RESUMEN
The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.
Asunto(s)
Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales/uso terapéutico , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/inmunología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Resina de Colestiramina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Íleon/efectos de los fármacos , Íleon/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacologíaRESUMEN
Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.
Asunto(s)
Hipertrofia Ventricular Izquierda/patología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Calcineurina/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Glucuronidasa/genética , Glucuronidasa/metabolismo , Células HEK293 , Humanos , Hipertrofia Ventricular Izquierda/metabolismo , Proteínas Klotho , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosfolipasa C gamma/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/deficiencia , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Transducción de SeñalRESUMEN
BACKGROUND: During early clinical development, prospective identification of a predictive biomarker and validation of an assay method may not always be feasible. Dichotomizing a continuous biomarker measure to classify responders also leads to challenges. We present a case study of a prospective-retrospective approach for a continuous biomarker identified after patient enrollment but defined prospectively before the unblinding of data. An analysis of the strengths and weaknesses of this approach and the challenges encountered in its practical application are also provided. METHODS: HERALD (NCT02134015) was a double-blind, phase 2 study in patients with non-small cell lung cancer (NSCLC) randomized to erlotinib with placebo or with high or low doses of patritumab, a monoclonal antibody targeted against human epidermal growth factor receptor 3 (HER3). While the primary objective was to assess safety and progression-free survival (PFS), a secondary objective was to determine a single predictive biomarker hypothesis to identify subjects most likely to benefit from the addition of patritumab. Although not identified as the primary biomarker in the study protocol, on the basis of preclinical results from 2 independent laboratories, expression levels of the HER3 ligand heregulin (HRG) were prospectively declared the predictive biomarker before data unblinding but after subject enrollment. An assay to measure HRG mRNA was developed and validated. Other biomarkers, such as epidermal growth factor receptor (EGFR) mutation status, were also evaluated in an exploratory fashion. The cutoff value for high vs. low HRG mRNA levels was set at the median delta threshold cycle. A maximum likelihood analysis was performed to evaluate the provisional cutoff. The relationship of HRG values to PFS hazard ratios (HRs) was assessed as a measure of internal validation. Additional NSCLC samples were analyzed to characterize HRG mRNA distribution. RESULTS: The subgroup of patients with high HRG mRNA levels ("HRG-high") demonstrated clinical benefit from patritumab treatment with HRs of 0.37 (P = 0.0283) and 0.29 (P = 0.0027) in the high- and low-dose patritumab arms, respectively. However, only 102 of the 215 randomized patients (47.4%) had sufficient tumor samples for HRG mRNA measurement. Maximum likelihood analysis showed that the provisional cutoff was within the optimal range. In the placebo arm, the HRG-high subgroup demonstrated worse prognosis compared with HRG-low. A continuous relationship was observed between increased HRG mRNA levels and lower HR. Additional NSCLC samples (N = 300) demonstrated a similar unimodal distribution to that observed in this study, suggesting that the defined cutoff may be applicable to future NSCLC studies. CONCLUSIONS: The prospective-retrospective approach was successful in clinically validating a probable predictive biomarker. Post hoc in vitro studies and statistical analyses permitted further testing of the underlying assumptions. However, limitations of this analysis include the incomplete collection of adequate tumor tissue and a lack of stratification. In a phase 3 study, findings are being confirmed, and the HRG cutoff value is being further refined. CLINICALTRIALSGOV NUMBER: NCT02134015.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neurregulina-1/genética , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Anticuerpos ampliamente neutralizantes , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Supervivencia sin Enfermedad , Método Doble Ciego , Receptores ErbB/genética , Clorhidrato de Erlotinib/administración & dosificación , Clorhidrato de Erlotinib/uso terapéutico , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Neurregulina-1/sangre , Estudios Prospectivos , Receptor ErbB-3/sangre , Receptor ErbB-3/inmunología , Estudios Retrospectivos , Investigación Biomédica Traslacional , Resultado del TratamientoRESUMEN
