Comparative transcriptional analyses of preclinical models and patient samples reveal MYC and RELA driven expression patterns that define the molecular landscape of IBC.

Inflammatory breast cancer (IBC) is an aggressive disease for which the spectrum of preclinical models was rather limited in the past. More recently, novel cell lines and xenografts have been developed. This study evaluates the transcriptome of an extended series of IBC preclinical models and performed a comparative analysis with patient samples to determine the extent to which the current models recapitulate the molecular characteristics of IBC observed clinically. We demonstrate that the IBC preclinical models are exclusively estrogen receptor (ER)-negative and of the basal-like subtype, which reflects to some extent the predominance of these subtypes in patient samples. The IBC-specific 79-signature we previously reported was retrained and discriminated between IBC and non-IBC preclinical models, but with a relatively high rate of false positive predictions. Further analyses of gene expression profiles revealed important roles for cell proliferation, MYC transcriptional activity, and TNFɑ/NFκB in the biology of IBC. Patterns of MYC expression and transcriptional activity were further explored in patient samples, which revealed interactions with ESR1 expression that are contrasting in IBC and nIBC and notable given the comparatively poor outcomes of ER+ IBC. Our analyses also suggest important roles for NMYC, MXD3, MAX, and MLX in shaping MYC signaling in IBC. Overall, we demonstrate that the IBC preclinical models can be used to unravel cancer cell intrinsic molecular features, and thus constitute valuable research tools. Nevertheless, the current lack of ER-positive IBC models remains a major hurdle, particularly since interactions with the ER pathway appear to be relevant for IBC.

Targeting SH2 domains in breast cancer.

Breast cancer is among the most commonly diagnosed cancer types in women worldwide and is the second leading cause of cancer-related disease in the USA. SH2 domains recruit signaling proteins to phosphotyrosine residues on aberrantly activated growth factor and cytokine receptors and contribute to cancer cell cycling, metastasis, angiogenesis and so on. Herein we review phosphopeptide mimetic and small-molecule approaches targeting the SH2 domains of Grb2, Grb7 and STAT3 that inhibit their targets and reduce proliferation in in vitro breast cancer models. Only STAT3 inhibitors have been evaluated in in vivo models and have led to tumor reduction. Taken together, these studies suggest that targeting SH2 domains is an important approach to the treatment of breast cancer.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer.

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKß in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 µM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK(+) IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.

Inflammatory breast cancer (IBC): clues for targeted therapies.

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, ß-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10-3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.

Genome wide proteomics of ERBB2 and EGFR and other oncogenic pathways in inflammatory breast cancer.

In this study we selected three breast cancer cell lines (SKBR3, SUM149 and SUM190) with different oncogene expression levels involved in ERBB2 and EGFR signaling pathways as a model system for the evaluation of selective integration of subsets of transcriptomic and proteomic data. We assessed the oncogene status with reads per kilobase per million mapped reads (RPKM) values for ERBB2 (14.4, 400, and 300 for SUM149, SUM190, and SKBR3, respectively) and for EGFR (60.1, not detected, and 1.4 for the same 3 cell lines). We then used RNA-Seq data to identify those oncogenes with significant transcript levels in these cell lines (total 31) and interrogated the corresponding proteomics data sets for proteins with significant interaction values with these oncogenes. The number of observed interactors for each oncogene showed a significant range, e.g., 4.2% (JAK1) to 27.3% (MYC). The percentage is measured as a fraction of the total protein interactions in a given data set vs total interactors for that oncogene in STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 9.0) and I2D (Interologous Interaction Database, version 1.95). This approach allowed us to focus on 4 main oncogenes, ERBB2, EGFR, MYC, and GRB2, for pathway analysis. We used bioinformatics sites GeneGo, PathwayCommons and NCI receptor signaling networks to identify pathways that contained the four main oncogenes and had good coverage in the transcriptomic and proteomic data sets as well as a significant number of oncogene interactors. The four pathways identified were ERBB signaling, EGFR1 signaling, integrin outside-in signaling, and validated targets of C-MYC transcriptional activation. The greater dynamic range of the RNA-Seq values allowed the use of transcript ratios to correlate observed protein values with the relative levels of the ERBB2 and EGFR transcripts in each of the four pathways. This provided us with potential proteomic signatures for the SUM149 and 190 cell lines, growth factor receptor-bound protein 7 (GRB7), Crk-like protein (CRKL) and Catenin delta-1 (CTNND1) for ERBB signaling; caveolin 1 (CAV1), plectin (PLEC) for EGFR signaling; filamin A (FLNA) and actinin alpha1 (ACTN1) (associated with high levels of EGFR transcript) for integrin signalings; branched chain amino-acid transaminase 1 (BCAT1), carbamoyl-phosphate synthetase (CAD), nucleolin (NCL) (high levels of EGFR transcript); transferrin receptor (TFRC), metadherin (MTDH) (high levels of ERBB2 transcript) for MYC signaling; S100-A2 protein (S100A2), caveolin 1 (CAV1), Serpin B5 (SERPINB5), stratifin (SFN), PYD and CARD domain containing (PYCARD), and EPH receptor A2 (EPHA2) for PI3K signaling, p53 subpathway. Future studies of inflammatory breast cancer (IBC), from which the cell lines were derived, will be used to explore the significance of these observations.

