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H. Osada



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    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O04.01 - Identification of CD74-NRG1, a new recurrent fusion gene in invasive mucinous lung adenocarcinomas of never smokers (ID 4022)

      10:30 - 12:00  |  Author(s): H. Osada

      • Abstract
      • Presentation
      • Slides

      Background
      Lung adenocarcinoma (AD) of patients who have never smoked frequently bear targetable genome kinase alterations, such as EGFR mutations and translocations affecting ALK, ROS1, and RET genes. These mutations correlate with kinase inhibitor sensitivity in mouse models or in patients. Unfortunately, therapeutically relevant kinase alterations are not present in all lung cancer specimens. Thus, additional genome alterations need to be discovered in order to provide a therapeutic opportunity for the remaining patients.

      Methods
      We collected a cohort of 25 AD specimens of never smokers lacking mutations in KRAS or EGFR, in which we performed transcriptome sequencing with the aim of identifying new oncogenic driver genes.

      Results
      We were able to identify known kinase fusions affecting ALK, ROS1 and RET genes in 3 cases each. Moreover, we detected one sample carrying a novel chimeric transcript fusing the first six exons of CD74 to the EGF-like domain of the NRG1 III-β3 isoform, leading to the expression of its EGF-like domain in an otherwise NRG1-negative tumor tissue. The fusion gene was further detected in four additional cases out of 94 pan-negative* ADs of never smokers. In total, all 5 cases were identified in stage I invasive mucinous lung adenocarcinomas (IMA) of never smoker females. This tumor type frequently presents with multifocal unresectable disease, for which no effective treatment has been yet established. IMA is highly associated with KRAS mutations; indeed, out of 15 IMA analysed, 6 carried a KRAS mutation (40%), and 4 the CD74-NRG1 fusion (27%). Given the fact that NRG1 signals through ERBB3 and ERBB4 receptors, we aimed to determine which receptor CD74-NRG1 provides the ligand for. We observed that ERBB4 was not expressed in the index case, while ERBB3 was relatively highly expressed and this expression also correlated with a positive phospho-ERBB3 (p-ERBB3) signal in the tumoral tissue of all 5 CD74-NRG1 positive cases. In order to test if this phosphorylation of ERBB3 was statistically significant, we stained a cohort of 241 ADs and found that p-ERBB3 was only positive in 6 of them (p-value<0.0001). Additionally, although both EGFR and ERBB2 were expressed in the index case, only ERBB2 expression correlated with a p-ERBB2 positive signal. These data suggest that CD74-NRG1 might provide the ligand for ERBB3, which may form heterodimers with ERBB2, since ERBB3 is devoid of intrinsic kinase activity and cannot support linear signaling in isolation. This is in line with previous studies showing that NRG1 induces an oncogenic signal through ERBB2-ERBB3 heterodimers engaging the PI3K-AKT pathway. This was further supported by the activation of the PI3K-AKT, but not the MAPK pathway, in CD74-NRG1 transduced H2052 lung cells, after 24h starvation. *pan-negative: EGFR, KRAS, ALK, HER2, BRAF, ROS1 and RET wild-type

      Conclusion
      Altogether, these data shows that CD74-NRG1 is a new recurrent oncogenic fusion gene, highly associated with IMA of never smokers. It also suggests that CD74-NRG1 fusion protein signals through the ERBB2-ERBB3 receptors complex leading to the activation of the PI3K-AKT pathway, providing a therapeutic opportunity for a tumor type with, so far, no effective treatment.

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    P3.02 - Poster Session 3 - Novel Cancer Genes and Pathways (ID 149)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.02-004 - Dysregulation of Hippo tumor-suppressive pathway in malignant mesothelioma (ID 1039)

      09:30 - 16:30  |  Author(s): H. Osada

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a highly aggressive tumor caused by asbestos exposure after a long latency. The neurofibromatosis type 2 (NF2) tumor suppressor gene is mutated in around 50% of MPM cases, which encodes Merlin that regulates the Hippo tumor-suppressive signaling pathway. We previously reported occasional genetic alteration in the LATS2 and SAV1 genes in MMs, which encode components of the Hippo pathway. The LATS2 inactivation was shown to lead to constitutive activation of YAP, a prooncogenic protein and transcriptional coactivator, which enhances multiple cell cycle regulation genes including cyclin D1 (CCDN1). To further delineate the exact inactivation mechanism of this pathway in MMs, we analyzed MM cell lines for other multiple components that regulate activation or inactivation of this pathway.

      Methods
      We studied several new components that have been identified to be involved in the Hippo signaling pathway including a LIM protein Ajuba. Expression analyses such as conventional western blot and real time RT-PCR were performed. Using MPM cell lines and their transplanted mouse models, biological assays were conducted to study the effects of cell proliferation, motility and invasion after the induction of overexpression or knockdown of candidate genes. Immunohistochemical analysis with primary MPM tissues and xenografts was also carried out.

      Results
      We found that the expression of Ajuba was significantly down-regulated in 6 of 20 MM cell lines compared to an immortalized normal mesothelial cell line, MeT-5A. Interestingly, the 6 cell lines with low Ajuba expression showed decreased phosphorylation (activation) levels of YAP. We infected the MM cells with Ajuba-expressing lentivirus and found that exogenous Ajuba increased phosphorylation (inactivation) of YAP and inhibited cell proliferation. Dual-luciferase reporter assays demonstrated that Ajuba suppressed the promoter activities of YAP-transcriptional targets including CCND1. Knockdown of LATS2 effectively increased these promoter activities, suggesting the mediation of LATS2 for Ajuba to inhibit this cascade. Immunohistochemical analysis also showed frequent downregulation of Ajuba in primary MMs.

      Conclusion
      Inactivation of Hippo signaling is a key mechanism for MPM cell proliferation and progression. Our findings suggest that Ajuba is also one of the target components for Hippo signaling inactivation; Ajuba negatively regulates YAP in the presence of LATS2, and thus the downregulation of Ajuba serves to enhance constitutive activation of YAP in MM cells. Our study also indicates that a strategy to normalize this signaling cascade may be the rationale for developing a new target therapy against MPMs.