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1.
JAMA Oncol ; 4(4): e175245, 2018 Apr 12.
Article in English | MEDLINE | ID: mdl-29423521

ABSTRACT

IMPORTANCE: Acquired resistance to anti-EGFR therapy (epidermal growth factor receptor) is frequently due to RAS and EGFR extracellular domain (ECD) mutations in metastatic colorectal cancer (mCRC). Some anti-EGFR-refractory patients retain tumor EGFR dependency potentially targetable by agents such as Sym004, which is a mixture of 2 nonoverlapping monoclonal antibodies targeting EGFR. OBJECTIVE: To determine if continuous blockade of EGFR by Sym004 has survival benefit. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, phase 2, randomized, clinical trial comparing 2 regimens of Sym004 with investigator's choice from March 6, 2014, through October 15, 2015. Circulating tumor DNA (ctDNA) was analyzed for biomarker and tracking clonal dynamics during treatment. Participants had wild-type KRAS exon 2 mCRC refractory to standard chemotherapy and acquired resistance to anti-EGFR monoclonal antibodies. INTERVENTIONS: Participants were randomly assigned in a 1:1:1 ratio to Sym004, 12 mg/kg/wk (arm A), Sym004, 9 mg/kg loading dose followed by 6 mg/kg/wk (arm B), or investigator's choice of treatment (arm C). MAIN OUTCOMES AND MEASURES: Overall survival (OS). Secondary end points included preplanned exploratory biomarker analysis in ctDNA. RESULTS: A total of 254 patients were randomized (intent-to-treat [ITT] population) (median age, 63 [range, 34-91] years; 63% male; n = 160). Median OS in the ITT population was 7.9 months (95% CI, 6.5-9.9 months), 10.3 months (95% CI, 9.0-12.9 months), and 9.6 months (95% CI, 8.3-12.2 months) for arms A, B, and C, respectively (hazard ratio [HR], 1.31; 95% CI, 0.92-1.87 for A vs C; and HR, 0.97; 95% CI, 0.68-1.40 for B vs C). The ctDNA revealed high intrapatient genomic heterogeneity following anti-EGFR therapy. Sym004 effectively targeted EGFR ECD-mutated cancer cells, and a decrease in EGFR ECD ctDNA occurred in Sym004-treated patients. However, this did not translate into clinical benefit in patients with EGFR ECD mutations, likely owing to co-occurring resistance mechanisms. A subgroup of patients was defined by ctDNA (RAS/BRAF/EGFR ECD-mutation negative) associated with improved OS in Sym004-treated patients in arm B compared with arm C (median OS, 12.8 and 7.3 months, respectively). CONCLUSIONS AND RELEVANCE: Sym004 did not improve OS in an unselected population of patients with mCRC and acquired anti-EGFR resistance. A prospective clinical validation of Sym004 efficacy in a ctDNA molecularly defined subgroup of patients with refractory mCRC is warranted. TRIAL REGISTRATION: clinicaltrialsregister.eu Identifier: 2013-003829-29.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Circulating Tumor DNA/blood , Colorectal Neoplasms/blood , Colorectal Neoplasms/drug therapy , Drug Resistance, Neoplasm , Patient Selection , Protein Kinase Inhibitors/therapeutic use , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Circulating Tumor DNA/analysis , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , Female , Humans , Male , Middle Aged , Neoplasm Metastasis , Survival Analysis , Treatment Outcome
2.
Neurotoxicology ; 56: 55-63, 2016 09.
Article in English | MEDLINE | ID: mdl-27392949

ABSTRACT

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic agent with high persistency that induces neurotoxic effects altering neurodevelopment and behavior. The molecular mechanisms and the signaling pathways involved in TCDD-mediated neurotoxicity, together with the search of its molecular targets in neurons are under intense study. We have previously shown that high nanomolar concentrations of TCDD for incubation times of minutes induce apoptosis in SHSY5Y human neuroblastoma cells by the disruption of calcium homeostasis, affecting membrane structural integrity. In this work, we have analyzed the effect of low nanomolar concentrations of TCDD for incubation times of hours to define the role of aryl hydrocarbon receptor which can be activated at those concentrations. TCDD induces toxicity in SHSY5Y human neuroblastoma cells under these experimental conditions with an EC50 value of approximately 3nM at 24h of incubation time. Transient transfection of a hairpin RNA for AhR protects against TCDD neurotoxicity, suggesting that AhR is mediating the dioxin effect. Altogether, these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells depends on dioxin concentration and time of incubation, with a main role of aryl hydrocarbon receptor at low nanomolar TCDD concentrations.


