RESUMEN
AIMS: Previous studies have shown that the activin-binding protein follistatin reduces inflammation in several mouse models of colitis. To determine whether follistatin also has a beneficial effect following bladder inflammation, we induced cystitis in mice using cyclophosphamide (CYP) and examined the relationship between bladder hypersensitivity and bladder follistatin expression. METHODS: Adult female C57BL/6 mice were treated with CYP (100 mg/kg) or vehicle (saline) three times over 5 days. Bladder hypersensitivity was assessed by recording the visceromotor response (VMR) to urinary bladder distension and in vitro single-fiber bladder afferent recording. Follistatin gene expression was measured using qRT-PCR. Immunohistochemistry was employed for further characterization. RESULTS: Bladder hypersensitivity was established by day 6 and persisted to day 14 in CYP-treated mice. On day 14, hypersensitivity was accompanied by increases in follistatin gene expression in the bladder. Follistatin-like immunoreactivity colocalized with laminin, and the percentage of structures in the lamina propria that were follistatin-positive was increased in CYP-treated mice. Exogenous follistatin increased VMR and afferent responses to bladder distension in CYP- but not vehicle-treated mice. CONCLUSIONS: Chronic bladder pain following CYP treatment is associated with increased follistatin expression in the bladder. These results suggest a novel, pro-nociceptive role for follistatin in cystitis, in contrast with its proposed therapeutic role in colitis. This protein has exciting potential as a biomarker and therapeutic target for bladder hypersensitivity. Neurourol. Urodynam. 36:286-292, 2017. © 2015 Wiley Periodicals, Inc.
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Cistitis/genética , Folistatina/genética , Vejiga Urinaria/metabolismo , Animales , Biomarcadores/metabolismo , Ciclofosfamida , Cistitis/inducido químicamente , Cistitis/metabolismo , Femenino , Folistatina/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
The sensory innervation of the distal colorectum includes mechanically insensitive afferents (MIAs; â¼25%), which acquire mechanosensitivity in persistent visceral hypersensitivity and thus generate de novo input to the central nervous system. We utilized an optogenetic approach to bypass the process of transduction (generator potential) and focus on transformation (spike initiation) at colorectal MIA sensory terminals, which is otherwise not possible in typical functional studies. From channelrhodopsin2-expressing mice (driven by Advillin-Cre), the distal colorectum with attached pelvic nerve was harvested for ex vivo single-fiber recordings. Afferent receptive fields (RFs) were identified by electrical stimulation and tested for response to mechanical stimuli (probing, stroking, and stretch), and afferents were classified as either MIAs or mechanosensitive afferents (MSAs). All MIA and MSA RFs were subsequently stimulated optically and MIAs were also tested for activation/sensitization with inflammatory soup (IS), acidic hypertonic solution (AHS), and/or bile salts (BS). Responses to pulsed optical stimuli (1-10 Hz) were comparable between MSAs and MIAs whereas 43% of MIAs compared with 86% of MSAs responded tonically to stepped optical stimuli. Tonic-spiking MIAs responded preferentially to AHS (an osmotic stimulus) whereas non-tonic-spiking MIAs responded to IS (an inflammatory stimulus). A significant proportion of MIAs were also sensitized by BS. These results reveal transformation as a critical factor underlying the differences between MIAs (osmosensors vs. inflammatory sensors), revealing a previously unappreciated heterogeneity of MIA endings. The current study draws attention to the sensory encoding of MIA nerve endings that likely contribute to afferent sensitization and thus have important roles in visceral pain.
