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Supplementary Figures 1-5 from PUMA Induction by FoxO3a Mediates the Anticancer Activities of the Broad-Range Kinase Inhibitor UCN-01

Purpose: Overexpression of inhibitors of apoptosis proteins (IAP) contributes to therapeutic resistance. Second mitochondria-derived activator of caspase (Smac) promotes caspase activation by binding to IAPs upon release from the mitochondria. IAP antagonists, also called SMAC mimetics, are promising anticancer agents modeled after this mechanism. We investigated the role and mechanisms of Smac- and Smac mimetic–mediated chemosensitization in head and neck squamous cell carcinoma (HNSCC) cells.Experimental Design: The effects of SMAC knockdown, SMAC overexpression, and a small molecule Smac mimetic on the chemosensitivities of HNSCC cells were determined. The mechanisms of Smac- and Smac mimetic–mediated chemosensitization were investigated by analyzing growth suppression, the mitochondrial apoptotic pathway, caspase activation, and IAP proteins. The therapeutic responses of HNSCC cells with different levels of Smac were compared in xenograft models.Results:We found that Smac mediates apoptosis induced by several classes of therapeutic agents through the mitochondrial pathway. SMAC knockdown led to impaired caspase activation, mitochondrial membrane depolarization, and release of cytochrome c. A small molecule Smac mimetic, at nanomolar concentrations, significantly sensitized HNSCC cells to gemcitabine-induced apoptosis and restored gemcitabine sensitivity in SMAC knockdown cells, through caspase activation, X-linked IAP dissociation, and mitochondria-associated events, but not the TNF-α pathway. Furthermore, Smac levels modulated the therapeutic response of HNSCC cells to gemcitabine in xenograft models.Conclusions: Our results establish a critical role of Smac in mediating therapeutic responses of HNSCC cells and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC. Clin Cancer Res; 17(8); 2361–72. ©2011 AACR.

Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer.Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth.Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2′-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor–containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation.Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.

Supplementary Figures S1-S7; Supplementary Methods.

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Supplementary Figures 1-6 from Fibulin-5 Suppresses Lung Cancer Invasion by Inhibiting Matrix Metalloproteinase-7 Expression

Supplementary Figures 1-5, Table 1 from Following Cytochrome c Release, Autophagy Is Inhibited during Chemotherapy-Induced Apoptosis by Caspase 8–Mediated Cleavage of Beclin 1

Supplementary Methods from Following Cytochrome c Release, Autophagy Is Inhibited during Chemotherapy-Induced Apoptosis by Caspase 8–Mediated Cleavage of Beclin 1

Supplementary Tables 1-5 from Fibulin-5 Suppresses Lung Cancer Invasion by Inhibiting Matrix Metalloproteinase-7 Expression

Supplementary Methods, Figures 1-6 from Fibulin-5 Suppresses Lung Cancer Invasion by Inhibiting Matrix Metalloproteinase-7 Expression

The high mortality rate of lung cancer is largely due to the spread of disease to other organs. However, the molecular changes driving lung cancer invasion and metastasis remain unclear. In this study, we identified fibulin-5, a vascular ligand for integrin receptors, as a suppressor of lung cancer invasion and metastasis. Fibulin-5 was silenced by promoter hypermethylation in a majority of lung cancer cell lines and primary tumors. It inhibited lung cancer cell invasion and down-regulated matrix metalloproteinase-7 (MMP-7), which promoted lung cancer cell invasion. Knockdown of fibulin-5 was sufficient to stimulate cell invasion and MMP-7 expression. The expression levels of fibulin-5 and MMP-7 were inversely correlated in lung tumors. Suppression of MMP-7 expression by fibulin-5 was mediated by an integrin-binding RGD motif via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, overexpression of fibulin-5 in H460 lung cancer cells inhibited metastasis in mice. Collectively, these results suggest that epigenetic silencing of fibulin-5 promotes lung cancer invasion and metastasis by activating MMP-7 expression through the ERK pathway. [Cancer Res 2009;69(15):6339–46]

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis

Key osteoclast (OCL) regulatory gene promoters in bone marrow-derived monocytes harbor bivalent histone modifications that combine activating Histone 3 lysine 4 tri-methyl (H3K4me3) and repressive H3K27me3 marks, which upon RANKL stimulation resolve into repressive or activating architecture. Enhancer of zeste homologue 2 (EZH2) is the histone methyltransferase component of the polycomb repressive complex 2, which catalyzes H3K27me3 modifications. Immunofluorescence microscopy reveals that EZH2 localization during murine osteoclastogenesis is dynamically regulated. Using EZH2 knockdown and small molecule EZH2 inhibitor GSK126, we show that EZH2 plays a critical epigenetic role in OCL precursors (OCLp) during the first 24 hours of RANKL activation. RANKL triggers EZH2 translocation into the nucleus where it represses OCL-negative regulators MafB, Irf8, and Arg1. Consistent with its cytoplasmic localization in OCLp, EZH2 methyltransferase activity is required during early RANKL signaling for phosphorylation of AKT, resulting in downstream activation of the mTOR complex, which is essential for induction of OCL differentiation. Inhibition of RANKL-induced pmTOR-pS6RP signaling by GSK126 altered the translation ratio of the C/EBPβ-LAP and C/EBPβ-LIP isoforms and reduced nuclear translocation of the inhibitory C/EBPβ-LIP, which is necessary for transcriptional repression of the OCL negative-regulatory transcription factor MafB. EZH2 in multinucleated OCL is primarily cytoplasmic and mature OCL cultured on bone segments in the presence of GSK126 exhibit defective cytoskeletal architecture and reduced resorptive activity. Here we present new evidence that EZH2 plays epigenetic and cytoplasmic roles during OCL differentiation by suppressing MafB transcription and regulating early phases of PI3K-AKT-mTOR-mediated RANKL signaling, respectively. Consistent with its cytoplasmic localization, EZH2 is required for cytoskeletal dynamics during resorption by mature OCL. Thus, EZH2 exhibits complex roles in supporting osteoclast differentiation and function. © 2019 American Society for Bone and Mineral Research.

