FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1984072004> ?p ?o ?g. }

• W1984072004 endingPage "92" @default.
• W1984072004 startingPage "77" @default.
• W1984072004 abstract "Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies. Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies. Malignant B cells induce the expression of PKC-βII in bone marrow stromal cells The activation of NF-κB in tumor stromal cells strictly depends on PKC-βII The PKC-βII-NF-κB pathway is indispensable for survival of malignant B cells in vivo The PKC-βII-NF-κB pathway is activated by ALL and mantle cell lymphoma cells Tumor-host interactions are crucial for the survival and progression of cancer cells. Specific targeting of the tumor microenvironment may therefore constitute an alternative to cytotoxic therapies. Here, we show that the expression of PKC-βII in the tumor microenvironment is induced by malignant cells from patients with CLL, ALL, and mantle cell lymphoma and required for the activation of NF-κB in bone marrow stromal cells. Interference with PKC-βII induction critically impairs the survival of CLL cells in vitro and in vivo, demonstrating that therapeutic targeting of the PKC-βII-NF-κB signaling pathway activated in the tumor microenvironment may be a meaningful treatment option. Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in adults, characterized by an accumulation of monoclonal CD5+ mature B cells in lymphoid tissues and the peripheral blood. The deletion of chromosome 13q14.3 represents the most common genetic alteration in CLL, causing autonomous B cell proliferation by affecting the expression of the microRNA cluster 15a/16-1 (Döhner et al., 2000Döhner H. Stilgenbauer S. Benner A. Leupolt E. Kröber A. Bullinger L. Döhner K. Bentz M. Lichter P. Genomic aberrations and survival in chronic lymphocytic leukemia.N. Engl. J. Med. 2000; 343: 1910-1916Crossref PubMed Scopus (2715) Google Scholar; Klein et al., 2010Klein U. Lia M. Crespo M. Siegel R. Shen Q. Mo T. Ambesi-Impiombato A. Califano A. Migliazza A. Bhagat G. Dalla-Favera R. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia.Cancer Cell. 2010; 17: 28-40Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Whole-genome sequencing recently identified recurrent mutations in NOTCH1, MYD88, and SF3B1 in CLL, opening up insights in the mechanisms of clonal evolution (Fabbri et al., 2011Fabbri G. Rasi S. Rossi D. Trifonov V. Khiabanian H. Ma J. Grunn A. Fangazio M. Capello D. Monti S. et al.Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational activation.J. Exp. Med. 2011; 208: 1389-1401Crossref PubMed Scopus (506) Google Scholar; Puente et al., 2011Puente X.S. Pinyol M. Quesada V. Conde L. Ordóñez G.R. Villamor N. Escaramis G. Jares P. Beà S. González-Díaz M. et al.Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.Nature. 2011; 475: 101-105Crossref PubMed Scopus (1224) Google Scholar; Quesada et al., 2012Quesada V. Conde L. Villamor N. Ordóñez G.R. Jares P. Bassaganyas L. Ramsay A.J. Beà S. Pinyol M. Martínez-Trillos A. et al.Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.Nat. Genet. 2012; 44: 47-52Crossref Scopus (783) Google Scholar; Wang et al., 2011Wang L. Lawrence M.S. Wan Y. Stojanov P. Sougnez C. Stevenson K. Werner L. Sivachenko A. DeLuca D.S. Zhang L. et al.SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.N. Engl. J. Med. 2011; 365: 2497-2506Crossref PubMed Scopus (891) Google Scholar). Increased expression levels of antiapoptotic proteins have reinforced the hypothesis that a cell intrinsic defect of apoptosis is causative for B cell longevity and a steady increase in the number of malignant B cells over time (Cimmino et al., 2005Cimmino A. Calin G.A. Fabbri M. Iorio M.V. Ferracin M. Shimizu M. Wojcik S.E. Aqeilan R.I. Zupo S. Dono M. et al.miR-15 and miR-16 induce apoptosis by targeting BCL2.Proc. Natl. Acad. Sci. USA. 2005; 102: 13944-13949Crossref PubMed Scopus (3023) Google Scholar; Kitada et al., 1998Kitada S. Andersen J. Akar S. Zapata J.M. Takayama S. Krajewski S. Wang H.G. Zhang X. Bullrich F. Croce C.M. et al.Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with In vitro and In vivo chemoresponses.Blood. 1998; 91: 3379-3389Crossref PubMed Google Scholar). However, primary CLL cells rapidly die ex vivo despite high levels of Bcl2 but can be cultured for weeks in the presence of different types of stromal cells (Burger et al., 2000Burger J.A. Tsukada N. Burger M. Zvaifler N.J. Dell’Aquila M. Kipps T.J. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1.Blood. 2000; 96: 2655-2663Crossref PubMed Google Scholar; Ding et al., 2009Ding W. Nowakowski G.S. Knox T.R. Boysen J.C. Maas M.L. Schwager S.M. Wu W. Wellik L.E. Dietz A.B. Ghosh A.K. et al.Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression.Br. J. Haematol. 2009; 147: 471-483Crossref PubMed Scopus (63) Google Scholar; Pedersen et al., 2002Pedersen I.M. Kitada S. Leoni L.M. Zapata J.M. Karras J.G. Tsukada N. Kipps T.J. Choi Y.S. Bennett F. Reed J.C. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1.Blood. 2002; 100: 1795-1801Crossref PubMed Scopus (110) Google Scholar). This indicates that the apoptosis defect in CLL is not cell autonomous but highly dependent on extrinsic signals derived from their microenvironment. Notably, this is not a static interaction in which stromal cells constitutively provide prosurvival signals to malignant cells but a dynamic process driven by bidirectional communications between the two. In the present study, we sought to investigate how CLL cells activate bone marrow stromal cells (BMSCs) and to characterize the signaling pathways and their functional consequences underlying this cell-cell communication. To study heterotypic cell-cell communications between stromal and CLL cells, we established a coculture system using primary leukemic B cells derived from patients’ blood and the murine cell line EL08-1D2 (Figure S1A available online), which has been carefully characterized as a stromal cell line able to maintain hematopoietic progenitor and stem cells ex vivo (Oostendorp et al., 2002Oostendorp R.A. Harvey K.N. Kusadasi N. de Bruijn M.F. Saris C. Ploemacher R.E. Medvinsky A.L. Dzierzak E.A. Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.Blood. 2002; 99: 1183-1189Crossref PubMed Scopus (143) Google Scholar). Analysis of apoptotic CLL cells after 5 days of coculture demonstrated that they were protected from spontaneous apoptosis. This antiapoptotic effect of stromal cells could not be recapitulated with CD19+ peripheral blood B cells. Notably, stromal cells provided little protection from spontaneous apoptosis of CD5+ B1 cells derived from blood of healthy donors (Figures 1A and S1B). To define cytokines induced in CLL-stroma cocultures, supernatants from these cocultures were analyzed using a mouse-cytokine antibody array. Of the 62 cytokines measured in this assay, 6 were significantly upregulated in CLL-stroma cocultures: SDF-1α, IL-6, G-CSF, GM-CSF, MIP-3α, and CXCL16 (Figure 1B). Because all the antibodies used in this analysis, with the exception of anti-SDF-1α, were specific to mouse cytokines, the detected cytokines must have been produced by the murine stroma and not the human CLL cells. The consistent upregulation of proinflammatory cytokines by stromal cells in response to contact with leukemic B cells suggested that EL08-1D2 cells share properties akin to so-called cancer-associated fibroblasts (CAFs). CAFs are characterized by promoting growth and invasion of epithelial tumors (Kalluri and Zeisberg, 2006Kalluri R. Zeisberg M. Fibroblasts in cancer.Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3524) Google Scholar), but their role in the pathogenesis of CLL is less clear. Immunofluorescence of EL08-1D2 cells demonstrated that α-SMA and stress fibers, both of which have been used to identify CAFs (Kalluri and Zeisberg, 2006Kalluri R. Zeisberg M. Fibroblasts in cancer.Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3524) Google Scholar; Tlsty and Coussens, 2006Tlsty T.D. Coussens L.M. Tumor stroma and regulation of cancer development.Annu. Rev. Pathol. 2006; 1: 119-150Crossref PubMed Scopus (803) Google Scholar), were induced by contact with CLL cells (Figure 1C, a–d). This remodeling of the actin skeleton depends on the GTP-binding protein RhoA (Ridley and Hall, 1992Ridley A.J. Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar). Correlating with the formation of stress fibers, RhoA was expressed in EL08-1D2 cells upon CLL contact (Figure 1C, e and f). For further characterization of EL08-1D2 cells, we compared transcriptomes of EL08-1D2 cells before and after 5 days of contact with CLL cells, derived from multiple donors. We found 474 genes consistently increased (Table S1) and 347 decreased (Table S2) in stromal cells, of which 129 and 40 genes showed greater than 2-fold change, respectively. Subjecting these 821 consistently altered target probe sets to overrepresentation analysis using GeneTrail (http://genetrail.bioinf.uni-sb.de/index.php) revealed significant enrichments for the gene ontology terms “inflammatory response” (p = 6.0 × 10−5) and “response to wounding” (p = 7.2 × 10−9) (Figure 1D). It was recently reported that CAFs from skin, mammary, and pancreatic tumors in mice are characterized by a proinflammatory gene signature (Erez et al., 2010Erez N. Truitt M. Olson P. Arron S.T. Hanahan D. Cancer-associated fibroblasts are activated in incipient neoplasia to orchestrate tumor-promoting inflammation in an NF-kappaB-dependent manner.Cancer Cell. 2010; 17: 135-147Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar). Comparison of those genes to our results revealed significant overlap of target genes (Figure 1D, lower panel). Therefore, in coculture, EL08-1D2 cells support the survival of malignant B cells and undergo genetic and morphologic alterations reminiscent of CAFs. Interactions between fibroblasts and tumor cells can contribute to drug resistance by increasing the expression of antiapoptotic proteins in tumor cells (Meads et al., 2009Meads M.B. Gatenby R.A. Dalton W.S. Environment-mediated drug resistance: a major contributor to minimal residual disease.Nat. Rev. Cancer. 2009; 9: 665-674Crossref PubMed Scopus (630) Google Scholar). We previously reported that the intrinsic activation of protein kinase C (PKC)-βII in CLL cells mediates apoptosis resistance due to posttranslational modifications of Bcl2 and BimEL (zum Büschenfelde et al., 2010zum Büschenfelde C.M. Wagner M. Lutzny G. Oelsner M. Feuerstacke Y. Decker T. Bogner C. Peschel C. Ringshausen I. Recruitment of PKC-betaII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70.Leukemia. 2010; 24: 141-152Crossref PubMed Scopus (34) Google Scholar). Therefore, we tested whether the PKC-β inhibitor enzastaurin could overcome the protective effect of EL08-1D2 cells on CLL cells. CLL cells cultured on EL08-1D2 cells were not protected from the cytotoxic effect of enzastaurin, whereas the cytotoxicity of doxorubicin was mitigated by contact with stromal cells (Figure 2A). Importantly, neither enzastaurin nor doxorubicin significantly influenced the viability of EL08-1D2 cells (Figure S2A). These data imply that PKC-β plays an important role in stroma-CLL interactions. To further analyze this effect, we assessed the expression of classical PKC isoforms in either cell compartment before and after coculture. CLL cells predominantly express PKC-βII (Abrams et al., 2007Abrams S.T. Lakum T. Lin K. Jones G.M. Treweeke A.T. Farahani M. Hughes M. Zuzel M. Slupsky J.R. B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CbetaII.Blood. 