Basal Breast Cancer Cell Research Articles

Abstract 5213: HRH1 inhibition enhances antitumor therapeutic responses through ERK activation in breast cancer

Abstract Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine, a ubiquitous messenger molecule released mostly from mast cells, has been demonstrated to be involved in cell proliferation, embryonic development, and tumor growth. In particular, high levels of HRH1 have been associated with dual behavior in the regulation of several tumors. Most of the physiological and pathological actions of HRH1 have been shown to depend strictly on its level of expression and the tumor cell type under study. We recently established that HRH1 is upregulated in patients with basal and HER2-enriched breast tumors and that its expression correlates with a worse prognosis. Despite this, the functional role of HRH1 basal and HER2 targeted therapies for resistant breast cancer (BC) progression has not so far been addressed. We assessed HRH1 expression in a wide panel of breast cancer cell lines and found HRH1 overexpressed in basal and HER2-targeted therapy resistant to BC cell lines at the protein and mRNA levels, corroborating data previously published using patient databases. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that inhibition of HRH1 activity in basal BC cells leads to sub-G0 accumulation, suppresses proliferation, promotes cell motility and triggers the activation of ERK signaling, initiating the mitochondrial pathway of caspase cascade activation, inducing apoptosis. Moreover, in vivo experiments showed that terfenadine therapy reduced basal and HER2-targeted therapies for resistant BC cell tumor growth but does not affect luminal and parental tumor growth. In conclusion, our results suggest that targeting HRH1 might be an effective therapeutic treatment in patients with basal and HER2-targeted resistant BC tumors. Citation Format: Patricia Fernandez-Nogueira, Aleix Noguera Castells, Gemma Fuster Orellana, Leire Recalde Percaz, Nuria Moragas Garcia, Anna Lopez Plana, Patricia Jauregui Jimenez, Maria Neus Carbó Carbó, Pedro Gascon Villaplana, Paloma Bragado Domingo, Mario Mancino. HRH1 inhibition enhances antitumor therapeutic responses through ERK activation in breast cancer [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 5213.

Abstract 5179: Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing PRPF4B as a metastasis driver

Abstract Metastasis is the major cause of death in cancer patients and migration of cancer cells from the primary tumor to distant sites is the prerequisite of metastasis formation. Here we applied an imaging-based RNAi phenotypic cell migration screen uing two highly migratory basal breast cancer cell lines (Hs578T and MDA-MB-231 to provide a repository of candidate metastasis drug targets. We screened ~4,200 individual target genes covering most cell signaling components. We discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, of which 45 genes were common denominators of cell migration. Interaction networks of candidate migratory modulators were in common with networks of different clinical breast cancer prognostic and metastasis classifiers. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF were amplified in human primary breast tumors and the expression was associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF caused primarily downregulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B was essential for TNBC cell migration and critical for breast cancer metastasis formation in vivo, making PRPF4B a candidate for further drug development. Our systematic phenotypic screen provides an important repository for signaling determinants that functionally drive cancer cell migration. Citation Format: Bob van de Water, Erik Danen, Esmee Koedoot, Maria Rogkoti, Michiel Fokkelman, Sylvia Le Devedec, John Martens, Peter Stoilov, John Foekens. Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing PRPF4B as a metastasis driver [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 5179.

FOXO Transcription Factors Rewire Metabolism in U87MG Glioblastoma Cells

Evolutionarily‐conserved FOXO transcription factors reside in the nucleus of glioblastoma and basal breast cancer cells that harbor constitutive PI3K Pathway output. Nuclear localization of FOXO factors in this setting challenges canonical PI3K models, as this pathway typically induces FOXO localization to the cytoplasm. Simultaneous PI3K activation and nuclear FOXO have also been observed in a set of diffuse large B cell lymphomas and embryonic stem cells. To interrogate the function of nuclear‐localized FOXO transcription factors in glioblastoma and basal breast cancer cell lines, we have constructed genetic models using CRISPR Cas9 genome editing technology. Our evidence indicates that FOXO factors drive metabolic changes in these cancers. FOXO disruption mutants showed increased mitochondrial membrane potential by TMRE staining and subsequent flow cytometric analyses. FOXO factors promote PEPCK gene expression to likely impact carbon utilization. These findings suggest that the nuclear localization of FOXO transcription factors causes metabolic rewiring in glioblastoma and basal breast cancer.Support or Funding InformationThis work was supported by HHMI 52007568 (N.V.), USDA Step 2 2015‐38422‐24061(A.L., R.G,), USDA H.S.I. 2016‐38422‐25760 (M.K. and E.M), NIH 1SC3GM11666901 (R.G.), UTRGV College of Sciences (COS) Seed Grant (M.K.), and NSF Advance 1209210 (M.K).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

