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INTRODUCTION: Subgroup analyses from clinical studies have suggested that among patients with metastatic NSCLC receiving chemotherapy, females may derive less benefit from the addition of the vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab (BV) than males. This has raised the question of whether estrogen may affect the response to antiangiogenic therapy. METHODS: To address this, we investigated the effects of estrogen on tumor growth, angiogenesis, and the response to BV in human xenograft models of NSCLC. RESULTS: We observed that estrogen induced marked resistance to BV, which was accompanied by a 2.3-fold increase in tumor vascular pericyte coverage (p = 0.01) and an up-regulation of proangiogenic factors, VEGF and platelet-derived growth factor-BB. We also investigated the role of infiltrating myeloid cells, a population that has been associated with resistance to anti-VEGF therapies. We observed that estrogen induced a greater than twofold increase (p = 0.001) in the recruitment of tumor-infiltrating myeloid cells and concomitant increases in the myeloid recruitment factors, G-CSF and CXCL1. Blockade of the estrogen receptor pathway using fulvestrant resensitized tumors to VEGF targeting as evidenced by reduced tumor vasculature and an increase in overall survival in our NSCLC xenograft models. CONCLUSIONS: Collectively, these data provide evidence that estrogen may promote resistance to VEGF-targeted therapies, potentially by enhancing pericyte coverage and myeloid recruitment, and suggest that estrogen receptor blockade merits further investigation as an approach to enhance the effects of antiangiogenic therapy.
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Inhibidores de la Angiogénesis , Bevacizumab , Resistencia a Antineoplásicos , Estrógenos/farmacología , Neoplasias Pulmonares , Inhibidores de la Angiogénesis/farmacología , Animales , Bevacizumab/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Neovascularización Patológica/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
BACKGROUND: Radium-223 dichloride (Ra-223) is a targeted alpha therapy that induces localized cytotoxicity in bone metastases. We evaluated the efficacy and safety of Ra-223 plus hormonal therapy in hormone receptor-positive (HR+), bone-dominant metastatic breast cancer. METHODS: In this single-center phase II study, 36 patients received Ra-223 (55 kBq/kg intravenously every 4 weeks) up to 6 cycles with endocrine therapy. The primary objective was to determine the clinical disease control rate at 9 months. Secondary objectives were to determine (a) tumor response rate at 6 months, (b) progression-free survival (PFS) durations, and (c) safety. RESULTS: The median number of prior systemic treatments for metastatic disease was 1 (range, 0-4). The disease control rate at 9 months was 49%. The tumor response rate at 6 months was 54% (complete response, 21%; partial, 32%). The median PFS was 7.4 months (95% CI, 4.8-not reached [NR]). The median bone-PFS was 16 months (95% CI, 7.3-NR). There were no grade 3/4 adverse events. CONCLUSIONS: Ra-223 with hormonal therapy showed possible efficacy in HR+ bone-dominant breast cancer metastasis, and adverse events were tolerable. We plan to further investigate the clinical application of Ra-223 in these patients. (NCT02366130).
