RESUMEN
Rictor, an essential component of mTOR complex 2 (mTORC2), plays a pivotal role in regulating mTOR signaling and other biological functions. Posttranslational regulation of rictor (e.g. via degradation) and its underlying mechanism are largely undefined and thus are the focus of this study. Chemical inhibition of the proteasome increased rictor ubiquitination and levels. Consistently, inhibition of FBXW7 with various genetic means including knockdown, knock-out, and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor. Hence, rictor is degraded through an FBXW7-mediated ubiquitination/proteasome mechanism. We show that this process is dependent on glycogen synthase kinase 3 (GSK3): GSK3 was associated with rictor and directly phosphorylated the Thr-1695 site in a putative CDC4 phospho-degron motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination, and increased rictor stability. Finally, enforced activation of Akt enhanced rictor levels and increased mTORC2 activity as evidenced by increased formation of mTORC2 and elevated phosphorylation of Akt, SGK1, and PKCα. Hence we suggest that PI3K/Akt signaling may positively regulate mTORC2 signaling, likely through suppressing GSK3-dependent rictor degradation.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Proteína 7 que Contiene Repeticiones F-Box-WD , Células HEK293 , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
BACKGROUND: The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. RESULTS: API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or ß-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. CONCLUSIONS: API-1 induces GSK3-dependent, ß-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.
Asunto(s)
Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Nucleósidos/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Leupeptinas/farmacología , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidoresRESUMEN
The acquisition of invasive properties is a prerequisite for tumor progression and metastasis. Molecular subtypes of KRAS-driven lung cancer exhibit distinct modes of invasion that likely contribute to unique growth properties and therapeutic susceptibilities. Despite this, pre-clinical discovery strategies designed to exploit invasive phenotypes are lacking. To address this, we designed an experimental system to screen for targetable signaling pathways linked to active early invasion phenotypes in the two most prominent molecular subtypes, TP53 and LKB1, of KRAS-driven lung adenocarcinoma (LUAD). By combining live-cell imaging of human bronchial epithelial cells in a 3D invasion matrix with RNA transcriptome profiling, we identified the LKB1-specific upregulation of bone morphogenetic protein 6 (BMP6). Examination of early-stage lung cancer patients confirmed upregulation of BMP6 in LKB1-mutant lung tumors. At the molecular level, we find that the canonical iron regulatory hormone Hepcidin is induced via BMP6 signaling upon LKB1 loss, where intact LKB1 kinase activity is necessary to maintain signaling homeostasis. Furthermore, pre-clinical studies in a novel Kras/Lkb1-mutant syngeneic mouse model show that potent growth suppression was achieved by inhibiting the ALK2/BMP6 signaling axis with single agents that are currently in clinical trials. We show that alterations in the iron homeostasis pathway are accompanied by simultaneous upregulation of ferroptosis protection proteins. Thus, LKB1 is sufficient to regulate both the 'gas' and 'breaks' to finely tune iron-regulated tumor progression.
RESUMEN
Oncogenic RAS mutations drive aggressive cancers that are difficult to treat in the clinic, and while direct inhibition of the most common KRAS variant in lung adenocarcinoma (G12C) is undergoing clinical evaluation, a wide spectrum of oncogenic RAS variants together make up a large percentage of untargetable lung and GI cancers. Here we report that loss-of-function alterations (mutations and deep deletions) in the gene that encodes HD-PTP (PTPN23) occur in up to 14% of lung cancers in the ORIEN Avatar lung cancer cohort, associate with adenosquamous histology, and occur alongside an altered spectrum of KRAS alleles. Furthermore, we show that in publicly available early-stage NSCLC studies loss of HD-PTP is mutually exclusive with loss of LKB1, which suggests they restrict a common oncogenic pathway in early lung tumorigenesis. In support of this, knockdown of HD-PTP in RAS-transformed lung cancer cells is sufficient to promote FAK-dependent invasion. Lastly, knockdown of the Drosophila homolog of HD-PTP (dHD-PTP/Myopic) synergizes to promote RAS-dependent neoplastic progression. Our findings highlight a novel tumor suppressor that can restrict RAS-driven lung cancer oncogenesis and identify a targetable pathway for personalized therapeutic approaches for adenosquamous lung cancer.
RESUMEN
[This corrects the article DOI: 10.18632/oncotarget.3291.].
RESUMEN
Loss of LKB1 activity is prevalent in KRAS mutant lung adenocarcinoma and promotes aggressive and treatment-resistant tumors. Previous studies have shown that LKB1 is a negative regulator of the focal adhesion kinase (FAK), but in vivo studies testing the efficacy of FAK inhibition in LKB1 mutant cancers are lacking. Here, we took a pharmacologic approach to show that FAK inhibition is an effective early-treatment strategy for this high-risk molecular subtype. We established a lenti-Cre-induced Kras and Lkb1 mutant genetically engineered mouse model (KLLenti) that develops 100% lung adenocarcinoma and showed that high spatiotemporal FAK activation occurs in collective invasive cells that are surrounded by high levels of collagen. Modeling invasion in 3D, loss of Lkb1, but not p53, was sufficient to drive collective invasion and collagen alignment that was highly sensitive to FAK inhibition. Treatment of early, stage-matched KLLenti tumors with FAK inhibitor monotherapy resulted in a striking effect on tumor progression, invasion, and tumor-associated collagen. Chronic treatment extended survival and impeded local lymph node spread. Lastly, we identified focally upregulated FAK and collagen-associated collective invasion in KRAS and LKB1 comutated human lung adenocarcinoma patients. Our results suggest that patients with LKB1 mutant tumors should be stratified for early treatment with FAK inhibitors.