FYN promotes mesenchymal phenotypes of basal type breast cancer cells through STAT5/NOTCH2 signaling node.

Basal type breast cancer is the most aggressive and has mesenchymal features with a high metastatic ability. However, the signaling node that determines the basal type features in breast cancer remains obscure. Here, we report that FYN among SRC family kinases is required for the maintenance of basal type breast cancer subtype. Importantly, FYN enhanced NOTCH2 activation in basal type breast cancer cells through STAT5-mediated upregulation of Jagged-1 and DLL4 NOTCH ligands, thereby contributed to mesenchymal phenotypes. In addition, we found that high levels of FYN persist in basal type breast cancer cells by a positive feedback loop between FYN and STAT5. FYN interacted directly with STAT5 and increased p-STAT5 that further acts as a transcription factor for FYN. Taken together, our findings demonstrate a pivotal role of FYN and its downstream effectors in maintaining the basal type features in breast cancer.

Mithramycin A suppresses basal triple-negative breast cancer cell survival partially via down-regulating Kr\xfcppel-like factor 5 transcription by Sp1

As the most malignant breast cancer subtype, triple-negative breast cancer (TNBC) does not have effective targeted therapies clinically to date. As a selective Sp1 inhibitor, Mithramycin A (MIT) has been reported to have anti-tumor activities in multiple cancers. However, the efficacy and the mechanism of MIT in breast cancer, especially TNBC, have not been studied. In this study, we demonstrated that MIT suppressed breast cancer cell survival in a dosage-dependent manner. Interestingly, TNBC cells were more sensitive to MIT than non-TNBC cells. MIT inhibited TNBC cell proliferation and promoted apoptosis in vitro in time- and dosage-dependent manners. MIT suppressed TNBC cell survival, at least partially, by transcriptionally down-regulating KLF5, an oncogenic transcription factor specifically expressed in basal TNBC. Finally, MIT suppressed TNBC cell growth in a xenograft mouse model. Taken together, our findings suggested that MIT inhibits basal TNBC via the Sp1/KLF5 axis and that MIT may be used for TNBC treatment.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

Epigenetic silencing of miR-296 and miR-512 ensures hTERT dependent apoptosis protection and telomere maintenance in basal-type breast cancer cells.

The catalytic subunit of the telomerase complex, hTERT, ensures unlimited proliferative potential of cancer cells by maintaining telomere function and protecting from apoptosis. Using a miRNA screening approach we identified miR-296-5p and miR-512-5p as miRNAs that target hTERT in breast cancer cells. Ectopic miR-296-5p and miR-512-5p reduce telomerase activity, drive telomere shortening and cause proliferation defects by enhancing senescence and apoptosis in breast cancer cells. In line with the relevance of hTERT expression for human cancer we found that miR-296-5p and miR-512-5p expression is reduced in human breast cancer. Accordingly, high expression of miR-296-5p and miR-512-5p target genes including hTERT is linked with significantly reduced distant metastasis free survival and relapse free survival of basal type breast cancer patients. This suggests relevance of the identified miRNAs in basal type breast cancer. Epigenetic silencing of miR-296 and miR-512 encoding genes is responsible for low levels of miR-296-5p and miR-512-5p expression in basal type breast cancer cells. Disrupting gene silencing results in a dramatic upregulation of miR-296-5p and miR-512-5p levels leading to reduced hTERT expression and increased sensitivity to the induction of apoptosis. Altogether, our data suggest that epigenetic regulatory circuits in basal type breast cancer may contribute to high hTERT levels by silencing miR-296-5p and miR-512-5p expression, thereby contributing to the aggressiveness of basal type breast cancer.