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Partículas alfa/efectos adversos , Antineoplásicos Hormonales/administración & dosificación , Neoplasias Óseas/terapia , Neoplasias de la Mama/terapia , Quimioradioterapia/métodos , Radio (Elemento)/administración & dosificación , Adulto , Anciano , Antineoplásicos Hormonales/efectos adversos , Neoplasias Óseas/mortalidad , Neoplasias Óseas/secundario , Huesos/diagnóstico por imagen , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Quimioradioterapia/efectos adversos , Denosumab/administración & dosificación , Denosumab/efectos adversos , Femenino , Fulvestrant/administración & dosificación , Fulvestrant/efectos adversos , Humanos , Inyecciones Intravenosas , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones , Supervivencia sin Progresión , Radioisótopos/administración & dosificación , Radioisótopos/efectos adversos , Radio (Elemento)/efectos adversos , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Tamoxifeno/administración & dosificación , Tamoxifeno/efectos adversosRESUMEN
Background: Inherited BRCA gene mutations (pathogenic variants) cause 10% of breast cancers. BRCA pathogenic variants predispose carriers to triple-negative breast cancer (TNBC); around 30% of patients with TNBC carry BRCA pathogenic variants. The 2018 NCCN Guidelines for Genetic/Familial High-Risk Assessment: Breast and Ovarian recommend genetic counseling referrals for patients with TNBC diagnosed at age ≤60 years. This study sought to describe genetic counseling referral patterns among long-term TNBC survivors at The University of Texas MD Anderson Cancer Center. Methods: This single-institution retrospective analysis of female long-term (disease-free for ≥5 years) TNBC survivors sought to determine the rate of genetic counseling referral among patients diagnosed at age ≤60 years between 1992 and 2008. Patients who underwent treatment and surveillance visits at our institution and were followed until 2017 were included. We collected BRCA pathogenic variant status among tested patients. Descriptive statistical methods and a univariate analysis were used to identify patient characteristics associated with genetic counseling referral. Results: We identified 646 female long-term TNBC survivors with a median age at diagnosis of 47 years. Of these, 245 (38%) received a recommendation for a genetic counseling referral. Among those referred, 156 (64%) underwent genetic testing, and 35% of those tested had BRCA pathogenic variants. Interestingly, among those referred, 20% declined genetic testing. The rate of genetic referrals improved over time, from 25% among TNBC survivors whose last surveillance visit was between 2011 and 2013 to 100% among those whose last surveillance visit was between 2014 or later. Younger age and premenopausal status at diagnosis and a family history of breast or ovarian cancer were associated with an increased rate of referral for genetic counseling. Conclusions: Among long-term TNBC survivors, the rate of referral to genetic counseling increased over time, and among those tested, 35% carried a BRCA pathogenic variant. Survivorship care provides an excellent opportunity to refer eligible patients for genetic counseling.
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Pruebas Genéticas/métodos , Adulto , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Sobrevivientes , Neoplasias de la Mama Triple Negativas/mortalidadRESUMEN
While several molecular targets have been identified for adenocarcinoma (ACA) of the lung, similar drivers with squamous cell carcinoma (SCC) are sparse. We compared signaling pathways and potential therapeutic targets in lung SCC and ACA tumors using reverse phase proteomic arrays (RPPA) from two independent cohorts of resected early stage NSCLC patients: a testing set using an MDACC cohort (N=140) and a validation set using the Cancer Genome Atlas (TCGA) cohorts. We identified multiple potentially targetable proteins upregulated in SCC, including NRF2, Keap1, PARP, TrkB, and Chk2. Of these potential targets, we found that TrkB also had significant increases in gene expression in SCC as compared to adenocarcinoma. Thus, we next validated the upregulation of TrkB both in vitro and in vivo and found that it was constitutively expressed at high levels in a subset of SCC cell lines. Furthermore, we found that TrkB inhibition suppressed tumor growth, invasiveness and sensitized SCC cells to tyrosine kinase EGFR inhibition in a cell-specific manner.
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BACKGROUND: A common class-specific toxicity of mammalian target of rapamycin (mTOR) inhibitors is stomatitis. Some patients experience a severe form of mTOR inhibitor-associated stomatitis (mIAS) that can have a negative effect on nutritional status, compromise quality of life, and potentially lead to nonadherence, reducing the efficacy of cancer therapy. OBJECTIVES: This article aims to address an unmet need for education about mIAS among oncology nurses and patients and to share findings about everolimus-related stomatitis from the SWISH trial. METHODS: The authors reviewed the literature on mIAS and selected a case series of experiences to illustrate successes and clinical challenges that an oncology nurse might encounter when caring for patients with advanced breast cancer who may develop everolimus-related stomatitis. FINDINGS: Recommendations are provided for oncology nurses to educate patients on prevention, early detection, monitoring, and management strategies to mitigate the incidence and severity of everolimus-related stomatitis.
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Neoplasias de la Mama/tratamiento farmacológico , Dexametasona/uso terapéutico , Everolimus/efectos adversos , Everolimus/uso terapéutico , Antisépticos Bucales/uso terapéutico , Estomatitis/tratamiento farmacológico , Estomatitis/prevención & control , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Femenino , Humanos , Persona de Mediana Edad , Enfermería Oncológica/normas , Guías de Práctica Clínica como Asunto , Resultado del TratamientoRESUMEN
Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Estimación de Kaplan-Meier , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Ratones , Terapia Molecular Dirigida , Estudios Multicéntricos como Asunto , Neovascularización Patológica/genética , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC50 ranging from 2 µM in the most sensitive CAKI-1 to 36 µM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway.