2007; 109: 1193-1201Crossref PubMed Scopus (69) Google Scholar), but its level of expression was unmodified after contact with EL08-1D2 cells (Figure 2B). In contrast, PKC-βII was not constitutively expressed in EL08-1D2 cells. However, contact with several different primary leukemic B cells induced the expression of PKC-βII in EL08-1D2 cells, but not PKC-α or PKC-βI, that were constitutively expressed in EL08-1D2 cells. The absence of ZAP70 in stromal cell lysates of coculture experiments with a ZAP70-positive CLL demonstrated that CLL contamination of stromal cell lysates is negligible in these experiments (Figures 2B and S2B). Of note, normal peripheral blood B cells from healthy donors did not induce PKC-βII in stromal cells (Figure 2C). Subcellular fractioning of stromal cell proteins before and after CLL contact indicated that induced PKC-βII was located in the cytoplasm, but not the nucleus (Figure 2D). Quantitative RT-PCR using mouse-specific primers showed that upregulation of PKC-β in EL08-1D2 cells was mostly due to transcriptional regulation (Figure 2E). To further exclude the possibility of cross-contamination with PKC-βII-positive CLL cells, we assessed PKC-βII expression in stromal cells by immunofluorescence. After 5 days of coculturing CLL cells on EL08-1D2 cells, CLL cells were removed and stromal cells analyzed for the expression of PKC-βII. Stromal cells could be identified by a positive staining for Sca-1 (Oostendorp et al., 2002Oostendorp R.A. Harvey K.N. Kusadasi N. de Bruijn M.F. Saris C. Ploemacher R.E. Medvinsky A.L. Dzierzak E.A. Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.Blood. 2002; 99: 1183-1189Crossref PubMed Scopus (143) Google Scholar), which is not expressed on CLL cells. Stromal PKC-βII was detected with a perinuclear expression pattern, characteristic of activated PKC-βII (Becker and Hannun, 2003Becker K.P. Hannun Y.A. cPKC-dependent sequestration of membrane-recycling components in a subset of recycling endosomes.J. Biol. Chem. 2003; 278: 52747-52754Crossref PubMed Scopus (54) Google Scholar) (Figure S2C, a–d and i). PKC-βII could not be detected in EL08-1D2 monocultures (Figure S2C, e–h). We next examined whether a direct contact between CLL cells and stromal cells was required to protect CLL cells from apoptosis and to induce the expression of PKC-βII in EL08-1D2 cells, and we found that both were lost if these cell compartments were separated in a transwell experiment (Figures 2F and 2G). Furthermore, conditioned medium from EL08-1D2/CLL cocultures failed to support survival of leukemic cells (Figure S2D). To eliminate the concern that the results seen so far were limited to coculturing human leukemic B cells on a murine cell line, we cocultured primary human CLL cells on primary human BMSCs (hBMSCs). Similar to EL08-1D2 cells, hBMSCs supported ex vivo survival of CLL cells, and accordingly, PKC-βII was induced (Figures 2H and 2I). Irradiation of EL08-1D2 cells or hBMSCs before coculturing abolished their antiapoptotic effect on CLL cells, indicating that only signaling competent stromal cells can protect CLL cells from cell death (Figure S2E). hBMSCs are a heterogeneous mixture of different cell types. To further define the subsets of cells that were responsible for these effects, we cocultured CLL cells on human umbilical vascular endothelial cells (HUVECs), primary human osteoblasts (hObs), and a murine osteoblast-cell line (MC3T3) under comparable conditions as used for EL08-1D2/CLL cocultures. Remarkably, all cell types supported CLL survival (Figure 2H), accompanied by PKC-βII induction (Figure 2J). Finally, extended cocultures of CLL cells and hBMSCs for 4 weeks demonstrated that stromal PKC-βII induction was not transient but persistent (Figure 2K). The strong correlation between PKC-βII upregulation and survival of CLL cells suggested that stromal PKC-βII was important for the antiapoptotic signals provided by EL08-1D2 cells. To test this hypothesis, PKC-βII induction in EL08-1D2 cells was suppressed using a siRNA directed against stromal PKC-βII. siRNA transfection of EL08-1D2 was performed 24 hr before coculturing with CLL cells to ensure that the PKC-βII of CLL cells was not targeted. PKC-α and PKC-β expression in EL08-1D2 cells was assessed by immunoblotting after 