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The 26S proteasome,a multicatalytic enzyme complex,is the main intracellular proteolytic system involved in the degradation of ubiquitinated proteins. The ability of proteasome inhibitors to induce apoptosis has been exploited in the recent development of chemotherapeutic agents. Here,we show that inhibition of proteasome by MG132 blocks DNA damage-induced apoptosis. Blockage of apoptosis by MG132 correlates with p53 stabilization and upregulation of p21/WAF1,a p53 transcriptional target. Surprisingly,in the absence of MG132,robust apoptosis induced by a high dose of UV irradiation correlate with rapid p53 degradation. This is in sharp contrast to p53 stabilization when cells were exposed to lower levels of UV irradiation. Our findings highlight a scenario in which severe UV damage can induce rapid p53 degradation by the proteasome. Importantly,these data suggest that the 26S proteasome plays a key role in promoting apoptosis induced by high doses of UV irradiation. View Publication

BACKGROUND: Previously,we enriched a subpopulation of head and neck cancer-derived tumor initiating cells (HNC-TICs) presented high tumorigenic,chemo-radioresistant,and coupled with epithelial-mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC-TICs. METHOD: ALDH1 activity of head and neck cancer cells with quercetin treatment was assessed by the Aldefluor assay flow cytometry analysis. Self-renewal,invasiveness,and EMT capability of HNC-TICs with different doses of quercetin was presented. RESULTS: We first observed that the treatment of quercetin significantly downregulated the ALDH1 activity of head and neck cancer cells in a dose-dependent manner (p textless .05). Moreover,quercetin reduced self-renewal property and stemness signatures expression in head and neck cancer-derived sphere cells. The migration ability of head and neck cancer-derived sphere cells was lessened under quercetin treatment partially due to the decreased productions of Twist,N-cadherin,and vimentin. CONCLUSION: Quercetin suppressing HNC-TICs characteristics may therefore be valuable therapeutics clinically in combination with standard treatment modalities. View Publication

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer,as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence,novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance,irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover,we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore,GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together,our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus,GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors,especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. View Publication

Breast cancer is a heterogenous disease,composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently,a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells,or alternatively,in the case of breast cancer,breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence� View Publication

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

BACKGROUND To evaluate the anticancer efficacy of the combination of epigenetic modifiers and cisplatin in human ovarian cancer. METHODS The effect of trichostatin A (TSA) and 5-aza-2'-deoxycytidine alone or in combination with low-dose cisplatin was evaluated on human ovarian cancer cell lines in vitro. We measured drug interaction by MTS assay,migration by transwell assay,expression of epithelial to mesenchymal transition (EMT) markers (Twist,Snail,Slug,E-cadherin,and N-cadherin),pluripotency markers (Oct4,Sox2,and Nanog),and epigenetic markers (DNMT3A,LSD1 and H3K4me2,H3K4me3,H3K9me2,and H3K9me3) by western blot,and the impact on and characteristics of spheroid growth when exposed to these drugs. Mouse xenografts were used to evaluate the anticancer effect of sequential drug treatment. RESULTS Combination treatment had greater efficacy than single drugs and significantly suppressed cell viability,migration,and spheroid formation and growth. Sequential treatment of cisplatin (1 mg kg(-1)) followed by TSA (0.3 mg kg(-1)) significantly suppressed tumorigenicity of HEY xenografts through inhibition of EMT and decreased pluripotency of ovarian cancer cells. CONCLUSION Epigenetic modifiers potentiate the anticancer efficacy of low-dose cisplatin in ovarian cancer through regulation of EMT and pluripotency,and may provide a promising treatment for ovarian cancer patients. View Publication

BACKGROUND Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors,our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin,chrysin,apigenin,emodin,aloe-emodin,rhein,cis-stilbene and trans-stilbene) were studied on cell proliferation,cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines,together with normal haematopoietic control cells. METHODS Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS Emodin,quercetin,and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 μM,8-33 μM,and 25-85 μM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 μM,19-50 μM,and 8-50 μM respectively). Generally,lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines,however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly,the differential sensitivity of emodin,quercetin,and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. View Publication

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment,low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82),little/no EpoR protein was detected and it was not functional. In contrast,EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20),and functional responses to rHuEpo were reported with MCF-7 cells. In another study,a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However,the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82,no EpoR protein was detectable in normal human and matching cancer tissues from breast,lung,colon,ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells,but no rHuEpo-induced phosphorylation of AKT,STAT3,pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However,tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs,especially those in cancer patients. To circumvent these issues,we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable,but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition,invasion,stemness,and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ,downstream protumor factors,and hyaluronan and its cofactor TSG6,which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf" therapies and bioengineering applications." 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