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Colon/inervación , Mecanotransducción Celular , Neuronas Aferentes/fisiología , Recto/inervación , Animales , Ácidos y Sales Biliares/farmacología , Colon/citología , Femenino , Fototransducción , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Optogenética , Presión Osmótica , Recto/citología , Rodopsina/genética , Rodopsina/metabolismoRESUMEN
PURPOSE: Chronic prostatitis/chronic pelvic pain syndrome causes symptoms that include the frequent and urgent need to urinate, pain or burning during urination and pain radiating to the back, abdomen and/or colorectum. These bladder symptoms suggest that chronic prostatitis/chronic pelvic pain syndrome is associated with sensitization of adjacent organs, termed cross-organ sensitization. The objective of this study was to determine the extent of 1) changes in immunomodulatory mediators in the prostate and bladder after inflammation of the prostate and 2) bladder function and bladder afferent sensitization. MATERIALS AND METHODS: Prostate and bladder histology, immunohistochemistry and expression of immunomodulatory targets were examined weekly after zymosan or vehicle was injected in the dorsal lobe of the mouse prostate. Cystometry, bladder and bladder afferent sensitivity were also assessed weekly. RESULTS: Prostate inflammation induced significant up-regulation in proinflammatory and anti-inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), growth factor NGF (nerve growth factor), and T-lymphocyte markers FoxP3, CD4 and CD8 in the prostate and the bladder. Notably, prostatitis significantly increased urinary voiding frequency, induced hypersensitivity to bladder distension and sensitized bladder afferents. We also examined sensory (afferent) co-innervation by injecting retrograde tracers DiI and Fast Blue in the bladder wall and the prostate, respectively. This showed that a significant proportion (approximately 17%) of dorsal root ganglion afferent somata contained tracers from the bladder and the prostate. CONCLUSIONS: These observations support an afferent contribution to chronic prostatitis/chronic pelvic pain syndrome and cross-organ sensitization from prostate to bladder.
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Ganglios Espinales/metabolismo , Prostatitis/complicaciones , Enfermedades de la Vejiga Urinaria/etiología , Vejiga Urinaria/inervación , Animales , Western Blotting , Enfermedad Crónica , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Ganglios Espinales/patología , Regulación de la Expresión Génica , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Prostatitis/diagnóstico , Prostatitis/genética , ARN/genética , Vejiga Urinaria/diagnóstico por imagen , Enfermedades de la Vejiga Urinaria/diagnóstico , Enfermedades de la Vejiga Urinaria/genéticaRESUMEN
There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Detección Precoz del Cáncer , Metilación de ADN/genética , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Neoadjuvant targeted therapy provides a brief, preoperative window of opportunity that can be exploited to individualize cancer care based on treatment response. We investigated whether response to neoadjuvant therapy during the preoperative window confers survival benefit in patients with operable head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: A pooled analysis of treatment-naïve patients with operable HNSCC enrolled in one of three clinical trials from 2009 to 2020 (NCT00779389, NCT01218048, NCT02473731). Neoadjuvant regimens consisted of EGFR inhibitors (n = 83) or anti-ErbB3 antibody therapy (n = 9) within 28 days of surgery. Clinical to pathologic stage migration was compared with disease-free survival (DFS) and overall survival (OS) while adjusting for confounding factors using multivariable Cox regression. Circulating tumor markers validated in other solid tumor models were analyzed. RESULTS: 92 of 118 patients were analyzed; all patients underwent surgery following neoadjuvant therapy. Clinical to pathologic downstaging was more frequent in patients undergoing neoadjuvant targeted therapy compared with control cohort (P = 0.048). Patients with pathologic downstage migration had the highest OS [89.5%; 95% confidence interval (CI), 75.7-100] compared with those with no stage change (58%; 95% CI, 46.2-69.8) or upstage (40%; 95% CI, 9.6-70.4; P = 0.003). Downstage migration remained a positive prognostic factor for OS (HR, 0.22; 95% CI, 0.05-0.90) while adjusting for measured confounders. Downstage migration correlated with decreased circulating tumor markers, SOX17 and TAC1 (P = 0.0078). CONCLUSIONS: Brief neoadjuvant therapy achieved pathologic downstaging in a subset of patients and was associated with significantly better DFS and OS as well as decreased circulating methylated SOX17 and TAC1.
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Neoplasias de Cabeza y Cuello , Terapia Neoadyuvante , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Supervivencia sin Enfermedad , Biomarcadores de TumorRESUMEN
Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.
