Breast Cancer-initiating Cells Research Articles

MAP7D3, a novel prognostic marker for triple-negative breast cancer, drives cell invasiveness and cancer-initiating cell properties to promote metastatic progression

BackgroundPatients with triple-negative breast cancer (TNBC) tend to develop visceral metastasis within five years, making them the most challenging BC patients to treat. The MAP7 protein family is a group of microtubule-binding proteins with a well-known role in microtubule-related cell migration, but its role in metastasis-related properties of TNBC remains unclear.MethodsqRT-PCR and western blot were used to validate mRNA and protein expression of the MAP7 family in the isogenic pairs of TNBC cell lines with low and high metastasis potential. Functional characterization of MAP7D3 was carried out using cell-based and mouse models. The clinical association between MAP7D3 and TNBC was established using datasets in the public domain.ResultsMAP7D3 expression was consistently upregulated in the metastatic subline IV2 and 468-LN at both mRNA and protein levels. Knockdown of MAP7D3 inhibited the 3D colony-forming ability, cell migration, and invasion ability of IV2 and 468-LN, indicating its significant contribution to the metastasis phenotypes. Mechanistically, inhibition of MAP7D3 could significantly increase the sensitivity of metastatic TNBC cells to docetaxel and gemcitabine treatment by reducing the expression of proteins related to breast cancer-initiating cells (BCICs) and drug resistance, as well as suppressing the activity of Rac1. The animal study showed that the depletion of MAP7D3 drastically reduced TNBC tumor growth and impaired the metastatic capability of TNBC cells. Elevated expression of MAP7D3 was found in the metastatic lymph nodes and was significantly associated with advanced stage and higher grade TNBC. Moreover, MAP7D3 expression was significantly correlated with the TNBC population, and its high expression was significantly associated with lymph node metastasis and poor survival outcomes of patients with TNBC.ConclusionOur study indicates that targeting MAP7D3 could be a promising therapeutic strategy for addressing the progression of TNBC, and MAP7D3 may serve as a novel predictive biomarker for the survival outcomes of triple-negative breast cancer.

Abstract 397: The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

Abstract Among women worldwide, breast cancer has the highest incidence and is the leading cause of cancer-related death. Patients with the triple-negative breast cancer (TNBC) subtype have an inferior prognosis in comparison to other breast cancers because the current therapies do not facilitate long-lasting responses. Thus, there is a critical need for novel therapeutic strategies that induce stronger responses. In our previous research, we discovered that augmenting the concentration of extracellular ATP (eATP) greatly enhances the chemotherapeutic response of TNBC cell lines by activating purinergic receptors (P2RXs), leading to cell death through the induction of non-selective membrane permeability. However, eATP levels are limited by several classes of extracellular ATPases. One endogenous molecule of interest that can inhibit multiple classes of extracellular ATPases is heparan sulfate. Heparan sulfate itself is degraded by heparanase, an enzyme that is known to be highly expressed in various cancers, including breast cancer. Heparan sulfate has previously been shown to regulate several cancer-related processes such as fibroblast growth factor signaling, neoangiogenesis by sequestering vascular endothelial growth factors in the extracellular matrix, hedgehog signaling and cell adhesion. In this project, we identified an additional mechanism for a tumor suppressor role of heparan sulfate: inhibition of extracellular ATPases, leading to augmented levels of eATP. Several heparanase inhibitors have been previously identified, including OGT 2115, suramin, PI-88, and PG 545. We hypothesized that heparanase inhibitors would augment eATP concentrations in TNBC by increasing heparan sulfate in the tumor microenvironment, resulting in increased cell death in response to chemotherapy. We treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with increasing concentrations of the chemotherapeutic agent paclitaxel in the presence of heparan sulfate and/or OGT 2115 with eATP content and cell viability being assessed. Moreover, to confirm that the effects of OGT 2115 are mediated through eATP, we utilized specific antagonists to the purinergic receptors P2RX4 and P2RX7. In addition, the protein expression of heparanase was compared in all the examined cell lines via Western blot analysis. We also evaluated the effects of this combination on the breast cancer-initiating cell population in the treated cells using flow cytometry and tumorsphere formation efficiency assays. These results demonstrate that inhibiting the degradation of heparan sulfate in the tumor microenvironment enhances the susceptibility of TNBC cell lines to chemotherapy by augmenting extracellular ATP concentrations. This strategy could potentially be used to induce deeper and more durable responses in triple-negative breast cancer patients. Citation Format: Jasmine M. Manouchehri, Jharna Datta, Mathew A. Cherian. The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative 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 397.

