Mammosphere Formation Efficiency Research Articles

Abstract PS11-38: Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer

Abstract Background and Aims: Chemotherapy is the main treatment for metastatic triple negative breast cancer (mTNBC) but many patients develop resistance and progress to metastatic disease, with a 5-year survival rate of 11%. Therefore, more effective and less toxic treatments are required. 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3) are two pharmacologically active enantiomers extracted from Panax ginseng. In our studies, we have shown that these two enantiomers have stereoselective activities in inhibition of proliferation, migration and invasion of triple-negative breast cancer (TNBC) cells, and hence, could be considered as two separate drugs that can be combined in a synergistic dose (S+R). Based on the inhibitory effects of single enantiomers on loop formation of human umbilical vein endothelial cells (HUVEC), we have optimized the concentrations of Rg3 enantiomers to be combined that have shown time- and dose-dependent inhibition of loop formation and migration of human and mouse endothelial cell lines. The aim of the current study was to show the mechanisms of S+R in inhibition of angiogenesis in vitro and its efficacy in inhibiting metastasis in vivo. Methodology: Molecular docking was used to study the Rg3- vascular endothelial growth factor receptor (VEGFR) 2 interaction, and the VEGF bioassay kit (Promega) was used to show this interaction in vitro. ELISA, quantitative-PCR and western blot (WB) were used to study the expression of transcript and protein levels of signalling molecules. Two TNBC cell lines (MDA-MB-231 and HCC1143) were cultured as mammospheres and the effects of S+R on formation of mammospheres, viability (trypan blue assay and flow cytometric analysis of apoptosis or cell cycle arrest) and expression of CD44+ cells (flow cytometry) were studied. Expression microarrays were used to determine the effect of the treatment on the PI3K signalling pathway in MDA-MB-231. Nod Scid gamma mice were used to develop a murine model of metastatic breast cancer model. Luciferase-tagged MDA-MB-231 cells were injected into the 4th mammary fat pad of the mice and upon primary tumour establishment, treatment started. Tumour growth and metastasis was monitored using IVIS Spectrum. Statistical analysis was performed using ANOVA (Prism v8). Results: According to molecular docking, binding score of Rg3-VEGFR2 was -9.0 cal/mol. The VEGF bioassay showed that Rg3 is an allosteric modulator of VEGFR2 receptor. In HUVEC, S+R treatment decreased the expression of VEGF (ELISA) and its receptor VEGFR2. It also decreased the expression of pAKT:AKT, aquaporin 1 (AQP1) and pFAK:FAK (WB). These proteins are involved in cell survival, migration, invasion and proliferation. In breast cancer cell lines, S+R decreased the expression of AQP1 and decreased mammosphere formation efficiency, not by decreasing cell viability but by decreasing the expression of CD44+ stem cell marker. In expression arrays, the treatment decreased the expression of BTK, a tyrosine kinase promoting cell death escape; increased the expression of both CASP9, promoting apoptosis, and INPPD5, favouring inhibition of metastasis. In vivo experiments showed that S+R lead to tumour shrinkage (p < .0001), decreased body burden of tumour (p < .0001) and decreased number of metastases (p = .002). Conclusion: These results support the anti-cancer effects of the combination of S+R for the treatment of mTNBC. Citation Format: Maryam Nakhjavani, Eric Smith, Timothy Price, Amanda R Townsend, Jennifer E Hardingham. Ginsenoside rg3 enantiomers in a defined ratio as a novel treatment for metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-38.