Subject(s)
Neurons/drug effects , Neurotoxins/pharmacology , Polychlorinated Dibenzodioxins/pharmacology , Receptors, Aryl Hydrocarbon/metabolism , Antioxidants/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Neuroblastoma/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Aryl Hydrocarbon/genetics , Resveratrol , Stilbenes/pharmacology , Time Factors , Transfection
3.
Clin Cancer Res ; 22(13): 3260-7, 2016 07 01.
Article in English | MEDLINE | ID: mdl-26888827

ABSTRACT

PURPOSE: Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. EXPERIMENTAL DESIGN: Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. RESULTS: Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. CONCLUSIONS: Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Cetuximab/therapeutic use , Colorectal Neoplasms/drug therapy , ErbB Receptors/antagonists & inhibitors , 3T3 Cells , Animals , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/genetics , ErbB Receptors/immunology , Humans , Male , Mice , Mice, Inbred BALB C , Mutation , Panitumumab , Signal Transduction/drug effects , Xenograft Model Antitumor Assays/methods
4.
Apoptosis ; 17(11): 1170-81, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22986482

ABSTRACT

The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P < 0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P < 0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.


Subject(s)
Apoptosis/drug effects , Calcium/metabolism , Homeostasis/drug effects , Intracellular Space/metabolism , Neurons/metabolism , Neurons/pathology , Polychlorinated Dibenzodioxins/toxicity , Anisotropy , Cell Line, Tumor , DNA/metabolism , DNA Fragmentation/drug effects , Diploidy , Humans , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Intracellular Space/drug effects , Membrane Potential, Mitochondrial/drug effects , Neuroblastoma/pathology , Neurons/drug effects , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism
5.
J Neurochem ; 118(1): 153-62, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21534955

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR-/-) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC(50) of 127±21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.


Subject(s)
Apoptosis/drug effects , Benzofurans/toxicity , Cerebellum/cytology , Environmental Pollutants/toxicity , Neurons/drug effects , Receptors, Aryl Hydrocarbon/physiology , Animals , Caspase 3/metabolism , Cells, Cultured , Cytochrome P-450 CYP1A2/metabolism , Dose-Response Relationship, Drug , Indoles , Mice , Mice, Knockout , Reactive Oxygen Species , Receptors, Aryl Hydrocarbon/deficiency , Tetrazolium Salts , Thiazoles , Trypan Blue
6.
Neurotoxicology ; 31(3): 267-76, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20302886

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that alters normal brain development and produces cognitive disability and motor dysfunction. However, after decades of intense study, the molecular mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and its molecular targets in neurons still remain unknown. TCDD acts as an exogenous ligand of the aryl hydrocarbon receptor (AhR) that becomes a key signaling molecule in the regulation of the toxic and carcinogenic properties of TCDD. We have used NGF-differentiated pheochromocytoma (dPC12) cells to determine the type of cell death that takes place by TCDD toxicity. TCDD induced cell death in dPC12 cultures with an EC(50) of 218+/-24 nM, similar to that obtained in undifferentiated PC12 cells, 171+/-31 nM. Nuclear fragmentation was observed after TCDD incubation in parallel to an increase in caspase-3 activity. Staurosporine, which readily induced apoptosis in dPC12 cells, showed a similar increase in caspase-3 activity and the characteristic pattern of nuclear fragmentation. Flow cytometry measurements showed that dPC12 cells in the presence of TCDD were positive for annexin V labeling but negative for propidium iodide staining. In addition, TCDD increased the area of the peak corresponding to hypodiploid (apoptotic) DNA content. All together these results support the hypothesis that TCDD toxicity in dPC12 cells takes place mainly through an apoptotic process.


