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Focal adhesion kinase (FAK) has been implicated in the development of cancers,including those of the breast. Nevertheless,the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here,we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers,including aldehyde dehydrogenase,CD24,CD29,and CD61,we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally,through transplantation in NOD-SCID mice,we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together,these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. View Publication

Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH(+) cells that were highly clonogenic. Moreover,ALDH(+) MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9,and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH(+) MCL cells,induced terminal plasma cell differentiation,and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH(+) cells as well as improve the activity of proteasome inhibitors in MCL. View Publication

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab,rituximab,and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs,and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast,rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab,with 19.2 and 23.5% cell death at 4 and 24 h,respectively,compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85,respectively). Interestingly,lysis by all three Abs at high concentrations was mostly complement dependent,because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion,it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays,but phagocytosis was not significant in whole blood due to excess Igs. Finally,GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101,but not rituximab,also mediated significant NK cell degranulation in whole blood samples. Thus,complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays. View Publication

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound,SGI-1776 inhibits Pim-1,Pim-2 and Pim-3,and was evaluated in AML-cell line,-xenograft model,and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets,c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47),were both decreased in actively cycling AML cell lines MV-4-11,MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2,Bcl-x(L),XIAP,and proapoptotic Bak and Bax were unchanged; however,a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data,xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly,SGI-1776 was also cytotoxic in AML primary cells,irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. View Publication

BACKGROUND Many cancers,including melanoma,exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast,mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules,we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1,MAGE-3,gp100,and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4),transfected with control siRNA (Arm B; n = 3),or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS Vaccination stimulated antigen-specific T cell responses in all subjects,which peaked after 3-4 vaccinations,but remained elevated in Arm C subjects. Also in Arm C,circulating melanoma cell levels (as detected by quantitative PCR) fell,and T cell lytic activity against autologous melanoma was induced. In HLA-A2 subjects,CD8 T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C),one had a partial clinical response,while the other,who exhibited diffuse dermal and soft tissue metastases,had a complete response. CONCLUSION These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION Clinicaltrials.gov NCT00672542. FUNDING Duke Clinical Research Institute/Duke Translational Medicine Institute,Duke Melanoma Consortium,and Duke University Department of Surgery. View Publication

Purpose: The bone marrow microenvironment is considered a critical component in the dissemination and fate of cancer cells in the metastatic process. We explored the possible correlation between bone marrow mesenchymal stem cells (BM-MSC) and disseminated breast cancer-initiating cells (BCIC) in primary breast cancer patients. Experimental design: Bone marrow mononuclear cells (BM-MNC) were collected at the time of primary surgery in 12 breast cancer patients. BM-MNC was immunophenotyped and BCIC was defined as epithelial cells (CD326+CD45-) with a stem-like" phenotype (CD44+CD24low/-� View Publication

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin,effective in treating type 2 diabetes and the insulin resistance syndromes,improves insulin resistance by reducing hepatic gluconeogenesis and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin use with decreased cancer incidence and cancer-related mortality. Furthermore,numerous preclinical and clinical studies have demonstrated anti-cancer effects of metformin,leading to an explosion of interest in evaluating this agent in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers associated with hyperinsulinaemia; however,metformin may also directly inhibit tumour growth. In this review,we describe the mechanism of action of metformin and summarise the epidemiological,clinical and preclinical evidence supporting a role for metformin in the treatment of cancer. In addition,the challenges associated with translating preclinical results into therapeutic benefit in the clinical setting will be discussed. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication

Treatment with arsenic trioxide (As(2)O(3)) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As(2)O(3) (iAs(III)) is methylated in the liver to mono- and dimethylated metabolites,including methylarsonic acid,methylarsonous acid,dimethylarsinic acid,and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins,genotoxins,and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAs(III). Therefore,we compared the potency of iAs(III) and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation,growth inhibition,and apoptosis. Methylarsine oxide (MAs(III)O) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAs(III) in NB4 cells,an APL cell line. This was also observed in K562 human leukemia,lymphoma cell lines,and in primary culture of chronic lymphocytic leukemia cells,but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAs(III)O,in contrast to iAs(III),did not induce PML-retinoic acid receptor alpha degradation,or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment,hepatoma-derived HepG2 cells,but not NB4 cells,methylate radiolabeled iAs(III). Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAs(III) may contribute to As(2)O(3)-induced apoptosis but not differentiation of APL cells. MAs(III)O as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma. View Publication

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA,followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample,whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles,we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP,CTCGAGCTGHHHHHAACTAC,where H is a mixture of base consisting of 50% A,25% T,and 25% C; and second 5' DOP,CTCGAGCTGDDDDDGTTTAG,where D is a mixture of base consisting of 50% T,25% G,and 25% A. Our results showed that bisulfite-modified DNAs from a cell line,cord blood cells,or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template. View Publication