Basal-like Breast Cancer Cells Research Articles

Sanguinarine suppresses basal-like breast cancer growth through dihydrofolate reductase inhibition

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

MELK is an oncogenic kinase essential for mitotic progression in basal-like breast cancer cells.

Despite marked advances in breast cancer therapy, basal-like breast cancer (BBC), an aggressive subtype of breast cancer usually lacking estrogen and progesterone receptors, remains difficult to treat. In this study, we report the identification of MELK as a novel oncogenic kinase from an in vivo tumorigenesis screen using a kinome-wide open reading frames (ORFs) library. Analysis of clinical data reveals a high level of MELK overexpression in BBC, a feature that is largely dependent on FoxM1, a master mitotic transcription factor that is also found to be highly overexpressed in BBC. Ablation of MELK selectively impairs proliferation of basal-like, but not luminal breast cancer cells both in vitro and in vivo. Mechanistically, depletion of MELK in BBC cells induces caspase-dependent cell death, preceded by defective mitosis. Finally, we find that Melk is not required for mouse development and physiology. Together, these data indicate that MELK is a normally non-essential kinase, but is critical for BBC and thus represents a promising selective therapeutic target for the most aggressive subtype of breast cancer.DOI: http://dx.doi.org/10.7554/eLife.01763.001.

T helper responses are maintained by basal-like breast cancer cells and confer to immune modulation via upregulation of PD-1 ligands.

A conspicuous T cell infiltration is frequently observed in triple-negative and/or basal-like breast cancers. Since the immunological course of breast cancer is explicitly directed by helper T cells, this study aims to determine the influence of basal-like breast cancer (BLBC) cells on CD4(+) T cell responses. Co-cultures were established with breast cancer cell lines and CD4(+) T cells under stimulatory conditions. Helper T cell activation, proliferation, cytokine secretion, and differentiation were assessed. Protein and mRNA expression of PD-1 ligands were determined on breast cancer cell lines. Blockade assays were performed in order to determine the functional assets of PD-1 ligation. In contrast to luminal breast cancer cells, BLBC cells allowed CD4(+) T cell activation, proliferation, and IFN-γ secretion, but only to a certain extent. A substantial population of CD25(+)CD127(low/-) regulatory T (Treg) cells was also induced in BLBC co-cultures. In return, IFN-γ stimulated the upregulation of PD-L1 (B7-H1) and/or PD-L2 (B7-DC) inhibitory molecules on the basal-like cells. In prolonged periods of co-culturing, blockade of PD-1 ligands on BLBC cell lines impaired Treg differentiation, restored IL-2 secretion, and increased CD8(+) T cell activation. In conclusion, T helper responses were maintained by BLBC cells. On the other hand, IFN-γ secreted from Th1 and other immune cells upregulated the expression of PD-1 ligands on BLBC cells and modulated the immune reactions. Our results indicate the capacity of BLBCs to adapt to IFN-γ-mediated anti-tumor immune responses and to evade immunity via upregulation of PD-1 ligands.

