Subpopulation Of Breast Cancer Cells Research Articles

Investigation of L-carnitine effects on CD44+ cancer stem cells from MDA-MB-231 breast cancer cell line as anti-cancer therapy

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells, capable of metastasis, recurrence, and drug resistance in breast cancer patients. Therefore, targeting BCSCs appears to be a promising strategy for the treatment and prevention of breast cancer metastasis. Mounting evidence supports the fact that carnitine, a potent antioxidant, modulates various mechanisms by enhancing cellular respiration, inducing apoptosis, and reducing proliferation and inflammatory responses in tumor cells. The objective of this study was to investigate the impact of L-carnitine (LC) on the rate of proliferation and induction of apoptosis in CD44+ CSCs. To achieve this, the CD44+ cells were enriched using the Magnetic-activated cell sorting (MACS) isolation method, followed by treatment with LC at various concentrations. Flow cytometry analysis was used to determine cell apoptosis and proliferation, and western blotting was performed to detect the expression levels of proteins. Treatment with LC resulted in a significant decrease in the levels of p-JAK2, p-STAT3, Leptin receptor, and components of the leptin pathway. Moreover, CD44+ CSCs-treated cells with LC exhibited a reduction in the proliferation rate, accompanied by an increase in the percentage of apoptotic cells. Hence, it was concluded that LC could potentially influence the proliferation and apoptosis of CD44+ CSC by modulating the expression levels of specific protein.

Kruppel-like factor 8 regulates triple negative breast cancer stem cell-like activity.

Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically.

Cooperative NF-κB and Notch1 signaling promotes macrophage-mediated MenaINV expression in breast cancer

Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and frequently to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin regulatory protein Mena, encoded by the ENAH gene, that endows tumor cells with transendothelial migration activity, allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. In this study, we discovered the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer, we further determined that for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but it enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling activation and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease.

Abstract 3598: Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer

Abstract Metastasis is a multistep process that leads to the formation of clinically detectable tumor foci at distant organs and, frequently, to patient demise. Only a subpopulation of breast cancer cells within the primary tumor can disseminate systemically and cause metastasis. To disseminate, cancer cells must express MenaINV, an isoform of the actin-regulatory protein, Mena, encoded by the ENAH gene that endows tumor cells with transendothelial migration activity allowing them to enter and exit the blood circulation. We have previously demonstrated that MenaINV mRNA and protein expression is induced in cancer cells by macrophage contact. Here, we report on the discovery of the precise mechanism by which macrophages induce MenaINV expression in tumor cells. We examined the promoter of the human and mouse ENAH gene and discovered a conserved NF-κB transcription factor binding site. Using live imaging of an NF-κB activity reporter and staining of fixed tissues from mouse and human breast cancer we further determined that, for maximal induction of MenaINV in cancer cells, NF-κB needs to cooperate with the Notch1 signaling pathway. Mechanistically, Notch1 signaling does not directly increase MenaINV expression, but instead enhances and sustains NF-κB signaling through retention of p65, an NF-κB transcription factor, in the nucleus of tumor cells, leading to increased MenaINV expression. In mice, these signals are augmented following chemotherapy treatment and abrogated upon macrophage depletion. Targeting Notch1 signaling in vivo decreased NF-κB signaling and MenaINV expression in the primary tumor and decreased metastasis. Altogether, these data uncover mechanistic targets for blocking MenaINV induction that should be explored clinically to decrease cancer cell dissemination and improve survival of patients with metastatic disease. Citation Format: Camille L. Duran, George S. Karagiannis, Xiaoming Chen, Ved P. Sharma, David Entenberg, John S. Condeelis, Maja H. Oktay. Cooperative NF-KappaB and Notch1 signaling promotes a macrophage-mediated metastatic phenotype in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3598.

KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells

One of the main challenges in treating breast cancer is the intra‐tumor heterogeneity, which, partly, is maintained and promoted by a sub‐population of breast cancer cells called breast cancer stem‐like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self‐renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O‐GlcNAc transferase (OGT). OGT is responsible to adding O‐GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP‐GlcNAc – final product of the hexosamine biosynthetic pathway. This modification is termed O‐GlcNAcylation and both OGT and O‐GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O‐GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA‐seq analysis of BCSC‐enriched mammospheres with OGT overexpression revealed Kruppel‐like‐factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O‐GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O‐GlcNAcylation, suggesting a possible feed‐forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple‐negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem‐like cells population.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Abstract 3084: KLF8 is a required downstream effector of OGT/O-GlcNAcylation in regulating breast cancer stem-like cells

Abstract One of the main challenges in treating breast cancer is the intra-tumor heterogeneity, which, partly, is maintained and promoted by a sub-population of breast cancer cells called breast cancer stem-like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self-renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O-GlcNAc transferase (OGT). OGT is responsible to adding O-GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP-GlcNAc - final product of the hexosamine biosynthetic pathway. This modification is termed O-GlcNAcylation and both OGT and O-GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O-GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA-seq analysis of BCSC-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we showed that KLF8 expression in breast cancer cells is regulated by OGT at protein and mRNA level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and CD44H/CD24L BCSC population. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O-GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, breast cancer patient survival was lower with high KLF8 or OGT levels. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer[LM1] . Together, our results suggested that KLF8 is a downstream target of OGT/O-GlcNAcylation in promoting breast cancer stem-like cells population. Citation Format: Giang Le Minh, Neha M. Akella, Tejsi Dhameliya, Mauricio J. Reginato. KLF8 is a required downstream effector of OGT/O-GlcNAcylation in regulating breast cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3084.

Phenotypic Heterogeneity and Metastasis of Breast Cancer Cells.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

Cell Cytoskeleton and Stiffness Are Mechanical Indicators of Organotropism in Breast Cancer.

Simple SummaryCancer cell dissemination exhibits organ preference or organotropism. Although the influence of intrinsic biochemical factors on organotropism has been intensely studied, little is known about the roles of mechanical properties of metastatic cancer cells. Our study suggests that there may be a correlation between cell cytoskeleton/stiffness and organotropism. We find that the cytoskeleton and stiffness of breast cancer cell subpopulations with different metastatic preference match the mechanics of the metastasized organs. The modification of cell cytoskeleton significantly influences the organotropism-related gene expression pattern and mechanoresponses on soft substrates which mimic brain tissue stiffness. These findings highlight the key role of cell cytoskeleton in specific organ metastasis, which may not only reflect but also impact the metastatic organ preference.Tumor metastasis involves the dissemination of tumor cells from the primary lesion to other organs and the subsequent formation of secondary tumors, which leads to the majority of cancer-related deaths. Clinical findings show that cancer cell dissemination is not random but exhibits organ preference or organotropism. While intrinsic biochemical factors of cancer cells have been extensively studied in organotropism, much less is known about the role of cell cytoskeleton and mechanics. Herein, we demonstrate that cell cytoskeleton and mechanics are correlated with organotropism. The result of cell stiffness measurements shows that breast cancer cells with bone tropism are much stiffer with enhanced F-actin, while tumor cells with brain tropism are softer with lower F-actin than their parental cells. The difference in cellular stiffness matches the difference in the rigidity of their metastasized organs. Further, disrupting the cytoskeleton of breast cancer cells with bone tropism not only elevates the expressions of brain metastasis-related genes but also increases cell spreading and proliferation on soft substrates mimicking the stiffness of brain tissue. Stabilizing the cytoskeleton of cancer cells with brain tropism upregulates bone metastasis-related genes while reduces the mechanoadaptation ability on soft substrates. Taken together, these findings demonstrate that cell cytoskeleton and biophysical properties of breast cancer subpopulations correlate with their metastatic preference in terms of gene expression pattern and mechanoadaptation ability, implying the potential role of cell cytoskeleton in organotropism.

Nucleotide de novo synthesis increases breast cancer stemness and metastasis via cGMP-PKG-MAPK signaling pathway.

