RESUMEN
Lung cancer is a major disease carrying heterogeneous molecular lesions and many of them remain to be analyzed functionally in vivo. Gain-of-function (GOF) SHP2 (PTPN11) mutations have been found in various types of human cancer, including lung cancer. However, the role of activating SHP2 mutants in lung cancer has not been established. We generated transgenic mice containing a doxycycline (Dox)-inducible activating SHP2 mutant (tetO-SHP2(E76K)) and analyzed the role of SHP2(E76K) in lung tumorigenesis in the Clara cell secretory protein (CCSP)-reverse tetracycline transactivator (rtTA)/tetO-SHP2(E76K) bitransgenic mice. SHP2(E76K) activated Erk1/Erk2 (Erk1/2) and Src, and upregulated c-Myc and Mdm2 in the lungs of bitransgenic mice. Atypical adenomatous hyperplasia and small adenomas were observed in CCSP-rtTA/tetO-SHP2(E76K) bitransgenic mice induced with Dox for 2-6 months and progressed to larger adenoma and adenocarcinoma by 9 months. Dox withdrawal from bitransgenic mice bearing magnetic resonance imaging-detectable lung tumors resulted in tumor regression. These results show that the activating SHP2 mutant promotes lung tumorigenesis and that the SHP2 mutant is required for tumor maintenance in this mouse model of non-small cell lung cancer. SHP2(E76K) was associated with Gab1 in the lung of transgenic mice. Elevated pGab1 was observed in the lung of Dox-induced CCSP-rtTA/tetO-SHP2(E76K) mice and in cell lines expressing SHP2(E76K), indicating that the activating SHP2 mutant autoregulates tyrosine phosphorylation of its own docking protein. Gab1 tyrosine phosphorylation is sensitive to inhibition by the Src inhibitor dasatinib in GOF SHP2-mutant-expressing cells, suggesting that Src family kinases are involved in SHP2 mutant-induced Gab1 tyrosine phosphorylation.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/patología , Mutación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The highly lethal nature of pancreatic cancer and the increasing recognition of high-risk individuals have made research into chemoprevention a high priority. Here, we tested the chemopreventive activity of δ-tocotrienol, a bioactive vitamin E derivative extracted from palm fruit, in the LSL-Kras(G12D/+);Pdx-1-Cre pancreatic cancer mouse model. At 10 weeks of age, mice (n = 92) were randomly allocated to three groups: (i) no treatment; (ii) vehicle and (iii) δ-tocotrienol (200mg/kg × 2/day, PO). Treatment was continued for 12 months. Mice treated with δ-tocotrienol showed increased median survival from the onset of treatment (11.1 months) compared with vehicle-treated mice (9.7 months) and non-treated mice (8.5 months; P < 0.025). Importantly, none of the mice treated with δ-tocotrienol harbored invasive cancer compared with 10% and 8% in vehicle-treated and non-treated mice, respectively. Furthermore, δ-tocotrienol treatment also resulted in significant suppression of mouse pancreatic intraepithelial neoplasm (mPanIN) progression compared with vehicle-treated and non-treated mice: mPanIN-1: 47-50% (P < 0.09), mPanIN-2: 6-11% (P < 0.001), mPanIN-3: 3-15% (P < 0.001) and invasive cancer: 0-10% (P < 0.001). δ-Tocotrienol treatment inhibited mutant Kras-driven pathways such as MEK/ERK, PI3K/AKT and NF-kB/p65, as well as Bcl-xL and induced p27. δ-Tocotrienol also induced biomarkers of apoptosis such as Bax and activated caspase 3 along with an increase in plasma levels of CK18. In summary, δ-tocotrienol's ability to interfere with oncogenic Kras pathways coupled with the observed increase in median survival and significant delay in PanIN progression highlights the chemopreventative potential of δ-tocotrienol and warrants further investigation of this micronutrient in individuals at high risk for pancreatic cancer.