PURPOSE: Establishment of antiapoptotic signaling pathways in tumor cells is a major cause for the failure of chemotherapy against cancer. To investigate the underlying mechanisms, we developed an experimental approach that is based on the genetic plasticity of cancer cells and the selection for cell survival on treatment with chemotherapeutic agents. EXPERIMENTAL DESIGN: Gene expression changes of surviving cell clones were analyzed by macroarrays. Involvement of fibroblast growth factor receptor 4 (FGFR4) in antiapoptotic pathways was elucidated by apoptosis assays, small interfering RNA experiments, and an antagonistic antibody. RESULTS: We show that FGFR4 gene expression is up-regulated in doxorubicin-treated, apoptosis-resistant cancer cell clones. Ectopic expression of FGFR4 in cancer cells led to reduced apoptosis sensitivity on treatment with doxorubicin or cyclophosphamide, whereas knockdown of endogenous FGFR4 expression in breast cancer cell lines had the opposite effect. FGFR4 overexpression resulted in Bcl-xl up-regulation at both mRNA and protein levels. Knockdown of FGFR4 expression by small interfering RNA caused a decrease in phospho-extracellular signal-regulated kinase 1/2 levels and reduced Bcl-xl expression. Moreover, an antagonistic FGFR4 antibody suppressed the resistance of cancer cells with endogenous FGFR4 expression against apoptosis-inducing chemotherapeutic agents. CONCLUSION: Based on these findings, we propose an antiapoptotic signaling pathway that is initiated by FGFR4 and regulating the expression of Bcl-xl through the mitogen-activated protein kinase cascade. Our findings are exemplary for a novel strategy toward the elucidation of diverse signaling pathways that define antiapoptotic potential in cancer cells. These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting FGFR4-overexpressing cancers.
Asunto(s)
Resistencia a Antineoplásicos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Línea Celular Tumoral , Doxorrubicina/farmacología , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Humanos , Sistema de Señalización de MAP Quinasas , ARN Mensajero/análisis , Regulación hacia Arriba , Proteína bcl-X/análisisRESUMEN
PURPOSE: A recent study presented first evidence that a single nucleotide polymorphism (SNP) at codon 388 of fibroblast growth factor receptor 4 (FGFR4) gene, causing a transmembrane domain missense mutation (Gly388Arg), is associated with disease outcome in node-positive breast cancer. This article addresses the clinical relevance of this SNP, FGFR4 genotype, phenotype, and HER2 regarding patient outcome and influence of adjuvant systemic therapy in a substantial primary breast cancer collective (n = 372; median follow-up, 94.5 months). METHODS: Polymerase chain reaction restriction fragment length polymorphism analysis of germ-line polymorphism was performed in uninvolved lymph nodes; FGFR4 and HER2 expression were assessed immunohistochemically in tissue microarrays. RESULTS: In 51% of patients, homo- or heterozygous Arg388 allele was present. No correlation existed between FGFR4 genotype and expression or HER2 status. In node-negative patients, FGFR4 genotype was not correlated with disease outcome. In node-positive patients, however, FGFR4 Arg388 was significantly associated with poor disease-free survival (DFS; P = .02) and overall survival (OS; P = .04). Notably, this association seems to be attributable to relatively poor therapy response in Arg388 carriers, reflected in their significantly shorter DFS (P = .02) and OS (P = .045) among patients receiving adjuvant systemic therapy. It is also seen as a significant interaction term in a multivariate proportional hazards model with Arg388 carriers having only about half as much benefit from adjuvant systemic therapy as wild-type carriers. CONCLUSION: According to this study, FGFR4 Arg388 genotype is a marker for breast cancer progression in patients with adjuvant systemic therapy, particularly chemotherapy, and thus may indicate therapy resistance.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Adulto , Anciano , Alelos , Arginina , Neoplasias de la Mama/metabolismo , Quimioterapia Adyuvante , Progresión de la Enfermedad , Femenino , Marcadores Genéticos , Genotipo , Glicina , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Análisis Multivariante , Mutación Missense , Fenotipo , Polimorfismo de Nucleótido Simple , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismoRESUMEN