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Antígenos CD/metabolismo , Interleucina-6 , Neoplasias , Linfocitos T CD8-positivos , Humanos , Inmunoterapia , Interleucina-6/metabolismo , Receptores Inmunológicos/metabolismo , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Transgenic (Tg) mice overexpressing human wild-type alpha-synuclein in oligodendrocytes under the control of the 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter are shown here to recapitulate features of multiple system atrophy (MSA), including the accumulation of filamentous human alpha-synuclein aggregates in oligodendrocytes linked to their degeneration and autophagocytosis of myelin. Significantly, endogenous mouse alpha-synuclein also accumulated in normal and degenerating axons and axon terminals in association with oligodendroglia and neuron loss and slowly progressive motor impairments. Our studies demonstrate that overexpression of alpha-synuclein in oligodendrocytes of mice results in MSA-like degeneration in the CNS and that alpha-synuclein inclusions in oligodendrocytes participate in the degeneration of neurons in MSA.
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Atrofia de Múltiples Sistemas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/patología , Neuronas/patología , Oligodendroglía/metabolismo , Degeneración Walleriana/metabolismo , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Animales , Axones/metabolismo , Axones/patología , Axones/ultraestructura , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Modelos Animales de Enfermedad , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Atrofia de Múltiples Sistemas/genética , Atrofia de Múltiples Sistemas/fisiopatología , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Vaina de Mielina/ultraestructura , Proteínas del Tejido Nervioso/genética , Oligodendroglía/patología , Oligodendroglía/ultraestructura , Fagocitosis/genética , Hidrolasas Diéster Fosfóricas/genética , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patología , Terminales Presinápticos/ultraestructura , Regiones Promotoras Genéticas/genética , Sinucleínas , Degeneración Walleriana/genética , Degeneración Walleriana/fisiopatología , alfa-SinucleínaRESUMEN
Treatment of human head and neck squamous cell carcinoma (HNSCC) cell lines with guggulsterone, a widely available, well-tolerated nutraceutical, demonstrated dose-dependent decreases in cell viability with EC(50)s ranging from 5 to 8 microM. Guggulsterone induced apoptosis and cell cycle arrest, inhibited invasion and enhanced the efficacy of erlotinib, cetuximab and cisplatin in HNSCC cell lines. Guggulsterone induced decreased expression of both phosphotyrosine and total signal transducer and activator of transcription (STAT)-3, which contributed to guggulsterone's growth inhibitory effect. Hypoxia-inducible factor (HIF)-1alpha was also decreased in response to guggulsterone treatment. In a xenograft model of HNSCC, guggulsterone treatment resulted in increased apoptosis and decreased expression of STAT3. In vivo treatment with a guggulsterone-containing natural product, Guggulipid, resulted in decreased rates of tumor growth and enhancement of cetuximab's activity. Our results suggest that guggulsterone-mediated inhibition of STAT3 and HIF-1alpha provide a biologic rationale for further clinical investigation of this compound in the treatment of HNSCC.
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Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Fitoterapia , Pregnenodionas/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cetuximab , Cisplatino/farmacología , Cisplatino/uso terapéutico , Commiphora , Sinergismo Farmacológico , Clorhidrato de Erlotinib , Femenino , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Preparaciones de Plantas/farmacología , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidoresRESUMEN
Studies indicate that elevated interleukin-6 (IL-6) levels engage IL6Rα-gp130 receptor complexes to activate signal transducer and activator of transcription 3 (STAT3) that is hyperactivated in many cancers including head and neck squamous cell carcinoma (HNSCC). Our previous HCS campaign identified several hits that selectively blocked IL-6-induced STAT3 activation. This study describes our investigation of the mechanism(s) of action of three of the four chemical series that progressed to lead activities: a triazolothiadiazine (864669), amino alcohol (856350), and an oxazole-piperazine (4248543). We demonstrated that all three blocked IL-6-induced upregulation of the cyclin D1 and Bcl-XL STAT3 target genes. None of the compounds exhibited direct binding interactions with STAT3 in surface plasmon resonance (SPR) binding assays; neither did they inhibit the recruitment and binding of a phospho-tyrosine-gp130 peptide to STAT3 in a fluorescence polarization assay. Furthermore, they exhibited little or no inhibition in a panel of 83 cancer-associated in vitro kinase profiling assays, including lack of inhibition of IL-6-induced Janus kinase (JAK 1, 2, and 3) activation. Further, 864669 and 4248543 selectively inhibited IL-6-induced STAT3 activation but not that induced by oncostatin M (OSM). The compounds 864669 and 4248543 abrogated IL-6-induced phosphorylation of the gp130 signaling subunit (phospho-gp130Y905) of the IL-6-receptor complex in HNSCC cell lines which generate docking sites for the SH2 domains of STAT3. Our data indicate that 864669 and 4248543 block IL-6-induced STAT activation by interfering with the recruitment, assembly, or activation of the hexamer-activated IL-6/IL-6Rα/gp130 signaling complex that occurs after IL-6 binding to IL-6Rα subunits.