Asunto(s)
Carcinoma in Situ/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Vitamina E/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Carcinoma in Situ/mortalidad , Carcinoma in Situ/prevención & control , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/prevención & control , Caspasa 3/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genotipo , Proteínas de Homeodominio/genética , Ratones , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/prevención & control , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/efectos de los fármacos , Proteínas Proto-Oncogénicas p21(ras)/genética , Sobrevida , Transactivadores/genética , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/metabolismo , Vitamina E/farmacología , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/metabolismoRESUMEN
Multiple sclerosis (MS) is a chronic, immune-mediated, demyelinating disease of the central nervous system (CNS). There is no known cure for MS, and currently available drugs for managing this disease are only effective early on and have many adverse side effects. Results from recent studies suggest that histone deacetylase (HDAC) inhibitors may be useful for the treatment of autoimmune and inflammatory diseases such as MS. However, the underlying mechanisms by which HDACs influence immune-mediated diseases such as MS are unclear. More importantly, the question of which specific HDAC(s) are suitable drug targets for the potential treatment of MS remains unanswered. Here, we investigate the functional role of HDAC11 in experimental autoimmune encephalomyelitis, a mouse model for MS. Our results indicate that the loss of HDAC11 in KO mice significantly reduces clinical severity and demyelination of the spinal cord in the post-acute phase of experimental autoimmune encephalomyelitis. The absence of HDAC11 leads to reduced immune cell infiltration into the CNS and decreased monocytes and myeloid DCs in the chronic progressive phase of the disease. Mechanistically, HDAC11 controls the expression of the pro-inflammatory chemokine C-C motif ligand 2 (CCL2) gene by enabling the binding of PU.1 transcription factor to the CCL2 promoter. Our results reveal a novel pathophysiological function for HDAC11 in CNS demyelinating diseases, and warrant further investigations into the potential use of HDAC11-specific inhibitors for the treatment of chronic progressive MS.
RESUMEN
RET fusions have been found in lung adenocarcinoma, of which KIF5B-RET is the most prevalent. We established inducible KIF5B-RET transgenic mice and KIF5B-RET-dependent cell lines for preclinical modeling of KIF5B-RET-associated lung adenocarcinoma. Doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET transgenic mice developed invasive lung adenocarcinoma with desmoplastic reaction. Tumors regressed upon suppression of KIF5B-RET expression. By culturing KIF5B-RET-dependent BaF3 (B/KR) cells with increasing concentrations of cabozantinib or vandetanib, we identified cabozantinib-resistant RETV804L mutation and vandetanib-resistant-RETG810A mutation. Among cabozantinib, lenvatinib, ponatinib, and vandetanib, ponatinib was identified as the most potent inhibitor against KIF5B-RET and its drug-resistant mutants. Interestingly, the vandetanib-resistant KIF5B-RETG810A mutant displayed gain-of-sensitivity (GOS) to ponatinib and lenvatinib. Treatment of doxycycline-induced CCSP-rtTA/tetO-KIF5B-RET bitransgenic mice with ponatinib effectively induced tumor regression. These results indicate that KIF5B-RET-associated lung tumors are addicted to the fusion oncogene and ponatinib is the most effective inhibitor for targeting KIF5B-RET in lung adenocarcinoma. Moreover, this study finds a novel vandetanib-resistant RETG810A mutation and identifies lenvatinib and ponatinib as the secondary drugs to overcome this vandetanib resistance mechanism. Mol Cancer Ther; 15(10); 2521-9. ©2016 AACR.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/farmacología , Biopsia , Línea Celular Tumoral , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Transgenes , Microtomografía por Rayos XRESUMEN
Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.