Immuno-oncology (IO) therapy is the focus of drug discovery in recent years. Unlike chemotherapy or targeted agents, the pharmacological efficacy of immune-oncology therapies needs to be evaluated in animal models with functional immune system. Syngeneic tumor models are established by inoculating mouse cancer cell lines to immunocompetent mice with the same genetic background. The host mice have complete immune activity and show histocompatibility with homograft tumor tissues, which can maximize the simulation of the real tumor microenvironment. Subcutaneous syngeneic models are easily established and have been widely used; however, the subcutaneous tumors lack organ-specific stromal-tumor interactions that are critical for disease progression in patients. Orthotopic tumor models are established in organ-specific sites, facilitating metastatic spread, supporting immune and stromal component interactions, and providing a more disease-relevant tumor microenvironment for IO therapy assessment. In order to facilitate the development of IO drug, we have established a panel of orthotopic syngeneic tumor models. We generated a series of luciferase-labeled bioluminescent mouse tumor cell lines, which were then engrafted into the organ of interest to establish orthotopic models. Bioluminescent imaging (BLI) enabled non-invasive in vivo imaging of orthotopic tumor burden, the real time tumor growth, and the response to therapies, monitored by quantitative bioluminescent signals. Overall, these established orthotopic syngeneic tumor models recapitulate the clinically relevant tumor microenvironment and serve as a useful tool in the pre-clinical evaluation of immunotherapies. Citation Format: Gaoxiang Liu, Quanhong Sun, Wenting Shi, Qingyang Gu, Qunsheng Ji. Developing a panel of orthotopic syngeneic tumor models for IO drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6024.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5’ end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

The use of cancer-relevant microRNA molecules as endogenous drug release stimuli is promising for personalized cancer treatment yet remains a great challenge because of their low abundance. Herein, we report a new type of microRNA-catalyzed drug release system based on DNA-programmed gold nanoparticle (GNP)-quantum dot (QD) complex. We show that a trace amount of miRNA-21 molecules could specifically catalyze the disassembly of doxorubicin (Dox)-loaded GNP-QDs complex through entropy driven process, during which the Dox-intercalating sites are destructed for drug release. This catalytic reaction could proceed both in fixed cells and live cells with miRNA-21 overexpression. Dox molecules could be efficiently released in the cells and translocate to cell nuclei. QD photoluminescence is simultaneously activated during catalytic disassembly process, thus providing a reliable feedback for microRNA-triggered drug release. The GNP-QDs-Dox complex exhibits much higher drug potency than free Dox molecules, and therefore represents a promising platform for accurate and effective cancer cell treatment.

Osteoclasts, the specialized cells that resorb bone, play a key role in normal bone homeostasis, and hence their dysregulation is involved in many skeletal diseases. Most often skeletal diseases involve excess osteoclast formation or activation that leads to pathological bone resorption such as observed in inflammatory disorders. The lack of osteoclasts or impaired osteoclast function causes osteopetrosis diseases. Understanding the dysregulated mechanisms affecting osteoclast differentiation and function in each disease will aid our understanding of the normal regulation and provide potential therapeutic targets for the pathological regulation.

Background In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown. Methods Human primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo. Results We found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth. Conclusions These results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM. Electronic supplementary material The online version of this article (10.1186/s13045-018-0666-5) contains supplementary material, which is available to authorized users.

Aiming at the problems in the management and sharing research of antique information, this paper discussed the necessity to build antique information distributed data warehouse. The data warehouse architecture and the multidi- mensional data model were put forward, by usingonline analysis processing (OLAP) the multidimensional analysis of an- tique distributed data warehouse was presented, and a multidimensional analysis process was given. By multidimensional analysis of the distributed data warehouse, the accessing and analyzing platform was provided for antique managers and researchers to provide strong support for the work of antique information management and sharing.

Paget's disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include increased sensitivity of osteoclast progenitors to 1,25(OH)2 D3 , receptor activator of NF-κB ligand (RANKL), and TNF-α; increased osteoclast numbers; and increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and IκB kinase-ϵ (IKKϵ) are IKK family members that complex with MVNP and activate both IRF3 and NF-κB pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKKϵ early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM owing to increased TBK1 protein stability. TBK1 overexpression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-κB, TAF12, and ATF7 proteins in several cell lines. Overexpression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2 D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation and on the expression of IL-6, a key contributor to the pagetic osteoclast phenotype.

