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Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted,yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence,in this study,we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2),mild dysplasia (DOK),severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44,p75(NTR),CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44,p75(NTR),CD24 antigens and ALDH activity (ALDEFLUOR(®) assay),with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer,increased FOXA1 and decreased FOXA2 expression correlated with disease severity,but OCT3/4,Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. View Publication

BACKGROUND AND OBJECTIVES: Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells,but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts. DESIGN AND METHODS: AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72,SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation,apoptosis and cytokine release. RESULTS: The cross-talk between these two cell populations,achieved via release of soluble mediators,resulted in increased AML blast proliferation,including increased proliferation of clonogenic progenitors,but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk,and normal osteoblasts could also increase the AML blast proliferation. Furthermore,co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8. INTERPRETATION AND CONCLUSIONS: Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels. View Publication

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations,gene expression profiles and response to treatment: WNT,Sonic Hedgehog (SHH),Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example,expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however,its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs),but not their normal counterparts (hENs),resemble Groups 3 and 4 MB in vitro and in vivo. Here,we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs,respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth,self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes,such as SOX2,and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast,OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. View Publication

The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study,constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML,expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression. View Publication

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study,we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs,it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples� View Publication

BACKGROUND Despite advances in early diagnosis and treatment of cancer patients,metastasis remains the major cause of mortality. TP53 is one of the most frequently mutated genes in human cancer,and these alterations can occur during the early stages of oncogenesis or as later events as tumors progress to more aggressive forms. Previous studies have suggested that p53 plays a role in cellular pathways that govern metastasis. To investigate how p53 deficiency contributes to late-stage tumor growth and metastasis,we developed paired isogenic patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC) differing only in p53 status for longitudinal analysis. METHODS Patient-derived isogenic human tumor lines differing only in p53 status were implanted into mouse mammary glands. Tumor growth and metastasis were monitored with bioluminescence imaging,and circulating tumor cells (CTCs) were quantified by flow cytometry. RNA-Seq was performed on p53-deficient and p53 wild-type tumors,and functional validation of a lead candidate gene was performed in vivo. RESULTS Isogenic p53 wild-type and p53-deficient tumors metastasized out of mammary glands and colonized distant sites with similar frequency. However,p53-deficient tumors metastasized earlier than p53 wild-type tumors and grew faster in both primary and metastatic sites as a result of increased proliferation and decreased apoptosis. In addition,greater numbers of CTCs were detected in the blood of mice engrafted with p53-deficient tumors. However,when normalized to tumor mass,the number of CTCs isolated from mice bearing parental and p53-deficient tumors was not significantly different. Gene expression profiling followed by functional validation identified B cell translocation gene 2 (BTG2),a downstream effector of p53,as a negative regulator of tumor growth both at primary and metastatic sites. BTG2 expression status correlated with survival of TNBC patients. CONCLUSIONS Using paired isogenic PDX-derived metastatic TNBC cells,loss of p53 promoted tumor growth and consequently increased tumor cell shedding into the blood,thus enhancing metastasis. Loss of BTG2 expression in p53-deficient tumors contributed to this metastatic potential by enhancing tumor growth in primary and metastatic sites. Furthermore,clinical data support conclusions generated from PDX models and indicate that BTG2 expression is a candidate prognostic biomarker for TNBC. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

PURPOSE: The molecular signal components essential to paclitaxel-dependent apoptosis in breast cancers are potential targets for combined therapy. However,the signal mechanisms underlying paclitaxel action still need to be better defined. EXPERIMENTAL DESIGN: In a breast cancer cell line,pharmacological agents and transient transfection with dominant interfering and constitutive active mutants were used to identify the signal transduction module involved in the regulation of paclitaxel-induced apoptosis and to evaluate its potential as a therapeutic target. RESULTS: In MDA-MB-435 cells,paclitaxel treatment stimulated the activity of both protein kinase A and p38,and inhibited the activity of the Na(+)/H(+) exchanger isoform 1 (NHE1) with similar IC(50) concentrations as for its activation of apoptosis. Activation and inhibition experiments demonstrated that protein kinase A and p38 participate sequentially upstream of the NHE1 in regulating the paclitaxel-induced apoptotic pathway. Importantly,concurrent specific inhibition of the NHE1 with paclitaxel treatment resulted in a synergistic induction of apoptosis and a reduction in the paclitaxel IC(50) for apoptosis. This sensitization of paclitaxel apoptotic action by specific inhibition of NHE1 was verified in breast cancer cell lines with different paclitaxel sensitivity. CONCLUSIONS: We have,for the first time,identified NHE1 as an essential component of paclitaxel-induced apoptosis in breast cancer cells and,importantly,identified that simultaneous inhibition of the NHE1 results in a synergistic potentiation of low-dose paclitaxel apoptotic action. As specific NHE1 inhibitors have finished Phase II/Phase III clinical trials for myocardial protection,there is the possibility for a rapid biological translation of this novel therapeutic strategy to a clinical setting. View Publication

BACKGROUND: Compelling evidence indicates that paclitaxel kills cancer cells through the induction of apoptosis. Paclitaxel binds microtubules and causes kinetic suppression (stabilization) of microtubule dynamics. The consequent arrest of the cell cycle at mitotic phase has been considered to be the cause of paclitaxel-induced cytotoxicity. However,the biochemical events,downstream from paclitaxel's binding to microtubules,that lead to apoptosis are not well understood. METHODS: The authors examined recent scientific literature about the mechanisms by which paclitaxel exerts cytotoxicity. RESULTS: In addition to an arrest of the cell cycle at the mitotic phase in paclitaxel-treated cells,recent discoveries of activation of signaling molecules by paclitaxel and paclitaxel-induced transcriptional activation of various genes indicate that paclitaxel initiates apoptosis through multiple mechanisms. The checkpoint of mitotic spindle assembly,aberrant activation of cyclin-dependent kinases,and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) are shown to be involved in paclitaxel-induced apoptosis. Consistent with observations that microtubules of different status (e.g.,cytoskeletal microtubules vs. mitotic spindles) have different sensitivity to paclitaxel,the concentration of paclitaxel appears to be the major determinant of its apoptogenic mechanisms. CONCLUSIONS: Advances in research of the cell cycle and apoptosis have extended our understanding of the mechanisms of paclitaxel-induced cell death. Further elucidation of resistance and enhancement of paclitaxel-induced apoptosis should expedite the development of better paclitaxel-based regimens for cancer therapy. View Publication

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4,which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast,at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia. View Publication