression, and might be a therapeutic target for breast cancer.

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs

BackgroundEstrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities.MethodsTo better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology.ResultsPhospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate.ConclusionsWe conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

Apoptosis and cell cycle regulatory effects of adenosine by modulation of GLI-1 and ERK1/2 pathways in CD44+ and CD24- breast cancer stem cells.

Breast cancer stem cells (CSCs) are a small population of tumour cells with the ability of self-renewal and resistance to chemotherapy. Targeting CSCs is a promising strategy for treatment of cancer. A recent study demonstrated that adenosine receptor agonists inhibit glioblastoma CSCs proliferation. At present, the effect of adenosine on breast CSCs has not been reported. Therefore, this study was designed to evaluate the effect of adenosine and its signalling pathways in breast CSCs. Anti-proliferative effect of adenosine on breast CSCs was evaluated by mammosphere formation and MTS assay. The effect of adenosine on cell cycle progression was examined using flow cytometry. Detection of apoptosis was conducted by Annexin V-FITC. The expression levels of cell cycle and apoptosis regulatory proteins as well as ERK1/2, and GLI-1 were measured by Western blot. Adenosine reduced CSCs population and mammosphere formation in breast CSCs. Adenosine induced G1 cell cycle arrest in breast CSCs in conjunction with a marked down-regulation of cyclin D1 and CDK4. Adenosine also induced apoptosis by regulation of Bax/Bcl-2 ratio, mitochondrial membrane potential depletion and activation of caspase-6. Moreover, adenosine inhibited ERK1/2 phosphorylation and GLI-1 protein expression. These findings indicated that adenosine induces cell cycle arrest and apoptosis through inhibition of GLI-1 and ERK1/2 pathways in breast CSCs.

Abstract P5-07-11: The ectopic FOXA1 expression correlates to the luminal breast cancer stem cells

Abstract Background: It is known that ER-positive breast cancer constitutes approximately 70% of all breast cancer and the particular population of these, called as luminal A breast cancer can lead to late recurrence, possibly due to the presence of dormant tumor cells. It is also argued that there are many similar biological characteristics between dormant cells and cancer stem cells (CSCs). So we hypothesized that some part of CSCs would be involved in the late recurrence of luminal type breast cancer and looked for the factors correlateing late recurrence by comparing CSC-enriched population and other population. Materials and Methods: We used the MCF-7 and HCC1500 human breast cell lines. Moreover, we isolated the BC#1 breast cancer cell from metastatic pleural effusion of a breast cancer patient (79 years old, ER+, PgR+, HER2 score 0). These cells are cultured in the adhesion culture condition or the mammosphere culture condition. The immunohistochemical characteristics such as ER, PgR, HER2, Cytokeratin (CK) 8, CK5/6 status of these cells were confirmed. The analysis of the ALDH activity and the CD44+/24- expression as the CSC marker were carried out using flow cytometry. The expression of target genes was analyzed using quantitative RT-PCR. Protein expression was determined by western-blotting. To assess the self-renewal potential of CSCs and the proliferation of immature cancer cells, we used mammosphere formation assay and colony formation assay. To investigate the effects of 4-hydroxytamoxifen(4-OHT) on mammosphere formation assay, 1µm of 4-OHT added to the medium in the particular experiment. Results: BC#1 was confirmed as luminal type by immunohistochemistry and qRT-PCR. The expression tendency between two CSC markers are almost same and correlated to the mammosphere formation capacity. We also confirmed that mammosphere culture have an increased ALDH-positive population compared with adherent culture cells. Using flow cytometry, we divided the cell lines and isolated cells into two populations, ALDH-positive and negative. By qRT-PCR analysis, ALDH-positive population showed higher expression of FOXA1 (ER expression related gene) than ALDH-negative population. In addition, we found the significantly higher expression of FOXA1 gene, and RPRM gene (p53 induced G2/M arrest related gene) in mammosphere culture samples than in adhesion culture samples. In terms of the effects of 4-OHT, 4-OHT resistant mammosphere samples showed significantly increased FOXA1 gene expression level. Finally, we established shFOXA1 MCF-7 cells to investigate the relationship between self-renewal potential and FOXA1 expression. We found that colony formation in shFOXA1 MCF-7 cells decreased compared with control while there is no significant difference in the number of mammospheres. Conclusion: CSC-enriched population showed higher FOXA1 expression and our results suggested that the expression of FOXA1 correlate to the proliferation of immature breast cancer cell rather than the induction of self-renewal potency of CSCs. Moreover, there is no report argued about the correlation between CSCs and G2/M arrest related gene like RPRM so that the biological mechanism should be investigated in future. Citation Format: Tachi K, Shiraishi A, Bando H, Yamashita T, Tsuboi I, Kato T, Ichioka E, Tsushima Y, Ikeda T, Iguchi A, Hara H, Ohneda O. The ectopic FOXA1 expression correlates to the luminal breast cancer stem cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-11.

Abstract P3-06-09: Selection of presumed CD49f antagonists and their biological evaluation in breast cancer cells

Abstract Breast cancer is the second cause of cancer death in women and the large majority of those deaths are due to drug resistance and/or recurrence. The cancer stem cell (CSC) model suggests that tumor stem-like cells play key roles in resistance and recurrence in breast cancer patients. Thus, CSCs have been pointed as targets for new anti-cancer therapies. CD49f is an integrin subunit that participates in the CSC-niche interaction. Knock-down of CD49f in breast cancer cells impairs mammosphere formation in vitro and tumorigenesis in vivo, suggesting that this protein is needed for maintenance of stemness. Aiming to target breast CSC, we used consensus docking to select potential CD49f antagonists from a collection of 13,000+ drugs with previous clinical evaluations. Seven compounds with the lowest consensus Z score were selected for in vitro biological validation. Cell adhesion assays showed that four of the selected drugs (5193, 1382, 7631, and 12723) decrease the binding of CD49f+ MDA-MB-231 breast cancer cells to laminin, suggesting that those compounds antagonize the receptor. Mammosphere formation assays showed that compounds 5193, 1382, and 12723 limit the clonogenic capability of CD49f+ breast cancer cells in vitro. Compound 5193 was highly toxic in 2D cell viability assays. On the other hand, the clonogenic impairment produced by compounds 1382 and 12723 is independent of bulk cell line cytotoxicity, suggesting a direct impact on the CSC pool. The analyses of the effect of these drugs on the expression of breast CSC markers and CD49f-activated pathways are on their way. Thus, we have identified some drugs that might be repurposed to target breast CSC. Since the pharmacokinetics and toxicology of those drugs is known, they could easily be ready for clinical trials. We demonstrated that in silico screening is useful to identify antagonists of receptors with relevant roles in breast CSC biology. This work was supported by CONACYT 221105, PAPIIT-UNAM IN228616, and Red temática de células troncales y medicina regenerativa. Citation Format: Velasco-Velázquez M, Velázquez-Quesada I, Aguirre-Alvarado C, Guerrero-Rodríguez S, Ruiz-Moreno A, Ramirez-Salinas G, Segura-Cabrera A, Pérez-Tapia M. Selection of presumed CD49f antagonists and their biological evaluation in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-09.

Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library

BackgroundCancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)’s Integrated Colony Enumerator (NICE) with a screening of protein kinase library.MethodsHuman breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated.ResultsAutomated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-β1-induced migration, colony formation and EMT of MCF-7 cells.ConclusionsWe have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.