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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
MINI35.11 - Mutant ARAF Drives Lung Carcinogenesis Through a Distinct Oncogenic Mechanism (ID 1016)
18:30 - 20:00 | Author(s): J.M. Amann
We recently identified a novel somatic mutation in ARAF in a lung adenocarcinoma from a patient that demonstrated a remarkable response to sorafenib. The S214C lies in a negative regulatory domain of ARAF, distinct from the catalytic domain mutations commonly found in BRAF. The aim herein was to characterize the biochemical and functional aspects of ARAF S214C.
ARAF constructs were generated and ectopically expressed in an immortalized bronchial epithelial cell line (BEAS-2B). We evaluated the acquisition of anchorage independence, MEK activation, and cell morphology. COS7 cells were used for co-immunoprecipitation (IP) and kinase assays.
Cells expressing ARAF S214C substantially increased soft agar colony formation relative to vector, wild-type, kinase-dead (D429A), and double-mutant (S214C+D429A) variants. Accordingly, ARAF S214C cells exhibited increased phospho-MEK levels, suggesting that the transforming potential is dependent on its kinase activity. Interestingly, ARAF S214C cells acquired an elongated, fibroblast-like shape, characteristic of MEK-active cells, whereas none of other variants presented this morphology. We also demonstrated that cells expressing ARAF S214C with an additional RAS-binding domain mutation (R52L) or dimerization interface mutation (R362H) lacked MEK activation, showing that RAS binding and RAF-RAF dimerization are essential for activity. To elucidate the role of BRAF and RAF1 as dimerization partners of ARAF S214C, we performed knockdowns of BRAF, RAF1, or both. ARAF S214C-induced MEK activation was not reversed by the BRAF knockdown, however both RAF1 and double knockdowns (BRAF and RAF1) led to loss of MEK activation, suggesting that RAF1 is required. Subsequently, COS7 cells were co-transfected with tagged constructs of ARAF and either BRAF or RAF1, followed by co-IP. We showed that mutant ARAF presents a higher rate of dimerization than wild-type ARAF in the presence of sorafenib. Importantly, sorafenib-induced heterodimers lacked kinase activity, compatible with the clinical response reported.
ARAF S214C demonstrates the in vitro features of a driver oncogene, and also a distinct mechanism of action. This oncogenic process can be successfully suppressed by RAF inhibitors like sorafenib, and could represent a new target for personalized therapy in advanced lung adenocarcinoma. Figure 1 Figure: Summary of the ARAF S214C oncogenic mechanism.
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P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
P1.08-004 - Aki1 as a Potential Therapeutics Target in CREB1 Signaling in Malignant Mesothelioma (ID 234)
09:30 - 17:00 | Author(s): J.M. Amann
Malignant pleural mesothelioma (MPM) is an aggressive tumor arising from the mesothelial cells of serosal membranes. Since current treatment options are largely ineffective, novel therapeutic strategies based on molecular mechanisms and the disease characteristics are needed to improve its prognosis. Akt kinase-interacting protein 1 (Aki1)/Freud-1/CC2D1A known as a scaffold protein of PI3K/PDK1/Akt that determines receptor signal selectivity for EGFR has been suggested as a therapeutic target in lung cancer. The aim of this study was to elucidate the role of Aki1 and its potential for treatment of MPM.
We tested the effects of the treatment with Aki1 or CREB1 siRNAs on cell viability by MTT assay, cell cycle by FACS analysis, cell signaling by WB, and CREB transcriptional activity in 7 MPM cells and 1 mesothelial cells using in vitro experiments. We investigated the efficacy of Aki1 siRNA against growth of 211H cells in an orthotropic implantation model using SCID mice. We further examined Aki1 and p-CREB1 expressions in MPM tumors from 35 patients by TMA specimens and from 33 patients by the tissues.
Cell based assay showed that silencing of Aki1 inhibited cell viability and caused cell arrest of some of MPM cells but not mesothelial cells. Importantly, we identified that the efficacy of Aki1 is regulated by CREB1 signaling which is involved in cell viability, cell cycle, and transcriptional activity. Aki1 and phosphorylated CREB1 were frequently expressed in MPM patients (65/68 cases) (30/35 cases), respectively. Furthermore, the expression of Aki1 correlated with phosphorylation of CREB1 (Spearman rank correlations = 0.521; p = 0.002). Furthermore, direct application of Aki1 siRNA into the pleural cavity significantly inhibited growth of 211H cells compared with that of control siRNA in an orthotropic implantation model using SCID mice.
Our data suggest an important role of Aki1/CREB axis in pathogenesis of MPM and provide a rationale for targeting Aki1 by intrathoracic therapy in locally advanced tumors.