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Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

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PURPOSE: To explore the expression of stem cell genes in breast cancer and the relationship between stem cell gene expression and clinical and pathological characteristics and prognosis of breast cancer. BACKGROUND: By now,stem cell differentiation-related genes and the relationship between the genes and clinic-pathological characteristics and prognosis of breast cancer are still unclear. MATERIALS AND METHODS: CD44+/CD24- tumor cells were selected by Flow cytometry. The differential expression of genes between CD44+/CD24- tumor cells and non-CD44+/CD24- tumor cells were detected by RT(2) Profiler™ PCR Array. The expression of stem cell gene Octamer-4 (Oct-4) was analyzed by immunohistochemistry staining and the relationship between Oct-4 and clinicopathological parameters of breast cancer was determined. RESULTS: Seven different genes including stem cell differentiation-related factors (CD44,Oct-4,and nestin),cell cycle regulators (APC and CDC2),and growth factors (HGF and TGF) were detected as significantly differently expressed between CD44+/CD24- tumor cells and non-CD44+/CD24- tumor cells. Oct-4 protein expressed significantly higher in cancerous tissues than adjacent-tumor tissues (P = 0.001). Moreover,we observed that the expression of Oct-4 protein was related to histological type,lymph node status and molecular type of breast cancer (P = 0.001,0.006,and 0.001,respectively). After survival analysis,the cases with highly expressed Oct-4 protein attained a significantly poorer postoperative disease-specific survival than those with none/low expressed Oct-4 protein (P = 0.001). In the Cox regression test,tumor size,histological type,disease stage,lymph node metastasis,Her-2 and Oct-4 were detected as the independent prognostic factors (P = 0.031,0.012,0.001,0.002,0.030,and 0.003,respectively). CONCLUSIONS: Oct-4 was highly expressed in CD44+/CD24- tumor cells,and may be a potential biomarker for the initiation,progression,and differentiation of breast cancer. View Publication

PURPOSE: Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy,many ultimately develop resistance to antiestrogens. However,mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped. EXPERIMENTAL DESIGN: We adapted four ER(+) human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy. RESULTS: The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole,each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally,knockdown of MYC inhibited LTED cell growth. CONCLUSIONS: A gene expression signature derived from ER(+) breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance. View Publication

OBJECTIVE: The role of semaphorins in tumor progression is still poorly understood. In this study,we aimed at elucidating the regulatory role of semaphorin 3A (SEMA3A) in primary tumor growth and metastatic dissemination. METHODS AND RESULTS: We used 3 different experimental approaches in mouse tumor models: (1) overexpression of SEMA3A in tumor cells,(2) systemic expression of SEMA3A following liver gene transfer in mice,and (3) tumor-targeted release of SEMA3A using gene modified Tie2-expressing monocytes as delivery vehicles. In each of these experimental settings,SEMA3A efficiently inhibited tumor growth by inhibiting vessel function and increasing tumor hypoxia and necrosis,without promoting metastasis. We further show that the expression of the receptor neuropilin-1 in tumor cells is required for SEMA3A-dependent inhibition of tumor cell migration in vitro and metastatic spreading in vivo. CONCLUSIONS: In sum,both systemic and tumor-targeted delivery of SEMA3A inhibits tumor angiogenesis and tumor growth in multiple mouse models; moreover,SEMA3A inhibits the metastatic spreading from primary tumors. These data support the rationale for further investigation of SEMA3A as an anticancer molecule. View Publication

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However,little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here,we used aldehyde dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin-) led to tumors in 13 (out of 15) mice,whereas 10,000 noncancer stem cells (ALDH-CD44-Lin-) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a subpopulation of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin- cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-μm radius) of blood vessels in human tumors,suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC,as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared with controls. Notably,selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively,these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck CSC. View Publication

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization,growth factor availability,cell adhesion,differentiation,and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study,we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity,whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably,we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly,SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings,increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together,our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC. View Publication

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. 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

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

PURPOSE: Genz-644282 [8,9-dimethoxy-5-(2-N-methylaminoethyl)-2,3-methylenedioxy-5H-dibenzo[c,h][1,6]naphthyridin-6-one] has emerged as a promising candidate for antitumor agents. This report describes the bone marrow colony-forming unit,granulocyte macrophage (CFU-GM) and tumor cell CFU activity of topoisomerase I (Top1) inhibitors,such as Genz-644282,topotecan,irinotecan/SN-38,and ARC-111,and examines their activity in several human tumor xenograft models. EXPERIMENTAL DESIGN: Colony-forming assays were conducted with mouse and human bone marrow and eight human tumor cell lines. In addition,29 human tumor cell lines representing a range of histology and potential resistance mechanisms were assayed for sensitivity to Genz-644282 in a 72-hour exposure assay. The efficacy of Genz-644282 was compared with standard anticancer drugs (i.e.,irinotecan,docetaxel,and dacarbazine) in human tumor xenografts of colon cancer,renal cell carcinoma,non-small cell lung cancer,and melanoma. RESULTS: Human bone marrow CFU-GM was more sensitive to the Top1 inhibitors than was mouse bone marrow CFU-GM. The ratio of mouse to human IC(90) values was more than 10 for the camptothecins and less than 10 for Genz-644282,which had more potency as a cytotoxic agent toward human tumor cells in culture than the camptothecins in the colony-forming and 72-hour proliferation assays. Genz-644282 has superior or equal antitumor activity in the human tumor xenografts than the standard drug comparators. CONCLUSIONS: On the basis of preclinical activity and safety,Genz-644282 was selected for development and is currently undergoing phase 1 clinical trial. View Publication

Cancer stem cells (CSCs) have been associated with metastasis and therapeutic resistance and can be generated via epithelial mesenchymal transition (EMT). Some studies suggest that the hormone melatonin acts in CSCs and may participate in the inhibition of the EMT. The objectives of this study were to evaluate the formation of mammospheres from the canine and human breast cancer cell lines,CMT-U229 and MCF-7,and the effects of melatonin treatment on the modulation of stem cell and EMT molecular markers: OCT4,E-cadherin,N-cadherin and vimentin,as well as on cell viability and invasiveness of the cells from mammospheres. The CMT-U229 and MCF-7 cell lines were subjected to three-dimensional culture in special medium for stem cells. The phenotype of mammospheres was first evaluated by flow cytometry (CD44+/CD24low/- marking). Cell viability was measured by MTT colorimetric assay and the expression of the proteins OCT4,E-cadherin,N-cadherin and vimentin was evaluated by immunofluorescence and quantified by optical densitometry. The analysis of cell migration and invasion was performed in Boyden Chamber. Flow cytometry proved the stem cell phenotype with CD44+/CD24low/- positive marking for both cell lines. Cell viability of CMT-U229 and MCF-7 cells was reduced after treatment with 1mM melatonin for 24 h (Ptextless0.05). Immunofluorescence staining showed increased E-cadherin expression (Ptextless0.05) and decreased expression of OCT4,N-cadherin and vimentin (Ptextless0.05) in both cell lines after treatment with 1 mM melatonin for 24 hours. Moreover,treatment with melatonin was able to reduce cell migration and invasion in both cell lines when compared to control group (Ptextless0.05). Our results demonstrate that melatonin shows an inhibitory role in the viability and invasiveness of breast cancer mammospheres as well as in modulating the expression of proteins related to EMT in breast CSCs,suggesting its potential anti-metastatic role in canine and human breast cancer cell lines. View Publication