The paradox of E-cadherin: role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism.

E-Cadherin is a cell:cell adhesion molecule critical for appropriate embryonic and mammary development. In cancer, E-Cadherin has been primarily viewed as being lost during the process of epithelial-mesenchymal transition (EMT), which occurs with a switch from E-Cadherin expression to a gain of N-Cadherin and other mesenchymal markers. EMT has been shown to play a role in the metastatic process while the reverse process, mesenchymal-epithelial transition (MET), is important for metastatic colonization. Here we report an unexpected role of E-Cadherin in regulating tumorigenicity and hypoxia responses of breast tumors in vivo. Reduced expression of E-Cadherin led to a dramatic reduction of the in vivo growth capability of SUM149, Mary-X and 4T1 tumor cells. Furthermore, over-expression of ZEB1, a known transcriptional repressor of E-Cadherin, led to reduced in vivo growth of SUM149 tumors. Gene set enrichment analysis identified the loss of hypoxia response genes as a major mechanism in mediating the lack of in vivo growth of SUM149 cells that lacked E-Cadherin or over-expressed ZEB1. The in vivo growth defect of SUM149 E-Cadherin knockdown tumors was rescued by the hypoxia-inducible 1α transcription factor (HIF-1α). Given the importance of HIF-1α in cellular metabolism, we observed reduced glycolytic capacity in SUM149 and 4T1 cells that had E-Cadherin knocked down. Our observations shed light on the complex functions of E-Cadherin in retention of an epithelial phenotype and as a mediator of survival of aggressive breast cancer under hypoxic conditions in vivo. Furthermore, we find that patients with basal subtype breast cancer and high E-Cadherin expression in their tumors had a poor clinical outcome. Our data suggests a novel function for E-Cadherin as a bona fide signaling molecule required for the in vivo growth of aggressive breast cancer tumor cells, that retain E-Cadherin expression, in mediating their metabolic function.

Uncovering the molecular secrets of inflammatory breast cancer biology: an integrated analysis of three distinct affymetrix gene expression datasets.