Metabolic reprogramming to fulfill the biosynthetic and bioenergetic demands of cancer cells has aroused great interest in recent years. However, metabolic reprogramming for cancer metastasis has not been well elucidated. Here, we screened a subpopulation of breast cancer cells with highly metastatic capacity to the lung in mice and investigated the metabolic alternations by analyzing the metabolome and the transcriptome, which were confirmed in breast cancer cells, mouse models, and patients’ tissues. The effects and the mechanisms of nucleotide de novo synthesis in cancer metastasis were further evaluated in vitro and in vivo. In our study, we report an increased nucleotide de novo synthesis as a key metabolic hallmark in metastatic breast cancer cells and revealed that enforced nucleotide de novo synthesis was enough to drive the metastasis of breast cancer cells. An increased key metabolite of de novo synthesis, guanosine-5'-triphosphate (GTP), is able to generate more cyclic guanosine monophosphate (cGMP) to activate cGMP-dependent protein kinases PKG and downstream MAPK pathway, resulting in the increased tumor cell stemness and metastasis. Blocking de novo synthesis by silencing phosphoribosylpyrophosphate synthetase 2 (PRPS2) can effectively decrease the stemness of breast cancer cells and reduce the lung metastasis. More interestingly, in breast cancer patients, the level of plasma uric acid (UA), a downstream metabolite of purine, is tightly correlated with patient’s survival. Our study uncovered that increased de novo synthesis is a metabolic hallmark of metastatic breast cancer cells and its metabolites can regulate the signaling pathway to promote the stemness and metastasis of breast cancer.

Fzd2 Contributes to Breast Cancer Cell Mesenchymal-Like Stemness and Drug Resistance.

Cancer cell stemness is responsible for cancer relapse, distal metastasis, and drug resistance. Here we identified that Frizzled 2 (Fzd2), one member of Wnt receptor Frizzled family, induced human breast cancer (BC) cell stemness via noncanonical Wnt pathways. Fzd2 was overexpressed in human BC tissues, and Fzd2 overexpression was associated with an unfavorable outcome. Fzd2 knockdown (KD) disturbed the mesenchymal-like phenotype, migration, and invasion of BC cells. Moreover, Fzd2 KD impaired BC cell mammosphere formation, reduced Lgr5+ BC cell subpopulation, and enhanced sensitivity of BC cells to chemical agents. Mechanistically, Fzd2 modulated and bound with Wnt5a/b and Wnt3 to activate several oncogenic pathways such as interleukin-6 (IL-6)/Stat3, Yes-associated protein 1 (Yap1), and transforming growth factor-β1 (TGF-β1)/Smad3. These data indicate that Fzd2 contributes to BC cell mesenchymal-like stemness; targeting Fzd2 may inhibit BC recurrence, metastasis, and chemoresistance.

Capicua restricts cancer stem cell-like properties in breast cancer cells.

Cancer stem cells (CSCs) play a central role in cancer initiation, progression, therapeutic resistance, and recurrence in patients. Here we present Capicua (CIC), a developmental transcriptional repressor, as a suppressor of CSC properties in breast cancer cells. CIC deficiency critically enhances CSC self-renewal and multiple CSC subpopulations of breast cancer cells without altering their growth rate or invasiveness. Loss of CIC relieves repression of ETV4 and ETV5 expression, consequently promoting self-renewal capability, EpCAM+/CD44+/CD24low/- expression, and ALDH activity. In xenograft models, CIC deficiency significantly increases CSC frequency and drives tumor initiation through derepression of ETV4. Consistent with the experimental data, the CD44high/CD24low CSC-like feature is inversely correlated with CIC levels in breast cancer patients. We also identify SOX2 as a downstream target gene of CIC that partly promotes CSC properties. Taken together, our study demonstrates that CIC suppresses breast cancer formation via restricting cancer stemness and proposes CIC as a potential regulator of stem cell maintenance.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

PD-L1 is overexpressed on breast cancer stem cells through notch3/mTOR axis

ABSTRACT The T-cell inhibitory molecule PD-L1 is expressed on a fraction of breast cancer cells. The distribution of PD-L1 on the different subpopulations of breast cancer cells is not well-defined. Our aim was to study the expression level of PD-L1 on breast cancer stem-like (CSC-like) cells and their differentiated-like counterparts. We used multi-parametric flow cytometry to measure PD-L1 expression in different subpopulations of breast cancer cells. Pathway inhibitors, quantitative immunofluorescence, cell sorting, and western blot were used to investigate the underlying mechanism of PD-L1 upregulation in CSC-like cells. Specifically, PD-L1 was overexpressed up to three folds on breast CSC-like cells compared with more differentiated-like cancer cells. Functional in vitro and in vivo assays show higher stemness of PD-L1hi as compared with PD-L1lo cells. Among different pathways examined, PD-L1 expression on CSCs was partly dependant on Notch, and/or PI3K/AKT pathway activation. The effect of Notch inhibitors on PD-L1 overexpression in CSCs was completely abrogated upon mTOR knockdown. Specific knockdown of different Notch receptors shows Notch3 as a mediator for PD-L1 overexpression on CSCs and important for maintaining their stemness. Indeed, Notch3 was found to be overexpressed on PD-L1hi cells and specific knockdown of Notch3 abolished the effect of notch inhibitors and ligands on PD-L1 expression as well as mTOR activation. Our data demonstrated that overexpression of PD-L1 on CSCs is partly mediated by the notch pathway through Notch3/mTOR axis. We propose that these findings will help in a better design of anti-PD-L1 combination therapies to treat breast cancer effectively.