Abstract 4959: Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness

Abstract Chemoresistance is a clinically significant issue in triple-negative breast cancer (TNBC) patients, leading to minimal residual disease. The surviving breast cancer stem cells (BCSCs) undergo multilineage differentiation and repopulate a more aggressive primary tumor leading to metastasis and subsequent mortality. Not only are BCSCs innately chemo/radioresistant but also contribute to therapy failure through interconversion between BCSCs and bulk tumor cells. Therefore, it is imperative to co-target BCSCs and bulk tumor cells simultaneously. Identifying novel molecular targets is key to eliminate both bulk tumor cells and BCSCs. The eukaryotic protein translation initiation machinery, the eIF4F complex, is a convergence point for many oncogenic signaling pathways. This complex plays a vital role in translation of many oncogenic mRNAs implicated in cell cycle, tumor progression, chemoresistance and metastasis. We have previously demonstrated that the mRNA helicase eIF4A1, of the eIF4F complex, might serve as a vulnerable node to kill TNBC cells. In the current study, we hypothesized that targeting of eIF4A1 would reduce/eliminate both bulk tumor cells and BCSCs and overcome chemoresistance. In order to evaluate the effects of targeting eIF4A1 in BCSCs, we isolated BCSCs based on aldehyde dehydrogenase activity and CD44 expression by Fluorescence Activated Cell Sorting and characterized for stemness. Through pharmacological targeting (Rocaglamide A) and genetic ablation (CRISPR-Cas9) of eIF4A1, we demonstrated that there was a statistically significant reduction in the self-renewal ability indicated by the reduction in primary and secondary mammosphere formation efficiency (MFE) of BCSCs, levels of the pluripotency transcription factors (SOX2, OCT4 and NANOG), expression of drug transporters (ABCG2, ABCB1 and ABCC1) and increased induction of apoptotic cell death. Overall, we demonstrated that targeting eIF4A1 is effective in eliminating BCSCs through reduction in the levels of stemness factors and drug transporters. This sets up a stage to employ small molecule inhibitors against eIF4A1 in co-targeting strategic therapies. Citation Format: Sangita Sridharan, Megan Robeson, Diwakar Tukaramrao Bastihalli, Cory Howard, Boopathi Subramaniyan, Augustus Tilley, Dayanidhi Raman. Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4959.

Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX‐01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX‐01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho‐STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months’ endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Obesity and Breast Cancer: Unraveling the Role of Adipocyte‐Derived Exosomes

Excessive adiposity strongly influences risk, prognosis and progression of breast cancer. The revised concept of adipose tissue from an inert depot for body energy to an endocrine and immunologically active organ placed growing interest on the potential role of adipocyte secreted soluble factors and vesicles, particularly exosomes, as important players in cancer biology. In the last decades, several reports demonstrated that exosomes, small membrane‐derived vesicles, are able to modify target cell phenotype, by transferring bioactive molecules such as proteins, mRNAs, microRNAs, DNAs, lipids and transcriptional factors. However, whether adipose tissue‐derived exosomes, acting as a mode of cell‐to‐cell communication, might have a role in mediating the adverse effects of obesity on breast cancer is still not completely understood.Here, we evaluated the effects of exosomes derived from differentiated 3T3‐L1 adipocytes (3T3‐L1 A), a well recognized ‘in vitro’ model of white adipocytes, on breast cancer phenotype, using as experimental models both estrogen receptor α (ERα)‐positive MCF‐7 and triple negative MDA‐MB‐231 breast cancer cells.Exosomes from 3T3‐L1 A were isolated by sequential ultracentrifugation method, and characterized by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Immunoblotting analyses. 3T3‐L1 A‐ derived exosome uptake in both breast cancer cell lines was tested by monitoring internalization of exosomes labeled with PKH‐26. Treatment of MCF‐7 and MDA‐MB‐231 breast cancer cells with adipocyte‐derived exosomes did not modify anchorage‐dependent cell growth, while it significantly increased the ability of cells to form colonies in anchorage‐independent soft agar growth assays. In addition, we found that both breast cancer cells treated with adipocyte‐derived exosomes displayed increased migration and invasion capabilities as evidenced by wound healing, and Boyden Chamber migration and invasion assays. Accordingly, adipocyte‐derived exosomes significantly induce the expression of genes involved in Epithelial to Mesenchymal Transition (EMT) in both cell lines. Since migratory potential and EMT represent important features for breast cancer stem cells (BCSCs), we also explored the effect of 3T3L‐1 A‐derived exosomes on MCF‐7 and MDA‐MB‐231 BCSC activity. Interestingly, we found that adipocyte‐derived exosomes were able to increase Mammosphere Forming Efficiency and Self Renewal in breast cancer cells. In conclusion, our data highlight how adipocyte‐derived exosomes may contribute to a more aggressive breast cancer phenotype, providing novel insight in the link between obesity and breast cancer. This may open a new molecular window for preventive and therapeutic strategies to fight breast cancer in obese patients.Support or Funding InformationThis research was funded by Fondazione AIRC: IG #11595 to SA and IG #21414 to SC, and PRIN2017 #2017EKMFTN to SC; PRIN2017 #2017WNKSLR to IB

Fetal Alcohol Exposure Alters Mammary Epithelial Cell Subpopulations and Promotes Tumorigenesis.

