RÉSUMÉ
BACKGROUND: Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. METHODS: We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector. RESULTS: A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, ß, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. CONCLUSIONS: Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.
Sujet(s)
Interleukines/génétique , Mélanome/génétique , Tumeurs cutanées/génétique , Biopsie , Dédifférenciation cellulaire/génétique , Lignée cellulaire tumorale , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes tumoraux , Humains , Interféron gamma/métabolisme , Lymphocytes TIL/métabolisme , Lymphocytes TIL/anatomopathologie , Mélanome/anatomopathologie , Analyse sur microréseau , Métastase tumorale , Tumeurs cutanées/anatomopathologie , Transcriptome , Microenvironnement tumoral/génétique , Facteur de nécrose tumorale alpha/métabolismeRÉSUMÉ
Precursor B acute lymphoblastic leukemias (pre-B ALLs) abnormally express a specific glycan structure, 9-O-acetylated sialic acid (9-O-Ac-Sia), on their cell surface, but glycoproteins that carry this modification have not been identified. Using three different lectins that specifically recognize this structure, we establish that nucleolin (NCL), a protein implicated in cancer, contains 9-O-Ac-Sia. Surprisingly, antibodies against the glycolipid 9-O-Ac-Sia GD3 also detected 9-O-Ac-Sia NCL. NCL is present on the surface of pre-B ALL cells as a sialoglycoprotein that is partly 9-O-acetylated and conversely, 9-O-Ac-Sia-containing structures other than NCL are present on these cells as well. Interestingly, NCL and the 9-O-Ac-Sia signal had less co-localization on normal pre-B cells. We also investigated regulation of NCL on the cell surface and found that sialidase treatment increased the percentage of cells positive for cell surface NCL, suggesting that sialylation of NCL promotes internalization. Treatment of pre-B ALL cells with the chemotherapy drug vincristine also increased the percentage of cells with surface NCL and correlated with increased 9-O-Ac-Sia expression. All tested leukemia cells including primary samples expressed NCL, suggesting it as a possible therapeutic target. We confirmed this by showing inhibition of cell proliferation in some pre-B ALLs by exposure to a NCL-specific aptamer AS1411.
Sujet(s)
Protéines membranaires/analyse , Phosphoprotéines/analyse , Leucémie-lymphome lymphoblastique à précurseurs B et T/anatomopathologie , Précurseurs lymphoïdes B/composition chimique , Protéines de liaison à l'ARN/analyse , Sialoglycoprotéines/analyse , Cellules cultivées , Humains ,RÉSUMÉ
BACKGROUND: Drug resistance of B-cell precursor acute lymphoblastic leukemia (BP-ALL) cells is conferred by both intrinsic and extrinsic factors, which could be targeted to promote chemo-sensitization. Our previous studies showed that Galectin-3, a lectin that clusters galactose-modified glycoproteins and that has both an intracellular and extracellular location, protects different subtypes of BP-ALL cells against chemotherapy. Galectin-1 is related to Galectin-3 and its expression was previously reported to be restricted to the MLL subtype of BP-ALL. METHODS AND RESULTS: Here, we report that Galectin-1 is expressed at different levels in and on different subclasses of BP-ALLs. Bone marrow plasma also contains high levels of Galectin-1. PTX008 is an allosteric inhibitor which inhibits Galectin-1 but not Galectin-3-mediated agglutination. The compound reduces migration of BP-ALL cells to CXCL12 and OP9 stromal cells and inhibits fibronectin-mediated adhesion. It also affects cell cycle progression of BCP-ALL cells. PTX008 is cytostatic for BP-ALL cells even when these are co-cultured with protective stroma, and can sensitize ALL cells to vincristine chemotherapy in vitro and in mice. CONCLUSIONS: PTX008 inhibits multiple functions that contribute to BP-ALL survival. The effects of Galectin-1 inhibition on both BP-ALL cell proliferation and migration suggest both the leukemia cells as well as the microenvironment that protects these cells may be targeted.