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2-Cloroadenosina/análogos & derivados , Proteínas Quinasas Activadas por AMP/metabolismo , Carcinoma de Células Renales/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Renales/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , 2-Cloroadenosina/farmacología , Western Blotting , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Análisis por Matrices de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales CultivadasRESUMEN
Epigenetic dysregulation has emerged as a major contributor to tumorigenesis. Histone methylation is a well-established mechanism of epigenetic regulation that is dynamically modulated by histone methyltransferases and demethylases. The pathogenic role of histone methylation modifiers in non-small cell lung cancer (NSCLC), which is the leading cause of cancer deaths worldwide, remains largely unknown. Here, we found that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is frequently overexpressed in NSCLC tumors and cell lines. KDM2A and its catalytic activity were required for in vitro proliferation and invasion of KDM2A-overexpressing NSCLC cells. KDM2A overexpression in NSCLC cells with low KDM2A levels increased cell proliferation and invasiveness. KDM2A knockdown abrogated tumor growth and invasive abilities of NSCLC cells in mouse xenograft models. We identified dual-specificity phosphatase 3 (DUSP3) as a key KDM2A target gene and found that DUSP3 dephosphorylates ERK1/2 in NSCLC cells. KDM2A activated ERK1/2 through epigenetic repression of DUSP3 expression via demethylation of dimethylated H3K36 at the DUSP3 locus. High KDM2A levels correlated with poor prognosis in NSCLC patients. These findings uncover an unexpected role for a histone methylation modifier in activating ERK1/2 in lung tumorigenesis and metastasis, suggesting that KDM2A may be a promising therapeutic target in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Epigénesis Genética/fisiología , Proteínas F-Box/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/fisiología , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , División Celular , Línea Celular Tumoral , Fosfatasa 3 de Especificidad Dual/biosíntesis , Fosfatasa 3 de Especificidad Dual/genética , Fosfatasa 3 de Especificidad Dual/fisiología , Epigénesis Genética/genética , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/biosíntesis , Proteínas F-Box/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Xenoinjertos , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/biosíntesis , Histona Demetilasas con Dominio de Jumonji/genética , Neoplasias Pulmonares/genética , Masculino , Metilación , Ratones , Ratones Desnudos , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Pronóstico , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genéticaRESUMEN
PURPOSE: c-Src inhibition in cancer cells leads to an abrogation of invasion but a variable effect on apoptosis. The pathways downstream of c-Src promoting survival are not well characterized. Because cancer therapy that both decreases invasion and induces significant apoptosis would be ideal, we sought to characterize the mechanisms of resistance to c-Src inhibition. EXPERIMENTAL DESIGN: c-Src was inhibited in a panel of oral cancer cell lines and subsequent survival and signaling measured. The interactions between c-Src and c-Met were evaluated using immunoprecitation and an in vitro kinase assay. Cytotoxicity was measured and the Chou-Talalay combination index calculated. An orthotopic model of oral cancer was used to assess the effects of c-Met and c-Src inhibitors. RESULTS: Inhibition of c-Src resulted in c-Met inhibition in sensitive cells lines, but not in resistant cell lines. Isolated c-Met was a c-Src substrate in both sensitive and resistant cells, but there was no interaction of c-Src and c-Met in intact resistant cells. To examine the biological consequences of this mechanism, we demonstrated synergistic cytotoxicity, enhanced apoptosis, and decreased tumor size with the combination of c-Src and c-Met inhibitors. CONCLUSIONS: Sustained c-Met activation can mediate resistance to c-Src inhibition. These data suggest that the differences between c-Met and c-Src signaling in sensitive and resistant cells are due to distinct factors promoting or inhibiting interactions, respectively, rather than to intrinsic structural changes in c-Src or c-Met. The synergistic cytotoxic effects of c-Src and c-Met inhibition may be important for the treatment of head and neck cancers.