Asunto(s)
Adenocarcinoma/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias Pulmonares/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Activación Enzimática , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
mTOR complex 2 (mTORC2) regulates cell survival and growth through undefined mechanisms. Mcl-1, a Bcl-2 family protein, functions as an oncogenic protein. The connection between mTORC2 and Mcl-1 stability has not been established and was thus the focus of this study. Mcl-1 levels in cancer cells were decreased by mTOR kinase inhibitors (TORKinibs), which inhibit both mTORCs, by knocking down rictor and by knocking out rictor or Sin1 but not by silencing raptor. TORKinib treatment and rictor knockdown did not alter Mcl-1 mRNA levels but rather decreased its protein stability. Moreover, TORKinib-induced Mcl-1 reduction was rescued by proteasome inhibition. Consistently, TORKinib increased Mcl-1 ubiquitination. Hence, it is clear that inhibition of mTORC2 enhances Mcl-1 degradation, resulting in Mcl-1 reduction. Suppression of glycogen synthase kinase 3 (GSK3) or FBXW7 rescued Mcl-1 reduction induced by TORKinibs or rictor knockdown. Thus, mTORC2 inhibition apparently induces Mcl-1 degradation through a GSK3-dependent and SCF-FBXW7-mediated mechanism. Intriguingly, we detected a direct association between mTORC2 and SCF-FBXW7; this association could be inhibited by TORKinib treatment, suggesting that mTORC2 may directly associate with and inhibit the SCF-FBXW7 complex, resulting in delayed Mcl-1 degradation. Collectively, our findings highlight a novel mechanism by which mTORC2 regulates cell survival and growth by stabilizing Mcl-1.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Neoplasias Pulmonares/metabolismo , Complejos Multiproteicos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/patología , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/análisis , Estabilidad Proteica , Proteolisis , ARN Mensajero/genética , UbiquitinaciónRESUMEN
The single-agent activity of rapalogs (rapamycin and its analogues) in most tumor types has been modest at best. The underlying mechanisms are largely unclear. In this report, we have uncovered a critical role of GSK3 in regulating degradation of some oncogenic proteins induced by rapalogs and cell sensitivity to rapalogs. The basal level of GSK3 activity was positively correlated with cell sensitivity of lung cancer cell lines to rapalogs. GSK3 inhibition antagonized rapamycin's growth inhibitory effects both in vitro and in vivo, while enforced activation of GSK3ß sensitized cells to rapamycin. GSK3 inhibition rescued rapamcyin-induced reduction of several oncogenic proteins such as cyclin D1, Mcl-1 and c-Myc, without interfering with the ability of rapamycin to suppress mTORC1 signaling and cap binding. Interestingly, rapamycin induces proteasomal degradation of these oncogenic proteins, as evidenced by their decreased stabilities induced by rapamcyin and rescue of their reduction by proteasomal inhibition. Moreover, acute or short-time rapamycin treatment dissociated not only raptor, but also rictor from mTOR in several tested cell lines, suggesting inhibition of both mTORC1 and mTORC2. Thus, induction of GSK3-dependent degradation of these oncogenic proteins is likely secondary to mTORC2 inhibition; this effect should be critical for rapamycin to exert its anticancer activity.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Glucógeno Sintasa Quinasa 3/fisiología , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Sirolimus/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antibióticos Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Proteínas Portadoras/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Interacciones Farmacológicas , Activación Enzimática , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Indoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Maleimidas/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteínas Recombinantes de Fusión/metabolismo , Proteína Reguladora Asociada a mTOR , Sirolimus/análogos & derivados , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3ß sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3ß-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Antineoplásicos/farmacología , Benzoxazoles/farmacología , Línea Celular Tumoral , Ciclina D1/metabolismo , Proteínas F-Box/metabolismo , Femenino , Expresión Génica , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Células HEK293 , Humanos , Indoles/farmacología , Concentración 50 Inhibidora , Maleimidas/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Morfolinas/farmacología , Complejos Multiproteicos/metabolismo , Naftiridinas/farmacología , Estabilidad Proteica , Proteolisis , Purinas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor for cap-dependent translation initiation, which is known to regulate oncogenesis. Elevated eIF4E and its negative impact on prognosis in human non-small cell lung cancer (NSCLC) have been reported previously. However, its potential as a therapeutic target and role in regulation of sensitivity to EGFR inhibitors is an area of ongoing investigations. In this study, we detected increased levels of eIF4E in 16 human NSCLC cell lines compared with their normal bronchial epithelial cells. Consistently, human tissue array analysis showed that eIF4E expression was significantly higher in human NSCLC tissues than normal tissues. Inhibition of eIF4E using eIF4E siRNA inhibited the growth and invasion of NSCLC cells. These data suggest that eIF4E overexpression plays a crucial role in positive regulation of the growth and invasion of NSCLC cells. By proteomics, we found that eIF4E levels were elevated in erlotinib-resistant cell lines compared with the sensitive parental cell line. In agreement, assembly of the eIF4F cap complex and several oncogenic proteins regulated by the cap-dependent translation mechanism, were also increased in erlotinib-resistant cells. Thus, erlotinib-resistant cells exhibit elevated eIF4E expression and cap-dependent translation. Inhibition of eIF4F with different means (e.g., gene knockdown) downregulated c-Met expression and partially restored cell sensitivity to erlotinib, suggesting that elevated eIF4E contributes to development of erlotinib resistance, likely through positive regulation of c-Met expression. Taken together, we suggest that elevated eIF4E in NSCLC cells is associated with proliferation, invasion and acquired erlotinib resistance.