Measles virus plays an important role as an environmental factor in the pathogenesis of Paget’s disease (PD). Previous studies have shown that IL-6 is increased in the bone marrow of Paget’s patients and that measles virus nucleocapsid protein (MVNP) induces IL-6 secretion by pagetic osteoclasts. Further, IL-6 plays a critical role in the development of pagetic osteoclasts and bone lesions induced by PD, but the mechanisms regulating IL-6 production by MVNP remain unclear. Our current studies revealed that MVNP expression in osteoclast precursors down-regulated Sirt1 mRNA and protein, a negative regulator of NF-κB activity, which is a key factor for IL-6 expression. MVNP expression in NIH3T3 cells also elevated Il-6 transcription and impaired the expression of Sirt1 mRNA both under basal conditions and upon activation of the Sirt1 upstream regulator FoxO3 by LY294002 (a PI3K/AKT inhibitor). Luciferase activity assays showed that constitutively active FoxO3 abolished the repressive effect of MVNP on reporters driven by either FoxO3 response elements or the Sirt1 promoter. Further, protein stability assays revealed that FoxO3 was degraded more rapidly in MVNP-expressing cells than in control cells following the addition of cycloheximide. Similarly, co-transfection of MVNP and FoxO3 into HEK293 cells demonstrated that MVNP decreased the protein levels of over-expressed FoxO3 in a dose-dependent manner. Treatment with the proteasome inhibitor, MG132, blocked the MVNP-triggered decrease of FoxO3, and the treatment with the serine/threonine phosphatase inhibitor, Calyculin A, revealed that MVNP increased phosphorylation of FoxO3. Further, over-expression of Sirt1 or treatment with the Sirt1 activator resveratrol blocked the increase in Il-6 transcription by MVNP. Finally, resveratrol reduced the numbers of TRAP positive multi-nuclear cells in bone marrow cultures from TRAP-MVNP transgenic mice to wild type levels. These results indicate that MVNP decreases FoxO3/Sirt1 signaling to enhance the levels of IL-6, which in part mediate MVNP’s contribution to the development of Paget’s disease.

In multiple myeloma, osteolytic lesions rarely heal because of persistent suppressed osteoblast differentiation resulting in a high fracture risk. Herein, chromatin immunoprecipitation analyses reveal that multiple myeloma cells induce repressive epigenetic histone changes at the Runx2 locus that prevent osteoblast differentiation. The most pronounced multiple myeloma–induced changes were at the Runx2-P1 promoter, converting it from a poised bivalent state to a repressed state. Previously, it was observed that multiple myeloma induces the transcription repressor GFI1 in osteoblast precursors, which correlates with decreased Runx2 expression, thus prompting detailed characterization of the multiple myeloma and TNFα-dependent GFI1 response element within the Runx2-P1 promoter. Further analyses reveal that multiple myeloma–induced GFI1 binding to Runx2 in osteoblast precursors and recruitment of the histone modifiers HDAC1, LSD1, and EZH2 is required to establish and maintain Runx2 repression in osteogenic conditions. These GFI1-mediated repressive chromatin changes persist even after removal of multiple myeloma. Ectopic GFI1 is sufficient to bind to Runx2, recruit HDAC1 and EZH2, increase H3K27me3 on the gene, and prevent osteogenic induction of endogenous Runx2 expression. Gfi1 knockdown in MC4 cells blocked multiple myeloma–induced recruitment of HDAC1 and EZH2 to Runx2, acquisition of repressive chromatin architecture, and suppression of osteoblast differentiation. Importantly, inhibition of EZH2 or HDAC1 activity in pre-osteoblasts after multiple myeloma exposure in vitro or in osteoblast precursors from patients with multiple myeloma reversed the repressive chromatin architecture at Runx2 and rescued osteoblast differentiation. Implications: This study suggests that therapeutically targeting EZH2 or HDAC1 activity may reverse the profound multiple myeloma–induced osteoblast suppression and allow repair of the lytic lesions. Mol Cancer Res; 15(4); 405–17. ©2017 AACR.

Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti–TNF-α and anti–IL-7 antibodies. Importantly, BMSCs isolated from Gfi1−/− mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