Highlights of This Issue

Due to the poor prognosis of glioblastoma patients, innovative treatment approaches are urgently needed. Here, Hyvönen and colleagues have identified a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in gliomas. This peptide was not only shown to be effective in SPECT/CT-imaging but also in selective delivery of chemotherapeutics that prolonged the lifespan of invasive brain tumor–bearing mice. Mammary-derived growth inhibitor (MDGI/H-FABP/FABP3), the expression of which is positively correlated with the histological grade of human brain tumors, was found to be the interacting partner for this novel peptide in gliomas.The presence of cancer stem cells (CSC) contributes to the chemoresistance and relapse of basal-like breast cancers (BLBC). Niclosamide inhibits the Wnt/β-catenin pathway involved in CSC regulation. TRA-8 antibody, specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and CSC. In this study, Londoño-Joshi, Arend and colleagues demonstrate that niclosamide in combination with TRA-8 reduced Wnt/β-catenin activity and produced increased cytotoxicity against BLBC cell lines, tumor xenografts, and patient-derived BLBC tumor cells. These results suggest that niclosamide or its analogs may be useful for the treatment of BLBC patients.Genetic alterations affecting the histone methyltransferase activity of EZH2 have been implicated as oncogenic drivers of a spectrum of human cancers; this includes heterozygous expression of point mutations, within EZH2, in a subset of non-Hodgkin lymphoma (NHL) patients. Knutson, Kawano and colleagues report detailed, preclinical characterization of a potent, selective, orally bioavailable EZH2 inhibitor, EPZ-6438 (also known as E7438), which is currently in phase I clinical testing. Twice daily oral administration of this compound to mice bearing human NHL xenografts, with different EZH2 mutations, resulted in profound and durable tumor regression, thus illustrating the potential clinical utility of this drug.The heterogeneous delivery of drugs in tumors has been a challenge for achieving effective treatment outcome. In the present study, Thurber and colleagues developed fluorescent PARP inhibitor olaparib drug conjugates that retain their target binding but are designed with different physiochemical and, thus, pharmacokinetic properties. The drug distribution in cell culture and tumor xenografts was followed with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents.

A time- and matrix-dependent TGFBR3-JUND-KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies.

Basal-like breast carcinoma is characterized by poor prognosis and high intratumor heterogeneity. In an immortalized basal-like breast epithelial cell line, we identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first contains multiple TGFβ-related genes including TGFBR3, whereas the second contains JUND and the basal-like marker, KRT5. TGFBR3 and JUND interconnect through four negative-feedback loops to form a circuit that exhibits spontaneous damped oscillations in 3D culture. The TGFBR3–JUND circuit appears conserved in some premalignant lesions that heterogeneously express KRT5. The circuit depends on ECM engagement, as detachment causes a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine tenascin C, which is critical for intraductal colonization of basal-like breast cancer cells in vivo. Intratumor heterogeneity need not stem from partial differentiation and could instead reflect dynamic toggling of cells between expression states that are not cell autonomous.

HOXC8 promotes breast tumorigenesis by transcriptionally facilitating cadherin-11 expression.

Cell-cell adhesion molecule cadherin-11(CDH11) is preferentially expressed in basal-like breast cancer cells and facilitates breast cancer cell migration by promoting small GTPase Rac activity. However, how the expression of CDH11 is regulated in breast cancer cells is not understood. Here, we show that CDH11 is transcriptionally controlled by homeobox C8 (HOXC8) in human breast cancer cells. HOXC8 serves as a CDH11-specific transcription factor and binds to the site of nucleotides -196 to -191 in the CDH11 promoter. Depletion of HOXC8 leads to the decrease in anchorage-independent cell growth, cell migration/invasion and spontaneous metastasis of breast cancer cells; however, suppressed tumorigenic events were fully rescued by ectopic CDH11 expression in HOXC8-knockdown cells. These results indicate that HOXC8 impacts breast tumorigenesis through CDH11. The analysis of publically available human breast tumor microarray gene expression database demonstrates a strong positive linear association between HOXC8 and CDH11 expression ( = 0.801, p < 0.001). Survival analysis (Kaplan-Meier method, log-rank test) show that both high HOXC8 and CDH11 expression correlate with poor recurrence-free survival rate of patients. Together, our study suggests that HOXC8 promotes breast tumorigenesis by maintaining high level of CDH11 expression in breast cancer cells.