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Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirimidinas/farmacología , Tiazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteína Tirosina Quinasa CSK , Carcinoma de Células Escamosas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crizotinib , Dasatinib , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Humanos , Ratones , Ratones Desnudos , Neoplasias de la Boca/patología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Familia-src QuinasasRESUMEN
Overexpression of the receptor tyrosine kinase EphA2 occurs in non-small cell lung cancer (NSCLC) and a number of other human cancers. This overexpression correlates with a poor prognosis, smoking, and the presence of Kirsten rat sarcoma (K-Ras) mutations in NSCLC. In other cancers, EphA2 has been implicated in migration and metastasis. To determine if EphA2 can promote NSCLC progression, we examined the relationship of EphA2 with proliferation and migration in cell lines and with metastases in patient tumors. We also examined potential mechanisms involving AKT, Src, focal adhesion kinase, Rho guanosine triphosphatases (GTPase), and extracellular signal-regulated kinase (ERK)-1/2. Knockdown of EphA2 in NSCLC cell lines decreased proliferation (colony size) by 20% to 70% in four of five cell lines (P < 0. 04) and cell migration by 7% to 75% in five of six cell lines (P < 0. 03). ERK1/2 activation correlated with effects on proliferation, and inhibition of ERK1/2 activation also suppressed proliferation. In accordance with the in vitro data, high tumor expression of EphA2 was an independent prognostic factor in time to recurrence (P = 0.057) and time to metastases (P = 0.046) of NSCLC patients. We also examined EphA2 expression in the putative premalignant lung lesion, atypical adenomatous hyperplasia, and the noninvasive bronchioloalveolar component of adenocarcinoma because K-Ras mutations occur in atypical adenomatous hyperplasia and are common in lung adenocarcinomas. Both preinvasive lesion types expressed EphA2, showing its expression in the early pathogenesis of lung adenocarcinoma. Our data suggest that EphA2 may be a promising target for treating and preventing NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/etiología , Neoplasias Pulmonares/etiología , Receptor EphA2/fisiología , Apoptosis , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Adhesión Celular , Ciclo Celular , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/fisiopatología , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Análisis de Matrices TisularesRESUMEN
PURPOSE: Signal transducer and activator of transcription-3 (STAT3) is downstream of growth factor and cytokine receptors, and regulates key oncogenic pathways in non-small cell lung cancer (NSCLC). Activation of STAT3 by cellular Src (c-Src) promotes tumor progression. We hypothesized that c-Src inhibition could activate STAT3 by inducing a homeostatic feedback loop, contributing to c-Src inhibitor resistance. EXPERIMENTAL DESIGN: The effects of c-Src inhibition on total and phosphorylated STAT3 were measured in NSCLC cell lines and in murine xenograft models by Western blotting. c-Src and STAT3 activity as indicated by phosphorylation was determined in 46 human tumors and paired normal lung by reverse phase protein array. Modulation of dasatinib (c-Src inhibitor) cytotoxicity by STAT3 knockdown was measured by MTT, cell cycle, and apoptosis assays. RESULTS: Depletion of c-Src by small interfering RNA or sustained inhibition by dasatinib increased pSTAT3, which could be blocked by inhibition of JAK. Similarly, in vivo pSTAT3 levels initially decreased but were strongly induced after sustained dasatinib treatment. In human tumors, phosphorylation of the autoinhibitory site of c-Src (Y527) correlated with STAT3 phosphorylation (r = 0.64; P = 2.5 x 10(-6)). STAT3 knockdown enhanced the cytotoxicity of dasatinib. CONCLUSIONS: c-Src inhibition leads to JAK-dependent STAT3 activation in vitro and in vivo. STAT3 knockdown enhances the cytotoxicity of dasatinib, suggesting a compensatory pathway that allows NSCLC survival. Data from human tumors showed a reciprocal regulation of c-Src and STAT3 activation, suggesting that this compensatory pathway functions in human NSCLC. These results provide a rationale for combining c-Src and STAT3 inhibition to improve clinical responses.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/biosíntesis , Anciano , Anciano de 80 o más Años , Animales , Proteína Tirosina Quinasa CSK , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Familia-src QuinasasRESUMEN
