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1.
Cancers (Basel) ; 16(9)2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38730619

ABSTRACT

Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) relapses are still associated with a dismal outcome, justifying the search for new therapeutic targets and relapse biomarkers. Using single-cell RNA sequencing (scRNAseq) data from three paired samples of pediatric T-ALL at diagnosis and relapse, we first conducted a high-dimensional weighted gene co-expression network analysis (hdWGCNA). This analysis highlighted several gene co-expression networks (GCNs) and identified relapse-associated hub genes, which are considered potential driver genes. Shared relapse-expressed genes were found to be related to antigen presentation (HLA, B2M), cytoskeleton remodeling (TUBB, TUBA1B), translation (ribosomal proteins, EIF1, EEF1B2), immune responses (MIF, EMP3), stress responses (UBC, HSP90AB1/AA1), metabolism (FTH1, NME1/2, ARCL4C), and transcriptional remodeling (NF-κB family genes, FOS-JUN, KLF2, or KLF6). We then utilized sparse partial least squares discriminant analysis to select from a pool of 481 unique leukemic hub genes, which are the genes most discriminant between diagnosis and relapse states (comprising 44, 35, and 31 genes, respectively, for each patient). Applying a Cox regression method to these patient-specific genes, along with transcriptomic and clinical data from the TARGET-ALL AALL0434 cohort, we generated three model gene signatures that efficiently identified relapsed patients within the cohort. Overall, our approach identified new potential relapse-associated genes and proposed three model gene signatures associated with lower survival rates for high-score patients.

3.
Cancers (Basel) ; 13(10)2021 May 20.
Article in English | MEDLINE | ID: mdl-34065348

ABSTRACT

Signaling, proliferation, and inflammation are dependent on K63-linked ubiquitination-conjugation of a chain of ubiquitin molecules linked via lysine 63. However, very little information is currently available about how K63-linked ubiquitination is subverted in cancer. The present study provides, for the first time, evidence that cadmium (Cd), a widespread environmental carcinogen, is a potent activator of K63-linked ubiquitination, independently of oxidative damage, activation of ubiquitin ligase, or proteasome impairment. We show that Cd induces the formation of protein aggregates that sequester and inactivate cylindromatosis (CYLD) and selective autophagy, two tumor suppressors that deubiquitinate and degrade K63-ubiquitinated proteins, respectively. The aggregates are constituted of substrates of selective autophagy-SQSTM1, K63-ubiquitinated proteins, and mitochondria. These protein aggregates also cluster double-membrane remnants, which suggests an impairment in autophagosome maturation. However, failure to eliminate these selective cargos is not due to alterations in the general autophagy process, as degradation of long-lived proteins occurs normally. We propose that the simultaneous disruption of CYLD and selective autophagy by Cd feeds a vicious cycle that further amplifies K63-linked ubiquitination and downstream activation of the NF-κB pathway, processes that support cancer progression. These novel findings link together impairment of selective autophagy, K63-linked ubiquitination, and carcinogenesis.

4.
Front Oncol ; 10: 557643, 2020.
Article in English | MEDLINE | ID: mdl-33240808

ABSTRACT

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive subtype of leukemia for which important progress in treatment efficiency have been made in the past decades to reach a cure rate of 75%-80% nowadays. It is nevertheless mandatory to find new targets and active molecules for innovative therapeutic strategies as relapse is associated with a very dismal outcome. We designed an experimental workflow to highlight the conserved core pathways associated with leukemogenesis by confronting the gene expression profiles (GEPs) of human T-ALL cases to the GEP of a murine T-ALL representative model, generated by the conditional deletion of the PTEN tumor suppressor gene in T cell precursors (tPTEN-/-). We identified 844 differentially expressed genes, common GEPs (cGEP) that were conserved between human T-ALL and murine signatures, and also similarly differentially expressed, compared to normal T cells. Using bioinformatic tools we highlighted in cGEPan upregulation of E2F, MYC and mTORC1. Next, using Connectivity Map (CMAP) and CMAPViz a visualization procedure for CMAP data that we developed, we selected in silico three FDA-approved, bioactive molecule candidates: α-estradiol (α-E), nordihydroguaiaretic acid (NDGA) and prochlorperazine dimaleate (PCZ). At a biological level, we showed that the three drugs triggered an apoptotic cell death in a panel of T-ALL cell lines, activated a DNA damage response and interfered with constitutive mTORC1 activation and c-MYC expression. This analysis shows that the investigation of conserved leukemogenesis pathways could be a strategy to reveal new avenues for pharmacological intervention.

5.
Cancer Cell ; 36(3): 268-287.e10, 2019 09 16.
Article in English | MEDLINE | ID: mdl-31447347

ABSTRACT

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.


Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Immunoblastic Lymphadenopathy/pathology , Lymphoma, T-Cell/pathology , NF-kappa B/metabolism , T-Lymphocytes/immunology , Aged , Animals , Cell Line, Tumor , Cell Lineage/immunology , Datasets as Topic , Disease Models, Animal , Female , Gene Knockdown Techniques , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , HEK293 Cells , Humans , Immunoblastic Lymphadenopathy/genetics , Lymphoma, T-Cell/drug therapy , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/immunology , Male , Mice, Transgenic , Middle Aged , NF-kappa B/genetics , Protein Kinase Inhibitors/administration & dosage , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , NF-kappaB-Inducing Kinase
6.
Theranostics ; 9(4): 1181-1199, 2019.
Article in English | MEDLINE | ID: mdl-30867824

ABSTRACT

Lysosomotropic agents such as sunitinib, lapatinib, and chloroquine belong to a drug family that is being used more frequently to treat advanced cancers. Sunitinib is standard care for metastatic renal cell carcinomas (mRCC) and lapatinib is used for trastuzumab/pertuzumab-refractory cancers. However, patients ineluctably relapse with a delay varying from a few months to a few years. To improve reactivity prior to relapse it is essential to identify the mechanisms leading to such variability. We showed previously that sunitinib became sequestered in lysosomes because of its basic pKa. Methods: Modifications to gene expression in response to sunitinib and in sunitinib resistant cells were analyzed by transcriptomic and proteomic analysis. ROS production was evaluated by FACS. Nuclear Factor kappa B (NFkB)-dependent transcriptional regulation of inflammatory gene expression was evaluated with a reporter gene. Correlation of CXCL5 with survival was analyzed with an online available data base (TCGA) and using a cohort of patients enrolled in the SUVEGIL clinical trial (NCT00943839). Results: We now show that sunitinib sequestration in lysosomes induced an incomplete autophagic process leading to activation of the NFkB inflammatory pathway. We defined a subset of inflammatory cytokines that were up-regulated by the drug either after an acute or chronic stimulus. One of the most up-regulated genes in sunitinib-resistant cells was the CXCL5 cytokine. CXCL5 was also induced in RCC by chloroquine and in a model of HER2 positive breast cancer cell lines after acute or chronic treatment with lapatinib. CXCL5 correlated to shorter survival in RCC and to the most aggressive forms of breast cancers. The levels of CXCL5 present in the plasma of patients treated with sunitinib were predictive of the efficacy of sunitinib but not of the VEGF-directed antibody bevacizumab. Conclusion: This translational study identified CXCL5 as a biomarker of efficacy of lysosomotropic drugs, a potential asset for personalized medicine.


Subject(s)
Antineoplastic Agents/pharmacology , Autophagy , Breast Neoplasms/drug therapy , Chemokine CXCL5/metabolism , Drug Resistance, Neoplasm , Kidney Neoplasms/drug therapy , Sunitinib/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Gene Expression Profiling , Humans , Inflammation , Proteomics , Sunitinib/therapeutic use , Survival Analysis
8.
Leuk Lymphoma ; 59(2): 460-468, 2018 02.
Article in English | MEDLINE | ID: mdl-28641473

ABSTRACT

Cancer cells reprogram their metabolism to optimize their growth and proliferation in the host microenvironment. For this purpose, they enhance the uptake of extracellular nutrients and deal with the metabolic waste products through the overexpression of numerous membrane proteins including amino-acid transporters (LAT1) and acid-base regulating enzymes, such as carbonic anhydrases (CAs). Here we describe the anti-tumoral effects of a new class of CAXII inhibitors, the glycosyl coumarins on T-ALL/LL cells. These effects appeared to be mediated through inhibition of mTOR/Akt pathway and c-myc downregulation. Interestingly, we show that the combined targeting of amino acid fluxes and pH regulators provides a promising therapeutic strategy in the future of T-ALL/LL management.


Subject(s)
Amino Acids, Essential/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Amino Acid Transport Systems/antagonists & inhibitors , Animals , Cell Death/drug effects , Cell Line, Tumor , Coumarins/pharmacology , Hydrogen-Ion Concentration , Intracellular Space/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/metabolism
9.
Biomedicines ; 5(2)2017 May 31.
Article in English | MEDLINE | ID: mdl-28561798

ABSTRACT

NF-κB (Nuclear Factor Κ-light-chain-enhancer of activated B cells) transcription factors are critical regulators of immunity, stress response, apoptosis, and differentiation. Molecular defects promoting the constitutive activation of canonical and non-canonical NF-κB signaling pathways contribute to many diseases, including cancer, diabetes, chronic inflammation, and autoimmunity. In the present review, we focus our attention on the mechanisms of NF-κB deregulation in hematological malignancies. Key positive regulators of NF-κB signaling can act as oncogenes that are often prone to chromosomal translocation, amplifications, or activating mutations. Negative regulators of NF-κB have tumor suppressor functions, and are frequently inactivated either by genomic deletions or point mutations. NF-κB activation in tumoral cells is also driven by the microenvironment or chronic signaling that does not rely on genetic alterations.