5 days of coculturing with CLL cells and showed that only PKC-βII was effectively targeted (Figure 3A). Analyzing the viability of CLL cells cultured on EL08-1D2 cells proficient or depleted of PKC-βII indicated that stromal PKC-βII was required for the survival of CLL cells (Figure 3B). Knockdown of stromal PKC-βII significantly impaired the survival of all samples tested from individual patients with CLL. This was particularly relevant because CLL is a heterogeneous disease with variations in the clinical course. The aberrant expression of ZAP70 in monoclonal B cells can be used as a prognostic marker of a more aggressive variant of CLL (Crespo et al., 2003Crespo M. Bosch F. Villamor N. Bellosillo B. Colomer D. Rozman M. Marcé S. López-Guillermo A. Campo E. Montserrat E. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia.N. Engl. J. Med. 2003; 348: 1764-1775Crossref PubMed Scopus (1173) Google Scholar). However, PKC-βII knockdown in stromal cells affected the survival of ZAP70-positive and -negative CLL, similarly (Figure 3C). To eliminate the possibility that off-target effects of the PKC-βII siRNA accounted for the impaired survival of CLL cells, we recapitulated our experiments with primary BMSCs from wild-type and PKC-β knockout (Prkcb−/−) mice. Prkcb−/− mice, which lack both βI and βII isoforms, are characterized by an impaired immune response but have no overt phenotype in the bone marrow stromal compartment (Leitges et al., 1996Leitges M. Schmedt C. Guinamard R. Davoust J. Schaal S. Stabel S. Tarakhovsky A. Immunodeficiency in protein kinase cbeta-deficient mice.Science. 1996; 273: 788-791Crossref PubMed Scopus (413) Google Scholar). CLL cells were effectively protected from spontaneous apoptosis when cocultured on wild-type BMSCs, accompanied by induction of PKC-β in stromal cells (Figures 3D and S3A). This protection effect was significantly impaired on Prkcb−/− stromal cells (Figures 3D and 3E). Characterization of the primary CLL cells used in these experiments, based on standard cytogenetic analyses and sequencing of TP53 (exons 4–11) and NOTCH (exons 25–34), did not identify any subset of patients being more dependent on PKC-β-mediated survival than others (Table S3). Ectopic expression of the wild-type PKC-β in Prkcb−/− BMSCs rescued their ability to provide survival cues to CLL cells (Figure 3F), confirming that PKC-β expressed in stromal cells is required for the survival of CLL cells. Moreover, expression of a kinase-dead mutant (K371R) of PKC-β (Feng and Hannun, 1998Feng X. Hannun Y.A. An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane.J. Biol. Chem. 1998; 273: 26870-26874Crossref PubMed Scopus (64) Google Scholar) failed to restore the survival functions of Prkcb−/− BMSCs, indicating that the kinase activity of PKC-β is essential for stroma-mediated protection of CLL cells from apoptosis (Figures 3F and S3B). The uniform upregulation of PKC-βII (Figures 2I and 2J) suggested that the antiapoptotic effect of different types of stromal cells on CLL cells was attributed to its expression. Consistent with this hypothesis, knockdown of PKC-βII in HUVECs abolished their prosurvival effects on CLL cells (Figure 3G). PKC-β connects the B cell receptor to canonical activation of NF-κB through a signaling complex, including Bcl10/MALT1 and NEMO/IKKγ (Zhou et al., 2004Zhou H. Wertz I. O’Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO.Nature. 