The exon 38-containing ARHGEF11 splice isoform is differentially expressed and is required for migration and growth in invasive breast cancer cells.

Breast cancer invasion involves the loss of cell-cell junctions and acquisition of an invasive, migratory phenotype, and breast cancer cells of the basal intrinsic subtype are more invasive and metastatic than breast cancer cells of other subtypes. ARHGEF11 is a RhoGEF that was previously shown to bind to the tight junction protein ZO-1 at perijunctional actomyosin ring (PJAR), a network of cortically organized actin and myosin filaments associated with junctional complexes that regulates cell-cell adhesion and polarization. We show here that ARHGEF11 shows splice isoform expression that differs according to the intrinsic subtype of breast cancer cells and that controls their invasive phenotype. Luminal subtype breast cancer cells express the isoform of ARHGEF11 lacking exon 38 (38-), which binds to ZO-1 at PJAR and is necessary for formation and maintenance of cell-cell junctions. Basal subtype breast cancer cells express the isoform of ARHGEF11 containing exon 38 (38+), which does not bind to ZO-1 and which drives cell migration and motility. Depletion of ARHGEF11 in basal subtype breast cancer cells is sufficient to alter cell morphology from a mesenchymal stellate form with extensive cell protrusions to a cobblestone-like epithelial form, and to suppress growth and survival both in vitro and in vivo. These findings show that the expression of the particular splice isoform of ARHGEF11 is critically linked to the malignant phenotype of breast cancer cells, identifying ARHGEF11 exon 38(+) as a biomarker and target for therapy of breast cancer.

Breast Cancer Spheroids Reveal a Differential Cancer Stem Cell Response to Chemotherapeutic Treatment

An abnormal multicellular architecture is a defining characteristic of breast cancer and, yet, most in vitro tumor models fail to recapitulate this architecture or accurately predict in vivo cellular responses to therapeutics. The efficacy of two front-line chemotherapeutic agents (paclitaxel and cisplatin) are described within three distinct in vitro models employing the triple-negative basal breast cancer cell line MDA-MB-231 and the luminal breast cancer cell line MCF7: a) a 3D collagen embedded multicellular spheroid tumor model, which reflects the architecture and cellular heterogeneity of tumors in vivo; b) a 3D collagen model with a single cell-type diffusely embedded; and c) a 2D monolayer. The MDA-MB-231 embedded spheroid tumor model exhibited the most robust response to chemotherapeutic treatment, and possessed the greatest cancer stem cell (CSC) content. CSC-related genes are elevated across all MDA-MB-231 in vitro models following paclitaxel treatment, indicating that paclitaxel enrichment of chemoresistant CSCs is less dependent on microenvironmental tumor structure, while cisplatin showed a more context-dependent response. In the MCF7 cell models a context-dependent response is observed with paclitaxel treatment increasing the CSC related genes in the 2D monolayer and 3D diffuse models while cisplatin treatment afforded an increase in ALDH1A3 expression in all three models.

Prognostic and functional role of subtype‐specific tumor–stroma interaction in breast cancer

None of the clinically relevant gene expression signatures available for breast cancer were specifically developed to capture the influence of the microenvironment on tumor cells. Here, we attempted to build subtype‐specific signatures derived from an in vitro model reproducing tumor cell modifications after interaction with activated or normal stromal cells. Gene expression signatures derived from HER2+, luminal, and basal breast cancer cell lines (treated by normal fibroblasts or cancer‐associated fibroblasts conditioned media) were evaluated in clinical tumors by in silico analysis on published gene expression profiles (GEPs). Patients were classified as microenvironment‐positive (μENV+ve), that is, with tumors showing molecular profiles suggesting activation by the stroma, or microenvironment‐negative (μENV−ve) based on correlation of their tumors' GEP with the respective subtype‐specific signature. Patients with estrogen receptor alpha (ER)+/HER2−/μENV+ve tumors were characterized by 2.5‐fold higher risk of developing distant metastases (HR = 2.546; 95% CI: 1.751–3.701, P = 9.84E‐07), while μENV status did not affect, or only suggested the risk of distant metastases, in women with HER2+ (HR = 1.541; 95% CI: 0.788–3.012, P = 0.206) or ER‐/HER2− tumors (HR = 1.894; 95% CI: 0.938–3.824; P = 0.0747), respectively. In ER+/HER2− tumors, the μENV status remained significantly associated with metastatic progression (HR = 2.098; CI: 1.214–3.624; P = 0.00791) in multivariable analysis including size, age, and Genomic Grade Index. Validity of our in vitro model was also supported by in vitro biological endpoints such as cell growth (MTT assay) and migration/invasion (Transwell assay). In vitro‐derived gene signatures tracing the bidirectional interaction with cancer activated fibroblasts are subtype‐specific and add independent prognostic information to classical prognostic variables in women with ER+/HER2− tumors.

