RESUMEN
Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1-S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1-S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1-S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727-38. ©2018 AACR.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Mutaciones Letales Sintéticas/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Although nanoparticle-based drug delivery formulations can improve the effectiveness and safety of certain anticancer drugs, many drugs, due to their chemical composition, are unsuitable for nanoparticle loading. Here, we describe a targeted nanogel drug delivery platform that can (i) encapsulate a wide range of drug chemotypes, including biological, small molecule, and cytotoxic agents; (ii) display targeting ligands and polymeric coatings on the surface; (iii) enhance drug retention within the nanogel core after photo-cross-linking; and (iv) retain therapeutic activity after lyophilization allowing for long-term storage. For therapeutic studies, we used integrin αvß3-targeted lipid-coated nanogels with cross-linked human serum albumin in the core for carrying therapeutic cargoes. These particles exhibited potent activity in tumor cell viability assays with drugs of distinct chemotype, including paclitaxel, docetaxel, bortezomib, 17-AAG, sorafenib, sunitinib, bosutinib, and dasatinib. Treatment of orthotopic breast and pancreas tumors in mice with taxane-loaded nanogels produced a 15-fold improvement in antitumor activity relative to Abraxane by blocking both primary tumor growth and spontaneous metastasis. With a modifiable surface and core, the lipid-coated nanogel represents a platform technology that can be easily adapted for specific drug delivery applications to treat a wide range of malignant diseases.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Polietileneimina/administración & dosificación , Polietileneimina/química , Paclitaxel Unido a Albúmina , Albúminas/administración & dosificación , Animales , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Liofilización/métodos , Humanos , Integrina alfaVbeta3/metabolismo , Lípidos/química , Ratones , Ratones Desnudos , Nanogeles , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/metabolismo , Polietilenglicoles/síntesis química , Polietileneimina/síntesis química , Polímeros/química , Taxoides/administración & dosificaciónRESUMEN
Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.
Asunto(s)
Carcinoma Ductal Pancreático/enzimología , Proteínas de Ciclo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/enzimología , Secuencia de Aminoácidos , Animales , Cadherinas/metabolismo , Carcinoma Ductal Pancreático/etiología , Carcinoma Ductal Pancreático/patología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/patología , Fosforilación , Proteómica , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteína Tirosina Quinasa CSK , Carcinoma/metabolismo , Carcinoma/patología , Adhesión Celular/fisiología , Proliferación Celular , Femenino , Fibronectinas/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Etiquetado Corte-Fin in Situ/métodos , Antígeno Ki-67/metabolismo , Metástasis Linfática , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , ARN Interferente Pequeño/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas , Familia-src QuinasasRESUMEN
Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.
Asunto(s)
Carcinoma/patología , Receptores ErbB/fisiología , Neoplasias Pancreáticas/patología , Receptor Cross-Talk/fisiología , Receptores de Vitronectina/fisiología , Animales , Carcinoma/genética , Movimiento Celular , Embrión de Pollo , Cartilla de ADN , Factor de Crecimiento Epidérmico/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Secuencias Invertidas Repetidas/genética , Pulmón/embriología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Pancreáticas/genética , Reacción en Cadena de la Polimerasa , ARN Neoplásico/genética , Receptores de Vitronectina/genética , Células Tumorales CultivadasRESUMEN
Integrin alphanubeta3 is found on a subset of tumor blood vessels where it is associated with angiogenesis and malignant tumor growth. We designed an alphanubeta3-targeted nanoparticle (NP) encapsulating the cytotoxic drug doxorubicin (Dox) for targeted drug delivery to the alphanubeta3-expressing tumor vasculature. We observed real-time targeting of this NP to tumor vessels and noted selective apoptosis in regions of the alphanubeta3-expressing tumor vasculature. In clinically relevant pancreatic and renal cell orthotopic models of spontaneous metastasis, targeted delivery of Dox produced an antimetastatic effect. In fact, alphanubeta3-mediated delivery of this drug to the tumor vasculature resulted in a 15-fold increase in antimetastatic activity without producing drug-associated weight loss as observed with systemic administration of the free drug. These findings reveal that NP-based delivery of cytotoxic drugs to the alphanubeta3-positive tumor vasculature represents an approach for treating metastatic disease.
Asunto(s)
Doxorrubicina/administración & dosificación , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Nanopartículas , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias/irrigación sanguínea , Neoplasias/patología , Inhibidores de la Angiogénesis/farmacología , Animales , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Endotelio/efectos de los fármacos , Endotelio/patología , Integrina alfaVbeta3/metabolismo , Neoplasias Renales/patología , Ratones , Neovascularización Patológica/patología , Oligopéptidos/farmacología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Focal adhesion kinase (FAK) plays a critical role during vascular development because knockout of FAK in endothelial cells (ECs) is embryonic lethal. Surprisingly, tamoxifen-inducible conditional knockout of FAK in adult blood vessels (inducible EC-specific FAK knockout [i-EC-FAK-KO]) produces no vascular phenotype, and these animals are capable of developing a robust growth factor-induced angiogenic response. Although angiogenesis in wild-type mice is suppressed by pharmacological inhibition of FAK, i-EC-FAK-KO mice are refractory to this treatment, which suggests that adult i-EC-FAK-KO mice develop a compensatory mechanism to bypass the requirement for FAK. Indeed, expression of the FAK-related proline-rich tyrosine kinase 2 (Pyk2) is elevated and phosphorylated in i-EC-FAK-KO blood vessels. In cultured ECs, FAK knockdown leads to increased Pyk2 expression and, surprisingly, FAK kinase inhibition leads to increased Pyk2 phosphorylation. Pyk2 can functionally compensate for the loss of FAK because knockdown or pharmacological inhibition of Pyk2 disrupts angiogenesis in i-EC-FAK-KO mice. These studies reveal the adaptive capacity of ECs to switch to Pyk2-dependent signaling after deletion or kinase inhibition of FAK.
Asunto(s)
Células Endoteliales/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Neovascularización Fisiológica , Animales , Aorta/citología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Humanos , Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
In the developing myocardium, vascular endothelial growth factor (VEGF)-dependent neovascularization occurs by division of existing vessels, a process that persists for several weeks following birth. During this remodeling phase, mRNA expression of beta3 integrin in the heart decreases significantly as vessel maturation progresses. However, in male mice lacking beta3, coronary capillaries fail to mature and continue to exhibit irregular endothelial thickness, endothelial protrusions into the lumen, and expanded cytoplasmic vacuoles. Surprisingly, this phenotype was not seen in female beta3-null mice. Enhanced VEGF signaling contributes to the beta3-null phenotype, because these vessels can be normalized by inhibitors of VEGF or Flk-1. Moreover, intravenous injection of VEGF induces a similar angiogenic phenotype in hearts of adult wild-type mice. These findings show a clear vascular phenotype in the hearts of mice lacking beta3 and suggest this integrin plays a critical role in coronary vascular development and the vascular response to VEGF.