Clinical investigations of an FGFR4 germline polymorphism, resulting in substitution of glycine by arginine at codon 388 (G388 to R388), have shown a correlation between FGFR4 R388 and aggressive disease progression in cancer patients. Here, we studied the differential effects of the two FGFR4 isotypes on cellular signalling and motility in the MDA-MB-231 human breast cancer cell model. cDNA array analysis showed the ability of FGFR4 G388 to suppress expression of specific genes involved in invasiveness and motility. Further investigations concentrating on cell signalling and motility revealed an abrogation of phosphatidylinositol-3-kinase-dependent LPA-induced Akt activation and cell migration due to downregulation of the LPA receptor Edg-2 in FGFR4 G388-expressing MDA-MB-231 cells. Moreover, FGFR4 G388 expression attenuated the invasivity of the breast cancer cell line and decreased small Rho GTPase activity. We conclude that FGFR4 G388 suppresses cell motility of invasive breast cancer cells by altering signalling pathways and the expression of genes that are required for metastasis. Therefore, the positive effect of FGFR4 R388 on disease progression appears to result from a loss of the tumour suppressor activity displayed by FGFR4 G388 rather than the acquisition or enhancement of oncogenic potential.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Movimiento Celular/genética , Polimorfismo de Nucleótido Simple , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Alelos , Sustitución de Aminoácidos , Arginina/genética , Neoplasias de la Mama/genética , Línea Celular , Células Cultivadas , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Glicina/genética , Humanos , Isoformas de Proteínas , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/fisiología , Transducción de SeñalRESUMEN
We have performed an in vitro selection for an anti-apoptotic phenotype that resembles the selection process that pre-malignant cells undergo in the initial phase of carcinogenesis in vivo. Using the cervical carcinoma cell line HeLa S3 as a model system, the selection procedure yielded cell clones that displayed increased resistance to apoptosis induced by Fas, tumor necrosis factor-related apoptosis-inducing ligand, and serum starvation. Gene expression profiling using gene family focused cDNA arrays revealed numerous genes that are differentially expressed in HeLa S3 and the resistant subclones and therefore are potentially involved in the definition of sensitivity to apoptotic stimuli. From the genes identified in this functional genomics approach we validated the anti-apoptotic activity of the membrane-anchored matrix metalloproteinase 15 (MMP-15) by means of small interfering RNA-mediated knock-down and ectopic expression in parental HeLa S3 cells and, to confirm a more general significance of our findings, in other cancer cell lines. The in vivo relevance of these findings is supported by the overexpression of MMP-15 in human lung adenocarcinoma compared with normal lung. Because MMP-15 is known to promote invasion, our results suggest that this protease connects metastasis and apoptosis resistance by an unknown regulatory mechanism. Our findings therefore strongly suggest that cancer characteristics such as metastatic potential, which are thought to evolve late in cancer progression, could be manifested early on by selection for an anti-apoptotic phenotype.
Asunto(s)
Apoptosis/genética , Apoptosis/fisiología , Metaloendopeptidasas/genética , Invasividad Neoplásica/genética , Selección Genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Transformación Celular Neoplásica , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Invasividad Neoplásica/fisiopatología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Interferente PequeñoRESUMEN
Mitogen-activated protein kinases (MAPKs) are important regulators of a vast number of biological functions that affect life and death of eukaryotic cells and are tightly regulated by the concerted action of several phosphatases. Among these is the human homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5) product, which dephosphorylates and thus inhibits members of the MAPK family. Here, we analyzed hVH-5 transcripts in mammary carcinoma cell lines and discovered a sequence with 88% similarity to hVH-5 transcripts. Because this variant of hVH-5 lacked intronic sequences in its genomic structure, we suggest it might be a processed pseudogene of hVH-5. psihVH-5 transcripts were detected in human peripheral tissues as well as in 11 of 14 breast cancer cell lines. In respect to the normal hVH-5 gene, the pseudogene contains several point mutations and a frame shift due to the deletion of 2 bases that would lead to the truncation of the putative psihVH-5 product.