Locoregional and distant recurrence remains common and usually fatal for patients with advanced head and neck squamous cell carcinoma (HNSCC). One promising molecular target in HNSCC is the Src family kinases (SFK). SFKs can affect cellular proliferation and survival by activating the signal transducer and activator of transcription (STAT) family of transcription factors, especially STAT3. Surprisingly, sustained SFK inhibition resulted in only transient inhibition of STAT3. We investigated the mechanism underlying STAT3 activation and its biological importance. Specific c-Src knockdown with small interfering RNA (siRNA) resulted in STAT3 activation showing specificity, which was inhibited by Janus-activated kinase (JAK; TYK2 and JAK2) depletion with siRNA. Sustained SFK inhibition also resulted in recovered JAK-STAT3 binding and JAK kinase activity after an initial reduction, although JAK phosphorylation paradoxically decreased. To determine the biological significance of STAT3 activation, we combined specific STAT3 depletion with a pharmacologic SFK inhibitor and observed increased cell cycle arrest and apoptosis. Likewise, the addition of STAT3- or JAK-specific siRNA to c-Src-depleted cells enhanced cytotoxicity relative to cells incubated with c-Src siRNA alone. These results show that reactivation of STAT3 after sustained, specific c-Src inhibition is mediated through altered JAK-STAT3 binding and JAK kinase activity and that this compensatory pathway allows for cancer cell survival and proliferation despite durable c-Src inhibition. To our knowledge, this novel feedback pathway has never been described previously. Given that pharmacologic SFK inhibitors are currently being evaluated in clinical trials, these results have potential clinical implications for cancer therapy.
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Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , TYK2 Quinasa/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dasatinib , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Fosforilación , Pirimidinas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Tiazoles/farmacologíaRESUMEN
The purpose of this study was to determine whether the efficacy of taxoid treatment combined with epidermal growth factor receptor (EGFR) inhibition is dose and sequence dependent in head and neck squamous cell carcinoma. Three head and neck squamous cell carcinoma cell lines, chosen on the basis of their diverse EGFR expression levels, were treated with docetaxel, erlotinib, or both. The combination index was calculated using the Chou-Talalay equation. Propidium iodide staining with fluorescence-activated cell sorting analysis was used to evaluate the effects of drugs on cell cycle changes. Western blot analysis was used to determine the effects of agents on cell signaling pathways. Administration of low-dose docetaxel (0.1-3 nmol/l) concurrently or before erlotinib had additive cytotoxic effects in two cell lines but was antagonistic in one line, whereas low-dose docetaxel after erlotinib was synergistic in all cell lines. In contrast, high-dose docetaxel (40 nmol/l) resulted in more apoptosis when given before, rather than after or concurrently with, erlotinib. Low-dose docetaxel induced an accumulation of cells in the sub-G0 phase of the cell cycle with no mitotic arrest or apoptosis, whereas high-dose docetaxel induced mitotic arrest and apoptosis. The low and high doses of docetaxel had opposite effects on EGFR expression: a decrease and an increase, respectively. The dose of docetaxel affects sequence-dependent cytotoxicity when docetaxel is combined with an EGFR inhibitor. The mechanism for this difference is a combination of the dose-dependent effects of docetaxel on the mode of cell death and on EGFR expression.
