RESUMEN
Rearranged during transfection (RET) rearrangements occur in 1% to 2% of lung adenocarcinomas as well as other malignancies and are now established targets for tyrosine kinase inhibitors. We developed three novel RET fusion-positive (RET+) patient-derived cancer cell lines, CUTO22 [kinesin 5B (KIF5B)-RET fusion], CUTO32 (KIF5B-RET fusion), and CUTO42 (echinoderm microtubule-associated protein-like 4-RET fusion), to study RET signaling and response to therapy. We confirmed each of our cell lines expresses the RET fusion protein and assessed their sensitivity to RET inhibitors. We found that the CUTO22 and CUTO42 cell lines were sensitive to multiple RET inhibitors, whereas the CUTO32 cell line was >10-fold more resistant to three RET inhibitors. We discovered that our RET+ cell lines had differential regulation of the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B (AKT) pathways. After inhibition of RET, the CUTO42 cells had robust inhibition of phosphorylated AKT (pAKT), whereas CUTO22 and CUTO32 cells had sustained AKT activation. Next, we performed a drug screen, which revealed that the CUTO32 cells were sensitive (<1 nM IC50) to inhibition of two cell cycle-regulating proteins, polo-like kinase 1 and Aurora kinase A. Finally, we show that two of these cell lines, CUTO32 and CUTO42, successfully establish xenografted tumors in nude mice. We demonstrated that the RET inhibitor BLU-667 was effective at inhibiting tumor growth in CUTO42 tumors but had a much less profound effect in CUTO32 tumors, consistent with our in vitro experiments. These data highlight the utility of new RET+ models to elucidate differences in response to tyrosine kinase inhibitors and downstream signaling regulation. Our RET+ cell lines effectively recapitulate the interpatient heterogeneity observed in response to RET inhibitors and reveal opportunities for alternative or combination therapies. SIGNIFICANCE STATEMENT: We have derived and characterized three novel rearranged during transfection (RET) fusion non-small cell lung cancer cell lines and demonstrated that they have differential responses to RET inhibition as well as regulation of downstream signaling, an area that has previously been limited by a lack of diverse cell line modes with endogenous RET fusions. These data offer important insight into regulation of response to RET tyrosine kinase inhibitors and other potential therapeutic targets.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Transducción de Señal , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.
Asunto(s)
Membrana Celular/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Proteínas/metabolismo , Anticuerpos/inmunología , Membrana Celular/efectos de los fármacos , Células Cultivadas , Humanos , Microscopía Confocal , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Proteínas/análisis , Proteínas/inmunología , Solubilidad , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citología , Proteína Inhibidora ATPasaRESUMEN
Nuclear factor of the Immunoglobulin Kappa chain of B cells (NF-kappaB) activation is an early event during cytokine-mediated endothelial activation related to increased adhesion of leucocytes. We report that soluble products secreted by two human lymphomas activate NF-kappaB, and increase the ability of endothelial cells to adhere U937 cells in vitro. Analysis of the tumor-derived products revealed the absence of tumor necrosis factor-alpha and interleukin-1beta. Interference of NF-kappaB activation prevented the increase in U937 cell adhesion, suggesting a potential role for endothelial NF-kappaB activation in the establishment of physical interactions between the vascular endothelium and tumor cells.