Fetal alcohol exposure (FAE) increases the risk of mammary tumorigenesis in adult offspring; however, the underlying mechanism remains unknown. This study tested the hypothesis that FAE shifts the mammary epithelial cell (MEC) composition toward one that promotes tumorigenesis. Pregnant Friend Virus BNIH Jacksondams bred to MMTV-Wnt1 male mice were given ad libitum access to 5% alcohol in 0.2% saccharin solution from GD9-10 and 10% alcohol in 0.2% saccharin from GD11-GD19 or 0.2% saccharin solution from GD9-GD19. Thoracic and inguinal mammary glands from wild-type (WT) and transgenic (Tg) female offspring were harvested at 5 and 10weeks of age and dissociated to yield a single cell suspension enriched for MECs for flow cytometry, mammosphere assay, and gene analysis. A subset of Tg offspring was followed for tumor formation. WT glands of FAE animals exhibited a decreased basal cell population and increased luminal: basal ratio at 10weeks of age. qRT-PCR analysis of total MECs found that Hey1 mRNA expression was increased in the WT FAE group at 10weeks of age. In Tg glands, FAE increased the luminal progenitor cell population at 5weeks of age but did not alter MEC composition at 10weeks of age. Tertiary mammosphere-forming efficiency was greater in the WT glands of FAE animals at 10weeks of age. Tumor latency was decreased in the FAE group. Flow cytometry analysis indicated that FAE females developed tumors with an increased basal cell population. These data indicate that FAE can shift MEC subpopulations, increasing the proportion of cells that are potentially vulnerable to transformation and affecting cancer risk.

Abstract P1-18-21: Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms

Abstract Background: Breast cancer stem cells (BCSCs) have been attributed to drug resistance and recurrence of disease following treatment. BCSCs employ various mechanisms to resist chemotherapy. One such mechanism is the increase in expression of the ATPase drug efflux pumps ABCB1 and ABCG2, reducing intracellular Paclitaxel and Mitoxantrone drug concentration, respectively. Neratinib a pan-HER tyrosine kinase inhibitor was reported to block both ABCB1 and ABCG2 pump activity. We aimed to determine the effect of Neratinib on CSC activity (primary and self-renewal) and determine if the mechanism included pump inhibition. Methods: The effects of 10nM Neratinib alone and in combination with either 1µM Tamoxifen, 0.1µM Paclitaxel or 0.1µM Mitoxantrone on anoikis resistance culture was evaluated in multiple breast cancer cell lines. The mammosphere forming efficiency (MFE) and generation of secondary mammospheres (self-renewal) were calculated. Blockage of the ABCG2 by Neratinib was assessed via FAC analysis. Statistical significance was determined using unpaired t-test and one-way ANOVA. Results: Neratinib reduced CSC activity assessed by MFE in BT474 cells by 50% (P < 0.01). Combining Neratinib with Tamoxifen further reduced CSC (by 75%) than either treatment alone (P < 0.05 and P < 0.01, respectively). The combination of Neratinib and Paclitaxel also reduced the MFE by a greater extent (90%, P < 0.0001) than either treatment alone (P < 0.05 and P < 0.0001, respectively). Neratinib alone had no effect on MFE in the triple negative breast cancer cell line MDA-MB-453, combining Neratinib with Paclitaxel reduced MFE by 85%, a greater reduction than paclitaxel alone (75%), P < 0.0001. Our data also indicates that Neratinib in combination with Paclitaxel reduces CSC self-renewal in MCF7, MCF7HER2 and MDA-MB-453 cell lines. Combining Neratinib with Mitoxantrone further decreases CSC activity than either treatment alone in MCF7, MCF7HER2, SKBR3 and MDA-MB-453 cells and completely inhibited CSC self-renewal in the triple negative cell line. Neratinib also reduced the side population in BT474 and SKBR3 cells in a dose response manner, demonstrating inhibition of ABCG2 activity. Conclusions: These findings support the idea that Neratinib has the ability to overcome drug resistance caused by the ABCB1 and ABCG2 pumps. Therefore, we are currently investigating the potential of Neratinib to block the activity of the ABCB1 and ABCG2 pumps using flow cytometry to analyse the side-population and drug accumulation. They also provide insight into a role for Neratinib to enhance paclitaxel chemotherapy in triple negative CSC. Citation Format: Nathan Hull, Robert Clarke, Nigel Bundred. Neratinib reduces HER2+/luminal B breast cancer stem cell activity and inhibits resistance by blocking efflux mechanisms [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-21.

CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro.

The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs.

Suppressing the metastatic properties of the breast cancer cells using STAT3 decoy oligodeoxynucleotides: A promising approach for eradication of cancer cells by differentiation therapy.

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple-negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3-labeled ODNs is in the cytoplasm and the nucleus of MDA-MB-231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA-MB-231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach.