BACKGROUND: Inflammatory breast cancer (IBC) is a poorly characterized form of breast cancer. So far, the results of expression profiling in IBC are inconclusive due to various reasons including limited sample size. Here, we present the integration of three Affymetrix expression datasets collected through the World IBC Consortium allowing us to interrogate the molecular profile of IBC using the largest series of IBC samples ever reported. EXPERIMENTAL DESIGN: Affymetrix profiles (HGU133-series) from 137 patients with IBC and 252 patients with non-IBC (nIBC) were analyzed using unsupervised and supervised techniques. Samples were classified according to the molecular subtypes using the PAM50-algorithm. Regression models were used to delineate IBC-specific and molecular subtype-independent changes in gene expression, pathway, and transcription factor activation. RESULTS: Four robust IBC-sample clusters were identified, associated with the different molecular subtypes (P<0.001), all of which were identified in IBC with a similar prevalence as in nIBC, except for the luminal A subtype (19% vs. 42%; P<0.001) and the HER2-enriched subtype (22% vs. 9%; P<0.001). Supervised analysis identified and validated an IBC-specific, molecular subtype-independent 79-gene signature, which held independent prognostic value in a series of 871 nIBCs. Functional analysis revealed attenuated TGF-ß signaling in IBC. CONCLUSION: We show that IBC is transcriptionally heterogeneous and that all molecular subtypes described in nIBC are detectable in IBC, albeit with a different frequency. The molecular profile of IBC, bearing molecular traits of aggressive breast tumor biology, shows attenuation of TGF-ß signaling, potentially explaining the metastatic potential of IBC tumor cells in an unexpected manner.

The class I HDAC inhibitor Romidepsin targets inflammatory breast cancer tumor emboli and synergizes with paclitaxel to inhibit metastasis.

Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells.

Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 µM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.

Early to intermediate steps of tumor embolic formation involve specific proteolytic processing of E-cadherin regulated by Rab7.

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. Using MARY-X, a human breast cancer xenograft that exhibits florid lymphovascular emboli in mice and spheroids in vitro, we established a model where the in vitro transition stages from minced tumoral aggregates to well-formed spheroids served as a surrogate for in vivo emboli formation. MARY-X well-formed spheroids and emboli exhibited strong similarity of expression. The aggregate-to-spheroid transition stages were characterized by increased ExoC5, decreased Hgs and Rab7, increased calpains, increased full-length E-cadherin (E-cad/FL), and the transient appearance of E-cad/NTF2, a 95 kDa E-cadherin fragment and increased Notch3icd (N3icd), the latter two fragments produced by increased γ-secretase. Both transient and permanent knockdowns of Rab7 in MCF-7 cells increased protein but not transcription of E-cad/FL and resulted in the de novo appearance of E-cad/NTF2, the presence of nuclear E-cad/CTF2, and increased Notch1icd (N1icd). Overexpression of Rab7 conversely decreased E-cad/FL, γ-secretase (PS1/NTF), and E-cad/NTF2. Overexpression of calpains did not alter PS1/NTF but decreased E-cad/FL and E-cad/NTF2 and increased N1icd. Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increased N3icd, the latter two fragments being the direct and indirect consequences, respectively, of increased calpains (calpain 1 and calpain 2). Inhibition of calpains decreased E-cad/NTF1 but increased E-cad/NTF2 showing that calpains compete with γ-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of γ-secretase to Notch 3, resulting in increased Notch 3 signaling in the well-formed spheroids.

A phosphopeptide mimetic prodrug targeting the SH2 domain of Stat3 inhibits tumor growth and angiogenesis.

Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a number of human cancers and cancer cell lines. Via its Src homology 2 (SH2) domain, Stat3 is recruited to phosphotyrosine residues on intracellular domains of cytokine and growth factor receptors, whereupon it is phosphorylated on Tyr705, dimerizes, translocates to the nucleus and is reported to participate in the expression of genes related to angiogenesis, metastasis, growth and survival. To block this process, we are developing cell-permeable, phosphatase-stable phosphopeptide mimics, targeted to the SH2 domain of Stat3, that inhibit the phosphorylation of Tyr705 of Stat3 in cultured tumor cells (Mandal et al., J. Med. Chem. 54, 3549-5463, 2011). At concentrations that inhibit tyrosine phosphorylation, these materials were not cytotoxic, similar to recent reports on JAK inhibitors. At higher concentrations, cytotoxicity was accompanied by off-target effects. We report that treatment of MDA-MB-468 human breast cancer xenografts in mice with peptidomimetic PM-73G significantly inhibited tumor growth, which was accompanied by reduction in VEGF production and microvessel density. No evidence of apoptosis or changes in the expression of the canonical genes cyclin D1 or survivin were observed. Thus selective inhibition of Stat3 Tyr705 phosphorylation may be a novel anti-angiogenesis strategy for the treatment of cancer.