2004; 427: 167-171Crossref PubMed Scopus (451) Google Scholar). Because stromal cells activated by CLL cells showed a proinflammatory phenotype (Figure 1), we investigated whether NF-κB activation could also occur following induction of PKC-βII in stromal cells and found that activation of NF-κB occurred in wild-type, but not in Prkcb−/− BMSCs in response to CLL contact (Figure 4A). These results were recapitulated in EL08-1D2 cells and in hBMSCs (Figures 4B and S4A). To test whether the loss of NF-κB activation was primarily dependent on PKC-β expression or a secondary event based on the impaired survival of CLL cells on PKC-β-deficient stromal cells, apoptosis of CLL cells was inhibited using the caspase inhibitor z.vad.fmk. Caspase inhibition rescued CLL cells cultured on PKC-β-deficient stromal cells from spontaneous apoptosis but did not result in activation of NF-κB in PKC-β-deficient stromal cells (Figures S4B and S4C), suggesting that PKC-β is directly involved in the activation of NF-κB. This activation is regulated by nuclear translocation and DNA binding of NF-κB subunits (Hayden and Ghosh, 2004Hayden M.S. Ghosh S. Signaling to NF-kappaB.Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3358) Google Scholar). Fractionation of EL08-1D2 cell lysates showed that the NF-κB subunits p50, p65, and c-REL accumulated in the nucleus upon contact with monoclonal B cells (Figures 4C and S4D). To further elaborate whether Bcl10 was linking PKC-βII to NF-κB activation in stromal cells, we used BMSCs from Bcl10−/− and wild-type mice for coculture experiments. Primary CLL cells survived equally well on Bcl10−/− and wild-type stromal cells (Figure 4D). Accordingly, CLL-dependent activation of NF-κB was not inhibited in Bcl10−/− stroma (Figure 4E). Notably, stromal PKC-βII was induced upon CLL contact regardless of Bcl10 (Figure 4F), excluding Bcl10 as a critical protein for PKC-βII-mediated activation of NF-κB in stroma cells. To assess whether stromal NF-κB was important for the prosurvival effects on malignant B cells, we analyzed the viability of CLL cells in cocultures with EL08-1D2 cells in the presence of an IKK2 inhibitor (Ziegelbauer et al., 2005Ziegelbauer K. Gantner F. Lukacs N.W. Berlin A. Fuchikami K. Niki T. Sakai K. Inbe H. Takeshita K. Ishimori M. et al.A selective novel low-molecular-weight inhibitor of IkappaB kinase-beta (IKK-beta) prevents pulmonary inflammation and shows broad anti-inflammatory activity.Br. J. Pharmacol. 2005; 145: 178-192Crossref PubMed Scopus (136) Google Scholar). IKK2 (IKKβ) is a member of the multiprotein IκB complex that also contains IKK1 (IKKα) and the essential regulatory subunit NEMO. Blockage of IKK2 significantly affected survival of CLL cells on stromal cells without affecting the viability of EL08-1D2 cells (Figures 4G and S2A). This impaired survival of CLL cells was associated with a decreased activation of NF-κB in stromal cells (Figure 4H). However, it was possible that the proapoptotic effect of the IKK2 inhibitor was exclusively related to NF-κB inhibition in CLL cells. To disentangle stroma from CLL-specific effects, we used BMSCs derived from conditional Nemo-knockout mice (NemoF) (Schmidt-Supprian et al., 2000Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israël A. Rajewsky K. Pasparakis M. NEMO/IKK gamma-deficient mice model incontinentia pigmenti.Mol. Cell. 2000; 5: 981-992Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar) and as controls BMSCs from YFP reporter mice (Srinivas et al., 2001Srinivas S. Watanabe T. Lin C.S. William C.M. Tanabe Y. Jessell T.M. Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev. Biol. 2001; 1: 4Crossref PubMed Scopus (2306) Google Scholar). Nemo was ablated from isolated monocultures of NemoF/F stromal cells by treatment with Cre protein (HTNC) (Figure 4J) (Peitz et al., 2002Peitz M. Pfannkuche K. Rajewsky K. Edenhofer F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes.Proc. Natl. Acad. Sci. USA. 2002; 99: 4489-4494Crossref PubMed Scopus (267) Google Scholar). NEMO-proficient and -deficient BMSCs were then propagated in the absence of HTNC for additional two passages before CLL cells were added. Activation of NF-κB in NEMO-deficient BMSCs was inhibited after CLL contact (Figure 4I), although PKC-βII was still induced (Figure 4J). These results indicated that PKC-βII was operating upstream of NEMO/NF-κB in BMSCs. Importantly, NEMO-deficient BMSCs failed to support the survival of primary CLL cells (Figure 4K), similar to loss of stromal PKC-βII (Figure 3). The