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Antineoplásicos/farmacología , Quinazolinas/farmacología , Taxoides/farmacología , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma de Células Escamosas , Línea Celular Tumoral , Docetaxel , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Antagonismo de Drogas , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib , Neoplasias de Cabeza y Cuello , Humanos , Quinazolinas/administración & dosificación , Taxoides/administración & dosificaciónRESUMEN
Malignant pleural mesothelioma (MPM) is a deadly disease with few systemic treatment options. One potential therapeutic target, the non-receptor tyrosine kinase c-Src, causes changes in proliferation, motility, invasion, survival, and angiogenesis in cancer cells and may be a valid therapeutic target in MPM. To test this hypothesis, we determined the effects of c-Src inhibition in MPM cell lines and examined c-Src expression and activation in tissue samples. We analyzed four MPM cell lines and found that all expressed total and activated c-Src. Three of the four cell lines were sensitive by in vitro cytotoxicity assays to the c-Src inhibitor dasatinib, which led to cell cycle arrest and increased apoptosis. Dasatinib also inhibited migration and invasion independent of the cytotoxic effects, and led to the rapid and durable inhibition of c-Src and its downstream pathways. We used immunohistochemical analysis to determine the levels of c-Src expression and activation in 46 archived MPM tumor specimens. The Src protein was highly expressed in tumor cells, but expression did not correlate with survival. However, expression of activated Src (p-Src Y419) on the tumor cell membrane was higher in patients with advanced-stage disease; the presence of metastasis correlated with higher membrane (P = 0.03) and cytoplasmic (P = 0.04) expression of p-Src Y419. Lower levels of membrane expression of inactive c-Src (p-Src Y530) correlated with advanced N stage (P = 0.02). Activated c-Src may play a role in survival, metastasis, and invasion of MPM, and targeting c-Src may be an important therapeutic strategy.
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Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Mesotelioma/patología , Neoplasias Pleurales/patología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Pirimidinas/farmacología , Tiazoles/farmacología , Línea Celular Tumoral , Dasatinib , Activación Enzimática/efectos de los fármacos , Exones/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Masculino , Mesotelioma/enzimología , Mutación/genética , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Pleurales/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: The Src family of kinases (SFKs) regulate multiple signal transduction cascades and influence proliferation, motility, survival, and angiogenesis. Dasatinib inhibits SFKs, which leads to cytotoxicity, cell cycle arrest, apoptosis, and decreased invasion of cancer cells. Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that regulates survival and proliferation. Dasatinib results in rapid and durable inhibition of c-Src, whereas STAT3 undergoes only transient inactivation. We hypothesized that the reactivation of STAT3 after dasatinib treatment represents the engagement of a compensatory signal for cell survival that blocks the antitumor effects of SFK inhibition. EXPERIMENTAL DESIGN: The effects of upstream inhibitors on STAT3 activation were assessed with western blotting and a quantitative bioplex phosphoprotein assay. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the cytotoxicity and propidium iodine/annexin V staining with fluorescence-activated cell sorting (FACS) analysis to evaluate cell cycle change and apoptosis. The combination index was calculated by the Chou-Talalay equation. Cytokines were quantitated using a multiplexed, particle-based FACS analysis. RESULTS: C-Src and several downstream molecules were rapidly and durably inhibited by dasatinib. However, STAT3 was reactivated by 24 h. The addition of JAK inhibitors during dasatinib incubation resulted in sustained inhibition of STAT3, although JAK activation by dasatinib was not shown. Combined SFK and JAK inhibition resulted in synergistic cytotoxicity due to increased apoptosis. CONCLUSIONS: The reactivation of STAT3 during dasatinib treatment is caused by the engagement of a compensatory pathway that suppresses the antitumor effects of SFK inhibition and allows cancer cell survival. Abrogation of this pathway resulted in synergistic cytotoxicity. Given that STAT3 reactivation occurred in 14 of 15 solid tumor cell lines, dasatinib combined with Janus-activated kinase inhibitors may have widespread application in cancer treatment.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Factor de Transcripción STAT3/metabolismo , Tiazoles/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citocinas/biosíntesis , Dasatinib , Activación Enzimática , Humanos , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/farmacología , Pirimidinas/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Tiazoles/uso terapéuticoRESUMEN