EZH2, the methyltransferase subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes H3K27me3 histone modifications and epigenetically regulates genes involved in cellular pluripotency and differentiation. EZH2 plays a role in myeloma (MM) cell proliferation, survival, stemness and its elevated expression correlates with poor prognosis in MM patients. We have previously shown that EZH2 plays a critical role in preventing osteoblast differentiation of myeloma-exposed bone marrow stromal cells (BMSCs). Here we show that GSK126 blocks MM-induced hyperactivation of osteoclast precursors (OCLp). RNA-seq profiling revealed that inhibition of EZH2 prevented RANKL-induced repression of genes associated with bivalent and/or H3K27me3 promoter signatures including OCL inhibitory factors MafB, Irf8, Bcl6b and Arg1. In contrast, we found that OCLp expansion in MM1.S-conditioned media induced significant gene expression changes, which correlated with TNF and IKK signaling, inflammatory responses and CXC-chemokine receptor pathways. Several classes of small molecule EZH2 inhibitors exhibited anti-MM effects, but their efficacy has been primarily studied in 2-dimensional (2D) cell culture systems or subcutaneous MM-tumor models in vivo. Therefore, we evaluated the effectiveness of GSK126 in the context of the bone microenvironment in a novel 3D model of MM co-cultures (3D-MM). We combined basement membrane extract (BME) hydrogels with devitalized bone slices to mimic the 3D setting of MM with the OCL-resorbing endosteal surface. This enabled us to test GSK126 alone or in combination with bortezomib simultaneously on MM survival and OCLp differentiation and resorption. Differentiating OCLp did not protect MM cells from GSK126 anti-MM effects, nor did the MM cells prevent GSK126 from blocking OCL differentiation. However, mature OCL added to 3D-MM co-cultures increased the IC50 MM inhibition dose of both bortezomib and GSK126 by 40% and 50%, respectively. Further their synergy on MM cells was reduced by 70%. 3D-MM co-cultures with total bone marrows harvested from mice of different ages (1-12 months old) showed selective protection from GSK126, but not bortezomib, on MM viability. Furthermore, the resistance to GSK126 was age-dependent. Cultures of bone marrows from older mice exhibited enhanced protection of MM cells from GSK126 as compared to younger marrows. Using confocal microscopy, we found that in addition to soluble factors, physical interaction between MM and bone marrow cells reduced the effectiveness of the epigenetic drug targeting against MM. Depletion of the BMSC population from the total bone marrows using CD45+ selection before establishing the 3D MM co-cultures resulted in diminished protection of MM cell survival from GSK126. Consistent with this, addition of both primary murine and MM-patient derived BMSCs to MM cultures significantly protected MM cells from EZH2 inhibition. In addition to cell-cell contacts, the pro-survival factor IL6 released by mature OCL and BMSCs, has been implicated in mediating chemo-resistance of MM cells. In agreement with this, addition of soluble IL6 to MM-3D cultures significantly protected MM cells from GSK126 inhibition. Here we show that various cell compartments of the bone microenvironment exhibit differential and drug-specific protection for MM cells from EZH2 inhibition. In addition to direct bone marrow-MM cell interactions, soluble IL6 also exhibits resistance to GSK126. Our novel 3D-MM system enables us to rapidly screen drug combinations, and simultaneously evaluate the influence of bone-microenvironmental interactions on MM drug resistance and bone marrow cell responses to the drugs. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients. Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support. These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. Disclosures Giuliani: Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown. Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio). We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1. Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Paget’s disease of bone (PDB) affects 1–2 million Americans. The primary lesion in PDB is the formation of abnormal osteoclasts (OCL), which leads to highly localized bone resorption accompanied by exuberant new bone formation that is of poor quality. The OCL are increased in number and size, including increased nuclei/OCL, and express a “pagetic phenotype” that distinguishes them from normal OCL. Pagetic OCL precursors form OCL more quickly and at lower levels of inducers of OCL differentiation, including receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor α, and 1,25-(OH)2D3. Another aspect of the pagetic OCL phenotype is the increased expression and secretion of the cytokine interleukin-6, which plays a large role in the development of pagetic OCL. Further, a number of key internal signaling molecules and transcription factors are altered in both pagetic OCL precursors and pagetic OCL. Lastly, pagetic OCL express increased coupling factors to promote osteoblastic differentiation. OCLs in PDB play a critical role in the enhanced osteoblast activity in PDB, since therapies targeting OCLs decrease new bone formation and induce clinical remission in PDB.

Although the oncogenic role of the Wnt/beta-catenin pathway is well defined, it remains unclear how this pathway is aberrantly activated in lung cancer. We found that Dickkopf (Dkk)-3, a member of Dkk family of Wnt antagonists, is frequently inactivated in lung cancer and plays a role in suppressing lung cancer cell growth through inhibition of beta-catenin/T-cell factor (TCF)-4 signaling. Dkk3 is the only Dkk family member abundantly expressed in normal lung, but silenced by promoter hypermethylation in a large fraction of lung cancer cell lines and lung tumors. Downregulation of Dkk3 was correlated with tumor progression and expression of nuclear beta-catenin in lung tumors. Ectopic expression of Dkk3 in lung cancer cells with Dkk3 hypermethylation induced apoptosis and inhibited TCF-4 activity as well as nuclear accumulation of beta-catenin and expression of TCF-4 targets c-Myc and cyclin D1. Furthermore, small interference RNA knock down of Dkk3 in cells lacking Dkk3 hypermethylation was sufficient to promote cell proliferation, beta-catenin nuclear translocation and expression of c-Myc. These observations suggested that epigenetic inactivation of Dkk3 activates the Wnt/beta-catenin pathway, thereby promoting the growth of lung cancer cells.

