RESUMEN
Notch ligands signal through one of four receptors on neighboring cells to mediate cell-cell communication and control cell fate, proliferation, and survival. Although aberrant Notch activation has been implicated in numerous malignancies, including breast cancer, the importance of individual receptors in distinct breast cancer subtypes and the mechanisms of receptor activation remain unclear. Using a novel antibody to detect active NOTCH3, we report here that NOTCH3 signals constitutively in a panel of basal breast cancer cell lines and in more than one third of basal tumors. Selective inhibition of individual ligands revealed that this signal does not require canonical ligand induction. A NOTCH3 antagonist antibody inhibited growth of basal lines, whereas a NOTCH3 agonist antibody enhanced the transformed phenotype in vitro and in tumor xenografts. Transcriptomic analyses generated a Notch gene signature that included Notch pathway components, the oncogene c-Myc, and the mammary stem cell regulator Id4 This signature drove clustering of breast cancer cell lines and tumors into the common subtypes and correlated with the basal classification. Our results highlight an unexpected ligand-independent induction mechanism and suggest that constitutive NOTCH3 signaling can drive an oncogenic program in a subset of basal breast cancers. Cancer Res; 77(6); 1439-52. ©2017 AACR.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Neoplasias Basocelulares/patología , Receptor Notch3/metabolismo , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Femenino , Humanos , Ratones , Ratones Noqueados , Ratones SCID , Neoplasias Basocelulares/metabolismo , Receptor Notch3/genética , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Quantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor micro-environment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines (19)F magnetic resonance imaging ((19)F-MRI) R 1 mapping with diffusion-based multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO2) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS (19)F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Oxígeno/metabolismo , Animales , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Imagen por Resonancia Magnética/métodos , Ratones , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Microambiente Tumoral/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing in vitro and in vivo.
Asunto(s)
Anticuerpos/uso terapéutico , Melanocitos/metabolismo , Melanoma/tratamiento farmacológico , Melanosomas/metabolismo , Antígeno gp100 del Melanoma/metabolismo , Animales , Anticuerpos/química , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Fluorescente , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Antígeno gp100 del Melanoma/genéticaRESUMEN
The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Modelos Animales de Enfermedad , Neoplasias Hepáticas/prevención & control , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Animales , Anticuerpos Neutralizantes/inmunología , Carcinoma Hepatocelular/patología , División Celular , Neoplasias Hepáticas/patología , Ratones , Ratones Transgénicos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/inmunologíaRESUMEN
The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.
Asunto(s)
Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores Notch/antagonistas & inhibidores , Inhibidores de la Angiogénesis/inmunología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos/efectos adversos , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Biblioteca de Péptidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/inmunología , Receptor Notch2/antagonistas & inhibidores , Receptor Notch2/inmunología , Receptores Notch/genética , Receptores Notch/inmunología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
Asunto(s)
Anticuerpos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Citotoxinas/farmacología , Inmunotoxinas/farmacocinética , Neoplasias Ováricas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/genética , Especificidad de Anticuerpos , Sitios de Unión , Antígeno Ca-125/inmunología , Línea Celular Tumoral , Química Farmacéutica/métodos , Cisteína/genética , Femenino , Humanos , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Mutagénesis Sitio-Dirigida , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.