Subject(s)
Apoptosis/drug effects , Cell Differentiation/drug effects , Dioxins/toxicity , Nerve Growth Factor/pharmacology , Neurotoxins/toxicity , Analysis of Variance , Animals , Annexin A5/metabolism , Caspase 3/metabolism , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Flow Cytometry/methods , Gene Expression Regulation/drug effects , Hippocampus/cytology , Indoles , Neuroglia/drug effects , Neurons/drug effects , PC12 Cells/drug effects , Rats , Reactive Oxygen Species/metabolism
7.
Brain Res Bull ; 81(4-5): 458-66, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-19931600

ABSTRACT

Zinc is a basic trace element that plays important roles in brain and, consequently, its homeostasis needs to be critically controlled. High zinc concentrations in the interneuron synaptic space may induce neuronal death through mechanisms still partially solved. Undifferentiated pheochromocytoma (PC12) cells have been used to study zinc toxicity. As these cells can be differentiated into neuronal-like cells, the results obtained from differentiated cultures are more useful to understand zinc toxicity in neurons. In this paper, we show by flow cytometry that nerve growth factor (NGF) induces PC12 cells differentiation characterized by cell cycle arrest in the G1/G0 phase, similarly to that observed in serum-deprived cultures. Zinc induces cell death in NGF-differentiated PC12 cultures with an EC(50) value of 143+/-14 microM, which reveals a higher sensitivity with respect to undifferentiated PC12 cultures (EC(50), 308+/-32 microM) and a similar response to that obtained in hippocampal neurons (134+/-12 microM). Thus, the differentiation process appeared responsible for such increase in sensitivity. To further support this tenet, when the NGF differentiation was impaired in presence of 10 microM MK-801, a selective blocker of the N-methyl-d-aspartate (NMDA) receptor that plays a role in the differentiation process, the higher sensitivity to zinc was reverted to an EC(50) value of 241+/-26 microM. Flow cytometry experiments showed that NGF-differentiated PC12 cells in presence of zinc were positive for propidium iodide but not for annexin-V labeling. These results, together with data from fluorescent labeling of nuclear fragmentation, caspase-3 activation, and reactive oxygen species generation, support the view that zinc toxicity in NGF-differentiated PC12 cells takes place mainly through a necrotic process.


Subject(s)
Cell Cycle/drug effects , Cell Cycle/physiology , Nerve Growth Factor/metabolism , Trace Elements/toxicity , Zinc Sulfate/toxicity , Animals , Caspase 3/metabolism , Cell Death/drug effects , Cell Death/physiology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Dizocilpine Maleate/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Metallothionein/metabolism , Necrosis/chemically induced , Necrosis/metabolism , Neurons/drug effects , Neurons/physiology , PC12 Cells , Rats , Reactive Oxygen Species/metabolism
8.
Neurochem Int ; 53(5): 148-54, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18694792

ABSTRACT

N-Methyl-d-aspartate (NMDA) receptors play a critical role in the brain stimulating synaptic plasticity and mediating neurodegeneration; a neuroprotective role has also been described, but its molecular mechanisms in hippocampus are under study. Here, we report that in primary cultures of rat hippocampal neurons exposure to low micromolar NMDA concentrations are neuroprotective against excitotoxic insults, while high micromolar NMDA concentrations provoke neuronal death. Molecular analysis reveals that a toxic concentration of NMDA induced a transient phosphorylation of cAMP-response element-binding protein (pCREB) in 2 min that rapidly decreased below basal levels. In contrast, a nontoxic NMDA concentration gave up to longer (20 min) rise of pCREB, suggesting that neuroprotection could be associated to a relatively prolonged presence of pCREB in the neurons. In support of this tenet, rolipram, an inhibitor of phosphodiesterase IV that increases the levels of cAMP and pCREB, protected against NMDA-induced neuronal death. Similar results were obtained with dibutyrate-cAMP (a cAMP analogue with membrane permeability) that also abrogated NMDA excitotoxicity. Conversely, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), an inhibitor of protein kinase A (PKA), that prevents the formation of pCREB induced by nontoxic NMDA concentrations, reverted the neuroprotection achieved by preincubation of low micromolar NMDA concentrations. These results substantiate the notion that induction of pCREB via PKA plays an important role in NMDA-mediated neuroprotection.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Hippocampus/cytology , N-Methylaspartate/physiology , Neurons/cytology , Receptors, N-Methyl-D-Aspartate/agonists , Animals , Cells, Cultured , Embryo, Nonmammalian/cytology , N-Methylaspartate/pharmacology , Phosphorylation , Rats , Signal Transduction
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