8039 Background: Myeloma bone disease is characterized by osteoclast activation and long-term osteoblast suppression. We investigated if Semaphorin 4D (Sema4D; CD100) plays a role in these processes. Sema4D has been shown to be a potent osteoblast inhibitor (Negishi-Koga T et al, Nat Med. 2011). A study recently identified that the breast cancer cell line MDA-MB-231 utilizes Sema4D to create osteolysis (Yang Y et al, PLOS One 2016). There have been previous data that Sema4D is increased in the serum of myeloma patients (Terpos et al, Blood 2012) and that co-culturing myeloma cell lines with osteocytes increases the expression of Sema4D mRNA in both (Suvannasankha et al, Blood 2016). We sought to investigate if myeloma cells are using Sema4D to suppress bone formation and if they affect the levels of Sema4D produced by osteoclasts. Methods: We used lentivirus carrying shRNA for Sema4D or control (Scr) to knock down the level of the protein in the 5TGM1 murine myeloma cell line. Knockdown was verified by qPCR and Western Blot. We subsequently co-cultured the 5TGM1 cells with the MC3T3-subclone M4 (MC4) murine stromal cell line for 2 days, removed the myeloma cells and then differentiated the MC4 cells using ascorbic acid and β-glycerolphosphate. At day 5, we analyzed the cells for Runx2 (a critical gene for the differentiation of stromal cells into osteoblasts) expression utilizing qPCR. Also, we performed qPCR in primary osteoclast (OCL) mouse cells differentiating into OCL with RANKL with or without pre-treatment with myeloma-conditioned media for 3 days before the addition of RANKL. Results: When 5TGM1-Scr were co-cultured with MC4 cells the expression of Runx2 on day 5 was decreased (p=0.02). Strikingly, the 5TGM1-shSema4D cells when co-cultured with MC4s did not have the same effect and allowed the upregulation of Runx2 expression on day 5 (p=0.01). Myeloma-conditioned media increased Sema4D expression by OCL throughout the 5 days of differentiation 2 to 3-fold (p=0.01 for day 5). Conclusions: The myeloma cells seem to be utilizing Sema4D both directly and indirectly to inhibit bone formation. Targeted therapy against Sema4D may improve outcomes and fracture-free survival for multiple myeloma patients.

With the development of distributed computing technology, there remain some important problems of collaborative control in distributed multi-user data sharing: heterogeneous environment, data consistency and concurrent operation. To solve the sharing issues in drilling engineering, this paper introduces a method of realizing information sharing by combining the advantages of multi-agent collaborative working mechanism and data warehouse technology. Firstly, the structure of multi-agent collaborative working mechanism is put forward based on distributed data warehouse, in which the underlying data layer adopts the distributed data warehouse to organize the information, and the model of that is set up by dimensional modeling technology. Then the information sharing platform and multi-user access controlling method are analyzed, and the collaborative model is put forward. Finally through the multi-agent interaction, the management and sharing of all kinds of information in DDW can be realized. So the application of multi-agent and DDW technology can maximize the value of drilling information and play an important role in improving data sharing degree. DOI : http://dx.doi.org/10.11591/telkomnika.v12i2.4411

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts (OCL) resulting in severe bone pain, pathological fractures and enhanced mortality. These bone lesions rarely heal even after therapeutic remission due to MM-induced suppression of bone marrow stromal cells (BMSC) differentiation into functional osteoblasts (OB), enhancing their support of MM growth and survival. Although new therapies for MM have greatly improved progression-free survival and overall survival, MM remains incurable for most patients. EZH2is the methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2) catalyzing the tri-methylation of histone-3 lysine-27 (H3K27me3), which induces gene repression. We previously reported that MM cells induce increased recruitment of EZH2 to the Runx2 gene in the murine pre-OB cell line MC4 resulting in H3K27me3-mediated repression of Runx2, thereby causing repression of OB differentiation. In the present study, we show that the repressive H3K27me3 levels are elevated with reduced H3K9 acetylation on the Runx2 promoter in MM patient derived BMSC. Using the selective small molecule inhibitor GSK126 to block EZH2 activity, we investigated if the MM-induced epigenetic repression of Runx2 is reversible. We found that GSK126 enhances the active architecture at the Runx2 promoter in both BMSC from MM patients and MM-treated MC4 cells, and rescues the osteogenic potential and mineralization capability. 5TGM1 MM cells exhibited reduced adhesion to MC4 pre-OB pre-treated with GSK126 as compared to vehicle in an in vitro adhesion assay, suggesting that EZH2 inhibition will decrease pre-OB support for MM cells. In addition to the direct osteo-anabolic effects of EZH2 inhibition on OB differentiation, we investigated if GSK126 treatment affected the pro-inflammatory, myeloma-amplified OCL differentiation that results in enhanced osteolysis in MM. We reported that expansion of bone marrow monocytes (BMM), which are OCL precursors, in the presence of MM1.S-conditioned media, significantly enhanced formation of TRAP-positive multinucleated OCL. Here we show that MM-conditioned media significantly increased EZH2 and OCL marker genes NFATc1, RANK, OSCAR, Cathepsin K, and DC-STAMP mRNA and enhanced RANKL-induced formation of OCL, which was blocked by GSK126. We found that EZH2 and the corresponding H3K27me3 levels increase during the initial 24h of RANKL-induced osteoclastogenesis and that GSK126 inhibition during the first 24h of RANKL treatment was necessary and sufficient to decrease formation of mature OCL. Thus, GSK126 is likely to both protect the bone from new lytic lesions and help heal the existing osteolytic lesions in MM. We found that GSK126 treatment (48h) inhibited proliferation of human (MM1.S, RPMI, U266, H929, and JJN3) and murine (5TGM1) MM cell lines, and primary 138+MM patient cells. We analyzed the interaction of GSK126 and bortezomib using IC50 constant ratio drug combinations based on the Chou & Talalay method using the CompuSyn program. We found that GSK126 synergizes with bortezomib to inhibit cell viability of these MM cell lines and activated apoptosis via induction of activated caspase-3. We used 5TGM1-GLuc-GFP cells, which secrete Guassia luciferase (GLuc), to measure MM cell viability in a longitudinal drug treatment study. GLuc levels in media correlated with cell proliferation, which was inhibited by single and combination doses of GSK126 and bortezomib, further demonstrating the synergism of these drugs. GSK126 decreased EZH2 mRNA and protein levels in MM cells, which is a cell-type specific effect, since we did not observe EZH2 protein alteration in GSK126 treated OCL progenitors. Our in vitro study suggests that selective inhibition of the epigenetic modifier EZH2 using GSK126 would positively regulate the bone microenvironment by improving the osteogenic potential of MM-exposed BMSC and suppressing OCL formation, and would directly decrease MM cell survival, particularly in combination with bortezomib. These data suggest that targeting EZH2 activity alone or in combination with bortezomib may prove a valuable therapeutic strategy to improve bone health and limit disease progression in MM patients. Disclosures Roodman: Amgen: Consultancy.