NSD3-Induced Methylation of H3K36 Activates NOTCH Signaling to Drive Breast Tumor Initiation and Metastatic Progression

Histone methyltransferase NSD3 is frequently dysregulated in human cancers, yet the epigenetic role of NSD3 during cancer development remains elusive. Here we report that NSD3-induced methylation of H3K36 is crucial for breast tumor initiation and metastasis. In patients with breast cancer, elevated expression of NSD3 was associated with recurrence, distant metastasis, and poor survival. In vivo, NSD3 promoted malignant transformation of mammary epithelial cells, a function comparable to that of HRAS. Furthermore, NSD3 expanded breast cancer-initiating cells and promoted epithelial-mesenchymal transition to trigger tumor invasion and metastasis. Mechanistically, the long isoform (full-length transcript) of NSD3, but not its shorter isoform lacking a catalytic domain, cooperated with EZH2 and RNA polymerase II to stimulate H3K36me2/3-dependent transactivation of genes associated with NOTCH receptor cleavage, leading to nuclear accumulation of NICD and NICD-mediated transcriptional repression of E-cadherin. Furthermore, mice harboring primary and metastatic breast tumors with overexpressed NSD3 showed sensitivity to NOTCH inhibition. Together, our findings uncover the critical epigenetic role of NSD3 in the modulation of NOTCH-dependent breast tumor progression, providing a rationale for targeting the NSD3-NOTCH signaling regulatory axis in aggressive breast cancer. SIGNIFICANCE: This study demonstrates the functional significance of histone methyltransferase NSD3 in epigenetic regulation of breast cancer stemness, EMT, and metastasis, suggesting NSD3 as an actionable therapeutic target in metastatic breast cancer.

Eradicating the Roots: Advanced Therapeutic Approaches Targeting Breast Cancer Stem Cells

Accumulating evidences have demonstrated that the existence of breast cancer-initiating cells, which drives the original tumorigenicity, local invasion and migration propensity of breast cancer. These cells, termed as breast cancer stem cells (BCSCs), possess properties including self-renewal, multidirectional differentiation and proliferative potential, and are believed to play important roles in the intrinsic drug resistance of breast cancer. One of the reasons why BCBCs cause difficulties in breast cancer treating is that BCBCs can control both genetic and non-genetic elements to keep their niches safe and sound, which allows BCSCs for constant self-renewal and differentiation. Therapeutic strategies designed to target BCSCs may ultimately result in effective interventions for the treatment of breast cancer. Novel strategies including nanomedicine, oncolytic virus therapy, immunotherapy and induced differentiation therapy are emerging and proved to be efficient in anti-BCSCs therapy. In this review, we summarized breast tumor biology and the current challenges of breast cancer therapies, focused on breast cancer stem cells, and introduced promising therapeutic strategies targeting BCSCs.

Serum erythropoietin levels, breast cancer and breast cancer-initiating cells

BackgroundCancer is frequently associated with tumor-related anemia, and many chemotherapeutic agents impair hematopoiesis, leading to impaired quality of life for affected patients. The use of erythropoiesis-stimulating agents has come under scrutiny after prospective clinical trials using recombinant erythropoietin to correct anemia reported increased incidence of thromboembolic events and cancer-related deaths. Furthermore, previous preclinical reports indicated expansion of the pool of breast cancer-initiating cells when erythropoietin was combined with ionizing radiation.MethodsUsing four established breast cancer cell lines, we test the effects of recombinant human erythropoietin and the number of breast cancer-initiating cells in vitro and in vivo and study if recombinant human erythropoietin promotes the phenotype conversion of non-tumorigenic breast cancer cells into breast cancer-initiating cells. In a prospective study, we evaluate whether elevated endogenous serum erythropoietin levels correlate with increased numbers of tumor-initiating cells in a cohort of breast cancer patients who were scheduled to undergo radiation treatment.ResultsOur results indicate that recombinant erythropoietin increased the number of tumor-initiating cells in established breast cancer lines in vitro. Irradiation of breast cancer xenografts caused a phenotype conversion of non-stem breast cancer cells into induced breast cancer-initiating cells. This effect coincided with re-expression of the pluripotency factors c-Myc, Sox2, and Oct4 and was enhanced by recombinant erythropoietin. Hemoglobin levels were inversely correlated with serum erythropoietin levels, and the latter were correlated with disease stage. However, tumor sections revealed a negative correlation between serum erythropoietin levels and the number of ALDH1A3-positive cells, a marker for breast cancer-initiating cells.ConclusionsWe conclude that physiologically slow-rising serum erythropoietin levels in response to tumor-related or chemotherapy-induced anemia, as opposed to large doses of recombinant erythropoietin, do not increase the pool of breast cancer-initiating cells.

Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer

The lack of a molecular target in triple-negative breast cancer (TNBC) makes it one of the most challenging breast cancers to treat. Radiation therapy (RT) is an important treatment modality for managing breast cancer; however, we previously showed that RT can also reprogram a fraction of the surviving breast cancer cells into breast cancer-initiating cells (BCICs), which are thought to contribute to disease recurrence. In this study, we characterize mebendazole (MBZ) as a drug with potential to prevent the occurrence of radiation-induced reprogramming and improve the effect of RT in patients with TNBC. A high-throughput screen was used to identify drugs that prevented radiation-induced conversion of TNBC cells into cells with a cancer-initiating phenotype and exhibited significant toxicity toward TNBC cells. MBZ was one of the drug hits that fulfilled these criteria. In additional studies, we used BCIC markers and mammosphere-forming assays to investigate the effect of MBZ on the BCIC population. Staining with propidium iodide, annexin-V, and γ-H2AX was used to determine the effect of MBZ on cell cycle, apoptosis, and double-strand breaks. Finally, the potential for MBZ to enhance the effect of RT in TNBC was evaluated invitro and invivo. MBZ efficiently depletes the BCIC pool and prevents the ionizing radiation-induced conversion of breast cancer cells into therapy-resistant BCICs. In addition, MBZ arrests cells in the G2/M phase of the cell cycle and causes double-strand breaks and apoptosis. MBZ sensitizes TNBC cells to ionizing radiation invitro and invivo, resulting in improved tumor control in a human xenograft model of TNBC. The data presented in this study support the repurposing of MBZ as a combination treatment with RT in patients with TNBC.

Abstract A77: Cripto-1 vaccination elicits protective immune response to metastatic breast cancer and breast cancer stem cells

Abstract Metastatic breast cancer is a fatal disease responding poorly to classical treatments. Cancer vaccines targeting antigens expressed by metastatic breast cancer and cancer stem cells (CSC) have the potential to become potent anti-cancer therapies. Cripto-1 is an onco-fetal protein frequently overexpressed in invasive breast cancer and cancer-initiating cells. It is involved in cellular processes such as cell migration, epithelial-mesenchymal transition, and invasion, hallmarks of aggressive cancers. In this study we explored the potential of a Cripto-1 encoding DNA vaccination to target metastatic breast cancer and breast cancer stem cells in preclinical models. Mice were vaccinated with a DNA vaccine encoding for mouse Cripto-1 (mCR) prior tumor challenge. In the 4T1 metastatic breast cancer model vaccination against mCR resulted in reduced primary tumor growth and significantly reduced metastatic burden in the lung. We further identified Cripto-1 specific antibodies in this model generated by the vaccination. TUBO is a Cripto-1 negative breast cancer cell line and vaccination against mCR in the transplantable BALB/c TUBO model did not result in protection. We show that spheroid culture of TUBO results in up-regulation of Cripto-1 on the cell surface. In addition, TUBO spheres display cancer stem cell phenotype. Consequently, tumor progression was inhibited in anti-mCR vaccinated mice challenged with TUBO spheres but not in control vaccinated mice. Our data indicates that DNA vaccination against Cripto-1 results in a protective immune response against mCR expressing tumors. Targeting Cripto-1 by vaccination is a promising potential immunotherapy for treatment of metastatic breast cancer. Citation Format: Kristina Witt, Maarten Alexander Ligtenberg, Laura Conti, Stefania Lanzardo, Roberto Ruiu, Helena Tufvesson-Stiller, Jeanette Ostling, Charlotte Rolny, Alvaro Lladser, Andreas Lundqvist, Federica Cavallo, Rolf Kiessling. Cripto-1 vaccination elicits protective immune response to metastatic breast cancer and breast cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A77.