RESUMEN
Intracellular accumulations of filamentous tau inclusions are neuropathological hallmarks of neurodegenerative diseases known as tauopathies. The discovery of multiple pathogenic tau gene mutations in many kindreds with familial frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) unequivocally confirmed the central role of tau abnormalities in the etiology of neurodegenerative disorders. To examine the effects of tau gene mutations and the role of tau abnormalities in neurodegenerative tauopathies, transgenic (Tg) mice were engineered to express the longest human tau isoform (T40) with or without the R406W mutation (RW and hWT Tg mice, respectively) that is pathogenic for FTDP-17 in several kindreds. RW but not hWT tau Tg mice developed an age-dependent accumulation of insoluble filamentous tau aggregates in neuronal perikarya of the cerebral cortex, hippocampus, cerebellum, and spinal cord. Significantly, CNS axons in RW mice contained reduced levels of tau when compared with hWT mice, and this was linked to retarded axonal transport and increased accumulation of an insoluble pool of RW but not hWT tau. Furthermore, RW but not hWT mice demonstrated neurodegeneration and a reduced lifespan. These data indicate that the R406W mutation causes reduced binding of this mutant tau to microtubules, resulting in slower axonal transport. This altered tau function caused by the RW mutation leads to increased accumulation and reduced solubility of RW tau in an age-dependent manner, culminating in the formation of filamentous intraneuronal tau aggregates similar to that observed in tauopathy patients.
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Transporte Axonal/genética , Tauopatías/genética , Tauopatías/fisiopatología , Proteínas tau/genética , Proteínas tau/metabolismo , Envejecimiento/patología , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exones/genética , Humanos , Cuerpos de Inclusión/patología , Ligadura , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Neuronas/patología , Unión Proteica/genética , Transporte de Proteínas/genética , Radiculopatía/fisiopatología , Solubilidad , Tasa de Supervivencia , Tauopatías/patologíaRESUMEN
EGFR polymorphisms have not been thoroughly evaluated for association with head and neck squamous cell carcinoma (HNSCC) risk. We genotyped 578 HNSCC patients and 588 cancer-free controls for 60 EGFR single nucleotide polymorphisms (SNPs) and tested associations with HNSCC risk. EGFR intronic SNPs rs12535536, rs2075110, rs1253871, rs845561 and rs6970262 and synonymous SNP rs2072454 were associated with HNSCC risk among all subjects (p < 0.05). SNPs rs12538371, rs845561, and rs6970262 were significantly associated with HNSCC risk (p < 0.05) among never tobacco users. We identified EGFR variants that likely modify risk for HNSCC including three variants that contribute to tobacco-independent risk.
