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Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless,a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways,particularly human epidermal growth factor receptor 2 (EGFR2/HER2),have been implicated in AI resistance. However,the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers,such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells,as well as from in vivo xenograft tumors,indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall,these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP,HER2,EGFR/HER1,and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance. View Publication

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-,G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ,intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly,treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs,lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (ptextless0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture. View Publication

The mammary gland undergoes significant remodeling during pregnancy and lactation,which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here,we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4,SOX2,NANOG,known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts,a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium,they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore,hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer,with potential use in bioengineering and tissue regeneration. View Publication

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist,Noggin,and the small molecule SB431542,respectively,induces efficient neuralization of hiPSCs,a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost,consistent activity,and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1,a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration,we could selectively modulate the number of SOX1 expressing cells,whereas PAX6,another neural precursor marker,remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations,therefore,suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons,a subset of which also express tyrosine hydroxylase. Thus,the combined use of DMH1,a highly specific BMP-pathway inhibitor,and SB431542,a TGF-β1-pathway specific inhibitor,provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. View Publication

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However,how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8(+) T cells towards stem cell-like memory (T(SCM)) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here,we evaluated if T(SCM) can be obtained from human mature CD8(+) T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119),which inhibits the glycogen synthase kinase-3β (GSK-3β),key inhibitor of the Wnt pathway. Human CD8(+) T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL),and treated with TWS119 gave rise to CD62L(+)CD45RA(+) cells,indicative of early differentiated stage,also expressing CD127 which is normally found on memory cells,and CD133,an hematopoietic stem cell marker. T(SCM) cells raised from either TIL or blood secreted numerous inflammatory mediators,but in lower amounts than those measured without TWS119. Finally,generated T(SCM) CD8(+) T cells expressed elevated Bcl-2 and no detectable caspase-3 activity,suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T(SCM) subset in human CD8(+) T cells from TIL and the periphery,which are relevant for ACT. View Publication

The possibility of using cell-based therapeutics to treat cardiac failure has generated significant interest since the initial introduction of stem cell-based technologies. However,the methods to quickly and robustly direct stem cell differentiation towards cardiac cell types have been limited by a reliance on recombinant growth factors to provide necessary biological cues. We report here the use of dorsomorphin homologue 1 (DMH1),a second-generation small molecule BMP inhibitor based on dorsomorphin,to efficiently induce beating cardiomyocyte formation in mouse embryonic stem cells (ESCs) and to specifically upregulate canonical transcriptional markers associated with cardiac development. DMH1 differs significantly from its predecessor by its ability to enrich for pro-cardiac progenitor cells that respond to late-stage Wnt inhibition using XAV939 and produce secondary beating cardiomyocytes. Our study demonstrates the utility of small molecules to complement existing in vitro cardiac differentiation protocols and highlights the role of transient BMP inhibition in cardiomyogenesis. View Publication

The combination of chronic hypoxia and treatment of rats with the vascular endothelial growth factor (VEGF) receptor blocker,SU5416,induces pulmonary angio-obliteration,resulting in severe pulmonary arterial hypertension (PAH). Inflammation is thought to contribute to the pathology of PAH. Allergic inflammation caused by ovalbumin (OVA) immunization causes muscularization of pulmonary arteries,but not severe PAH. Whether disturbance of the immune system and allergic inflammation in the setting of lung endothelial cell apoptosis causes PAH is unknown. We investigated the effects of OVA-allergic inflammation on the development of PAH initiated by VEGF blockade-induced lung endothelial cell apoptosis. OVA-immunized rats were treated with SU5416 to induce pulmonary vascular endothelial cell apoptosis. The combination of OVA and SU5416 treatment resulted in severe angio-obilterative PAH,accompanied by increased IL-6 expression in the lungs. c-Kit(+) and Sca-1(+) cells were found in and around the lung vascular lesions. Pan-caspase inhibiton,dexamethasone treatment,and depletion of B-lymphocytes using an anti-CD20 antibody suppressed this remodeling. OVA immunization also increased lung tissue hypoxia-induced factor-1α and VEGF expression. Our results also suggest that the increased expression of hypoxia-induced factor-1α and IL-6 induced by the allergic lung inflammation may be a component of the pathogenesis of PAH. View Publication

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and its efficacy has been tested in pre-clinical models by delivering it systemically as a purified ligand or via engineered stem cells (SC). However,about 50% of tumor lines are resistant to TRAIL and overcoming TRAIL resistance in aggressive tumors,such as glioblastoma-multiforme (GBM),and understanding the molecular dynamics of TRAIL-based combination therapies are critical to broadly use TRAIL as a therapeutic agent. In this study,we developed death receptor (DR)4/5-reporters that offer an imaging-based platform to identify agents that act in concert with a potent,secretable variant of TRAIL (S-TRAIL) by monitoring changes in DR4/5 expression. Utilizing these reporters,we show a differential regulation of DR4/5 when exposed to a panel of clinically relevant agents. A histone deacetylase inhibitor,MS-275,resulted in upregulation of DR4/5 in all GBM cell lines,and these changes could be followed in real time both in vitro and in vivo in mice bearing tumors and they correlated with increased TRAIL sensitivity. To further assess the dynamics of combinatorial strategies that overcome resistance of tumors to SC released S-TRAIL,we also engineered tumor cells to express live-cell caspase-reporters and SCs to express S-TRAIL. Utilizing DR4/5 and caspase reporters in parallel,we show that MS-275 sensitizes TRAIL-resistant GBM cells to stem cell (SC) delivered S-TRAIL by changing the time-to-death in vitro and in vivo. This study demonstrates the effectiveness of a combination of real-time reporters of TRAIL-induced apoptosis pathway in evaluating the efficacy of SC-TRAIL-based therapeutics and may have implications in targeting a broad range of cancers. View Publication

Introduction: Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However,hESC are difficult to adapt to 96-well plate screening assays,because they survive best when plated as colonies,which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. Methods: We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy,and the method was tested using known toxicants. Results: The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable,and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products,which contain a variety of chemicals. The screen was done using single wells/dose,and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement,and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied,and methods to monitor and avoid vapor effects were incorporated into the assay. Discussion: Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development. ?? 2012 Elsevier Inc. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions,the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4,Nanog,Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (ptextless0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC,E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2),at concentrations that selectively block I(K1) (50-100 µM),failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I(K1) in 53% (20/38) of the cells studied,but presence of I(Kr) in all (11/11). Consistent with the electrophysiological data,RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1) in the majority of cells,but robust expression of I(Kr.) In contrast to recently reported studies,our data point to major deficiencies of hiPSC-CM,with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I(Kr) due to the absence of I(K1). Thus,efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications. View Publication

PURPOSE We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS In vitro,PF-03084014 exhibited activity against tumor cell migration,endothelial cell tube formation,and mammosphere formation. In vivo,we observed apoptosis,antiproliferation,reduced tumor cell self-renewal ability,impaired tumor vasculature,and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However,the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines,suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models,the baseline expressions of the Notch receptors,ligands,and the cleaved Notch1 failed to predict the antitumor response to PF-03084014,whereas several Notch pathway target genes,including HEY2,HES4,and HES3,strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients. View Publication