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The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here,we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells,and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16,but not doxorubicin,transiently induced NF-kappaB activity in all cell lines,whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells,but rather high in Capan-1 and A818-4 cells,as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin,MG132 and Sulfasalazine),or transfection with the IkappaBalpha super-repressor,strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study,we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs),since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity,but 9-cis-retinoic acid (RA),13-cis-RA,and all-trans-RA (ATRA) were markedly more efficacious than Ro40-8757,Ro13-6298,and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs,thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1,a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems,the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact,retinoid-induced modifications of cell morphology,phenotype (downregulation of CD19,HLA-DR,and s-Ig,and increased expression of CD38 and c-Ig),and IgM production were late events,highly heterogeneous,and often slightly relevant,being therefore only partially indicative of a drug-related differentiative process. Moreover,EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids,indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients. View Publication

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055,an ATP-competitive mTOR inhibitor,by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors,including RMS. Therefore,in the present study,we searched for AZD8055-based combination therapies. Here,we identify a new synergistic lethality of AZD8055 together with ABT-737,a BH3 mimetic that antagonizes Bcl-2,Bcl-xL,and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index textless 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level,as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential,activation of caspases,and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055,the PI3K/mTOR inhibitor NVP-BEZ235,the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis,whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly,molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly,knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737,our findings have important implications for the development of molecular targeted therapies for RMS. View Publication

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced by adipocytes,but paradoxically decreased in obesity,that has been implicated in MM progression. Herein,we evaluated how prolonged exposure to adiponectin affected the survival of MM cells as well as putative signaling mechanisms. Adiponectin activates protein kinase A (PKA),which leads to decreased AKT activity and increased AMP-activated protein kinase (AMPK) activation. AMPK,in turn,induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated,at least in part,by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC),which is essential to lipogenesis. Supplementation with palmitic acid,the preliminary end product of fatty acid synthesis,rescues MM cells from adiponectin-induced apoptosis. Furthermore,5-(tetradecyloxy)-2-furancarboxylic acid (TOFA),an ACC inhibitor,exhibited potent antiproliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus,adiponectin's ability to reduce survival of MM cells appears to be mediated through its ability to suppress lipogenesis. Our findings suggest that PKA/AMPK pathway activators,or inhibitors of ACC,may be useful adjuvants to treat MM. Moreover,the antimyeloma effect of adiponectin supports the concept that hypoadiponectinemia,as occurs in obesity,promotes MM tumor progression. View Publication

The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013),and for six of these drugs,the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers,suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors,and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR,ABL1 and BRAF(V600E)-driven cell growth,and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors,we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action. View Publication

Gastric diseases,including peptic ulcer disease and gastric cancer,affect 10% of the world's population and are largely due to chronic Helicobacter pylori infection. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis,and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells. We show that temporal manipulation of the FGF,WNT,BMP,retinoic acid and EGF signalling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains,proliferative zones containing LGR5-expressing cells,surface and antral mucous cells,and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signalling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease,we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor,activation of signalling and induction of epithelial proliferation. Together,these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. View Publication

TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides,the most common type of cutaneous T-cell lymphoma (CTCL),as compared to normal skin or benign inflammatory dermatoses. However,its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study,we demonstrate that TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome,a leukemic variant of CTCL,and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression,leading to less cell viability and colony-forming ability in vitro and to reduced tumor growth in vivo. Furthermore,TOX knockdown significantly increased 2 cyclin-dependent kinase (CDK) inhibitors,CDKN1B and CDKN1C. Lastly,blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively,these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL. View Publication