Traditional method to select pumps on the basis of several operating parameters is the cause of problems with air conditioning water system pumps and piping, through simulation of the pump performance curve equation, this paper intuitively shows the data of combination operation point of pump and pipe to facilitate the designer timely to adjust scheme and revise design, and writes the corresponding computer program to use the computer to select the pump to achieve the water system design optimization, through checking contrast with a numerical example, the satisfactory result is achieved.

Constitutive activation of pro-survival kinases has become a promising target of small molecules with an increasing interest in developing multi-targeted agents. The mechanisms underlying the responsiveness to most agents targeting cancer specific survival pathways are still poorly understood but critical for their clinical application. In this study, we found that sunitinib, a small molecule inhibitor of multiple tyrosine kinases including VEGFRs and PDGFRs induces apoptosis and inhibits cell growth in colon cancer cells in cell culture and xenograft models via the BH3-only protein PUMA. Sunitinib treatment induced PUMA transcription via the AKT/FoxO3a axis. PUMA, BH3 mimetics, or 5-Flurourical sensitized colon cancer cells to sunitinib-induced apoptosis. Furthermore, PUMA was induced by sunitinib treatment in xenograft tumors, and deficiency in PUMA significantly suppressed the anti-tumor effects of sunitinib. Our study suggests that PUMA-mediated apoptosis is important for the therapeutic responses to sunitinib, and activation of the mitochondrial pathway by BH3 mimetics or PUMA manipulation may be useful for improving the antitumor activity of sunitinib. Modulation of PUMA and selective Bcl-2 family members might be potential biomarkers for predicting sunitinib responses.

Aiming at the current situation of information management and sharing in all Fossil Power Plant, this paper outlines the basic concepts of DW, introduces a method using Data Warehouse (DW) technology in the platform. This paper has analyzed subjects of DW in Fossil Power. Basing on DW, the model has been developed with dimensional modeling technology, a model of information sharing platform has been introduced based on this. It can realize information sharing through the storage and visiting for data warehouse. The application of DW technology can maximize the value of information in Fossil Power Plant and has all important role in improving data sharing degree.

Autophagy is an evolutionarily conserved stress response mechanism that often occurs in apoptosis-defective cancer cells and can protect against cell death. In this study, we investigated how apoptosis and autophagy affect each other in cancer cells in response to chemotherapeutic treatment. We found that specific ablation of the proapoptotic function of cytochrome c, a key regulator of mitochondria-mediated apoptosis, enhanced autophagy following chemotherapeutic treatment. Induction of autophagy required Beclin 1 and was associated with blockage of Beclin 1 cleavage by caspase 8 at two sites. To investigate the role of Beclin 1 cleavage in the suppression of autophagy and cell survival, a caspase-resistant mutant of Beclin 1 was knocked into HCT116 colon cancer cells. Beclin 1 mutant knockin resulted in markedly increased autophagy and improved long-term cell survival after chemotherapeutic treatment but without affecting apoptosis and caspase activation. Furthermore, Beclin 1 mutant tumors were significantly less responsive to chemotherapeutic treatment than were wild-type tumors. These results show that chemotherapy-induced apoptosis inhibits autophagy at the execution stage subsequent to cytochrome c release through caspase 8–mediated cleavage of Beclin 1. If apoptosis fails to execute, autophagy is unleashed due to lack of Beclin 1 cleavage by caspases and can contribute to cancer cell survival and therapeutic resistance. Therefore, Beclin 1 may be a useful target for inhibiting autophagy to sensitize chemotherapy. Cancer Res; 71(10); 3625–34. ©2011 AACR.