Asunto(s)
Carcinoma de Células Escamosas/genética , Receptores ErbB/genética , Predisposición Genética a la Enfermedad/genética , Neoplasias de Cabeza y Cuello/genética , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Femenino , Frecuencia de los Genes , Genotipo , Neoplasias de Cabeza y Cuello/patología , Humanos , Intrones/genética , Desequilibrio de Ligamiento , Modelos Logísticos , Masculino , Persona de Mediana Edad , Infecciones por Papillomavirus/virología , Factores de Riesgo , Fumar , Adulto JovenRESUMEN
Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 has been implicated in cell proliferation and survival of many cancers including head and neck squamous cell carcinoma (HNSCC). AZD1480, an orally active pharmacologic inhibitor of JAK1/JAK2, has been tested in several cancer models. In the present study, the in vitro and in vivo effects of AZD1480 were evaluated in HNSCC preclinical models to test the potential use of JAK kinase inhibition for HNSCC therapy. AZD1480 treatment decreased HNSCC proliferation in HNSCC cell lines with half maximal effective concentration (EC50) values ranging from 0.9 to 4 µM in conjunction with reduction of pSTAT3(Tyr705) expression. In vivo antitumor efficacy of AZD1480 was demonstrated in patient-derived xenograft (PDX) models derived from two independent HNSCC tumors. Oral administration of AZD1480 reduced tumor growth in conjunction with decreased pSTAT3(Tyr705) expression that was observed in both PDX models. These findings suggest that the JAK1/2 inhibitors abrogate STAT3 signaling and may be effective in HNSCC treatment approaches.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Quinasas Janus/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Dosificación de Gen , Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/genética , Ratones , Fosforilación , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y Cuello , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Strategies to inhibit the EGF receptor (EGFR) using the tyrosine kinase inhibitor erlotinib have been associated with limited clinical efficacy in head and neck squamous cell carcinoma (HNSCC). Co-activation of alternative kinases may contribute to erlotinib resistance. EXPERIMENTAL DESIGN: We generated HNSCC cells expressing dominant-active c-Src (DA-Src) to determine the contribution of c-Src activation to erlotinib response. RESULTS: Expression of DA-Src conferred resistance to erlotinib in vitro and in vivo compared with vector-transfected control cells. Phospho-Met was strongly upregulated by DA-Src, and DA-Src cells did not produce hepatocyte growth factor (HGF). Knockdown of c-Met enhanced sensitivity to erlotinib in DA-Src cells in vitro, as did combining a c-Met or c-Src inhibitor with erlotinib. Inhibiting EGFR resulted in minimal reduction of phospho-Met in DA-Src cells, whereas complete phospho-Met inhibition was achieved by inhibiting c-Src. A c-Met inhibitor significantly sensitized DA-Src tumors to erlotinib in vivo, resulting in reduced Ki67 labeling and increased apoptosis. In parental cells, knockdown of endogenous c-Src enhanced sensitivity to erlotinib, whereas treatment with HGF to directly induce phospho-Met resulted in erlotinib resistance. The level of endogenous phospho-c-Src in HNSCC cell lines was also significantly correlated with erlotinib resistance. CONCLUSIONS: Ligand-independent activation of c-Met contributes specifically to erlotinib resistance, not cetuximab resistance, in HNSCC with activated c-Src, where c-Met activation is more dependent on c-Src than on EGFR, providing an alternate survival pathway. Addition of a c-Met or c-Src inhibitor to erlotinib may increase efficacy of EGFR inhibition in patients with activated c-Src.
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Neoplasias de Cabeza y Cuello/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Quinazolinas/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Activación Enzimática , Clorhidrato de Erlotinib , Expresión Génica , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/genética , Quinazolinas/uso terapéutico , Activación TranscripcionalRESUMEN
Noninvasive imaging methodologies are needed to assess treatment responses to novel molecular targeting approaches for the treatment of squamous cell carcinoma of the head and neck (SCCHN). Computer tomography and magnetic resonance imaging do not effectively distinguish tumors from fibrotic tissue commonly associated with SCCHN tumors. Positron emission tomography (PET) offers functional non-invasive imaging of tumors. We determined the uptake of the PET tracers 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-[(18)F]Fluoro-3'-deoxythymidine ([(18)F]FLT) in several SCCHN xenograft models. In addition, we evaluated the utility of [(18)F]FLT microPET imaging in monitoring treatment response to an EGFR antisense approach targeted therapy that has shown safety and efficacy in a phase I trial. Two of the 3 SCCHN xenograft models tested demonstrated no appreciable uptake or retention of [(18)F]FDG, but consistent accumulation of [(18)F]FLT. The third tumor xenograft SCCHN model (Cal33) demonstrated variable uptake of both tracers. SCCHN xenografts (1483) treated with EGFR antisense gene therapy decreased tumor volumes in 4/6 mice. Reduced uptake of [(18)F]FLT was observed in tumors that responded to epidermal growth factor antisense (EGFRAS) gene therapy compared to non-responding tumors or tumors treated with control sense plasmid DNA. These findings indicate that [(18)F]FLT PET imaging may be useful in monitoring SCCHN response to molecular targeted therapies, while [(18)F]FDG uptake in SCCHN xenografts may not be reflective of the level of metabolic activity characteristic of human SCCHN tumors.