The consequences of selective inhibition of signal transducer and activator of transcription 3 (STAT3) tyrosine705 phosphorylation by phosphopeptide mimetic prodrugs targeting the Src homology 2 (SH2) domain.

Herein we review our progress on the development of phosphopeptide-based prodrugs targeting the SH2 domain of STAT3 to prevent recruitment to cytokine and growth factor receptors, activation, nuclear translocation and transcription of genes involved in cancer. We developed high affinity phosphopeptides (K I = 46-200 nM). Corresponding prodrugs inhibited constitutive and IL-6 induced Tyr705 phosphorylation at 0.5-1 µM in a variety of human cancer cell lines. They were not cytotoxic at 5 µM in vitro but they inhibited tumor growth in a human xenograft breast cancer model in mice, accompanied by reduced VEGF expression and angiogenesis.

Efficient synthesis of apricoxib, CS-706, a selective cyclooxygenase-2 inhibitor, and evaluation of inhibition of prostaglandin E2 production in inflammatory breast cancer cells.

An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using copper catalyzed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis to an aldehyde, and condensation with sulfanilamide. This method provided multi-gram access of aprocoxib in good yield. Apricoxib exhibited potency equal to celecoxib at inhibition of prostaglandin E2 synthesis in two inflammatory breast cancer cell lines.

Different gene expressions are associated with the different molecular subtypes of inflammatory breast cancer.

The goal of this study was to determine whether gene expression differences exist between inflammatory breast cancers (IBC) and T stage-matched non-IBC patients stratified by hormone receptor and HER2 status. We used Affymetrix GeneChips to analyze 82 tumor samples (25 T4d patients, and 57 T4a-c patients) of newly diagnosed breast cancers. Genes that were differentially expressed between the IBC and non-IBC specimens were identified using the t test, and differential expression of gene sets was assessed using gene set analysis. Three distinct clinical subtypes of IBC and non-IBC were compared: ER-positive/HER2-normal, HER2-amplified, and ER-negative/HER2-normal. When we compared expression data from all IBC with all non-IBC, we found no significant differences after adjusting for multiple testing. When IBC and non-IBC tumors were compared by clinical subtype, however, significant differences emerged. Complement and immune system-related pathways were overexpressed in ER-positive/HER2-normal IBC. Protein translation and mTOR signaling were overexpressed in HER2-amplified IBC. Apoptosis-, neural-, and lipid metabolism-related pathways were overexpressed in ER-negative/HER2-normal IBC compared with non-IBC of the same receptor phenotype. In this T stage-matched case-control study, the survival curves of patients with IBC and non-IBC were similar for all three clinical subtypes. IBC tumors can be divided into molecular and clinical subtypes similar to those of non-IBC. Clinical subtypes of IBC show molecular differences compared with similar subtypes of non-IBC.

A global approach to inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer. In spite of the comprehensive multidisciplinary approach to the management of this disease, the prognosis remains dismal. Moreover, there have been no major advancements in understanding the etiology and biology of IBC and no significant improvements in the diagnosis of the disease. The International Inflammatory Breast Cancer Conference was established in 2008 with the intention of creating a forum for the discussion, collaboration and development of proposals and working hypotheses. Furthermore, the conference represented an opportunity to raise awareness regarding IBC. The second international conference reported on several new exciting projects based on work from investigators and research teams devoted to making a difference in the fight against this disease.

Inflammatory breast cancer: the disease, the biology, the treatment.

Inflammatory breast cancer (IBC) is a rare and aggressive form of invasive breast cancer accounting for 2.5% of all breast cancer cases. It is characterized by rapid progression, local and distant metastases, younger age of onset, and lower overall survival compared with other breast cancers. Historically, IBC is a lethal disease with less than a 5% survival rate beyond 5 years when treated with surgery or radiation therapy. Because of its rarity, IBC is often misdiagnosed as mastitis or generalized dermatitis. This review examines IBC's unique clinical presentation, pathology, epidemiology, imaging, and biology and details current multidisciplinary management of the disease, which comprises systemic therapy, surgery, and radiation therapy.