10.
Leuk Lymphoma ; 58(6): 1433-1445, 2017 06.
Article in English | MEDLINE | ID: mdl-27736268

ABSTRACT

Iron is an essential nutrient, acting as a catalyst for metabolic reactions that are fundamental to cell survival and proliferation. Iron complexed to transferrin is delivered to the metabolism after endocytosis via the CD71 surface receptor. We found that transformed cells from a murine PTEN-deficient T-cell lymphoma model and from T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LL) cell lines overexpress CD71. As a consequence, the cells developed an addiction toward iron whose chelation by deferoxamine (DFO) dramatically affected their survival to induce apoptosis. Interestingly, DFO displayed synergistic activity with three ALL-specific drugs: dexamethasone, doxorubicin, and L-asparaginase. DFO appeared to act through a reactive oxygen species-dependent DNA damage response and potentiated the action of an inhibitor of the PARP pathway of DNA repair. Our results demonstrate that targeting iron metabolism could be an interesting adjuvant therapy for acute lymphoblastic leukemia.


Subject(s)
Iron Chelating Agents/pharmacology , Iron/metabolism , Lymphoma, T-Cell/genetics , Lymphoma, T-Cell/metabolism , PTEN Phosphohydrolase/deficiency , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Apoptosis/drug effects , Asparaginase/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Chemotherapy, Adjuvant , DNA Damage , Deferoxamine/pharmacology , Disease Models, Animal , Drug Synergism , Gene Expression , Humans , Iron Chelating Agents/therapeutic use , Lymphoma, T-Cell/drug therapy , Lymphoma, T-Cell/mortality , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Reactive Oxygen Species/metabolism , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism
11.
Nat Commun ; 7: 12856, 2016 Sep 26.
Article in English | MEDLINE | ID: mdl-27665925

ABSTRACT

Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target.

12.
Blood ; 128(2): 253-64, 2016 07 14.
Article in English | MEDLINE | ID: mdl-27257182

ABSTRACT

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.


Subject(s)
Bone Marrow Cells/metabolism , Leukemia, Myeloid, Acute/metabolism , Mitochondria/metabolism , Animals , Bone Marrow Cells/pathology , Coculture Techniques , HL-60 Cells , Heterografts , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Nude , Mitochondria/pathology , Neoplasm Transplantation , Stromal Cells/metabolism , Stromal Cells/pathology , U937 Cells
13.
Cancer Res ; 76(8): 2082-6, 2016 04 15.
Article in English | MEDLINE | ID: mdl-26960976

ABSTRACT

Acute myeloid leukemia (AML) is sustained by a subpopulation of rare leukemia-initiating cells (LIC) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in vivo The xenotransplantation model captures functional properties of LICs that have clinical prognostic value. However, the long duration of this in vivo assay has hampered its use as a prognostic tool. Here, we show, using an ex vivo coculture system, that intermediate and poor risk AML patient samples at diagnosis have a 5 to 7 times higher frequency of leukemic long-term culture-initiating cells (L-LTC-IC) compared with the good risk group. We defined a fluorescence dilution factor (FDF) parameter that monitors sample proliferation over 1 week and established a strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor prognostic AMLs or for samples capable of engrafting NSG mice compared with good risk AMLs or nonengrafters. Importantly, FDF could classify normal karyotype intermediate risk patients into two groups with a significant difference in their overall survival, thus making this nongenetic and non-in vivo approach a new clinically relevant tool for better diagnosis of AML patients. Cancer Res; 76(8); 2082-6. ©2016 AACR.