α-catenin acts as a tumour suppressor in E-cadherin-negative basal-like breast cancer by inhibiting NF-κB signalling

Basal-like breast cancer is a highly aggressive tumor subtype associated with poor prognosis. Aberrant activation of NF-κB signaling is frequently found in triple-negative basal-like breast cancer cells, but the cause of this activation has remained elusive. Here we report that α-catenin functions as a tumor suppressor in E-cadherin-negative basal-like breast cancer cells by inhibiting NF-κB signaling. Mechanistically, α-catenin interacts with IκBα protein and stabilizes IκBα by inhibiting its ubiquitination and its association with the proteasome. This stabilization in turn prevents nuclear localization of RelA and p50, leading to decreased expression of TNFα, IL-8 and RelB. In human breast cancer, CTNNA1 expression is specifically downregulated in the basal-like subtype, correlates with clinical outcome and inversely correlates with TNF and RELB expression. Taken together, these results uncover a previously undescribed mechanism by which the NF-κB pathway is activated in E-cadherin-negative basal-like breast cancer.

S100A2 is a BRCA1/p63 coregulated tumour suppressor gene with roles in the regulation of mutant p53 stability

Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.

Disrupting the Interaction of BRD4 with Diacetylated Twist Suppresses Tumorigenesis in Basal-like Breast Cancer

Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we report a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a "histone H4-mimic" GK-X-GK motif that is diacetylated by Tip60. The diacetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructing an activated Twist/BRD4/P-TEFb/RNA-Pol II complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirmed that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies, and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

Epigenetic silencing of ARRDC3 expression in basal-like breast cancer cells

Arrestin domain-containing 3 (ARRDC3) is a tumor suppressor whose expression is either lost or suppressed in basal-like breast cancer (BLBC). However, the mechanism by which BLBC suppresses ARRDC3 expression is not established. Here, we show that expression of ARRDC3 in BLBC cells is suppressed at the transcriptional level. Suppression of ARRDC3 expression in BLBC cells involves epigenetic silencing as inhibitors of class III histone deacetylases (HDACs) significantly restores ARRDC3 levels in BLBC cells. SIRT2, among class III HDACs, plays a major role in epigenetic silencing of ARRDC3 in MDA-MB-231 cells. Acetylation levels of the ARRDC3 promoter in BLBC cells is significantly lower than that of other sub-types of BC cells. Chromatin immunopreciptitation analysis established SIRT2 binding at ARRDC3 promoter in BLBC cells. Our studies indicate that SIRT2 dependent epigenetic silencing of ARRDC3 is one of the important events that may contribute to the aggressive nature of BLBC cells.

P-cadherin signals through the laminin receptor α6β4 integrin to induce stem cell and invasive properties in basal-like breast cancer cells

P-cadherin is a classical cell-cell adhesion molecule that, in contrast to E-cadherin, has a positive role in breast cancer progression, being considered a poor prognostic factor in this disease. In previous reports, we have shown that this protein induces cancer stem cell and invasive properties to basal-like breast cancer cells. Here, we clarify the downstream signaling pathways that are triggered by P-cadherin to mediate these effects. We demonstrated that P-cadherin inhibition led to a significant decreased adhesion of cancer cells to the basement membrane substrate laminin, as well as to a major reduction in the expression of the laminin receptor α6β4 integrin. Remarkably, the expression of this heterodimer was required for the invasive capacity and increased mammosphere forming efficiency induced by P-cadherin expression. Moreover, we showed that P-cadherin transcriptionally up-regulates the α6 integrin subunit expression and directly interacts with the β4 integrin subunit. We still showed that P-cadherin downstream signaling, in response to laminin, involves the activation of focal adhesion (FAK), Src and AKT kinases. The association between the expression of P-cadherin, α6β4 heterodimer and the active FAK and Src phosphorylated forms was validated in vivo. Our data establish that there is a crosstalk between P-cadherin and the laminin receptor α6β4 integrin signaling pathway, which link has never been previously described. The activation of this heterodimer explains the stem cell and invasive properties induced by P-cadherin to breast cancer cells, pointing to a new molecular mechanism that may be targeted to counteract the effects induced by this adhesion molecule.