Purpose: Overexpression of inhibitors of apoptosis proteins (IAP) contributes to therapeutic resistance. Second mitochondria-derived activator of caspase (Smac) promotes caspase activation by binding to IAPs upon release from the mitochondria. IAP antagonists, also called SMAC mimetics, are promising anticancer agents modeled after this mechanism. We investigated the role and mechanisms of Smac- and Smac mimetic–mediated chemosensitization in head and neck squamous cell carcinoma (HNSCC) cells. Experimental Design: The effects of SMAC knockdown, SMAC overexpression, and a small molecule Smac mimetic on the chemosensitivities of HNSCC cells were determined. The mechanisms of Smac- and Smac mimetic–mediated chemosensitization were investigated by analyzing growth suppression, the mitochondrial apoptotic pathway, caspase activation, and IAP proteins. The therapeutic responses of HNSCC cells with different levels of Smac were compared in xenograft models. Results:We found that Smac mediates apoptosis induced by several classes of therapeutic agents through the mitochondrial pathway. SMAC knockdown led to impaired caspase activation, mitochondrial membrane depolarization, and release of cytochrome c. A small molecule Smac mimetic, at nanomolar concentrations, significantly sensitized HNSCC cells to gemcitabine-induced apoptosis and restored gemcitabine sensitivity in SMAC knockdown cells, through caspase activation, X-linked IAP dissociation, and mitochondria-associated events, but not the TNF-α pathway. Furthermore, Smac levels modulated the therapeutic response of HNSCC cells to gemcitabine in xenograft models. Conclusions: Our results establish a critical role of Smac in mediating therapeutic responses of HNSCC cells and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC. Clin Cancer Res; 17(8); 2361–72. ©2011 AACR.

Most targeted anticancer drugs are inhibitors of kinases that are aberrantly activated in cancer cells. However, the mechanisms by which kinase inhibitors suppress tumor growth remain unclear. In this study, we found that UCN-01, a staurosporine analogue and broad-range kinase inhibitor used in clinical trials, inhibits colon cancer cell growth by inducing apoptosis via PUMA, a BH3-only Bcl-2 family member and a p53 target. PUMA expression was markedly elevated in a p53-independent fashion following UCN-01 treatment. The induction of PUMA by UCN-01 was mediated by direct binding of FoxO3a to the PUMA promoter following inhibition of AKT signaling. Deficiency in PUMA abrogated UCN-01–induced apoptosis, caspase activation, and mitochondrial dysfunction, and rendered UCN-01 resistance in a clonogenic assay, whereas elevated PUMA expression or a BH3 mimetic sensitized UCN-01 induced apoptosis. Chemosensitization by UCN-01 seemed to involve simultaneous PUMA induction through both p53-dependent and p53-independent mechanisms. Furthermore, deficiency in PUMA suppressed the antitumor effects of UCN-01 in a xenograft model, concurrent with reduced apoptosis and caspase activation in vivo. These results suggest that PUMA-mediated apoptosis is pivotal for the anticancer activities of UCN-01, and possibly other clinically used kinase inhibitor drugs, and that PUMA manipulation may be useful for improving their anticancer activities. Mol Cancer Ther; 9(11); 2893–902. ©2010 AACR.

The high mortality rate of lung cancer is largely due to the spread of disease to other organs. However, the molecular changes driving lung cancer invasion and metastasis remain unclear. In this study, we identified fibulin-5, a vascular ligand for integrin receptors, as a suppressor of lung cancer invasion and metastasis. Fibulin-5 was silenced by promoter hypermethylation in a majority of lung cancer cell lines and primary tumors. It inhibited lung cancer cell invasion and down-regulated matrix metalloproteinase-7 (MMP-7), which promoted lung cancer cell invasion. Knockdown of fibulin-5 was sufficient to stimulate cell invasion and MMP-7 expression. The expression levels of fibulin-5 and MMP-7 were inversely correlated in lung tumors. Suppression of MMP-7 expression by fibulin-5 was mediated by an integrin-binding RGD motif via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, overexpression of fibulin-5 in H460 lung cancer cells inhibited metastasis in mice. Collectively, these results suggest that epigenetic silencing of fibulin-5 promotes lung cancer invasion and metastasis by activating MMP-7 expression through the ERK pathway. [Cancer Res 2009;69(15):6339–46]

Apoptosis is tightly regulated at the transcriptional level through a number of transcription factors. In response to different stimuli, these transcription factors bind to specific DNA sequences in the promoters of the genes that are involved in apoptosis regulation to stimulate or suppress their expression, leading to apoptosis initiation. Transcriptional regulation of apoptosis is essential for tissue homeostasis and normal development and serves as a barrier against tumorigenesis.

Head and neck squamous cell carcinoma (HNSCC) ranks the eighth most common cancer worldwide. The patients often present with advanced disease, which responds poorly to chemoradiation therapy. PUMA is a BH3-only Bcl-2 family protein and a p53 target that is required for apoptosis induced by p53 and various chemotherapeutic agents. In this study, we found that PUMA induction by chemotherapeutic agents is abrogated in most HNSCC cell lines. Adenoviral gene delivery of PUMA induced apoptosis and chemosensitization more potently than did adenoviral delivery of p53 in HNSCC cells. Finally, we showed that PUMA suppressed the growth of HNSCC xenograft tumors and sensitized them to cisplatin through induction of apoptosis. Our data suggest that absence of PUMA activation in HNSCC cells contributes to chemoresistance and that gene therapy with PUMA might be an efficient substitute for p53 to enhance the responses of HNSCC cells to chemotherapy. [Mol Cancer Ther 2007;6(12):3180–8]