Targeted Toxin Gene Therapy Of Breast Cancer Stem Cells Using CXCR1 Promoter And bFGF 5'UTR.

BackgroundBreast cancer stem cells (BCSCs) are cells with a higher ability to metastasis and resistance to conventional treatments. They have a phenotype of (CD44high/CD24low) and the unlimited ability for proliferation. Development of strategies to target the BCSC population may lead to the establishment of more effective cancer therapies. Pseudomonas exotoxin A (PE) is a potent cytotoxic protein. CXCR1 promoter provides BCSC and HER2 specificity on transcription level. 5′UTR of the basic fibroblast growth factor-2 (bFGF 5ʹUTR) provides tumor specificity on translation level. Here, we utilized a mutant form of PE encoding DNA “PE38”, CXCR1 promoter and bFGF 5ʹUTR to target BCSCs.MethodsThe stemness of SK-BR-3, MDA-MB-231 and MCF10A cell lines were evaluated based on the expression of the CD44high/CD24low stem cell signature and the ability to form mammospheres. Then, the cell lines were transfected with constructs encoding luciferase/PE38 under the control of the CMV/CXCR1 promoter with or without bFGF 5′UTR. Luciferase protein expression was evaluated using dual-luciferase reporter assay. PE38 transcript expression was measured by real-time PCR, and the cytotoxic effect of PE38 protein expression was determined by MTT assay.ResultsThe percentage of CD44high/CD24low population did not correlate to mammosphere forming efficiency (MFE). Given that the percentage of CD44 high/CD24 low is not a conclusive BCSC profile, we based our work on the mammosphere assay. However, in comparison with MCF10A, the two tumorigenic cell lines had higher MFE, probably due to their higher BCSC content. Reporter assay and real-time PCR results demonstrated that CXCR1 promoter combined with bFGF 5ʹUTR increased BCSC-specific gene expression. Meanwhile, tightly regulated expression of PE38 using these two gene regulatory elements resulted in high levels of cell death in the two tumorigenic cell lines while having little toxicity toward normal MCF10A.ConclusionOur data show that PE38, CXCR1 promoter and bFGF 5ʹUTR in combination can be considered as a promising tool for killer gene therapy of breast cancer.

Characterization of the stemness potency of mammospheres isolated from the breast cancer cell lines.

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.

Abstract 5071: Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice

Abstract Palbociclib is the first CDK4/6-Cyclin D inhibitor approved by the FDA for ER+/Her2(erbB2)-breast cancer treatment. With the success of the drug in hormone-positive breast cancer, it is encouraging to explore more options of Palbociclib as an anticancer agent. erbB2 is a receptor tyrosine kinase (RTK) frequently overexpressed in breast cancer. The molecular hallmarks of erbB2+ breast cancer include the upregulation of cyclin D. The aim of this study was to explore the potential of Palbociclib as a preventive agent using MMTV-erbB2 transgenic mice, with a focus on the stemness of mammary epithelial cells (MECs) in the premalignant tissues. We first tested the in vivo effect of Palbociclib on tumor cells on MMTV-erbB2 mammary tumors using syngeneic tumor graft models. The animals bearing grafted tumors were treated with saline (control), low (75 mg/kg/day) and high (150 mg/kg/day) Palbociclib via oral gavage every 3 days for 29 days. We found that Palbociclib significantly inhibited tumor growth in a concentration dependent manner. To test the effect of Palbociclib on MEC stemness in premalignant mammary tissues, the animals at 9 wks of age were treated with saline, low and high (0, 75, 150 mg/kg/day) doses of Palbociclib every 3 days for 4 wks. We found the efficiency of both primary and secondary mammosphere formation of MECs from drug treated mice was significantly inhibited, suggesting the inhibition of mammary stemness in the premalignant tissues. Consistently, results from 3D cultures showed that colony numbers of the cells from drug treated mice were also lower than the control. Using CD24 and CD49f as markers, flow cytometry was performed to analyze the effect of Palbociclib on MEC subpopulations, including luminal, basal, and stromal populations. We showed that the percentage of luminal cells and putative mammary reconstitution units (MRUs) in drug treated mice was significantly inhibited. We also found that the percentage of CD61+/CD49+ cells, which are enriched with luminal progenitor cells and the origin of tumor initiation cells of this model, in Palbociclib treated tissues were also inhibited. The data suggest that Palbociclib induces MEC reprogramming in the premalignant tissues. Moreover, we found that the protein levels of pRb, E2F1, cyclin D1, c-myc, and cdc2 in cell cycle regulation, and pAKT, pERK, pmTOR, and p4EBP1 in the RTK pathway, pER, and DVL2 and LRP6 in the Wnt pathway were significantly downregulated in Palbociclib treated tissues, which was more evident in the high dose group. Taken together, our data indicate that Palbociclib inhibits MEC proliferation and stemness in the premalignant mammary tissues. Downregulation of cell cycle regulators and the signaling in ER, RTK, and Wnt pathway plays a critical role in this process. Our data support further investigation of Palbociclib in the prevention of ER+/ErbB2+ breast cancer. Citation Format: Amanda B. Parris, Yongxuan Liu, Zhikun Ma, Erin W. Howard, Xiaoshan Feng, Xiaohe Yang. Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5071.

