[Show abstract][Hide abstract]ABSTRACT: Multiple myeloma (MM) is incurable once osteocytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in MM cell lines and primary bone marrow (BM) cells from patients. CD166+ MM cells homed more efficiently than CD166- cells to the BM of engrafted immunodeficient NSG mice. CD166 silencing in MM cells enabled longer survival, a smaller tumor burden and less osteolytic lesions, as compared to mice bearing control cells. CD166 deficiency in MM cell lines or CD138+ BM cells from MM patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Further, CD166 deficiency in MM cells also reduced formation of osteolytic disease in vivo after intra-tibial engraftment. Mechanistic investigation revealed that CD166 expression in MM cells inhibited osteoblastogenesis of BM-derived osteoblast progenitors by suppressing RUNX2 gene expression. Conversely, CD166 expression in MM cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in MM cell homing to the BM and MM progression, rationalizing its further study as a candidate therapeutic target for MM treatment.
Full-text available · Article · Sep 2016 · Cancer Research
[Show abstract][Hide abstract]ABSTRACT: Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.
Full-text available · Article · Aug 2016 · Scientific Reports
[Show abstract][Hide abstract]ABSTRACT: Toll-like receptor 4 (TLR4) plays a central role in host responses to bacterial infection, but the precise mechanism(s) by which its downstream signaling components coordinate the bone marrow response to sepsis is poorly understood. Using mice deficient in TLR4 downstream adapters MYD88 or TRIF, we demonstrate that both cell-autonomous and non-cell-autonomous MYD88 activation are major causes of myelosuppression during sepsis, while having a modest impact on hematopoietic stem cell (HSC) functions. In contrast, cell-intrinsic TRIF activation severely compromises HSC self-renewal without directly affecting myeloid cells. Lipopolysaccharide-induced activation of MYD88 or TRIF contributes to cell-cycle activation of HSC and induces rapid and permanent changes in transcriptional programs, as indicated by persistent downregulation of Spi1 and CebpA expression after transplantation. Thus, distinct mechanisms downstream of TLR4 signaling mediate myelosuppression and HSC exhaustion during sepsis through unique effects of MyD88 and TRIF.
Full-text available · Article · Jun 2016 · Stem Cell Reports
[Show abstract][Hide abstract]ABSTRACT: The microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) niche alters hematopoietic homeostasis and leads to lethal myeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ(-/-) mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders.
[Show abstract][Hide abstract]ABSTRACT: We previously showed that immature CD166+ osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48-CD166+CD150+ and LSKCD48-CD166+CD150+CD9+ cells as well as human Lin-CD34+CD38-CD49f+CD166+ cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166- cells. CD166(-/-) (KO) LSK cells engrafted poorly in wild type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short- but not long-term WT HSC engraftment confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions and suggest that CD166 expression can be modulated to enhance HSC function.
[Show abstract][Hide abstract]ABSTRACT: Introduction: Medulloblastoma (MB) is defined at molecular level into at least four major subtypes: WNT, SHH, Group C and Group D. Previously, we have demonstrated that levels of HOX transcripts in medulloblastoma specimens correlate with their malignant phenotype. HOX gene regulation involves long noncoding RNAs (lncRNAs), the epigenetic activator of the Trithorax group (TrxG) and the epigenetic repressors of the polycomb group (PcG). Therefore, we performed transcriptoma profiling in adult MBs and normal cerebellum samples to elucidate potential epigenetic regulatory mechanisms involved in tumor progression.
Methodology: We analyzed frozen tumor samples from 5 MB adult patients with different tumor subtypes and with ages ranging from 18-33 years and 3 control cerebellar tissues obtained from infants with ages from 23 weeks to six months. RNA was isolated and integrity was assessed using the Agilent 2100 Bioanalyzer. Two colors microarray-based gene expression analysis was performed according to the manufacture's instructions. Quality control and quantile normalization were done using R. Hierarchical clustering was performed using subsets of genes identified in previous studies analysis to classify the tumor samples. There was also considerable variability among the control samples which was separately studied using bootstrap analysis and rank based tests.
Results The hierarchical clustering of samples considering the 200 most variable genes, followed by molecular analyses suggests the existence of at least three groups among the tumor specimens analyzed. Hierarchical clustering of samples using the Hox genes demonstrates that the HOX pattern expression might be related to defined medulloblastoma molecular subgroups. For example, in SHH, group C and group D, HOXA3, HOXA6 and HOXB4, are upregulated while HOXC4 is increased in the Wnt group. Interestingly, in control samples these HOX genes showed little variation. Hierarchical clustering of tumor samples using 1414 long intergenic noncoding RNAs (lincRNAs) from the chromosomal (7, 17, 12 and 2) of HOX genes shows similar grouping between control and tumor samples. Moreover five lincRNAs, whose distance from HOX is comparable with the distance from HOTAIR to HOX, were identified: three of these five are in HOX C, one in HOX B9 and another close to HOX D3 genes with the distance varying from 3,800 - 11,819 bp from HOX genes. In our analysis, HOTAIR showed little expression across the samples and HOX regulators (TrxG and PcG complexes) displayed variation in expression level among samples. We found that there is considerable heterogeneity among the control samples used in this study.