High-mobility group box 1 released by autophagic cancer-associated fibroblasts maintains the stemness of luminal breast cancer cells.

Cancer stem cells/cancer-initiating cells (CICs) and their microenvironmental niche play a vital role in malignant tumour recurrence and metastasis. Cancer-associated fibroblasts (CAFs) are major components of the niche of breast cancer-initiating cells (BCICs), and their interactions may profoundly affect breast cancer progression. Autophagy has been considered to be a critical process for CIC maintenance, but whether it is involved in the cross-talk between CAFs and CICs to affect tumourigenesis and pathological significance has not been determined. In this study, we found that the presence of CAFs containing high levels of microtubule-associated protein 1 light chain 3 (LC3II), a marker of autophagosomes, was associated with more aggressive luminal human breast cancer. CAFs in human luminal breast cancer tissues with high autophagy activity enriched BCICs with increased tumourigenicity. Mechanistically, autophagic CAFs released high-mobility group box 1 (HMGB1), which activated its receptor, Toll-like receptor (TLR) 4, expressed by luminal breast cancer cells, to enhance their stemness and tumourigenicity. Furthermore, immunohistochemistry of 180 luminal breast cancers revealed that high LC3II/TLR4 levels predicted an increased relapse rate and a poorer prognosis. Our findings demonstrate that autophagic CAFs play a critical role in promoting the progression of luminal breast cancer through an HMGB1-TLR4 axis, and that both autophagy in CAFs and TLR4 on breast cancer cells constitute potential therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

BRCA1 haploinsufficiency cell-autonomously activates RANKL expression and generates denosumab-responsive breast cancer-initiating cells.

Denosumab, a monoclonal antibody to the receptor activator of nuclear factor-κB ligand (RANKL), might be a novel preventative therapy for BRCA1-mutation carriers at high risk of developing breast cancer. Beyond its well-recognized bone-targeted activity impeding osteoclastogenesis, denosumab has been proposed to interfere with the cross-talk between RANKL-producing sensor cells and cancer-initiating RANK+ responder cells that reside within premalignant tissues of BRCA1-mutation carriers. We herein tested the alternative but not mutually exclusive hypothesis that BRCA1 deficiency might cell-autonomously activate RANKL expression to generate cellular states with cancer stem cell (CSC)-like properties. Using isogenic pairs of normal-like human breast epithelial cells in which the inactivation of a single BRCA1 allele results in genomic instability, we assessed the impact of BRCA1 haploinsufficiency on the expression status of RANK and RANKL. RANK expression remained unaltered but RANKL was dramatically up-regulated in BRCA1mut/+ haploinsufficient cells relative to isogenic BRCA1+/+ parental cells. Neutralizing RANKL with denosumab significantly abrogated the ability of BRCA1 haploinsufficient cells to survive and proliferate as floating microtumors or “mammospheres” under non-adherent/non-differentiating conditions, an accepted surrogate of the relative proportion and survival of CSCs. Intriguingly, CSC-like states driven by epithelial-to-mesenchymal transition or HER2 overexpression traits responded to some extent to denosumab. We propose that breast epithelium-specific mono-allelic inactivation of BRCA1 might suffice to cell-autonomously generate RANKL-addicted, denosumab-responsive CSC-like states. The convergent addiction to a hyperactive RANKL/RANK axis of CSC-like states from genetically diverse breast cancer subtypes might inaugurate a new era of cancer prevention and treatment based on denosumab as a CSC-targeted agent.

Membrane proteome functional characterization of breast cancer-initiating cells subjected to bone morphogenetic protein signaling inhibition by dorsomorphin

In this study, A17 cells, which are an invasive mesenchymal cell line with cancer stem cell properties, were exploited for the study of the role of bone morphogenetic protein pathways in cancer-initiating cells employing a proteomics-based approach. A17 cells were treated with the bone morphogenetic protein signaling inhibitor dorsomorphin for 3 days. After that, subcellular fractionation of cell samples was performed and the membrane fraction analyzed by shotgun proteomics. The extracted membrane proteins were enzymatically digested and the resulting peptide mixture was analyzed by nano liquid chromatography coupled to tandem mass spectrometry and relative label-free quantitation. Protein profiles of A17 membrane fractions before and after dorsomorphin treatment were compared, and further mined by Gene Ontology search. The protein profile of untreated A17 samples correlated with the mesenchymal phenotype, whereas changes were observed in dorsomorphin-treated samples, further supporting a mesenchymal to epithelial transition upon bone morphogenetic protein signaling pathway inhibition and the importance of this pathway in breast cancer cell malignancy.