Analysis of the Density and Distribution of HER2 In Breast Cancer Cell Subpopulations and Their Response to Anti-Cancer Drugs on the Single Cell Level Using Liquid-Phase Electron Microscopy

Journal Article Analysis of the Density and Distribution of HER2 In Breast Cancer Cell Subpopulations and Their Response to Anti-Cancer Drugs on the Single Cell Level Using Liquid-Phase Electron Microscopy Get access Patricia Blach, Patricia Blach INM – Leibniz Institute for New Materials, D-66123 Saarbrücken, GermanyDepartment of Physics, Saarland University, D-66123 Saarbrücken, Germany Corresponding author: Patricia.Blach@leibniz-inm.de Search for other works by this author on: Oxford Academic Google Scholar Florian Weinberg, Florian Weinberg INM – Leibniz Institute for New Materials, D-66123 Saarbrücken, Germany Search for other works by this author on: Oxford Academic Google Scholar Diana Peckys, Diana Peckys Department of Biophysics, Saarland University, D-66421 Homburg, Germany Search for other works by this author on: Oxford Academic Google Scholar Niels de Jonge Niels de Jonge INM – Leibniz Institute for New Materials, D-66123 Saarbrücken, GermanyDepartment of Physics, Saarland University, D-66123 Saarbrücken, Germany Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 25, Issue S2, 1 August 2019, Pages 1192–1193, https://doi.org/10.1017/S143192761900669X Published: 01 August 2019

Abstract 1989: Breast cancer-derived exosomes modulate the endocytic pathway in brain endothelial cells

Abstract Breast cancer brain metastasis is associated with high mortality and has shown a rising trend in incidence during the recent years. Given the rapid progression of brain metastasis, elucidation of the early events that lead to brain metastasis will pave the way to identifying potential diagnostic and therapeutic targets for early intervention. To explore the early mechanisms of breast cancer brain metastasis, we focused on the role of breast cancer-derived exosomes in this process. While extensive studies have explored the role of tumor-derived exosomes in tumor progression, the current understanding of the contribution of tumor-derived exosomes to brain metastasis remains limited. Using a mouse model of brain metastasis, we showed that pre-treatment of mice with exosomes derived from the brain-seeking MDA-MB-231 breast cancer cell line (Br-Ex) increases brain metastasis growth. This facilitation of brain metastasis was not observed with exosomes derived from the parental MDA-MB-231 cells (P-Ex). It is widely acknowledged that during early stages of brain metastasis, tumor cells grow along the brain vasculature, the blood brain barrier (BBB). To understand the mechanisms of the exosome-derived facilitation of brain metastasis, we studied the interactions between exosomes and the BBB. Using state-of-the-art models of the BBB and high-resolution microscopy, we have identified, for the first time, the mechanisms by which Br-Ex modulate the endocytic pathway in brain endothelial cells to decrease exosome degradation. Interestingly, our mechanistic studies showed that these effects are induced through the transfer of exosomal miRNAs enriched in Br-EX. Moreover, we have shown that Br-Ex can exclusively change the expression of integrins in brain endothelial cells in a way that it could alter the microenvironment around the BBB in favor of metastatic tumor cells. These findings indicate that exosomes derived from a brain-seeking subpopulation of breast cancer cells can exclusively modify the physiological regulation of the BBB at multiple levels to accelerate metastatic growth in the brain microenvironment. Taken together, these studies increase our understanding of the early events that facilitate brain metastasis and provide multiple potential targets for diagnostic and therapeutic applications in brain metastasis. (This work was supported by the Breast Cancer Research Foundation and NIH R01CA185530.) Citation Format: Golnaz Morad, Christopher V. Carman, Marsha A. Moses. Breast cancer-derived exosomes modulate the endocytic pathway in brain endothelial cells [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 1989.

