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Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells,presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study,20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02),number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02),tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01),and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. View Publication

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS,we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3),including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also,GSK3 phosphorylated PAX3-FKHR in vitro,suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. View Publication

Adipose-derived stem cells (ASCs) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix (ECM) composition and stiffness,migration,and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A,ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225,and MCF10A overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly,ASCs promoted the self-renewal of all cell types except SUM225. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs,IFN$$ and HGF displayed unique actions on epithelial cell hierarchy. IFN$$ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres,whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF,whereas adipocytes expressed higher levels of IFN$$. Since luminal progenitor cells are believed to be prone for transformation,IFN$$ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. IMPLICATIONS This study suggests that the ratio of adipose-derived stem cells to adipocytes influences cancer cell hierarchy,which may impact incidence and progression. View Publication

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor,cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first,and randomized,after tumor regression to continuing treatment with gemcitabine,a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24,CD44,ALDH,nestin,and the hedgehog pathway. After induction with gemcitabine,treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently,a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer. View Publication

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus,inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study,we investigated the effects of imetelstat (GRN163L),a potent telomerase inhibitor,on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro,telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally,imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat,but not control oligonucleotides,also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after textless4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice,concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat,suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. View Publication

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However,NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study,we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation,apoptosis,gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay,flow cytometry,terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining,Western Blot and a xenograft mouse model,respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells,stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR,p-EGFR,p-STAT3,Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover,the combined treatment of TG101348 and erlotinib induced apoptosis,inhibited the activation of p-EGFR and p-STAT3,and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment. View Publication

Cancer stem cells (CSCs) or tumor initiating cells (TICs) make up only a small fraction of total tumor cell population,but recent evidence suggests that they are responsible for tumor initiation and the maintenance of tumor growth. Whether CSCs/TICs originate from normal stem cells or result from the dedifferentiation of terminally differentiated cells remains unknown. Here we provide evidence that sustained expression of the proinflammatory protein tissue transglutaminase (TG2) confers stem cell like properties in non-transformed and transformed mammary epithelial cells. Sustained expression of TG2 was associated with increase in CD44(high)/CD24(low/-) subpopulation,increased ability of cells to form mammospheres,and acquisition of self-renewal ability. Mammospheres derived from TG2-transfected mammary epithelial cells (MCF10A) differentiated into complex secondary structures when grown in Matrigel cultures. Cells in these secondary structures differentiated into Muc1-positive (luminal marker) and integrin α6-positive (basal marker) cells in response to prolactin treatment. Highly aggressive MDA-231 and drug-resistant MCF-7/RT breast cancer cells,which express high basal levels of TG2,shared many traits with TG2-transfected MCF10A stem cells but unlike MCF10A-derived stem cells they failed to form the secondary structures and to differentiate into Muc1-positive luminal cells when grown in Matrigel culture. Downregulation of TG2 attenuated stem cell properties in both non-transformed and transformed mammary epithelial cells. Taken together,these results suggested a new function for TG2 and revealed a novel mechanism responsible for promoting the stem cell characteristics in adult mammary epithelial cells. View Publication

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication

Melanoma,a potentially lethal skin cancer,is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses,limited knowledge exists on the role of mature B cells. We describe an approach,including a cell-based ELISA,to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (Ptextless0.0001). Interestingly,we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (Ptextless0.0001). Overall,28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly,a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients,which is reduced with disease progression,adding to previous reports of tumor-reactive antibodies in patient sera,and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. View Publication

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However,the role of PKCiota in lung tumor development has not been investigated. To address this question,we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene,Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore,the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus,Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore,aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment. View Publication

BACKGROUND Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases,such as cancer. The lncRNA,CCDC26,is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26,gene knockdown (KD) was performed using short hairpin RNAs (shRNAs),and four KD clones,in which CCDC26 expression was suppressed to 1% of its normal level,were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA),indicating that transcriptional gene suppression (TGS),not post-transcriptional suppression,was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant,which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast,enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor,KIT,hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03,a KIT-specific inhibitor,eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered. View Publication

Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha,Akt2/PKBbeta,and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent,isozyme-specific activity. In this report,we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds,maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin,a downstream substrate of Akt,is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally,we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells,but not normal cells,to apoptotic stimuli. View Publication