The role of Src-family kinases (SFKs) in non-small cell lung cancer (NSCLC) has not been fully defined. Here we addressed this question by examining SFK phosphorylation in NSCLC biopsy samples and using genetic and pharmacological approaches to inhibit SFK expression and activity in cultured NSCLC cells. Immunohistochemical analysis of NSCLC biopsy samples using a Tyr416 phosphorylation-specific, pan-SFK antibody revealed staining in 123 (33%) of 370 tumors. Because c-Src is known to be both an upstream activator and downstream mediator of epidermal growth factor receptor (EGFR), we next investigated SFK phosphorylation in a panel of NSCLC cell lines, including ones that depend on EGFR for survival. The EGFR-dependent NSCLC cell lines HCC827 and H3255 had increased phosphorylation of SFKs, and treatment of these cells with an SFK inhibitor (PP1 or SKI-606) induced apoptosis. PP1 decreased phosphorylation of EGFR, ErbB2, and ErbB3 and strikingly enhanced apoptosis by gefitinib, an EGFR inhibitor. HCC827 cells transfected with c-Src short hairpin RNA exhibited diminished phosphorylation of EGFR and ErbB2 and decreased sensitivity to apoptosis by PP1 or gefitinib. We conclude that SFKs are activated in NSCLC biopsy samples, promote the survival of EGFR-dependent NSCLC cells, and should be investigated as therapeutic targets in NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Receptores ErbB/metabolismo , Neoplasias Pulmonares/enzimología , Familia-src Quinasas/metabolismo , Compuestos de Anilina/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Supervivencia Celular , Activación Enzimática , Humanos , Neoplasias Pulmonares/patología , Nitrilos/farmacología , Fosforilación , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
PURPOSE: Epithelial tumors, including non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), present clinical challenges. One potential target for systemic therapy is Src family nonreceptor tyrosine kinases, which are overexpressed in these tumors and induce pleiotropic effects, including increased proliferation, enhanced survival, stimulation of angiogenesis, and changes in motility. Dasatinib (BMS-354825), an ATP-competitive, small molecule tyrosine kinase inhibitor, suppresses the activity of these kinases at subnanomolar concentrations. Therefore, we tested the antitumor effects of this inhibitor in vitro to determine whether in vivo analyses were warranted. EXPERIMENTAL DESIGN: The antitumor effects of dasatinib on HNSCC and NSCLC cells were evaluated using assays to measure cell cycle progression, apoptosis, migration, and invasion. Western blotting was used to monitor its effects on cell signaling. RESULTS: Dasatinib inhibited migration and invasion in all cell lines and induced cell cycle arrest (blocking the G1-S transition) and apoptosis in some lines. The effects on migration and invasion correlated with the inhibition of Src and downstream mediators of adhesion [e.g., focal adhesion kinase (FAK), p130, and paxillin], and the cell cycle effects and apoptosis correlated with the induction of p27 and the dephosphorylation of Rb. Dasatinib also induced morphologic changes that were consistent with an upstream role for Src in regulating focal adhesion complexes. CONCLUSIONS: This study showed that Src inhibition in HNSCC and NSCLC has antitumor effects in vitro. This suggests that dasatinib would have therapeutic activity against these tumors. Clinical studies in these tumor types are warranted.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Tiazoles/farmacología , Adenosina Trifosfato/química , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Colágeno/farmacología , Proteína Sustrato Asociada a CrK/metabolismo , Dasatinib , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Fase G1 , Violeta de Genciana/farmacología , Humanos , Inmunoprecipitación , Concentración 50 Inhibidora , Laminina/farmacología , Invasividad Neoplásica , Neovascularización Patológica , Paxillin/metabolismo , Fosforilación , Proteoglicanos/farmacología , Proteína de Retinoblastoma/metabolismo , Fase S , Transducción de Señal , Factores de Tiempo , Azul de Tripano/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and c-kit accounts for its clinical activity in leukemia and sarcoma, respectively. In this report, we describe other cellular targets for imatinib. Treatment of head and neck squamous carcinoma cells with clinically relevant concentrations of imatinib-induced changes in cell morphology and growth similar to changes associated with epidermal growth factor receptor (EGFR) activation. Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which suggested direct involvement of EGFR in this process. Western blot analysis of cells incubated with imatinib demonstrated activation of EGFR and downstream signaling that was reduced by inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly affect EGFR kinase activity, suggesting involvement of EGFR-activating molecules. Inhibitors and neutralizing antibodies against heparin-binding epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent transforming growth factor-alpha, reduced imatinib-mediated mitogen activated protein kinase (MAPK) activation. Imatinib stimulated the rapid release of soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which correlated with biphasic MAPK activation. Together, these results suggested that imatinib affects EGFR activation and signaling pathways through rapid release and increased expression of endogenous EGFR-activating ligands. Although, imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates the need for careful use of this drug in cancer patients.