slightly reduced induction of PKC-βII in NEMO-deficient BMSCs compared to untreated NemoF/F cells is most likely related to an impaired survival of and therefore reduced “induction signal” by CLL cells on these BMSCs (Figure 4J). These findings provide evidence that induction of PKC-βII in stromal cells precedes the activation of NF-κB and that the PKC-βII/NF-κB pathway is required for stromal cell-mediated survival of malignant B cells. Comparing the transcriptome of HTNC-treated and -untreated NemoF/F murine bone marrow stroma cells (mBMSCs) after 5 days of coculturing with CLL revealed that 372 genes were significantly increased (≥2-fold) in NEMO-proficient BMSCs (Table S4). Accordingly, 388 genes were downregulated (≤0.5-fold) in BMSCs expressing NEMO (Table S5). Notably, BMSCs from NemoF/F mice were HTNC treated at least 8 days before the coculture with CLL cells, making it unlikely that changes in gene expression were merely related to the addition of HTNC. Using the GeneTrail software and the KEGG database to further analyze these genes, we identified a gene cluster of “cytokine and cytokine-receptors” (p = 0.029) and a cluster of “cell adhesion molecules” (p = 0.004), which were decreased in NEMO-deficient mBMSCs (Figure 5A). To validate the differential regulation of these genes, we analyzed the levels of IL-1α and IL-15, both of which have been described to support CLL survival in the absence of stromal cells (de Totero et al., 2008de Totero D. Meazza R. Capaia M. Fabbi M. Azzarone B. Balleari E. Gobbi M. Cutrona G. Ferrarini M. Ferrini S. The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways.Blood. 2008; 111: 517-524Crossref PubMed Scopus (94) Google Scholar; Jewell et al., 1995Jewell A.P. Lydyard P.M. Worman C.P. Giles F.J. Goldstone A.H. Growth factors can protect B-chronic lymphocytic leukaemia cells against programmed cell death without stimulating proliferation.Leuk. Lymphoma. 1995; 18: 159-162Crossref PubMed Scopus (21) Google Scholar), in supernatants from CLL and BMSC cocultures after 5 days. CLL contact induced the secretion of IL-1α by mBMSCs, which was completely abolished in NEMO-deficient BMSCs (Figure 5B). Similar to IL-1α, IL-15 was induced in a NEMO-dependent manner in stromal cells by CLL contact, although to greater variations (Figure 5C). Importantly, antibodies used in these ELISAs were specific for murine IL-1α and IL-15 and not cross-reactive to human cytokines, excluding the possibility that the detected factors were produced by human CLL cells. Upregulation of IL-1α and IL-15 mRNA in hBMSCs upon CLL contact (Figure S5A) indicated that these results were not limited to xenogeneic culture conditions. Finally, to test that murine IL-1α, IL-1β, and IL-15 could rescue primary CLL cells from apoptosis, increasing doses of these cytokines were supplemented to CLL monocultures or CLL/NEMO-deficient BMSC cocultures. The analysis of apoptotic CLL cells after 5 days indicated that IL-1 and IL-15 partially rescued NEMO deficiency in BMSCs (Figures 5D, 5E, and S5B). Combining IL-1 and IL-15 demonstrated a stronger prosurvival effect on CLL cells (Figure S5C). Notably, cytokine doses required to partly compensate for NEMO deficiency in stromal cells exceeded the concentrations of IL-1 and IL-15 produced by stromal cells after CLL" @default.
• W1984072004 created "2016-06-24" @default.
• W1984072004 creator A5003682222 @default.
• W1984072004 creator A5005029408 @default.
• W1984072004 creator A5007123700 @default.
• W1984072004 creator A5010533875 @default.
• W1984072004 creator A5012540791 @default.
• W1984072004 creator A5020841765 @default.
• W1984072004 creator A5021480569 @default.
• W1984072004 creator A5022268863 @default.
• W1984072004 creator A5023137678 @default.
• W1984072004 creator A5029499905 @default.
• W1984072004 creator A5034944592 @default.
• W1984072004 creator A5039437432 @default.
• W1984072004 creator A5039762705 @default.
• W1984072004 creator A5043836556 @default.