Subject(s)
Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Cell Proliferation/physiology , Female , Humans , Male , Prognosis , Tumor Cells, Cultured
14.
Cell Discov ; 1: 15030, 2015.
Article in English | MEDLINE | ID: mdl-27462428

ABSTRACT

Specific BRAFV600E inhibitors (BRAFi) are highly effective in the treatment of melanoma. However, acquired drug resistances invariably develop after the initial response. Therefore, the identification of new mechanisms of acquired resistance gives important clues towards the development of therapies that could elicit long lasting responses. Here we report that CD271 confers resistance to BRAFi in melanoma cells. The expression of CD271 is increased by BRAFi through a stimulation of tumor necrosis factor-alpha (TNFα) secretion that leads to NF-κB signaling pathway activation. CD271 is upregulated in a subset of BRAFi-resistant melanoma cells. The inhibition of TNFα/NF-κB pathway and CD271 silencing restore the BRAFi sensitivity of resistant melanoma cells. Finally, increase of CD271 expression is validated in BRAFi-resistant xenografts tumors and also in tumors from the patients who relapsed under BRAFi. In summary, these results reveal a novel TNFα/NF-κB/CD271 axis whose activation contributes to the acquisition of resistance to BRAFi and therefore may represent a novel therapeutic target to improve the efficacy of therapy in melanoma.

15.
PLoS One ; 9(8): e103069, 2014.
Article in English | MEDLINE | ID: mdl-25118595

ABSTRACT

Salmonella enterica serovar Typhimurium (ST) is an enteropathogenic Gram-negative bacterium that causes infection following oral ingestion. ST spreads rapidly along the gastrointestinal tract (GIT) and invades the intestinal epithelium to ultimately reach internal body organs. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is prescribed for prophylaxis of diarrheal infectious diseases. We previously showed that S.b-B prevents weight loss in ST-infected mice and significantly decreases bacterial translocation to the spleen and liver. This study was designed to investigate the effect of S.b-B on ST migration along the GIT and the impact of the yeast on the host's early innate immune responses. Bioluminescent imaging (BLI) was used to evaluate the effect of S.b-B on the progression of luminescent Salmonella Typhimurium (ST-lux) in the GIT of mice pretreated with streptomycin. Photonic emission (PE) was measured in GIT extracts (stomach, small intestine, cecum and colon) at various time periods post-infection (PI). PE analysis revealed that, 45 min PI, ST-lux had migrated slightly faster in the mice treated with S.b-B than in the untreated infected animals. At 90 min PI, ST-lux had reached the cecum in both groups of mice. Adhesion of ST to S.b-B was visualized in the intestines of the mice and probably accounts for (1) the faster elimination of ST-lux in the feces, and (2) reduced translocation of ST to the spleen and liver. In the early phase of infection, S.b-B also modifies the host's immune responses by (1) increasing IFN-γ gene expression and decreasing IL-10 gene expression in the small intestine, and (2) elevating both IFN-γ, and IL-10 mRNA levels in the cecum. BLI revealed that S.b-B modifies ST migration and the host immune response along the GIT. Study findings shed new light on the protective mechanisms of S.b-B during the early phase of Salmonella pathogenesis.


Subject(s)
Host-Pathogen Interactions/drug effects , Intestines/microbiology , Probiotics/pharmacology , Saccharomyces/physiology , Salmonella typhimurium/physiology , Animals , Bacterial Adhesion , Female , Gene Expression Regulation , Immunity, Innate/drug effects , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Intestines/immunology , Luminescent Measurements , Mice, Inbred C57BL , Salmonella typhimurium/immunology
16.
Cancer Lett ; 336(1): 114-26, 2013 Aug 09.
Article in English | MEDLINE | ID: mdl-23612073

ABSTRACT

We show here that the antidiabetic agents metformin and phenformin and the AMPK activator AICAR exert strong anti-tumoural effects on tPTEN-/- lymphoma cells and on human T-ALL cell lines and primary samples. The compounds act by inhibiting tumour metabolism and proliferation and by inducing apoptosis in parallel with an activation of AMPK and an inhibition of constitutive mTOR. In tPTEN-/- cells, the drugs potentiated the anti-leukaemic effects of dexamethasone, and metformin and phenformin synergised with 2-deoxyglucose (2DG) to impair tumour cell survival. In vivo, metformin and AICAR strongly decreased the growth of luciferase-expressing tPTEN-/- cells xenografted in Nude mice, demonstrating that metabolism targeting could be a potent adjuvant strategy for lymphoma/leukaemia treatment.


Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Gene Expression Regulation, Neoplastic , Lymphoma, T-Cell/metabolism , Metformin/pharmacology , PTEN Phosphohydrolase/metabolism , Phenformin/pharmacology , Ribonucleotides/pharmacology , Alleles , Aminoimidazole Carboxamide/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival , Enzyme Activation , Humans , Jurkat Cells , Mice , Mice, Nude , Neoplasm Transplantation , Transgenes
17.
Cancer Lett ; 333(1): 76-88, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23348702

ABSTRACT

The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN-/- mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.