Overexpression of miR-146a in basal-like breast cancer cells confers enhanced tumorigenic potential in association with altered p53 status

Overexpression of miR-146a in basal-like breast cancer cells confers enhanced tumorigenic potential in association with altered p53 status

Genomic Analyses across Six Cancer Types Identify Basal-like Breast Cancer as a Unique Molecular Entity

To improve our understanding of the biological relationships among different types of cancer, we have characterized variation in gene expression patterns in a set of 1,707 samples representing 6 human cancer types (breast, ovarian, brain, colorectal, lung adenocarcinoma and squamous cell lung cancer). In the unified dataset, breast tumors of the Basal-like subtype were found to represent a unique molecular entity as any other cancer type, including the rest of breast tumors, while showing striking similarities with squamous cell lung cancers. Moreover, gene signatures tracking various cancer- and stromal-related biological processes such as proliferation, hypoxia and immune activation were found expressed similarly in different proportions of tumors across the various cancer types. These data suggest that clinical trials focusing on tumors with common profiles and/or biomarker expression rather than their tissue of origin are warranted with a special focus on Basal-like breast cancer and squamous cell lung carcinoma.

Abstract S6-01: JAK2 amplifications are enriched in triple negative breast cancers (TNBCs) after neoadjuvant chemotherapy and predict poor prognosis

Abstract Janus-kinase 2 (JAK2) is a receptor-coupled tyrosine kinase which transmits cytokine-mediated signals to the STAT pathway to drive signals of proliferation and differentiation. JAK2/STAT signaling has been shown to play a role in promoting breast cancer ‘stem-ness’ and driving the proliferation of CD44+/CD24- basal-like breast cancer cells. We performed targeted next-generation sequencing (tNGS) using HiSeq2000 at a CLIA-certified laboratory on 68 residual TNBCs after neoadjuvant chemotherapy (NAC), which are putatively enriched for drug-resistant cells. JAK2 amplifications were identified in 7/68 (10.2%) post-NAC tumors. Gene copy estimation in amplified cases ranged from 7-10 copies. All cases were confirmed by a novel JAK2-FISH assay that we developed using BAC-derived probes targeting the JAK2 locus on chromosome 9, using CEN9 as a normalization factor. Using JAK2-FISH, we identified only one amplified tumor in an independent cohort of 30 untreated TNBCs (3%). Rates of JAK2 amplification in post-NAC residual cancers in this study were also higher than those reported by TCGA (&amp;lt;1%), although their methodology differed. However, low level gains were more commonly identified in basal-like (46%) vs. luminal A/B tumors (4%) in the TCGA cohort, suggesting a subtype-specific copy number alteration pattern in breast cancer. Furthermore, drawing from a large database of breast tumors sequenced by identical methods, the frequency of JAK2 amplifications was 0.9%; the majority of these were reported as metastatic tumors, post-systemic therapy. These data suggest that JAK2 amplifications are enriched in frequency in drug-resistant and potentially metastatic TNBCs. JAK2 amplifications in the post-NAC residual cancer were associated with poor RFS (median: 7 mo. vs 17 mo; HR: 3.36; p = 0.006) and OS (median: 11.3 mo. vs 27.6 mo; HR: 4.16; p = 0.002), and were significantly associated with higher gene expression of IL6, CXCR1 and SNAI1, among others. The coexistence of these alterations suggests an association of amplified JAK2 with an epithelial-to-mesenchymal transition and cancer-stem cell programs. To explore the clonal evolution of JAK2 lesions in breast cancer, we sequenced sequential samples (diagnostic biopsy, post-NAC residual disease and metastatic recurrence) of two TNBCs where JAK2 amplifications were detected in the residual disease. In each of these, JAK2 gain was noted at levels below the amplification threshold at diagnosis, but was focally amplified in both the residual tumor at the time of definitive surgery as well as in the subsequent metastatic recurrence. To our knowledge, this is the first report of JAK2 gene amplification in breast cancer. These data suggest a role for JAK2 amplifications in driving chemotherapeutic resistance and disease progression. We hypothesize these lesions are therapeutically targetable with currently approved JAK2 or pan-JAK inhibitors. Additional data to be presented will include analysis of tumor-infiltrating lymphocytes in JAK2-amplified tumors and molecular studies of the role of JAK2 overexpression in breast cancer cell lines as well as their sensitivity to JAK2 inhibitors in clinical development. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-01.