• W1984072004 creator A5045993698 @default.
• W1984072004 creator A5066200247 @default.
• W1984072004 creator A5067987771 @default.
• W1984072004 creator A5068920358 @default.
• W1984072004 creator A5070000759 @default.
• W1984072004 creator A5078763289 @default.
• W1984072004 creator A5082142875 @default.
• W1984072004 creator A5088022766 @default.
• W1984072004 creator A5089219567 @default.
• W1984072004 date "2013-01-01" @default.
• W1984072004 modified "2023-10-18" @default.
• W1984072004 title "Protein Kinase C-β-Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo" @default.
• W1984072004 cites W1488755326 @default.
• W1984072004 cites W1531083786 @default.
• W1984072004 cites W1537427513 @default.
• W1984072004 cites W180295822 @default.
• W1984072004 cites W1939497685 @default.
• W1984072004 cites W1968730445 @default.
• W1984072004 cites W1975627280 @default.
• W1984072004 cites W1981308197 @default.
• W1984072004 cites W1985563073 @default.
• W1984072004 cites W1991131833 @default.
• W1984072004 cites W1991895418 @default.
• W1984072004 cites W1992451339 @default.
• W1984072004 cites W1993355130 @default.
• W1984072004 cites W1998562562 @default.
• W1984072004 cites W2007660746 @default.
• W1984072004 cites W2007850471 @default.
• W1984072004 cites W2012074391 @default.
• W1984072004 cites W2013964637 @default.
• W1984072004 cites W2018531061 @default.
• W1984072004 cites W2031121711 @default.
• W1984072004 cites W2031604577 @default.
• W1984072004 cites W2032335304 @default.
• W1984072004 cites W2035918677 @default.
• W1984072004 cites W2035985010 @default.
• W1984072004 cites W2037198742 @default.
• W1984072004 cites W2058376258 @default.
• W1984072004 cites W2060303297 @default.
• W1984072004 cites W2060390249 @default.
• W1984072004 cites W2061222562 @default.
• W1984072004 cites W2062809449 @default.
• W1984072004 cites W2066616821 @default.
• W1984072004 cites W2075083870 @default.
• W1984072004 cites W2079849140 @default.
• W1984072004 cites W2086572638 @default.
• W1984072004 cites W2086637735 @default.
• W1984072004 cites W2099042353 @default.
• W1984072004 cites W2109627278 @default.
• W1984072004 cites W2116431837 @default.
• W1984072004 cites W2117510681 @default.
• W1984072004 cites W2120601121 @default.
• W1984072004 cites W2143599742 @default.
• W1984072004 cites W2145842706 @default.
• W1984072004 cites W2148119611 @default.
• W1984072004 cites W2161744213 @default.
• W1984072004 cites W2169281418 @default.
• W1984072004 cites W2172290514 @default.
• W1984072004 cites W2310521729 @default.
• W1984072004 cites W2316531329 @default.
• W1984072004 cites W2318933236 @default.
• W1984072004 cites W4250894669 @default.
• W1984072004 cites W4252517229 @default.
• W1984072004 cites W76370582 @default.
• W1984072004 doi "https://doi.org/10.1016/j.ccr.2012.12.003" @default.
• W1984072004 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3546417" @default.
• W1984072004 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23328482" @default.
• W1984072004 hasPublicationYear "2013" @default.
• W1984072004 type Work @default.
• W1984072004 sameAs 1984072004 @default.
• W1984072004 citedByCount "129" @default.
• W1984072004 countsByYear W19840720042013 @default.
• W1984072004 countsByYear W19840720042014 @default.
• W1984072004 countsByYear W19840720042015 @default.
• W1984072004 countsByYear W19840720042016 @default.
• W1984072004 countsByYear W19840720042017 @default.
• W1984072004 countsByYear W19840720042018 @default.
• W1984072004 countsByYear W19840720042019 @default.
• W1984072004 countsByYear W19840720042020 @default.
• W1984072004 countsByYear W19840720042021 @default.
• W1984072004 countsByYear W19840720042022 @default.
• W1984072004 countsByYear W19840720042023 @default.

• next