Transcriptome-based identification of lovastatin as a breast cancer stem cell-targeting drug.

Breast cancer is a neoplastic disease with high morbidity and mortality in women worldwide. Breast cancer stem cells (CSCs) have a significant function in tumor growth, recurrence, and therapeutic resistance. Thus, CSCs have been pointed as targets of new therapies for breast cancer. Herein, we aimed to repurpose certain drugs as breast CSC-targeting agents. We compared a consensus breast CSC signature with the transcriptomic changes that were induced by over 1300 bioactive compounds using Connectivity Map. The effects of the selected drugs on SOX2 promoter transactivation, SOX2 expression, viability, clonogenicity, and ALDH activity in breast cancer cells were analyzed by luciferase assay, western blot, MTT assay, mammosphere formation assay, and ALDEFLUOR® test, respectively. Gene Set Enrichment Analysis (GSEA) was performed using the gene expression data from mammary tumors of mice that were treated with lovastatin. Five drugs (fasudil, pivmecillinam, ursolic acid, 16,16-dimethylprostaglandin E2, and lovastatin) induced signatures that correlated negatively with the query CSC signature. In vitro, lovastatin inhibited SOX2 promoter transactivation, and reduced the efficiency of mammosphere formation and the percentage of ALDH+ cells. Mevalonate mitigated the effects of lovastatin, suggesting that the targeting of CSCs by lovastatin was mediated by the inhibition of its reported target, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR). By GSEA, lovastatin downregulated genes that are involved in stemness and invasiveness in mammary tumors, corroborating our in vitro findings. Lovastatin is a breast CSC-targeting drug. The inhibition of HMGCR might develop new adjuvant therapeutic strategies for breast tumors.

Abstract P5-08-09: Tannic acid inhibited TGFβ-induced phenotype transition and mammosphere formation of breast cancer cells via modulation of NF-κB signaling

Abstract BACKGROUND: Cancer stem cells (CSCs) play a crucial role in tumor initiation, metastasis and recurrence, which are therefore regarded as promising therapeutic target. Aberrant NF-κB signaling has been identified in many cancers, and proven to promote CSC formation via several mechanisms, including an induction of epithelial-to-mesenchymal transition (EMT) of cancer cells. Tannic acid (C76H52O46, TA) was reported to inhibit the proliferation of cancer cells, however there was no data regarding the effect of TA on CSCs. The present study investigated the effects of TA on CSC formation, NF-κB signaling, and EMT in breast cancer cells. MATERIALS AND METHODS: The effect of TA on mammosphere formation and the activity of aldehyde dehydrogenase 1 (ALDH1) was investigated in MCF7 cells. IKK phosphorylation, nuclear translocation of p65 and the expression of IκBα and CSCs markers (CD44/CD24, ALDH1) were assessed by western blotting. Role of NF-κB signaling in EMT of MCF7 cells were evaluated using p65 siRNA (sip65) and NF-κB specific inhibitor (PDTC) or IKK inhibitor (Bay11-7082). The effect of TA on tumor growth was also examined in a mouse xenograft model established by subcutaneous implantation of MCF7 cells (1.5 x 107) in 6-week-old Balb/c nude mice with intraperitoneal injection of TGFβ (40 ng/kg, 3 times/week). In 4 weeks of intraperitoneal administration of TA (2 mg/kg, twice/week), tumor volume was measured with an evaluation of the alteration of CSC markers and NF-κB signaling. RESULTS: TA (10 μM) inhibited the formation and growth of mammosphere in MCF7 cells expressed as a decrease in mammosphere formation efficiency (MFE) and ALDH1 activity. An activation of NF-κB pathway was observed in MCF7-derived mammosphere shown as an up-regulation of p65, a degradation of IκBα and an increased IL-6. Blocking of NF-κB signaling by sip65, PDTC or Bay11-7082 resulted in a decrease in the MFE, the expression of ALDH1, and an increase in CD44high/CD24low ratio. TA alleviated the markers of NF-κB activation in MCF7-derived mammosphere. TGFβ (10 ng/mL)-induced EMT, increase in MFE and NF-κB activation was alleviated by TA. In murine xenograft model, tumor volume was decreased by TA with a decrease in CD44 expression and IKK phosphorylation in xenograft. CONCLUSIONS: The present study showed that TA inhibited mammosphere formation in breast cancer cells, which was associated with an alleviation of both NF-kB signaling and EMT. Moreover, TA treatment ameliorated tumor growth in an in-vivo mouse xenograft of breast cancer. These data suggest TA may serve as an effective therapeutic agent for breast cancer patients. Citation Format: Kim D-A, Ryu E-S, Kang H-J, Kang D-H. Tannic acid inhibited TGFβ-induced phenotype transition and mammosphere formation of breast cancer cells via modulation of NF-κB signaling [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-09.