Oncosis and apoptosis induction by activation of an overexpressed ion channel in breast cancer cells

The critical role of calcium signalling in processes related to cancer cell proliferation and invasion has seen a focus on pharmacological inhibition of overexpressed ion channels in specific cancer subtypes as a potential therapeutic approach. However, despite the critical role of calcium in cell death pathways, pharmacological activation of overexpressed ion channels has not been extensively evaluated in breast cancer. Here we define the overexpression of transient receptor potential vanilloid 4 (TRPV4) in a subgroup of breast cancers of the basal molecular subtype. We also report that pharmacological activation of TRPV4 with GSK1016790A reduced viability of two basal breast cancer cell lines with pronounced endogenous overexpression of TRPV4, MDA-MB-468 and HCC1569. Pharmacological activation of TRPV4 produced pronounced cell death through two mechanisms: apoptosis and oncosis in MDA-MB-468 cells. Apoptosis was associated with PARP-1 cleavage and oncosis was associated with a rapid decline in intracellular ATP levels, which was a consequence of, rather than the cause of, the intracellular ion increase. TRPV4 activation also resulted in reduced tumour growth in vivo. These studies define a novel therapeutic strategy for breast cancers that overexpress specific calcium permeable plasmalemmal ion channels with available selective pharmacological activators.

Down-regulation of the Tumor Suppressor CYLD Enhances the Transformed Phenotype of Human Breast Cancer Cells.

The cylindromatosis tumor suppressor (CYLD) has been implicated in the inhibition of human breast cancer development by virtue of the poor prognosis of patients with down-regulated CYLD expression. In order to investigate the mechanism of breast cancer suppression by CYLD, in the present study, cellular and molecular aspects of CYLD-dependent phenotypic regulation of different types of human breast cancer cell lines were analyzed. CYLD expression was down-regulated by RNA interference in human breast cancer cell lines. Parental and CYLD-deficient cell lines were evaluated for their viability, migratory capacity, anchorage-independent growth and chemoresistance. Wild-type and mutated forms of CYLD were also evaluated for their ability to suppress the clonogenic potential of breast cancer cells. CYLD down-regulation enhanced the survival and migratory properties of basal and luminal breast cancer cell lines. In addition, down-regulation of CYLD expression enhanced the ability of human breast cancer cells to grow in an anchorage-independent manner and could be associated with resistance to chemotherapeutic drugs. The growth-suppressive properties of CYLD on breast cancer cell lines were dependent on its de-ubiquitinating activity and its amino terminal cytoskeleton-interacting region. Our results establish a broad range of tumor-suppressive properties that are conferred by CYLD in basal and luminal human breast cancer cells and support the significance of targeted de-ubiquitination by CYLD in breast cancer cell growth suppression.

Abstract 4486: Redox regulation of the biosynthesis of mitronic tsmiR miR6855 in the basal like breast cancer cells