AurkA controls self-renewal of breast cancer-initiating cells promoting wnt3a stabilization through suppression of miR-128.

AurkA overexpression was previously found in breast cancer and associated to its ability in controlling chromosome segregation during mitosis, however whether it may affect breast cancer cells, endorsed with stem properties (BCICs), is still unclear. Surprisingly, a strong correlation between AurkA expression and β-catenin localization in breast cancer tissues suggested a link between AurkA and Wnt signaling. In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells. As a consequence, the amount of BCICs and their migratory capability dramatically decreased. Conversely, wnt3a mRNA stabilization and increased CD44+/CD24low/− subpopulation was found in AurkA-overexpressing MCF7 cells. In vivo, AurkA-overexpressing primary breast cancer cells showed higher tumorigenic properties. Interestingly, we found that AurkA suppressed the expression of miR-128, inhibitor of wnt3a mRNA stabilization. Namely, miR-128 suppression realized after AurkA binding to Snail. Remarkably, a strong correlation between AurkA and miR-128 expression in breast cancer tissues confirmed our findings. This study provides novel insights into an undisclosed role for the kinase AurkA in self-renewal and migration of BCICs affecting response to cancer therapies, metastatic spread and recurrence. In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

Targeting the epigenetics of the DNA damage response in breast cancer.

Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.

Abstract 2314: d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells

Abstract The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-driven activation of BCICs. In this context, our preliminary analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from spontaneous transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, including CD44, Wnt, Notch and Bmi1, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing breast cancer cell lines BT474 and MDA-MB-361, which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions, as indicated by qPCR analyses. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a consistently higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a greater role than WTHER2 in regulating BCICs of HER2-driven mammary tumors. Supported by Minister of Health and AIRC Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314

Abstract 1528: GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo

Abstract Recurrence of breast cancer after treatment could be caused by breast cancer stem-like cells (BCSCs) or breast cancer-initiating cells. Therefore, targeting BCSCs is an important strategy that could complement standard chemotherapy. We recently reported that the ganglioside GD2 is exclusively expressed on BCSCs and that targeting its metabolic enzyme (GD3 synthase, GD3S), reduced breast tumor growth and metastasis in immuno-deficient mice (Battula et al., JCI, 2012). To identify signaling pathways that are activated in GD2+ BCSCs, we performed proteomic analysis in GD2+/- cells from breast cancer cell lines MDA-MB-231 and SUM159 cells using Kinexus® antibody arrays. Data analysis employing Ingenuity® pathway analysis revealed activation of NFκB signaling in GD2+ cells in both cell lines. To validate the array data, we tested activation of NFκB (phospho p65) by CyTOF mass cytometry using metal tagged antibodies and found that NFκB was indeed activated in GD2+ but not in GD2- cells, suggesting activation of canonical NFκB signaling in GD2+ BCSCs. Systemic phospho- and total-protein analyses by immuno blotting revealed that proteins involved in non-canonical signaling, including RelB, NFκB2, and Traf2, were also up-regulated (2-3 fold) in GD2+ compared to GD2- cells. These data suggest that both canonical and non-canonical NFκB signaling are active in GD2+ cells and that it may require inhibition of both pathways to block GD2+ BCSC growth. We found that BMS345541 (IC50 for IKKβ = 0.4μM (canonical) and IC50 for IKKα = 4μM (canonical and non-canonical) reduced GD2+ cells and inhibited GD3S expression (determined by qRT-PCR) in a dose- and time-dependent fashion. Treatment with BMS345541 at 5μM for 72hrs reduced GD2+ cell frequency from 18.5±2.5% to 1.7±0.4% (P <0.001) which is >90% reduction of GD2+ cells in a dose-dependent manner. In contrast, treatment with doxorubicin increased the percentage of GD2+ cells, from 13.5±2.5% to 21±2.6% in MDA-MB-231 cells, suggesting that GD2+ cells are resistant to doxorubicin. BCSCs generate mammospheres under low adhesion conditions. Treatment with the BMS compound inhibited the ability of breast cancer cells to form mammospheres app.12 fold in vitro. To test if BMS345541 inhibits in vivo tumor growth, NOD/SCID mice were transplanted with MDA-MB-231 cells (0.5×106) injected into the mammary fat pad. Following engraftment (2 weeks after transplantation), mice were treated with 50mg/kg of BMS345541 or with DMSO daily for only 5 days (IP, n = 5). The BMS345541-treated group showed a significant decrease (P <0.01) in tumor volume, and prolonged survival compared to the control group: median survival was 78 days for the BMS345541-treated group vs. 58 days for controls (P<0.002). Our data suggest that NFκB inhibition by BMS345541 reduces tumor growth in vivo and prolongs survival of tumor-bearing mice by inhibiting NFκB-mediated GD2+ BCSC growth. Citation Format: Venkata Lokesh Battula, Jeffrey Sun, Gabriel N. Hortobagyi, Michael Andreeff. GD2+ breast cancer stem cell growth is dependent on NFκB signaling and suppressed by the IKK inhibitor BMS345541 in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1528. doi:10.1158/1538-7445.AM2015-1528