Abstract P6-04-17: The irreversible c-Jun N-terminal kinase (JNK) inhibitor, JNK-IN-8, sensitizes basal-like breast cancer cells to lapatinib

Abstract Basal-like breast cancers represent 15-20% of breast cancers diagnosed each year. These tumors appear earlier in life and have the worst prognosis due to high prevalence of metastasis. Endocrine and molecularly targeted therapies such as trastuzumab or lapatinib are ineffective against this tumor subtype. A high frequency of p53 mutations, low BRCA1 expression along with high epidermal growth factor receptor (EGFR) expression provide some insight into what types of targeted therapies may be useful for basal-like tumors in the future. We have recently shown that JNK2 mediates the expression of p53 and BRCA1 in response to EGFR activation to promote epithelial to mesenchymal transition and metastasis. Further, high JNK2 expression is associated with poorer survival in patients with basal-like breast cancer. These findings suggest that the JNK pathway may be a promising target for basal-type breast cancer treatment. Commonly used competitive ATP inhibitors of JNK have suffered from lack of specificity for JNK. However, the Gray laboratory recently developed highly specific covalent inhibitors of JNK (Zhang, et al. 2012). The agent showing the best properties is JNK-IN-8. Our data show that treatment of human and mouse basal-like, breast cancer cell lines with JNK-IN-8 sensitizes them to lapatinib. Single agents had minor affects on cell viability and cell cycle regulation, but combination treatment led to G2/M arrest and endoreduplication along with synergistic apoptotic responses. Further, the expression and electrophoretic mobility of mitotic arrest deficient 2 (MAD2) (a protein mediating chromosomal segregation) decreased after combination treatment which could explain the chromosomal duplication abnormalities observed. Combination treatment using lapatinib and JNK-IN-8 modulates extracellular signal-regulated kinase (ERK) phosphorylation unlike either drug alone suggesting that both JNK and ERK target G2/M check point proteins, and when perturbed, sensitize basal-type breast cancer cells to targeted therapies. We conclude that use of specific JNK inhibitors in tumors that are resistant to lapatinib, or perhaps trastuzumab, may sensitize them to treatments with these drugs, and may also prevent resistance to these drugs in tumors that were initially responsive. Considering that JNKs are nodes in receptor tyrosine kinase and many other signaling pathways, JNK-IN-8 treatment may improve the efficacy of other targeted therapies as well. Zhang, T. et al. “Discovery of potent and selective covalent inhibitors of JNK”. Chem Biol. 2012 Jan 27;19(1):140-54. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-17.

Abstract P1-06-13: Periostin controls cytokine production to maintain breast cancer stem cells