Sujet(s)
Antinéoplasiques/pharmacologie , Galectine 1/antagonistes et inhibiteurs , Leucémie-lymphome lymphoblastique à précurseurs B/métabolisme , Animaux , Apoptose/effets des médicaments et des substances chimiques , Apoptose/génétique , Adhérence cellulaire/génétique , Cycle cellulaire/effets des médicaments et des substances chimiques , Cycle cellulaire/génétique , Lignée cellulaire tumorale , Membrane cellulaire/métabolisme , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Techniques de coculture , Modèles animaux de maladie humaine , Galectine 1/génétique , Galectine 1/métabolisme , Expression des gènes , Humains , Souris , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B/génétique , Leucémie-lymphome lymphoblastique à précurseurs B/anatomopathologie , Liaison aux protéines , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.
Sujet(s)
Antinéoplasiques/pharmacologie , Mitogen-Activated Protein Kinase 1/antagonistes et inhibiteurs , Mitogen-Activated Protein Kinase 3/antagonistes et inhibiteurs , Leucémie-lymphome lymphoblastique à précurseurs B/métabolisme , Inhibiteurs de protéines kinases/pharmacologie , Animaux , Antinéoplasiques/usage thérapeutique , Benzimidazoles/pharmacologie , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Activation enzymatique , Cytométrie en flux , Humains , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Souris , Mutation , Phosphorylation , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B/génétique , Inhibiteurs de protéines kinases/usage thérapeutique , Protéines proto-oncogènes p21(ras)/génétique , Cellules stromales/effets des médicaments et des substances chimiques , Cellules stromales/métabolismeRÉSUMÉ
The molecular interactions between B-cell precursor acute lymphoblastic leukemia (pre-B ALL) cells and stromal cells in the bone marrow that provide microenvironmentally-mediated protection against therapeutic drugs are not well-defined. Galectin-3 (Lgals3) is a multifunctional galactose-binding lectin with reported location in the nucleus, cytoplasm and extracellular space in different cell types. We previously reported that ALL cells co-cultured with stroma contain high levels of Galectin-3. We here establish that, in contrast to more mature B-lineage cancers, Galectin-3 detected in and on the ALL cells originates from stromal cells, which express it on their surface, secrete it as soluble protein and also in exosomes. Soluble and stromal-bound Galectin-3 is internalized by ALL cells, transported to the nucleus and stimulates transcription of endogenous LGALS3 mRNA. When human and mouse ALL cells develop tolerance to different drugs while in contact with protective stromal cells, Galectin-3 protein levels are consistently increased. This correlates with induction of Galectin-3 transcription in the ALL cells. Thus Galectin-3 sourced from stroma becomes supplemented by endogenous Galectin-3 production in the pre-B ALL cells that are under continuous stress from drug treatment. Our data suggest that stromal Galectin-3 may protect ALL cells through auto-induction of Galectin-3 mRNA and tonic NFκB pathway activation. Since endogenously synthesized Galectin-3 protects pre-B ALL cells against drug treatment, we identify Galectin-3 as one possible target to counteract the protective effects of stroma.
Sujet(s)
Galectine -3/métabolisme , Communication paracrine , Leucémie-lymphome lymphoblastique à précurseurs B/métabolisme , Transduction du signal , Cellules stromales/métabolisme , Transport nucléaire actif , Animaux , Antinéoplasiques/pharmacologie , Protéines du sang , Lignée cellulaire tumorale , Endocytose , Exosomes/métabolisme , Galectine -3/déficit , Galectine -3/génétique , Galectines , Régulation de l'expression des gènes dans la leucémie , Humains , Souris de lignée C57BL , Souris knockout , Facteur de transcription NF-kappa B/métabolisme , Communication paracrine/effets des médicaments et des substances chimiques , Leucémie-lymphome lymphoblastique à précurseurs B/traitement médicamenteux , Leucémie-lymphome lymphoblastique à précurseurs B/génétique , Leucémie-lymphome lymphoblastique à précurseurs B/anatomopathologie , ARN messager/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , Cellules stromales/anatomopathologie , Facteurs temps , Transcription génétique , Activation de la transcription , Transfection , Microenvironnement tumoralRÉSUMÉ
Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.