A novel peptide-based recognition probe for the sensitive detection of CD44 on breast cancer stem cells

Metastasis and recurrence of breast cancer remain significant clinical problems. The expression level of CD44 protein is higher in breast cancer-initiating cancer stem cells; therefore, the early detection of CD44 using a sensitive diagnostic probe is important for breast cancer diagnosis and therapeutic purposes. In this study, we fabricated a polyvalent directed peptide polymer (PDPP) that specifically recognized the CD44 biomarker, as confirmed by immunocytochemistry tests and fluorescence-activated cell sorting assessment. Our results indicate that PDPP is useful as a novel tool for the sensitive detection of breast cancer stem cells.

Abstract 3897: Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells

Abstract Background: Breast cancer is the leading worldwide cause of death among women due to the high metastatic spread of this disease. As by definition, Cancer Initiating Cells (CICs) are a fraction of primary tumor cells harboring tumorigenic potential and successful outgrowth at metastatic sites. Targeting molecular events affecting CICs peculiarities, as self-renewal and an innate chemoresistance, could improve the ineffectiveness of current therapies. Sam68, the major CD44 splicing regulator, is a multifunctional RNA-binding protein involved in multiple steps of RNA metabolism and its expression is aberrant in breast cancer. Herein, we highlight novel implications of Sam68 in the mammary tumor onset and progression. Material and methods: Tissue microarrays (TMA) were composed of n=155 histologically confirmed invasive breast carcinoma (T1-T2, N0, M0) and Sam68 expression was quantified as percent of immunoreactive tumor cells, a cut-off of 90% positive cells was chosen. Breast CICs were obtained from mechanically and enzimatically digestion of human breast tissues. Stable Sam68 knockdown was produced by lentiviral transduction and puromicin selection. Invasion assay was performed in 8µm pore size transwell coated with GF-depleted matrigel 1:3 in serum free medium. 3x104 cells were plated and 10% FBS medium was used as chemoattractant. Breast CICs (3 x 105) were suspended in 1:6 matrigel and orthotopically injected in NOD/SCID mice. Results: Univariate analysis of the TMA data showed that invasive breast carcinoma over-expressing Sam68 (≥ 90% positive cells) were associated with a significantly higher incidence of distant relapse (P = 0.011). Notably, in a cohort of screened breast cancer patients, breast CICs expressed higher Sam68 protein levels than tumor bulk, highlighting that Sam68 expression was likely restricted to cells with self-renewal activity. Stable knockdown of Sam68 in breast CICs significantly curtailed proliferation rate and was coupled with an increase in p27kip1 and reduction of Bcl-xL. Compared to control cells, downregulation of Sam68 was correlated with an attenuation of cell motility capability and Twist, Snail, CD44v6 and Met levels. Moreover, Sam68 modulated the expression of an oncogenic alternative splice variant of Met (Met-SM), probably through the ASF/SF2 splicing factor. In addition, depletion of Sam68 sensitized breast CICs to standard chemotherapy. Finally, Sam68 silencing ablated breast cancer xenograft formation in immunocompromised mice. Conclusions: Based on these results, we deem that Sam68 may promote pro-invasive signals by inducing and modulating the alternative splicing of oncogenic variants of several genes, such as CD44 and Met. Thus, further investigations on signaling pathways affecting CICs self-renewal and invasiveness could represent a crucial key to improve selective cancer treatment. Citation Format: Alice Turdo, Miriam Gaggianesi, Antonina Benfante, Mauro Piantelli, Matilde Todaro, Giorgio Stassi. Sam68 sustains self-renewal and invasiveness of breast cancer initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2014-3897

VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2.