Characterization of breast cancer stem cells isolated from 4T1 cell line cultured in vitro

Breast cancer is the most common cancer in women of which the main causes of death are its relapse and drug resistance. Recently, researchers clarified a small population of cancer cells holding the abilities of self-renewal, unlimited propagation responsible for tumor progression, metastasis, therapy resistance, and tumor recurrence. These cells are termed as cancer sterm cells (CSCs), possessing characteristics of stem cells. Thus, cancer stem cells have become to be a potential target for cancerous drug discovery. They can be used in a new research model for establishing novel therapy as well as sreening new compounds that target CSCs. In our studies, we isolated cancer stem cells from 4T1 cell line using serum-free medium and non-adherent culture surface. Those CSCs clones were formed after 10 culturing days from 1 initial cell. The clones could initiate subtumorsphere better than 4T1 cells themselves. There were 3 selected clones named as Đ3G5, Đ3C3 and Đ4E7 which had mammosphere formation efficiency over 30% (31.67%, 36.33%, and 35.33%, respectively). These clones showed the significant withstanding against ellipticine and/or doxorobicin stronger than that of 4T1 cells (P<0.05). The IC50 values (inhibition of cell growth at 50%) of elliticine was 0.89 ± 0.07 μg/mL and of doxorobicin was 0.58 ± 0.04 μg/mL toward 4T1 cell growth while those values were 2 - 3 folds higher in our isolated cancer stem cell clones (P<0.05). Those clones also exhibited strong expression of several typical genes of stem cells such as nanog and Sox2.

Hydroxytyrosol inhibits cancer stem cells and the metastatic capacity of triple-negative breast cancer cell lines by the simultaneous targeting of epithelial-to-mesenchymal transition, Wnt/β-catenin and TGFβ signaling pathways.

This study was aimed to determine the impact of hydroxytyrosol (HT), a minor compound found in olive oil, on breast cancer stem cells (BCSCs) and the migration capacity of triple-negative breast cancer (TNBC) cell lines through the alteration of epithelial-to-mesenchymal transition (EMT) and embryonic signaling pathways. BCSCs self-renewal was determined by the mammosphere-forming efficiency in SUM159PT, BT549, MDA-MB-231 and Hs578T TNBC cell lines. Flow cytometric analysis of CD44+/CD24-/low and aldehyde dehydrogenase positive (ALDH+) subpopulations, migration by the "wound healing assay", invasion and Western blot of EMT markers and TGFβ signaling were investigated in SUM159PT, BT549 and MDA-MB-231 cell lines. Wnt/β-catenin signaling was assessed by Western blot in BT549 cells expressing WNT1 and MDA-MB-231 cells. Changes in TGFβ activity was determined by SMAD Binding Element (SBE) reporter assay. HT reduced BCSCs self-renewal, ALDH+ (aldehyde dehydrogenase) and CD44+/CD24-/low subpopulations, tumor cell migration and invasion. Consistently, HT suppressed Wnt/β-catenin signaling by decreasing p-LRP6, LRP6, β-catenin and cyclin D1 protein expression and the EMT markers SLUG, ZEB1, SNAIL and VIMENTIN. Finally, HT inhibited p-SMAD2/3 and SMAD2/3 in SUM159PT, BT549 and MDA-MB-231 cells, what was correlated with a less TGFβ activity. In conclusion, we report for the first time the inhibitory role of HT on BCSCs and tumor cell migration by targeting EMT, Wnt/β-catenin and TGFβ signaling pathways. Our findings highlight the importance of the chemopreventive compound HT as a novel candidate to be investigated as an alternative targeted therapy for TNBC.