Conclusion: This study suggests that lncRNAs might affect the expression of HOX gene products in adult medulloblastoma. Validation analyses and genetic studies are undergoing to elucidate the possible mechanisms of action of this class of ncRNAs during MB progression.
[Show abstract][Hide abstract]ABSTRACT: HOX genes are a family of homeodomain-containing transcription factors defined as master genes of development, altered in cancer cells and thus having implications for tumorigenesis. Developmental genes have been recognized as one of the keys to understanding the tumor progression in some type of cancers. This study aimed to correlate gene expression profile of some HOX genes with the tumorigenic potential of medulloblastoma cell lines (MCL).
We used three human MCL, the UW473, UW472 and DAOY and two human cerebellum primary cultures (CPC). The MCL and CPC were characterized morphologically by light microscopy and immunophenotypically by flow cytometry. MCL were assessed by tumorigenic potential infusing 3x106 cells subcutaneously in NUDE mice. MCL and CPC were evaluated for gene expression profile of HOXA3, HOXA10, HOXB3, HOXB4 and HOXB6 genes by quantitative real time PCR.
MCL are morphologically heterogeneous (polygonal and fibroblastoid morphology) and the CPC present fibroblastoid morphology. Immunophenotypically, MCL and CPC were similar for some CD markers and showed a high percentage (70-99%) for CD44, CD73, CD105, CD166 and CD29 and low or absence (0-5.3%) for CD144, CD31, CD34, CD45 and CD133. Some differences were observed for CD140b (0.28±0.11%; 6.2±8.3%; 0.78±1.1%; 0.44%), CD24 (52.5±1.7%; 64.6±6.4%; 20.5±6.4%; 1.9%), CD146 (60.4±8.8%; 90.6±3.8%; 34.6±12%; 98.4%), CD73 (77.2±6.9%; 81.4±8.98%; 52,97±12.4%; 99.1%), CD271 (3.3±3.7%; 26.9±16.22%; 0.6±0.8%; 0,26%) and CD90 (3,7±1%; 99.3±0.8%; 88.5±3.3%; 77%) in the UW472, UW473 and DAOY MCL, and CPC respectively. Regarding to tumorigenic potential, among the UW402, UW473 and DAOY MCL, only the DAOY cell line gave rise to tumor nodules that presented histology features similar to medulloblastoma. About gene expression, the HOXA3 gene was 4,027.7±430.9, 283.1±3.8 and 1.4±0.8 times higher expressed in DAOY, UW473 and UW402 MCL respectively when compared to CPC (p<0.0001, p<0.0001, p=0.32), the HOXA10 gene was 22,462.78±26.9, 0.89±0.6 and 1.18±0.7 times (p<0.0001, p=0.82, p=0.77), the HOXB3 gene was 2,867.2±1,318.6, 115.9±12.6 and 67.8±21.9 times (p=0.0074, p<0.0001, p=0.0022), the HOXB4 gene was 5,647.8±567.4, 53.6±24.1 and 60.6±34.6 times (p<0.0001, p=0.0021, p=0.0065), the HOXB6 gene was 2,422.4±579.6, 722.4±58.6 and 1.5±1.2 times (p<0.0001, p<0.0001, p=0.30).
Taken together, this study demonstrates that MCL are morphologically heterogeneous and CPC present fibroblastoid morphology. MCL and CPC showed an immunophenotype somewhat different for some markers and DAOY cell line was the only one that gave rise to tumor nodules in NUDE mice. Correlating the tumorigenic potential with HOX gene expression level, the HOXA10 is strongly expressed in DAOY tumorigenic cell line and low expressed in UW402 and UW473 cell lines, suggesting that HOXA10 gene can be related to tumor development in medulloblastoma.
[Show abstract][Hide abstract]ABSTRACT: STAT3 activity is inhibited by APE1 knockdown, however STAT3 mRNA and protein levels do not change. Representative experiment of Panc-1 cells transduced with pGF-STAT3-Luc clones #3 (A) and #9 (B) following transfection with scrambled or APE1 siRNA (50 nM) and induced with IL-6 (50 ng/mL, 6 hr). C) Quantitation of Western blot of total STAT3 protein levels after APE1/Ref-1 knockdown in PaCa-2 cells. Total STAT3 levels were normalized to Tubulin. D) The amount of mRNA for STAT3 was analyzed by qPCR, using RPLP0 as the internal control for patient-derived lines (black bars) and Actin mRNA as the internal control for PaCa-2 (gray bars). For the patient-derived lines, the mRNA from three specimens was measured separately, in triplicate, and then averaged. PaCa-2 was done in three separate experiments in triplicate and the data averaged. E) Quantitation of Western blot for p-STAT3 levels following APE1 knockdown in PaCa-2 cells. p-STAT3 levels were normalized to total STAT3. Data represent average ± SD and are expressed as treated to scrambled (SC) control (n = 4–6).
[Show abstract][Hide abstract]ABSTRACT: Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3-APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3-APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.
Full-text available · Article · Oct 2012 · PLoS ONE
[Show abstract][Hide abstract]ABSTRACT: STAT3-APE1 dual targeting effectively inhibits PDAC cell proliferation. MTS assay was used to determine cell survival. Both drugs were added and were present for 72 h. Panc-1 and PaCa-2 were treated with 50 µM E3330. DMSO was tested as vehicle control. Data shown as mean ± SE of at least four independent experiments.