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3.

Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention.

The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway.

Targeting breast cancer initiating cells: advances in breast cancer research and therapy.

Over the past 10 years there have been significant advances in our understanding of breast cancer and the important roles that breast cancer initiating cells (CICs) play in the development and resistance of breast cancer. Breast CICs endowed with self-renewing and tumor-initiating capacities are believed to be responsible for the relapses which often occur after various breast cancer therapies. In this review, we will summarize some of the key developments in breast CICs which will include discussion of some of the key genes implicated: estrogen receptor (ER), HER2, BRCA1, TP53, PIK3CA, RB, P16INK1 and various miRs as well some drugs which are showing promise in targeting CICs. In addition, the concept of combined therapies will be discussed. Basic and clinical research is resulting in novel approaches to improve breast cancer therapy by targeting the breast CICs.

Abstract P5-07-03: The nuclear coactivator amplified in breast cancer 1 maintains tumor initiating cells during development of ductal carcinoma in situ

Abstract Introduction: Ductal Carcinoma In Situ (DCIS) has become the most diagnosed breast tumor type in North America. Amplified In Breast Cancer 1 (AIB1) is amplified in 5 to 10% of primary breast tumors. We have recently demonstrated that AIB1 causes development of DCIS lesions in the mammary gland of 30% of the AIB1 transgenic mice. Our laboratory has also shown that deletion of one allele of AIB1 prevents HER2-induced mammary tumor development in mice, suggesting a pivotal role of AIB1 during earlier stage of breast cancer. We hypothesize that AIB1-mediated pathways promote the transformation of the mammary epithelial cells and the alterations of the functional integrity of the mammary gland. By contrast, decrease in AIB1 functionality may result in prevented DCIS initiation and development. Results: We show by immunohistochemistry that AIB1 is expressed at low levels in normal breast but is highly expressed in DCIS lesions. We investigated the impact of decreasing AIB1 expression on the early transformed MCFDCIS cell line. MCFDCIS cells generate multiacinar disorganized structures with filling of the lumen, loss of polarization and escape from the proliferative suppression. Reduction of AIB1 in human MCFDCIS cells restored a more normal mammary acinar structure with basement membrane in 3D growth in Matrigel. Then, we studied the effect of regulating AIB1 on the histopathology development of the DCIS lesions both prior to DCIS development or in existing MCFDCIS lesions in a xenograft mouse model by subcutaneously injecting MCFDCIS cells deficient or not in AIB1 into nude mice. The MCFDCIS xenografted tumors obtained reproduced the human breast cancer progression and displayed multiple comedo DCIS-like lesions. Decreased levels of AIB1 in MCFDCIS cells, inhibited tumor growth and led to smaller, necrotic lesions. To investigate how AIB1 affects initiation and progression of DCIS, we compared global gene expression changes in MCFDCIS cells +/-AIB1 shRNA in vitro using cDNA array analysis. Among the most significant expression changes observed were genes that are differentially expressed in Breast Cancer Initiating Cells (BCIC) including CD24 and members of the NOTCH signaling pathway (DLL1 and DLL3). We demonstrated by flow cytometric and immunohistochemical analysis that AIB1 reduction in MCFDCIS cells was correlated with significant reduction in the CD24-/CD44+ BCIC population, and a decrease in myoepithelial progenitor cells in the DCIS lesions in vitro and in vivo. Loss of AIB1 in MCFDCIS cells was also accompanied by a loss of expression of NOTCH 2, 3 and 4, JAG2, HES1, GATA3, HER2 and HER3 in vivo. Conclusions: These data indicate that AIB1 plays a central role in the initiation and maintenance of DCIS and that reduction of AIB1 causes loss of BCIC, loss of components of the NOTCH, HER2 and HER3 signaling pathways and fewer DCIS myoepithelial progenitor cells in vivo. We propose that increased expression of AIB1, through maintenance of BCIC, facilitates formation of DCIS, a necessary step prior to development of invasive disease. Thus, targeting AIB1 may represent a new therapeutic paradigm especially for women with AIB1-positive primary tumor. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-03.