Sujet(s)
Protéines de Drosophila/composition chimique , Protéines de Drosophila/métabolisme , Modèles moléculaires , Protéine-1 apparentée à Twist/composition chimique , Protéine-1 apparentée à Twist/métabolisme , Séquence d'acides aminés , Animaux , Évolution biologique , Séquence conservée , Drosophila/composition chimique , Drosophila/métabolisme , Régulation de l'expression des gènes , Humains , Liaison aux protéines , Stabilité protéique , Structure tertiaire des protéines , Spécificité du substratRÉSUMÉ
The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.
Sujet(s)
Techniques de coculture/méthodes , Cellules souches embryonnaires/cytologie , Cellules souches hématopoïétiques/cytologie , Animaux , Différenciation cellulaire/physiologie , Hématopoïèse/physiologie , SourisRÉSUMÉ
The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.
Sujet(s)
Embryon de mammifère/cytologie , Cellules souches embryonnaires/métabolisme , Régulation de l'expression des gènes/physiologie , Hématopoïèse , Protéines à homéodomaine/génétique , Facteurs de transcription/génétique , Animaux , Apoptose , Technique de Western , Cycle cellulaire , Différenciation cellulaire , Lignage cellulaire , Prolifération cellulaire , Cellules cultivées , Test clonogénique , Embryon de mammifère/métabolisme , Antigènes CD45/métabolisme , Souris , Souris knockout , Glycoprotéine-IIb de membrane plaquettaire/métabolisme , ARN messager/génétique , RT-PCR , Cellules stromales/métabolismeRÉSUMÉ
We have previously demonstrated recombinant adeno-associated viral (rAAV) transduction of human CD34(+) hematopoietic stem cells (HSCs) capable of serial engraftment in vivo. Here we evaluated the capacity of rAAV2 to mediate gene transfer into nondividing, quiescent, primitive CD34(+) cells subdivided on the basis of metabolic, mitotic, and phenotypic properties. Results revealed that CD34(+)CD38() marrow cells are the most quiescent, exist primarily in G(0) at isolation and are the only population to remain nondividing during the entire exposure to free rAAV. Despite significant differences in the extended clonogenic capacities of CD34(+) subsets in stromal cultures, the frequency of rAAV marking of colonies derived from primitive progenitors was similar in all three populations, suggesting that both primitive and more differentiated progenitors were initially transduced at equal levels. After transduction, episomal and integrated rAAV genomes were detected in all CD34(+) subsets. However, the more quiescent cells displayed higher levels of integrated rAAV than did rapidly dividing cells. Importantly, stable long-term integration was observed only in the most primitive, quiescent CD34(+)CD38(-) subset, indicating that this HSC compartment comprises the preferred substrate for stable rAAV2 transduction. Previously described rate limitations to transgene expression were observed in transduced CD34(+) cells and could be overcome by tyrphostin pretreatment, which resulted in augmented second-strand synthesis. These results represent the first demonstration of rAAV-mediated gene transfer to primitive, quiescent human CD34(+)CD38(-) stem cells and reveal that nondividing CD34(+)CD38(-) HSCs are the optimal CD34(+) targets for rAAV transduction.
Sujet(s)
Antigènes CD34 , Dependovirus/génétique , Thérapie génétique/méthodes , Vecteurs génétiques , Cellules souches hématopoïétiques/immunologie , Antigènes CD34/biosynthèse , Cellules de la moelle osseuse/immunologie , Cellules de la moelle osseuse/métabolisme , Cycle cellulaire , Prolifération cellulaire , Cellules cultivées , Expression des gènes , Cellules souches hématopoïétiques/virologie , Humains , Protéines recombinantes/génétique , Phase G0 , Transduction génétique , TransgènesRÉSUMÉ
A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in vivo demonstration of efficient rAAV2 transduction of human HSC.