Abstract Radiation and chemotherapeutic agents eradicate tumors by inducing irreparable DNA damage. However, cancer cells often develop resistance to therapy by manipulating the DNA repair machinery. Conversely, a dividing cell constantly exposed to environmental and endogenous DNA damaging agents can transform into a tumor due to incorrect repair. Therefore the expression level of DNA repair proteins is critical both for cancer therapy and tumorigenesis. Our preliminary studies implied a unique mechanism involving an miRNA miR-6855-3p that promotes the biosynthesis of the transcriptional regulator protein SPRDX5 which enhances the expression of the tumor suppressor protein BRCA2 leading to the genotoxin and radiation-resistance of aggressive SLUG-high basal-like breast cancer (BLBC) cells. The miRNA miR-6855-3p thus acts as a tumor suppressor miRNA (tsmiR). Interestingly, the gene for the miRNA miR-6855-3p is mitronic and is nested within the intron#13 of the USP20 gene which is a deubiquitinase. Thus, the transcription of miR-6855-3p gene is regulated through the USP20 gene promoter. We present evidence here that USP20 and miR-6855 gene transcriptions are up regulated by oxidative stress. Tert-butylhydroperoxide was used for oxidative stress. Under the oxidative stress conditions tested, NRF2 level and the levels of several NRF2-regulated proteins are increased, indicating activation of NRF2 in the stressed BLBC cells. Under the stress conditions the levels of USP20 and miR-6855-3p are increased significantly. Knockdown of NRF2 prevented stress-induced elevation of USP20 and miR-6855-3p RNA level in the BLBC cells indicating the involvement of NRF2 in this process. The binding of NRF2 to the USP20 gene promoter and its function were characterized by ChIP analysis, site-directed mutagenesis and using the promoter-luciferase reporter constructs. We conclude that the biosynthesis of the tsmiR miR-6855-3p in the BLBC cells is regulated by oxidative stress and involves NRF2. Since oxidative stress is a hallmark in the etiology and progression of metastatic breast cancer, understanding the redox regulation of a tsmiR that positively influences BRCA2 gene expression in the BLBC cells could be clinically significant. Supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM. Citation Format: Smita Misra, Gautam Chaudhuri. Redox regulation of the biosynthesis of mitronic tsmiR miR6855 in the basal like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2017-4486

Abstract 2149A: CRISPR/Cas9 mutagenesis invalidates a genetic target of clinical trials in cancer

Abstract The Maternal Embryonic Leucine Zipper Kinase MELK has been described as a genetic dependency in several cancer types, most notably in the highly-aggressive basal subtype of breast cancer; MELK inhibition through the use of both RNAi and small-molecule approaches appears to block the growth of cancer types with such dependency. Based on these results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in multiple clinical trials. Here, however, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from other cancer types. Through seven guide RNAs targeting the kinase and kinase-associated domains of MELK, we demonstrate that mutagenesis of MELK causes no defect in proliferative ability or anchorage independent growth in these cancer types. Additionally, cells with mutagenized MELK remain sensitive to OTS167, suggesting that this drug blocks proliferation through an off-target mechanism. Finally, the patient tumor gene expression data that initially identified MELK as being significantly upregulated in patients with poor survival was reexamined. As MELK is thought to play a role in mitosis, we compared MELK expression to a set of well-known cell proliferation markers and show significant correlations of MELK with the proliferation genes; this suggests a role of MELK in representing the mitotic activity of a tumor, rather than possessing a transformative role in itself. In total, our results undermine the rationale for a series of current clinical trials based on MELK inhibition and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied. Citation Format: Ann Lin, Christopher Giuliano, Nicole Sayles, Joan Smith, Jason Sheltzer. CRISPR/Cas9 mutagenesis invalidates a genetic target of clinical trials in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2149A. doi:10.1158/1538-7445.AM2017-2149A

