技术资料

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%,despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival,such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies,including AML,and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover,constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation,although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor,C188-9,inhibited G-CSF-induced Stat3 phosphorylation,induced apoptosis in AML cell lines and primary samples,and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore,Stat3 inhibition may be a valuable strategy for targeted therapies for AML. 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

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

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells,suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study,we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC),which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore,anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells,indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs,both as a monotherapy and in combination with lenalidomide,to improve MM patient outcome. 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

Inflammatory breast cancer (IBC) is an angioinvasive and most aggressive type of advanced breast cancer characterized by rapid proliferation,chemoresistance,early metastatic development and poor prognosis. IBC tumors display a triple-negative breast cancer (TNBC) phenotype characterized by centrosome amplification,high grade of chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα),progesterone receptor (PR) and HER-2 tyrosine kinase receptor. Since the TNBC cells lack these receptors necessary to promote tumor growth,common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer. To date,not a single targeted therapy has been approved for non-inflammatory and inflammatory TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents remains the standard therapy. IBC tumors generally display activation of epithelial to mesenchymal transition (EMT) that is functionally linked to a CD44+/CD24-/Low stem-like phenotype. Development of EMT and consequent activation of stemness programming is responsible for invasion,tumor self-renewal and drug resistance leading to breast cancer progression,distant metastases and poor prognosis. In this study,we employed the luminal ER+ MCF-7 and the IBC SUM149PT breast cancer cell lines to establish the extent to which high grade of CIN and chemoresistance were mechanistically linked to the enrichment of CD44+/CD24low/- CSCs. Here,we demonstrate that SUM149PT cells displayed higher CIN than MCF-7 cells characterized by higher percentage of structural and numerical chromosomal aberrations. Moreover,centrosome amplification,cyclin E overexpression and phosphorylation of retinoblastoma (Rb) were restricted to the stem-like CD44+/CD24-/Low subpopulation isolated from SUM149PT cells. Significantly,CD44+/CD24-/Low CSCs displayed resistance to conventional chemotherapy but higher sensitivity to SU9516,a specific cyclin-dependent kinase 2 (Cdk2) inhibitor,demonstrating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44+/CD24-/Low CSC subpopulation in IBC. In conclusion,our findings propose a novel therapeutic approach to restore chemosensitivity and delay recurrence of IBC tumors based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase. 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

OBJECTIVE The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However,the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. METHODS The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR,immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay. RESULTS The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (Ptextless0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (Ptextless0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486,P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza),the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased,the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. CONCLUSION KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis. View Publication

INTRODUCTION: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®),an allosteric mTOR inhibitor,is proving valuable in this setting; however,some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors,exemplified by AZD8055,by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth,cell proliferation (Ki67),viability and migration,with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 μM),rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055,which rapidly inhibited both mTORC1 and mTORC2/AKT activity,was a highly effective (P textless0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM),and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P textless0.05) inhibited resistant cell proliferation,increased cell death and reduced migration. Furthermore,dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P textless0.05). Co-treating with AZD8055 alongside tamoxifen (P textless0.01) or oestrogen deprivation (P textless0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X,it had no effect on ER ser118 activity or expression of several ER-regulated genes,suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC,even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease,and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance. View Publication

OBJECTIVE: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. DESIGN: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing,the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. RESULTS: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts,murine Hepa129 allografts,and MH3924A rat allografts. Additionally,tumor growth,ascites formation,and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7,Hep3B xenografts,and MH3924A allografts. On the signaling pathway level,the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA,inhibition of ERK phosphorylation,tumor cell proliferation,and microvessel density was observed in vivo. CONCLUSION: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC. 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

Tunneling nanotubes (TnTs) are long,non-adherent,actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study,we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48. h; and this effect was most prominent in media conditions (low-serum,hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs,in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs,which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation,and also lipid raft formation as a potential biomarker for TnT-forming cells. textcopyright 2014 Elsevier Inc. View Publication