Analysis of Breast Cancer Cell Subpopulations for the Expression and Distribution of HER2 and their Response to Anti-Cancer Drugs Using Electron Microscopy

Analysis of Breast Cancer Cell Subpopulations for the Expression and Distribution of HER2 and their Response to Anti-Cancer Drugs Using Electron Microscopy

New generation CPPs show distinct selectivity for cancer and noncancer cells.

In the last three decades, many new cell-penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini-crotamine and mini-maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV-TAT(47-57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N-terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG-peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image-guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.

Abstract 144: Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells

Abstract Many patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumor relapse and metastasis due to the resistance to conventional chemotherapy. These tumor recurrences have been attributed to the presence of breast cancer stem cells (BCSCs), a subpopulation of breast cancer cells that possesses stem-like properties and displays resistance to chemotherapy. Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. We generated chemotherapy-resistant TNBC cells through cyclic treatments with paclitaxel (pac). The pac-resistant cells displayed increased self-renewal potential compared to the parental TNBC cells, higher percentage BCSCs and increased phosphorylation of Src kinase at Tyr416. After performing a kinase library screening on BCSCs in chemotherapy-resistant cells, we identified dasatinib, a Src kinase family inhibitor, potently reduced BCSC self-renewal and two BCSC populations (ALDH+ and CD24lowCD44high) as well as phosphor-Src. We also demonstrated dasatinib to block pac-induced BCSC enrichment and Src activation in the parental TNBC cells. Interestingly, we found that dasatinib induced an epithelial differentiation of pac-resistant cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the proportion of BCSCs and their self-renewal capacity but also synergistically reduced cell viability of pac-resistant cells. In vivo studies further demonstrated the effectiveness of dasatinib/paclitaxel combination treatment in inhibiting breast tumor growth. All these data suggest that dasatinib is a promising anti-BCSC agent and can be used in combination with paclitaxel to overcome chemotherapy resistance in TNBCs. Citation Format: Jun Tian, Chieh Lo, Fatmah Al Raffa, Meiou Dai, Jean-Jacques Lebrun. Dasatinib enhances the effects of paclitaxel on chemotherapy-resistant breast cancer through targeting breast cancer stem cells [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 144.

Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells that have been proposed to be a primary cause of failure of therapies, including ionizing radiation (IR). Their embryonic stem-like signature is associated with poor clinical outcome. In the present study, the function of octamer-binding transcription factor 4 (Oct4), an embryonic stem cell factor, in the resistance of BCSCs to IR was investigated. Mammosphere cells exhibited increased expression of stemness-associated genes, including Oct4 and sex-determining region Y-box 2 (Sox2), and were more resistant to IR compared with serum-cultured monolayer cells. IR-resistant MCF7 cells also exhibited significantly increased expression of Oct4. To investigate the possible involvement of Oct4 in IR resistance of breast cancer cells, cells were transfected with Oct4. Ectopic expression of Oct4 increased the clonogenic survival of MCF7 cells following IR, which was reversed by treatment with small interfering RNA (siRNA) targeting Oct4. Oct4 expression decreased phosphorylated histone H2AX (γ-H2AX) focus formation and suppressed IR-induced premature senescence in these cells. Mammosphere, IR-resistant and Oct4-overexpressing MCF7 cells exhibited enhanced phosphorylation of signal transducer and activation of transcription 3 (STAT3) (Tyr705) and inhibitor of nuclear factor κB (NF-κB), and blockade of these pathways with siRNA against STAT3 and/or specific inhibitors of STAT3 and NF-κB significantly increased IR-induced senescence. Secretome analysis revealed that Oct4 upregulated interleukin 24 (IL-24) expression through STAT3 and NF-κB signaling, and siRNA against IL-24 increased IR-induced senescence, whereas recombinant human IL-24 suppressed it. The results of the present study indicated that Oct4 confers IR resistance on breast cancer cells by suppressing IR-induced premature senescence through STAT3- and NF-κB-mediated IL-24 production.