Asunto(s)
Adenocarcinoma/enzimología , Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Evidence links chronic inflammation with cancer, but cellular mechanisms involved in this process remain unclear. We have demonstrated that in humans, inflammatory conditions that predispose to development of skin and colon tumors are associated with accumulation in tissues of CD33+S100A9+ cells, the phenotype typical for myeloid-derived suppressor cells in cancer or immature myeloid cells (IMCs) in tumor-free hosts. To identify the direct role of these cells in tumor development, we used S100A9 transgenic mice to create the conditions for topical accumulation of these cells in the skin in the absence of infection or tissue damage. These mice demonstrated accumulation of granulocytic IMCs in the skin upon topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in a dramatic increase in the formation of papillomas during epidermal carcinogenesis. The effect of IMCs on tumorigenesis was not associated with immune suppression, but with CCL4 (chemokine [C-C motif] ligand 4)-mediated recruitment of IL-17-producing CD4+ T cells. This chemokine was released by activated IMCs. Elimination of CD4+ T cells or blockade of CCL4 or IL-17 abrogated the increase in tumor formation caused by myeloid cells. Thus, this study implicates accumulation of IMCs as an initial step in facilitation of tumor formation, followed by the recruitment of CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Células Mieloides/patología , Neoplasias Cutáneas/patología , Animales , Linfocitos T CD4-Positivos/inmunología , Calgranulina B/metabolismo , Transformación Celular Neoplásica/patología , Quimiocina CCL4/metabolismo , Colitis/metabolismo , Colitis/patología , Femenino , Humanos , Ratones Transgénicos , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
Accumulation of myeloid-derived suppressor cells (MDSCs) associated with inhibition of dendritic cell (DC) differentiation is one of the major immunological abnormalities in cancer and leads to suppression of antitumor immune responses. The molecular mechanism of this phenomenon remains unclear. We report here that STAT3-inducible up-regulation of the myeloid-related protein S100A9 enhances MDSC production in cancer. Mice lacking this protein mounted potent antitumor immune responses and rejected implanted tumors. This effect was reversed by administration of wild-type MDSCs from tumor-bearing mice to S100A9-null mice. Overexpression of S100A9 in cultured embryonic stem cells or transgenic mice inhibited the differentiation of DCs and macrophages and induced accumulation of MDSCs. This study demonstrates that tumor-induced up-regulation of S100A9 protein is critically important for accumulation of MDSCs and reveals a novel molecular mechanism of immunological abnormalities in cancer.
Asunto(s)
Calgranulina B/metabolismo , Diferenciación Celular/fisiología , Células Dendríticas/fisiología , Células Mieloides/fisiología , Neoplasias/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Calgranulina B/genética , Células Dendríticas/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Macrófagos/citología , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Mieloides/citología , Células 3T3 NIH , Neoplasias/patología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
BACKGROUND: Epidermal growth factor (EGF) markedly attenuates tubular apoptosis induced by unilateral ureteral obstruction (UUO) in the neonatal rat, and reduces apoptosis induced by mechanical stretch of cultured rat tubular cells. METHODS: To investigate the role of EGF in modulating apoptosis resulting from UUO, neonatal wild type and mutant mice lacking EGF (knockout), or with diminished EGF receptor activity (waved-2 mutant) were compared to control mice for tubular apoptosis and atrophy. Rat and mouse kidneys were compared for localization of the EGF receptor. Apoptosis was also measured in cultured mouse tubular cells subjected to stretch and exposed to EGF. RESULTS: UUO reduced endogenous renal EGF expression in wild-type mice. Unlike the rat, exogenous EGF did not decrease tubular apoptosis or atrophy in the obstructed kidney, and significantly increased stretch-induced apoptosis of cultured mouse tubular cells. Tubular apoptosis was 50% lower in the obstructed kidney of EGF knockout and waved-2 mice relative to wild type and heterozygous animals. Exogenous EGF increased tubular apoptosis and doubled atrophy in the obstructed kidney of waved-2 mice. Species differences in EGF receptor localization were detected in 3-day-old kidneys. CONCLUSION: EGF acts as a survival factor in the neonatal rat, but potentiates tubular cell death in the neonatal mouse. Species differences are maintained in cultured cells, suggesting that differences in EGF receptor signaling underlie these opposing effects.