Purpose: The goal of this study is to identify novel genes frequently silenced by promoter hypermethylation in lung cancer. Experimental Designs: Bioinformatic analysis was done to identify candidate genes significantly down-regulated in lung cancer. The effects of DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine on the expression of the candidate genes were determined. Methylated CpG sites in the promoters of the candidate genes were identified using bisulfite DNA sequencing. Methylation-specific PCR was developed and used to analyze DNA methylation in cell lines and clinical specimen. Pathologic and functional analyses were done to study the role of one candidate gene, receptor activity-modifying protein 2 (RAMP2), in suppressing lung cancer cell growth. Results: Among 54 candidate genes down-regulated in lung cancer, 31 were found to contain CpG islands in their promoters. Six of these 31 genes could be reactivated by 5-aza-2′-deoxycytidine in at least four of six lung cancer cell lines analyzed. Promoter hypermethylation of RAMP2, epidermal growth factor–containing fibulin-like extracellular matrix protein 1, and deleted in U Twenty Twenty cells was detected in 36% to 77% of 22 lung cancer cell lines and in 38% to 50% of 32 primary lung tumors, whereas hypermethylathion of these genes was rarely found in the matched normal samples. The methylation frequencies of these genes in lung cancer were similar to those of commonly used methylation markers, such as RAS association domain family protein 1A, p16, and methylguanine-DNA methyltransferase. Immunohistochemistry showed that RAMP2 was down-regulated in a majority of lung tumors, and RAMP2 down-regulation was correlated with high tumor grade. Ectopic expression of RAMP2 inhibited lung cancer cell growth and caused apoptotic cell death. Knockdown of RAMP2 by RNA interference stimulated cell proliferation. Conclusions: Studying the newly identified genes may provide new insight into lung tumorigenesis. These genes might be useful as molecular markers of lung cancer.

Multiple myeloma (MM) causes osteolytic bone lesions that rarely heal even after therapeutic remission. We reported that the MM-induced pro- inflammatory bone marrow microenvironment causes upregulation of the transcriptional repressor Gfi1 in bone marrow stromal cells (BMSC) via TNFα. Gfi1 represses the key osteoblast (OB) differentiation factor Runx2, resulting in impaired BMSC differentiation into OB. In this study, we explored the molecular mechanisms involved in Gfi1-mediated repression of Runx2 and the role of histone modifiers and possibly of cofactors, which co-operate with the MM-induced Gfi1 binding to epigenetically repress Runx2. The Runx2 promoter in MC4 pre-OB co-cultured with 5TGM1 MM cells (48 h) had reduced activating acetylation (H3K9ac) levels and enhanced heterochromatic methylation (H3K27me3), consistent with decreased mRNA expression that stayed refractory to OB differentiation. BMSC from MM patients compared to normals also had decreased levels of H3K9Ac at the Runx2 gene promoter. MM co-culture induced binding of Gfi1 to the Runx2 promoter in pre-OB with increased occupancy at 36 and 48 h, concomitantly with increased histone deacetylase HDAC1 (erases H3K9ac) and the PRC2 complex methyltransferase subunit EZH2 (adds H3K27me3). Further, ectopic Gfi1 also bound Runx2 and recruited these histone modifiers. We found that Gfi1 knockdown in pre-OB MC4 prevented MM-dependent HDAC1 and EZH2 recruitment, resulting in less Runx2 de-acetylation, lack of repressive H3K27 tri-methylation and rescue of differentiation-induced Runx2 mRNA expression. Additionally, the use of the selective inhibitors MC1293 (HDAC1i) and GSK126 (EZH2i) also prevented Runx2 mRNA suppression in MM treated pre-OB. The LIM-domain protein family member Ajuba was reported to serve as a Gfi1 corepressor on a subset of Gfi1 target genes in macrophages. Therefore, we investigated if Gfi1 requires Ajuba to repress Runx2 in pre-OB. After 36-48 h MM exposure of MC4 cells, enrichment of Ajuba occupancy was co-localized to the Gfi1 binding site in the Runx2 promoter concurrently with the recruitment of Gfi1. Biotin-oligo pulldown of Gfi1 brought down Ajuba as well. Further, co-transfection of Ajuba and Gfi1 revealed that Ajuba enhanced repression by suboptimal doses of Gfi1 of both a Runx2-luciferase reporter as well as the endogenous Runx2 gene in pre-OB. Studies using deletion constructs showed that the LIM region of Ajuba in conjunction with Gfi1 is necessary and sufficient for Runx2 repression, and the pre-LIM portion of Ajuba does not affect Runx2 luciferase expression. Transfected Ajuba exhibits cytoplasmic localization in MC4 cells unless co-expressed with full-length Gfi1, which brings it into the nucleus. Nuclear co-localization of Ajuba with Gfi1 was uncoupled in MC4 cells when Ajuba was co-transfected with Gfi1 containing only the DNA binding region (aa 239-423). Transfected 239-423 Gfi1 binds the endogenous Runx2 promoter, but fails to repress transcription, likely due to impaired recruitment of Ajuba and histone co-repressors. Importantly, knockdown of Ajuba caused decreased recruitment of Gfi1 to the Runx2 gene in pre-OB and prevented the Gfi1 repression of a Runx2 reporter. Collectively these data show that Ajuba functions as a required Gfi1 co-factor recruiting HDAC1 and EZH2 to establish long-termepigenetic suppression of Runx2 transcription in OB lineage cells in MM bone disease. Disclosures Roodman: Amgen: Consultancy; Eli Lilly: Research Funding.