Activating Transcription Factor 4 Modulates TGFβ-Induced Aggressiveness in Triple-Negative Breast Cancer via SMAD2/3/4 and mTORC2 Signaling.

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling-tailored gene signature.Experimental Design: Patient survival data were determined by Kaplan-Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX).Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial-mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1-RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer.Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway-based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697-709. ©2018 AACR.

Abstract 276: Targeting cancer stem cells with chemopreventive agents for breast cancer prevention

Abstract Breast cancer accounts for one of the most common causes of cancer-related deaths in women. Despite the available treatment modalities to cure breast cancer, therapeutic resistance and recurrence remain major challenges, owing to the culminating evidence of the presence of cancer stem cell subpopulations in breast tumors. The purpose of the study is to identify chemopreventive and dietary compounds that effectively target cancer stem cells to inhibit breast cancer development and progression. The mammosphere culture system has been proven to enrich cancer stem-like cells, and cells from mammospheres are shown to be more tumorigenic in vivo. By culturing mammospheres from breast cancer cell lines, we have shown that 1,25 vitamin D3, a vitamin D3 analog, BXL0124, pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), tocopherols and triterpenoids (CDDO-Im) decreased the mammosphere forming efficiency (MFE) in a dose-dependent manner. Vitamin D compounds (1,25D3 and BXL0124) decreased the MFE in SUM159, MCF10DCIS and MCF-7 breast cancer cell lines (dose range 1-100 nM). Pterostilbene, a bioactive component of blueberries, effectively decreased the MFE in the MCF-7 cell line. The size of the mammospheres was found to be smaller with increasing concentrations (10-100 μM). Tocopherols significantly decreased estrogen-stimulated MFE in MCF-7 cells (1-10 μM). CDDO-Im, a synthetic triterpenoid derived from natural oleanolic acid, has also been shown to decrease MFE and sphere size in MCF-7 cells (50-100 nM). The effect on the shape of the spheres was also observed, suggesting the differentiation-inducing properties of these compounds. Taken together, we demonstrate that naturally occurring compounds as well as synthetic analogs derived from natural and dietary compounds significantly inhibit sphere forming efficiency and cancer stemness in breast cancer cells. Our study suggests that chemopreventive compounds, 1,25D3, BXL0124, pterostilbene, tocopherols, and CDDO-Im, are agents with the potential to target cancer stem-like cells to prevent breast cancer. Citation Format: Naing Lin Shan, Jeffrey Yang, Min Ji Bak, Joseph Wahler, Nanjoo Suh. Targeting cancer stem cells with chemopreventive agents for breast cancer prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 276.

Abstract 2883: Repurposing atovaquone, an antiprotozoal drug for management of breast cancer by inhibiting HER2/β-catenin signaling

Abstract Breast cancer is the second highest cause of cancer-related mortality in women. HER2 is an oncogene overexpressed in about 30% of breast cancer patients and β-catenin, a proto-oncogene plays a pivotal role in breast cancer metastasis and drug resistance leading to poor prognosis. In the current study, we evaluated the anti-cancer effects of an anti-protozoal drug, atovaquone against several breast cancer cell lines such as MCF-7, 4T1, HCC1806, CI66, SKBR3 and T47D. Our results showed that atovaquone treatment induced apoptosis as evaluated by PI/Annexin assay and cleavage of caspase-3 and PARP, and inhibited the growth of all the breast cancer cell lines tested. Similar observations were made in patient-derived cancer cell lines. In addition, atovaquone treatment significantly reduced the expression of HER2, β-catenin and its downstream molecules such as pGSK-3β, TCF-4, cyclin D1 and c-Myc in these cell lines. Treatment of HCC1806 cells with 5 µM atovaquone resulted in significant reduction in mammosphere forming efficiency (MFE) of breast cancer cells. Mammosphere-forming cells are known to exhibit cancer stem cell (CSC)-like properties together with drug resistance. Efficacy of atovaquone was evaluated in the in-vivo tumor model by orthotopic implantation of two highly aggressive 4T1 and CI66, triple negative breast cancer cells in the mammary fats of female mice. Our results demonstrate that oral administration of atovaquone suppressed the growth of CI66 and 4T1 tumors by 70% and 60% respectively. Atovaquone treated tumors exhibited reduced HER2, β-catenin and increased apoptosis when analyzed by western blot. We also developed resistance in 4T1 breast cancer cells towards paclitaxel, a first line chemotherapeutic agent for treatment of metastatic breast cancer. 4T1 paclitaxel resistant cells were orthotopically implanted in female balb/c mice and mice were treated with atovaquone for 20 days. Our results showed that atovaquone was able to suppress the growth of paclitaxel resistant tumors by 42%. Conclusively, our results indicate that atovaquone effectively reduces the growth of primary breast tumors as well as paclitaxel resistant tumors by inhibiting HER2/β-catenin signaling. Most importantly, atovaquone is already in clinical use with an established safety record therefore, any positive findings from our studies will prompt further clinical investigation into repurposing atovaquone for the treatment of advanced breast cancer patients. [Supported in part by R01 grant CA 129038 awarded by National Cancer Institute, NIH]. Citation Format: Nehal Gupta, Sanjay Srivastava. Repurposing atovaquone, an antiprotozoal drug for management of breast cancer by inhibiting HER2/β-catenin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2883.