RESUMEN
Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Receptores ErbB/genética , Guanidina/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacología , Ácidos Nucleicos de Péptidos/farmacología , Animales , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Receptores ErbB/metabolismo , Guanidina/química , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Pulmonares/patología , Ratones , Oligonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/química , Carcinoma de Células Escamosas de Cabeza y Cuello , Relación Estructura-ActividadRESUMEN
PURPOSE: EGF receptor (EGFR) is upregulated in most epithelial cancers where signaling through EGFR contributes to cancer cell proliferation and survival. The limited clinical efficacy of EGFR inhibitors suggests that identification of resistance mechanisms may identify new pathways for therapeutic targeting. STAT3 is upregulated in many cancers and activated via both EGFR-dependent and -independent pathways. In the present study, we tested the consequences of STAT3 inhibition in EGFR inhibitor-resistant head and neck squamous cell carcinoma (HNSCC) and bladder cancer models to determine whether STAT3 blockade can enhance responses to EGFR targeting. EXPERIMENTAL DESIGN: pSTAT3 expression was assessed in human HNSCC tumors that recurred following cetuximab treatment. Cetuximab-sensitive and -resistant cell lines were treated with a STAT3 decoy to determine EC(50) concentrations and the effects on STAT3 target gene expression by Western blotting. In vivo assays included evaluation of antitumor efficacy of STAT3 decoy in cetuximab-sensitive and -resistant models followed by immunoblotting for STAT3 target protein expression. RESULTS: Targeting STAT3 with a STAT3 decoy reduced cellular viability and the expression of STAT3 target genes in EGFR inhibitor resistance models. The addition of a STAT3 inhibitor to EGFR blocking strategies significantly enhanced antitumor effects in vivo. Biopsies from HNSCC tumors that recurred following cetuximab treatment showed increased STAT3 activation compared with pretreatment biopsies. CONCLUSIONS: These results suggest that STAT3 activation contributes to EGFR inhibitor resistance both in HNSCC and bladder cancer where concomitant targeting of STAT3 may represent an effective treatment strategy.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Cetuximab , Resistencia a Antineoplásicos/genética , Clorhidrato de Erlotinib , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Humanos , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Quinazolinas/farmacología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
G-protein-coupled receptors (GPCR) activate the epidermal growth factor receptor (EGFR) and mediate EGFR-independent signaling pathways to promote the growth of a variety of cancers, including head and neck squamous cell carcinoma (HNSCC). Identification of the common signaling mechanisms involved in GPCR-induced EGFR-dependent and EGFR-independent processes will facilitate the development of more therapeutic strategies. In this study, we hypothesized that phosphoinositide-dependent kinase 1 (PDK1) contributes to GPCR-EGFR cross-talk and signaling in the absence of EGFR and suggests that inhibition of the PDK1 pathway may be effective in the treatment of HNSCC. The contribution of PDK1 to the EGFR-dependent and EGFR-independent signaling in HNSCC was determined using RNA interference, a kinase-dead mutant, and pharmacologic inhibition. In vivo xenografts studies were also carried out to determine the efficacy of targeting PDK1 alone or in combination with the U.S. Food and Drug Administration-approved EGFR inhibitor cetuximab. PDK1 contributed to both GPCR-induced EGFR activation and cell growth. PDK1 also mediated activation of p70S6K in the absence of EGFR. Blockade of PDK1 with a small molecule inhibitor (AR-12) abrogated HNSCC growth, induced apoptosis, and enhanced the antiproliferative effects of EGFR tyrosine kinase inhibitors in vitro. HNSCC xenografts expressing kinase-dead PDK1 showed increased sensitivity to cetuximab compared with vector-transfected controls. Administration of AR-12 substantially decreased HNSCC tumor growth in vivo. These cumulative results show that PDK1 is a common signaling intermediate in GPCR-EGFR cross-talk and EGFR-independent signaling, and in which targeting the PDK1 pathway may represent a rational therapeutic strategy to enhance clinical responses to EGFR inhibitors in HNSCC.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Anticuerpos Monoclonales Humanizados/uso terapéutico , Apoptosis , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Proliferación Celular , Cetuximab , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
UNLABELLED: Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as "undruggable." We developed a decoy targeting STAT3 and conducted a phase 0 trial. Expression levels of STAT3 target genes were decreased in head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexaethylene glycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development. SIGNIFICANCE: This is the fi rst study of a STAT3-selective inhibitor in humans and the fi rst evidence that a transcription factor decoy can be modifi ed to enable systemic delivery. These findings have therapeutic implications beyond STAT3 to other "undruggable" targets in human cancers.