Abstract 4036: Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel

Abstract Basal-like breast cancer has received considerable attention in recent years, but despite all efforts, conventional chemotherapy remains the main therapeutic option in the clinic with taxane therapy, the most frequently prescribed treatment. A significant reason for the lack of effective therapeutics may be related to the high degree of heterogeneity and aneuploidy which is characteristic for basal-like breast cancer. A new approach in this field is the concept that cancer cells may be targeted by the introduction of a detrimental level of aneuploidy. Inhibition of one of the main spindle assembly checkpoint kinases, MPS1 is a rational choice to achieve this goal. We report here the identification of a synergistic effect between the novel, oral, highly potent and selective MPS1 inhibitor CCT289346 and paclitaxel. We demonstrate robust synergism in multiple basal-like breast cancer cell lines and provide mechanistic insights into how both compounds’ individual properties work together to maximise mitotic defects thereby increasing aneuploidy and cell death in a single round of mitosis. Additionally, in vivo studies in basal-like breast cancer xenograft models, including patient derived xenografts and systemic metastatic models, using clinically relevant doses of paclitaxel with well-tolerated doses of CCT289346, demonstrate significant benefit of combination of the two agents in comparison to paclitaxel alone. Citation Format: Simon J. Anderhub, Grace WY Mak, Amir Faisal, Katie Walsh, Mark Gurden, Liz Day, Paolo Innocenti, Hannah L. Woodward, Isaac M. Westwood, Kwai-Ming J. Cheung, Sebastien Naud, Angela Hayes, Gary Box, Alexis de Haven Brandon, Melanie R. Valenti, Lisa O’Fee, Harry Saville, Rosemary Burke, Rob LM van Montfort, Florence I. Raynaud, Suzanne A. Eccles, Julian Blagg, Swen Hoelder, Spiros Linardopoulos. Induction of detrimental aneuploidy in basal breast cancer cells treated by MPS1 inhibitors in combination with paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4036. doi:10.1158/1538-7445.AM2017-4036

Abstract 2984: The chemokine CCL2/CCR2 signaling mediated fibroblasts-cancer cells crosstalk promotes basal like breast cancer progression

Abstract Cancer associated fibroblasts are the most abundant stromal cells in breast cancer, but their function in cancer progression has not been fully understood. We previously identified that the chemokine CCL2 was highly expressed in breast cancer associated fibroblasts, and high stromal CCl2 expression predicted poor outcome in basal like breast cancer. CCL2 is known to recruit monocyte/macrophage and promotes cancer progression. We previously found that recombinant CCL2 can directly signal to breast cancer cells and promote cell survival and invasion in vitro. In this study, we aimed to determine the functional importance of CCL2 signaling mediated fibroblast-cancer cell interactions in breast cancer progression. We used a fibroblast and cancer cell co-graft mouse model as the main functional assay. We generated primary fibroblasts from mouse mammary tumor and human breast cancer, and confirmed most of they expressed high level of CCL2. When co-grafted with the human basal breast cancer cell line MCF10A-CA1D into nude mice, fibroblasts enhanced xenograft growth. Stable knockdown of CCL2 expression from fibroblasts significantly reduced its ability in tumor growth promotion, while knockdown CCL2 from cancer cells did not. Decreased CCL2 production from fibroblasts resulted in increased apoptosis and autophagy in tumor samples. To determine the importance of direct CCL2 signaling to cancer cell, we generated the CCL2 receptor CCR2 mutant CA1D cancer cell lines by CRISPR-Cas9 targeting technology. Mutation of CCR2 in cancer cell significantly reduced tumor growth when co-grafted with CCL2 secreting fibroblasts. Lastly, we tested continuous delivery of CCL2 neutralizing antibody in the co-graft tumor model, but observed minimal therapeutic effect. Further examination of blood CCL2 level revealed an increased production of CCL2 from fibroblasts and mouse host after antibody treatment, which may contribute to the lack of therapeutic effect. In summary, our studies demonstrated the importance of CCL2/CCR2 signaling mediated fibroblasts-cancer cell interaction in basal like breast cancer progression. The CCL2 signaling pathway can be potentially served as therapeutic target, but requires development of efficient targeting strategy. Citation Format: Min Yao, Wei Bin Fang, Fang Fan, Nehemiah Alvarez, Patrick E. Fields, Nikki Cheng. The chemokine CCL2/CCR2 signaling mediated fibroblasts-cancer cells crosstalk promotes basal like breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2017-2984

Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling.

This corrects the article DOI: 10.1038/onc.2016.394.

PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA-MB-231 breast cancer cells.

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4‐bisphosphate (PI(3,4)P2) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P2 on focal adhesion dynamics in MDA‐MB‐231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5‐trisphosphatase (PIP 3) 5‐phosphatase that generates PI(3,4)P2, in MDA‐MB‐231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3‐phosphatase that de‐phosphorylates PIP 3 and PI(3,4)P2, induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P2, and knockdown of Lpd, a downstream effector of PI(3,4)P2, resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis.

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials.

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.