PO-010 Dual chemotherapy and photodynamic therapy: a synergistic strategy to improve cancer treatment

IntroductionPhotodynamic therapy (PDT) is a clinical-approved option in several diseases characterised by uncontrolled cell proliferation, as cancer. PDT is recognised as a minimally invasive and toxic treatment. It is based on the administration of light activatable molecule known as photosensitizer (PS). After irradiation, a photochemical reaction between the PS and molecular oxygen leads to the generation of reactive oxygen species (ROS), which results in tumour regression.Nowadays different strategies are being introduced in order to enhance PDT effectiveness, such as combination of PDT with chemotherapy or improvement of PS features.Material and methodsA new combined PDT-chemotherapy comprising two drugs widespread in clinical research -the hydrophobic Zinc(II)-phthalocyanine (ZnPc) as PS and the common chemotherapeutic agent doxorubicin (DOX)- was tested in tumour cell lines and primary cells from patients affected by metastatic breast cancer. ZnPc was incorporated into nanoliposomes to increase its solubility and uptake into tumoral cells. This dual-therapy was also assayed in vivo (intravenously administrated) in a breast cancer xenograft model following tumour growth by luciferase activity.Results and discussionsMTT cytotoxicity assay showed that combination of both therapies remarkably increases the effectiveness of the treatment by inducing a synergistic cell death effect when compared to DOX or ZnPc monotherapy. In addition, annexin-V detection by flow cytometry, analysis of active caspase-3 and cytochrome c by immunofluorescence and time-lapse videomicroscopy corroborated a fine-tunable effect depending on light dose, leading to apoptotic or necrotic mechanism of cell death.Subcellular location visualised by fluorescence microscopy confirmed internalisation of both drugs. Using DCFH-DA probe, we demonstrate that a significant higher ROS generation into cells was the main cause of the synergistic effect of this combined treatment. Further, mammosphere formation efficiency assay (MFE) showed a reduced breast cancer stem cell activity in established cell line and primary cells obtained from patients, even using DOX at much lower concentration than clinical level. Finally, studies in human breast cancer xenografts indicated a high efficiency also in vivo.ConclusionAll these results provide novel and valuable information that contribute to consider chemophototherapy as a promising tool in current antitumoral treatments, potentially overcoming resistance to cancer chemotherapy and targeting cancer stem cells.

Mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines

The detection of recurrent mutations affecting the hormone binding domain (HBD) of estrogen receptor alpha (ERα/ESR1) in endocrine therapy-resistant and metastatic breast cancers has prompted interest in functional characterization of these genetic alterations. Here, we explored the role of HBD-ESR1 mutations in influencing the behavior of breast cancer stem cells (BCSCs), using various BC cell lines stably expressing wild-type or mutant (Y537 N, Y537S, D538G) ERα. Compared to WT-ERα clones, mutant cells showed increased CD44+/CD24- ratio, mRNA levels of stemness genes, Mammosphere Forming Efficiency (MFE), Self-Renewal and migratory capabilities. Mutant clones exhibited high expression of NOTCH receptors/ligands/target genes and blockade of NOTCH signaling reduced MFE and migratory potential. Mutant BCSC activity was dependent on ERα phosphorylation at serine 118, since its inhibition decreased MFE and NOTCH4 activation only in mutant cells. Collectively, we demonstrate that the expression of HBD-ESR1 mutations may drive BC cells to acquire stem cell traits through ER/NOTCH4 interplay. We propose the early detection of HBD-ESR1 mutations as a challenge in precision medicine strategy, suggesting the development of tailored-approaches (i.e. NOTCH inhibitors) to prevent disease development and metastatic spread in BC mutant-positive patients.