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells (CSCs). Basal-like breast cancer represents a particularly aggressive disease subtype and the tumor cells share phenotypic similarities to breast cancer stem cells. However, the extracellular cues and intracellular pathways that CSCs rely on remain unclear. Working with a breast cancer progression model system we found the invasive breast cancer cell line to exhibit numerous stem-like characteristics. Gene expression profiling, along with a careful review of the literature, identified a secreted extracellular matrix molecule, periostin (POSTN), which we hypothesized to be required for the maintenance of a cancer stem cell state. POSTN expression was increased in numerous basal-like breast cancer cell lines, as well as in cells grown as mammospheres and in the CD44+/CD24- fraction of tumor cells. Furthermore, CSC-like lines displayed increased surface levels of the integrin alpha-v beta-3 heterodimer, a known receptor for POSTN, suggesting that hyperactivation of a POSTN signaling axis could potentially regulate the CSC state. Using stable cell lines expressing an shRNA directed at POSTN or ITGB3 we have found that POSTN signaling is required for efficient mammosphere formation and for the maintenance of an ALDH-positive CSC population. Disruption of this signaling axis resulted in decreased production of cytokines, including IL-6 and IL-8 - known regulators of CSCs - as well as downstream STAT3 signaling. Furthermore, high POSTN expression is associated with a worse clinical prognosis in ER-negative breast cancer. Therefore, we suggest that POSTN signaling through integrin receptors may enrich for highly tumorigenic breast CSCs in a subset of basal-like tumors, leading to aggressive and recurrent malignancies. This reveals a previously unknown link between POSTN signaling and cytokine production, and suggests that basal-like cancers can establish a microenvironmental niche supportive of cancer stem cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-13.

GATA3 Transcription Factor Abrogates Smad4 Transcription Factor-mediated Fascin Overexpression, Invadopodium Formation, and Breast Cancer Cell Invasion

Transforming growth factor β (TGFβ) is a potent and context-dependent regulator of tumor progression. TGFβ promotes the lung metastasis of basal-like (but not the luminal-like) breast cancer. Here, we demonstrated that fascin, a pro-metastasis actin bundling protein, was a direct target of the canonical TGFβ-Smad4 signaling pathway in basal-like breast cancer cells. TGFβ and Smad4 induced fascin overexpression by directly binding to a Smad binding element on the fascin promoter. We identified GATA3, a transcription factor crucial for mammary gland morphogenesis and luminal differentiation, as a negative regulator of TGFβ- and Smad4-induced fascin overexpression. When ectopically expressed in basal-like breast cancer cells, GATA-3 abrogated TGFβ- and Smad4-mediated overexpression of fascin and other TGFβ response genes, invadopodium formation, cell migration, and invasion, suggesting suppression of the canonical TGFβ-Smad signaling axis. Mechanistically, GATA3 abrogated the canonical TGFβ-Smad signaling by abolishing interactions between Smad4 and its DNA binding elements, potentially through physical interactions between the N-terminal of GATA3 and Smad3/4 proteins. Our findings provide mechanistic insight into how TGFβ-mediated cell motility and invasiveness are differentially regulated in breast cancer.

Asymmetric segregation of template DNA strands in basal-like human breast cancer cell lines

Background and methodsStem or progenitor cells from healthy tissues have the capacity to co-segregate their template DNA strands during mitosis. Here, we set out to test whether breast cancer cell lines also possess the ability to asymmetrically segregate their template DNA strands via non-random chromosome co-segregation, and whether this ability correlates with certain properties attributed to breast cancer stem cells (CSCs). We quantified the frequency of asymmetric segregation of template DNA strands in 12 human breast cancer cell lines, and correlated the frequency to molecular subtype, CD44+/CD24-/lo phenotype, and invasion/migration ability. We tested if co-culture with human mesenchymal stem cells, which are known to increase self-renewal, can alter the frequency of asymmetric segregation of template DNA in breast cancer.ResultsWe found a positive correlation between asymmetric segregation of template DNA and the breast cancer basal-like and claudin-low subtypes. There was an inverse correlation between asymmetric segregation of template DNA and Her2 expression. Breast cancer samples with evidence of asymmetric segregation of template DNA had significantly increased invasion and borderline significantly increased migration abilities. Samples with high CD44+/CD24-/lo surface expression were more likely to harbor a consistent population of cells that asymmetrically segregated its template DNA; however, symmetric self-renewal was enriched in the CD44+/CD24-/lo population. Co-culturing breast cancer cells with human mesenchymal stem cells expanded the breast CSC pool and decreased the frequency of asymmetric segregation of template DNA.ConclusionsBreast cancer cells within the basal-like subtype can asymmetrically segregate their template DNA strands through non-random chromosome segregation. The frequency of asymmetric segregation of template DNA can be modulated by external factors that influence expansion or self-renewal of CSC populations. Future studies to uncover the underlying mechanisms driving asymmetric segregation of template DNA and dictating cell fate at the time of cell division may explain how CSCs are maintained in tumors.