Asunto(s)
Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Factor de Transcripción STAT3/genética , Animales , Línea Celular Tumoral , Femenino , Expresión Génica , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Inyecciones Intralesiones , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Distribución Aleatoria , Factor de Transcripción STAT3/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Epidermal growth factor receptor (EGFR) overexpression is correlated with decreased survival in head and neck cancer (HNC) where the addition of EGFR inhibition to standard chemoradiation approaches has improved treatment responses. However, the basis for the limited efficacy of EGFR inhibitors in HNC is incompletely understood. G-protein-coupled receptors (GPCR) have been shown to be overexpressed in HNC where GPCR activation induces HNC growth via both EGFR-dependent and -independent pathways. We hypothesized that targeting GPCR-induced EGFR-independent signaling would improve the efficacy of EGFR inhibition. EXPERIMENTAL DESIGN: Using a high-throughput phosphoproteome array, we identified proteins that were phosphorylated in HNC cells where EGFR expression was downmodulated by RNA interference (RNAi) in the presence or absence of a GPCR ligand. We confirmed the findings from the array by Western blotting followed by in vitro and in vivo phenotypic assays. RESULTS: p70S6K phosphorylation was elevated approximately sixfold in EGFR siRNA-transfected cells treated with a GPCR ligand. In addition to RNAi-mediated EGFR downmodulation, GPCR-mediated phosphorylation of p70S6K was modestly increased by EGFR inhibitor cetuximab approved by the Food and Drug Administration. Biopsies from cetuximab-treated patients also displayed increased phospho-p70S6K staining compared with pretreatment biopsies. HNC cells were growth inhibited by both genetic and pharmacologic p70S6K targeting strategies. Furthermore, p70S6K targeting in combination with cetuximab resulted in enhanced antitumor effects in both in vitro and in vivo HNC models. CONCLUSIONS: These results indicate that increased phosphorylation of p70S6K in cetuximab-treated patients may be due to increased GPCR signaling. Therefore, the addition of p70S6K targeting strategies may improve treatment responses to EGFR inhibition.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Cetuximab , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Ligandos , Terapia Molecular Dirigida , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
Chemoprevention of head and neck squamous cell carcinoma (HNSCC), a disease associated with high mortality rates and frequent occurrence of second primary tumor (SPT), is an important clinical goal. The epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription (STAT)-3 signaling pathway is known to play a key role in HNSCC growth, survival, and prognosis, thereby serving as a potential therapeutic target in the treatment of HNSCC. In the current study, the 4-nitroquinoline-1-oxide (4-NQO)-induced murine model of oral carcinogenesis was utilized to investigate the chemopreventive activities of compounds that target the EGFR-STAT3 signaling pathway. This model mimics the process of oral carcinogenesis in humans. The drugs under investigation included erlotinib, a small molecule inhibitor of the EGFR, and guggulipid, the extract of an Ayurvedic medicinal plant, which contains guggulsterone, a compound known to inhibit STAT3. Dietary administration of guggulipid failed to confer protection against oral carcinogenesis. On the other hand, the mice placed on erlotinib-supplemented diet exhibited a 69% decrease (P < 0.001) in incidence of preneoplastic and neoplastic lesions compared with mice on the control diet. Immunostaining of dysplastic lesions demonstrated modest decreases in STAT3 levels, with both drug treatments, that were not statistically significant. The results of the present study provide the basis for exploring the efficacy of erlotinib for prevention of HNSCC in a clinical setting.