Subject(s)
Apoptosis/drug effects , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/physiology , Cell Proliferation/drug effects , Lymphoma, T-Cell/drug therapy , Animals , Carbonic Anhydrases/drug effects , Carbonic Anhydrases/genetics , Cell Line, Tumor , Humans , Hydrogen-Ion Concentration , Lymphoma, T-Cell/enzymology , Mice , PTEN Phosphohydrolase/physiology
18.
J Pathol ; 227(1): 118-29, 2012 May.
Article in English | MEDLINE | ID: mdl-22069124

ABSTRACT

CPT-11 (irinotecan), the first-line chemotherapy for advanced stage colorectal cancer, remains inactive in about half of patients (primary chemoresistance) and almost all initial responders develop secondary resistance after several courses of treatment (8 months on average). Nude mice bearing HT-29 colon cancer xenografts were treated with CPT-11 and/or an NF-κB inhibitor for two courses. We confirm that NF-κB inhibition potentiated CPT-11 anti-tumoural effect after the first course of treatment. However, tumours grew again at the end of the second course of treatment, generating resistant tumours. We observed an increase in the basal NF-κB activation in resistant tumours and in two resistant sublines, either obtained from resistant HT-29 tumours (HT-29R cells) or generated in vitro (RSN cells). The decrease of NF-κB activation in HT-29R and RSN cells by stable transfections with the super-repressor form of IκBα augmented their sensitivity to CPT-11. Comparing gene expression profiles of HT-29 and HT-29R cells, we identified the S100A10/Annexin A2 complex and calpain 2 as over-expressed potential NF-κB inducers. SiRNA silencing of calpain 2 but not of S100A10 and/or annexin A2, resulted in a decrease in NF-κB activation, an increase in cellular levels of IκBα and a partial restoration of the CPT-11 sensitivity in both HT-29R and RSN cells, suggesting that calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance. Thus, targeted therapies directed against calpain 2 may represent a novel strategy to enhance the anti-cancer efficacy of CPT-11.


Subject(s)
Antineoplastic Agents/pharmacology , Calpain/metabolism , Camptothecin/analogs & derivatives , Colorectal Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , I-kappa B Proteins/metabolism , Animals , Annexin A2/genetics , Annexin A2/metabolism , Apoptosis/drug effects , Camptothecin/pharmacology , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , I-kappa B Proteins/antagonists & inhibitors , Irinotecan , Mice , Mice, Inbred Strains , Mice, Nude , NF-kappa B/biosynthesis , Neoplasm Transplantation , Pyrimidines/pharmacology , S100 Proteins/genetics , S100 Proteins/metabolism , Transfection , Xenograft Model Antitumor Assays
19.
Genes Dev ; 25(12): 1245-61, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21646373

ABSTRACT

Melanoma cells can enter the process of senescence, but whether they express a secretory phenotype, as reported for other cells, is undetermined. This is of paramount importance, because this secretome can alter the tumor microenvironment and the response to chemotherapeutic drugs. More generally, the molecular events involved in formation of the senescent-associated secretome have yet to be determined. We reveal here that melanoma cells experiencing senescence in response to diverse stimuli, including anti-melanoma drugs, produce an inflammatory secretory profile, where the chemokine ligand-2 (CCL2) acts as a critical effector. Thus, we reveal how senescence induction might be involved in therapeutic failure in melanoma. We further provide a molecular relationship between senescence induction and secretome formation by revealing that the poly(ADP-ribose) polymerase-1 (PARP-1)/nuclear factor-κB (NF-κB) signaling cascade, activated during senescence, drives the formation of a secretome endowed with protumoral and prometastatic properties. Our findings also point to the existence of the PARP-1 and NF-κB-associated secretome, termed the PNAS, in nonmelanoma cells. Most importantly, inhibition of PARP-1 or NF-κB prevents the proinvasive properties of the secretome. Collectively, identification of the PARP-1/NF-κB axis in secretome formation opens new avenues for therapeutic intervention against cancers.


Subject(s)
NF-kappa B/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Cell Line, Tumor , Cellular Senescence , Chemokine CCL2/metabolism , DNA Damage , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Melanoma/physiopathology , Neoplasm Invasiveness/pathology , Poly (ADP-Ribose) Polymerase-1 , Signal Transduction
20.
FASEB J ; 25(9): 3092-105, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21676945

ABSTRACT

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.


Subject(s)
Cell Proliferation , Keratinocytes/metabolism , MicroRNAs/metabolism , Wounds and Injuries/metabolism , Animals , Antibodies , Epithelial Cells , Gene Silencing , Humans , Keratinocytes/cytology , Mice , MicroRNAs/genetics , Oligonucleotides , Skin/metabolism , Time Factors
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