Abstract B015: Knock-in of PIK3CA mutations in MCF10A mammary epithelial cells modifies their proteomic profile to resemble basal-like breast cancer and stimulate EGFR-dependent cell proliferation

Abstract PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol-3 kinase (PI3K), is frequently mutated in breast cancer. Activating mutations in PIK3CA are known to transform mammary epithelial cells (MECs). Genomic knock-in of the two most frequent hot-spot PIK3CA mutations (E545K or H1047R) into MCF10A MECs resulted in growth factor-independent proliferation. Shotgun LC-MS/MS mass spectrometry analysis of wild type, E545K and H1047R MCF10A cell lysates revealed 73 proteins uniquely altered in the both mutant cell lines compared to wild type cells. KEGG pathway analysis demonstrated that PIK3CA mutant cells have elevated levels of proteins involved in focal adhesion, ECM-receptor interactions and actin cytoskeleton regulation. Nearly half the proteins upregulated in the mutant cells are secreted or involved in extracellular matrix (ECM) processing or signaling. The EGFR ligand amphiregulin was five-fold higher in the conditioned media harvested from PIK3CA mutant cells as compared to wild type cells. The conditioned media of PIK3CA mutant cells, but not that of wild-type cells, was sufficient to stimulate proliferation and EGFR phosphorylation in wild type MCF10A cells. Proliferation and EGFR activation were inhibited with an amphiregulin-neutralizing antibody or with EGFR neutralizing antibodies and kinase inhibitors. PIK3CA mutant cells downregulated PTPRF, a receptor tyrosine phosphatase. EGFR signaling and proliferation were stimulated in wild type cells when PTPRF was downregulated with siRNA, suggesting that PIK3CA mutant MCF10A cells activate EGFR-dependent proliferation by suppression of a negative regulatory phosphatase and increased secretion of amphiregulin. The expression of transglutaminase 2, peroxidasin, fibronectin, integrin α5, laminin β3, laminin γ2, thrombospondin and EphA2, eight proteins upregulated in PIK3CA mutant MCF10A cells, was evaluated in a panel of breast cancer cell lines. All eight proteins were more highly expressed in basal-like compared to luminal-like breast cancer cell lines. The expression of PTPRF, which is decreased in PIK3CA mutant MCF10A cells, was lower in basal-like cell lines. Interrogation of microarray data demonstrated that the RNA signal of proteins upregulated in mutant PIK3CA MCF10A cells correlated with decreased relapse-free survival in basal-like, but not in luminal-like breast cancer. siRNA-mediated silencing of peroxidasin, laminin γ2, EphA2, integrin β1 or amphiregulin reduced the proliferation of PIK3CA mutant MCF10A cells, suggesting that these proteins are necessary for maintenance of the transformed phenotype. siRNA-mediated silencing of peroxidasin and EphA2 proteins also reduces the proliferation of basal-like breast cancer cells. Our proteomic analysis of PIK3CA mutant MCF10A cells revealed mechanisms of autocrine and paracrine induced proliferation and alterations mostly limited to basal-like breast cancer cells. These may serve as therapeutic targets in this subtype of breast cancer. Citation Format: Christian D. Young, Lisa J. Zimmerman, Corbin A. Whitwell, Ariella B. Hanker, Thomas Stricker, Dana M. Brantley-Sieders, Ben Ho Park, Daniel C. Liebler, Rebecca S. Cook, Carlos L. Arteaga. Knock-in of PIK3CA mutations in MCF10A mammary epithelial cells modifies their proteomic profile to resemble basal-like breast cancer and stimulate EGFR-dependent cell proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B015.