Imaging and analysis of 3D tumor spheroids enriched for a cancer stem cell phenotype.

Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as "label-retaining cells"; express specific surface markers such as CD44(+)/CD24(-/low) and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells.

BACKGROUND: Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced by serial xenograft passages through transplantation. Herein we fully characterize cancer stem cell-like features in 293T human embryonic kidney cells. RESULTS: 293T cells can be readily cultured and passaged as spheres in serum-free stem cell promoting culture conditions. Cells cultured in vitro as three-dimensional spheres (3D) were shown to contain higher ALDH1 and CD44+/CD24- population compared to monolayer cells. These cells were also resistant to radiation and upregulate stem cell survival signaling including beta-catenin, Notch1 and Survivin in response to radiation. Moreover, 3D spheres generated from the 293T cells have increased expression of mesenchymal genes including vimentin, n-cadherin, zeb1, snail and slug as well as pro-metastatic genes RhoC, Tenascin C and MTA1. In addition, microRNAs implicated in self-renewal and metastases were markedly reduced in 3D spheres. CONCLUSIONS: 293T cells exhibit a cancer stem cell-like phenotype when cultured as 3D spheres and represent an important research tool for studying the molecular and biological mechanisms of cancer stem cells and for testing and developing novel targets for cancer therapy.

Toxicological and pathophysiological roles of reactive oxygen and nitrogen species.

'Oxidative and Nitrative Stress in Toxicology and Disease' was the subject of a symposium held at the EUROTOX meeting in Dresden 15th September 2009. Reactive oxygen (ROS) and reactive nitrogen species (RNS) produced during tissue pathogenesis and in response to viral or chemical toxicants, induce a complex series of downstream adaptive and reparative events driven by the associated oxidative and nitrative stress. As highlighted by all the speakers, ROS and RNS can promote diverse biological responses associated with a spectrum of disorders including neurodegenerative/neuropsychiatric and cardiovascular diseases. Similar pathways are implicated during the process of liver and skin carcinogenesis. Mechanistically, reactive oxygen and nitrogen species drive sustained cell proliferation, cell death including both apoptosis and necrosis, formation of nuclear and mitochondrial DNA mutations, and in some cases stimulation of a pro-angiogenic environment. Here we illustrate the pivotal role played by oxidative and nitrative stress in cell death, inflammation and pain and its consequences for toxicology and disease pathogenesis. Examples are presented from five different perspectives ranging from in vitro model systems through to in vivo animal model systems and clinical outcomes.

Suberoylanilide hydroxamic acid blocks self-renewal and homotypic aggregation of inflammatory breast cancer spheroids.

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E-cadherin and exhibit the ability to undergo self-renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE-IBC and PE-LABC. METHODS: Methods used are as follows: culture of IBC cells from clonal density single cells in low adherence culture conditions that promote formation of IBC tumor spheroids; clonogenic assays; cell fractionation and Western blotting; confocal microscropy; and modified Boyden chamber invasion assays. RESULTS: SAHA inhibited self-renewal of IBC tumor spheroids from established IBC cell lines and PE-IBC and PE-LABC, as assessed by decreased clonogenic growth. SAHA blocked homotypic aggregation of the cells that comprised the IBC tumor spheroids leading to loss of their 3-dimensional (3D) structure, which was associated with a change in location of E-cadherin protein from the plasma membrane in untreated IBC tumor spheroids to the cytoplasm of cells within IBC tumor spheroids with SAHA treatment. In addition, SAHA blocked the robust invasion exhibited by IBC tumor spheroids of established cell lines as well as by tumor spheroids derived from PE-IBC and PE-LABC. CONCLUSIONS: SAHA targets the integrity and biological activities of IBC tumor spheroids and may be a promising agent to evaluate for its effectiveness in treatment of IBC.