Asunto(s)
Proteínas Tirosina Fosfatasas/genética , Seudogenes , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/genética , Línea Celular Tumoral , Mapeo Cromosómico , Fosfatasas de Especificidad Dual , Humanos , Mutación , Alineación de Secuencia , Distribución Tisular , Transcripción GenéticaRESUMEN
Fibroblast growth factor receptors (FGFRs) have been implicated in various forms of human hyperproliferative disorders such as cancers of the cervix and bladder. We investigated the expression pattern of FGFR4 and the clinical significance of the recently identified Gly/Arg polymorphism (388) in head and neck squamous cell carcinomas (HNSCCs) of the oral cavity and the oropharynx. Sections from 104 paraffin-embedded tumors were analyzed by a restriction fragment length polymorphism-based method to determine the FGFR4 genotypes. Protein expression was investigated immunohistochemically and graded into a low, intermediate, or high degree of staining. FGFR4 expression was scored as high in 17, as intermediate in 59 and as low in 28 cases. The FGFR4 Arg388 allele was found in 59 tumors, 46 of them having heterozygous and 13 homozygous genotypes. High expression of the FGFR4 Arg388 allele was significantly associated with reduced overall survival (p = 0.032) and with an advanced tumor stage (p = 0.023), whereas expression of the FGFR4 Gly388 had no impact on disease progression. Our findings indicate that high expression of FGFR4 in connection with the Arg388 allele is associated with poor clinical outcome and support the significance of FGFR4 as a diagnostic marker and a target for therapeutic intervention in human HNSCC.
Asunto(s)
Carcinoma de Células Escamosas/genética , Perfilación de la Expresión Génica , Marcadores Genéticos , Neoplasias de Cabeza y Cuello/genética , Polimorfismo Genético , Receptores de Factores de Crecimiento de Fibroblastos/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Factores de Crecimiento de Fibroblastos , Genotipo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo de Longitud del Fragmento de Restricción , Pronóstico , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Análisis de SupervivenciaRESUMEN
Expression analysis of genes encoding components of the phosphotyrosine signaling system by cDNA array hybridization revealed elevated levels of FGFR4 transcripts in several mammary carcinoma cell lines. In the FGFR4 gene transcript from MDA-MB-453 mammary carcinoma cells, a G to A conversion was discovered that results in the substitution of glycine by arginine at position 388 in the transmembrane domain of the receptor. The Arg(388) allele was also found in cell lines derived from a variety of other tumor types as well as in the germ-line of cancer patients and healthy individuals. Analysis of three geographically separated groups indicated that it occurs in approximately 50% of the human population. Investigation of the clinical data of 84 breast cancer patients revealed that homo- or heterozygous carriers of the Arg(388) allele had a significantly reduced disease-free survival time (P = 0.01) within a median follow-up of 62 months. Moreover, the FGFR4 Arg(388) allele was associated with early lymph node metastasis and advanced tumor-node-metastasis (TNM) stage in 82 colon cancer patients. Consistent with this finding, MDA-MB-231 mammary tumor cells expressing FGFR4 Arg(388) exhibited increased motility relative to cells expressing the FGFR4 Gly(388) isotype. Our results support the conclusion that the FGFR4 Arg(388) allele represents a determinant that is innocuous in healthy individuals but predisposes cancer patients for significantly accelerated disease progression.
Asunto(s)
Alelos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Arginina/genética , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Genotipo , Humanos , Datos de Secuencia Molecular , Mutación Missense , Polimorfismo de Nucleótido Simple , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Células Tumorales CultivadasRESUMEN
Receptor tyrosine kinases (RTKs) are the primary mediators of the signaling network that transmit extracellular signals into the cell. Gene amplification and/or overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal autocrine-paracrine growth factor loops contribute to constitutive RTK signaling, ultimately resulting in the manifestation of dysregulated cell growth and cancer. The mechanism of uncontrolled RTK signaling that leads to cancer has provided the rationale for anti-RTK drug development. Strategies towards the prevention and interception of RTK signaling include monoclonal antibodies, small-molecule inhibitors, immunotoxins and antisense oligonucleotides.