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Yoshitaka Sekido
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MA 19 - Mesothelioma: Bench to Bedside (ID 680)
- Event: WCLC 2017
- Type: Mini Oral
- Track: Mesothelioma
- Presentations: 1
- Moderators:Dean A Fennell, Hedy Lee Kindler
- Coordinates: 10/18/2017, 11:00 - 12:30, Room 315
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MA 19.08 - Discussant - MA 19.05, MA 19.06, MA 19.07 (ID 10830)
11:45 - 12:00 | Presenting Author(s): Yoshitaka Sekido
- Abstract
- Presentation
Abstract not provided
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MS 08 - Novel Treatment for Mesothelioma (ID 530)
- Event: WCLC 2017
- Type: Mini Symposium
- Track: Mesothelioma
- Presentations: 1
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MS 08.05 - New Biological Insights (ID 7679)
16:45 - 17:00 | Presenting Author(s): Yoshitaka Sekido
- Abstract
- Presentation
Abstract:
Malignant mesothelioma (MM) is a mesodermally derived, primarily pleural or peritoneal tumor with aggressive behavior. The incidence of this once-rare tumor is increasing rapidly because of the widespread use of asbestos. In Japan, the annual number of MM-related deaths in 2015 was approximately 1,400, which is 3-fold higher than that observed 20 years ago. The median survival of patients with malignant pleural mesothelioma is 9 to 18 months after diagnosis, which necessitates urgent development of more effective new therapeutic modalities against this aggressive disease. Neurofibromatosis type 2 (NF2) is a tumor suppressor gene that is deleted or mutated in approximately 40% of MM tumors. The gene product of NF2, Merlin, suppresses MM cell proliferation, at least in part, by regulating the Hippo signaling pathway. Hippo signaling is a tumor-suppressive pathway, and alterations in the components of this pathway, including LATS2, SAV1, and AJUBA, have been detected in MM cells. Inactivation of the Hippo signaling pathway leads to the constitutive activation of YAP and TAZ, downstream transcription coactivators that are regulated by this pathway. We previously reported that inhibition of YAP in Merlin-deficient MM cells reduces cell proliferation and inhibits anchorage-independent growth, whereas expression of an active YAP mutant in immortalized mesothelial cells induces oncogenic transformation. While the oncogenic roles of YAP has been extensively studied in MM cells, the possible pro-oncogenic functions of TAZ, a homolog of YAP, is not well understood. Using a panel of MM cell lines, we observed that approximately 65% of these cell lines show activation (underphosphorylation) of TAZ. We knocked down TAZ with shRNA-TAZ in MM cells with high TAZ activation and detected strong inhibition of cell proliferation, anchorage independent growth, cell motility, and invasion in vitro. Meanwhile, immortalized mesothelial cells transduced with a constitutive activated form of TAZ (TAZ S89A mutant) showed enhancement of these in vitro phenotypes and tumorigenicity in nude mice. Using microarray analyses, we identified that while most upregulated genes were common between the TAZ and YAP activated cells, TAZ induced transcription of genes encoding cytokines and their receptors more than YAP. Among the upregulated cytokines, we observed that TAZ binds to the promoter region of the gene encoding IL1-beta along with TEAD transcription factors, which increased IL1-beta transcription and subsequently cell proliferation of immortalized mesothelial cells. In contrast, IL1-beta knockdown or an IL1 receptor antagonist inhibited cell proliferation of MM cells, suggesting that IL-beta signaling suppression may have stronger inhibitory effects on MM cells with TAZ activation. The mevalonate pathway has recently been reported to play a pivotal role in regulating the downstream events of the Hippo pathway. We identified antitumor effects of statin on MM cells with Hippo signaling pathway inactivation. Statin attenuated proliferation and migration of MM cells harboring a NF2 mutation by accelerating YAP phosphorylation/inactivation. Interestingly, not all MM cells with NF2-Hippo pathway inactivation exhibited statin sensitivity. All the statin high-sensitive MM cell lines had increased p-YAP/YAP ratios (inactivation) after statin exposure, whereas in statin low-sensitive cells, the p-YAP/YAP ratio was generally low or unchanged. Genetically, the statin high-sensitive MM cells harbored NF2 and/or LATS2 mutations without BAP1 mutation, whereas BAP1 mutations were frequently identified in statin low-sensitive cells. Indeed, the Y-MESO-25 cell line, carrying both NF2 and BAP1 mutations, regained moderate statin-sensitivity after transfection with a wild-type BAP1 plasmid, indicating that BAP1 mutations interfered with the anti-proliferative effects of statins on MM cells with Hippo pathway inactivation. However, the interactions between BAP1 and the Hippo pathway remain to be elucidated. In conclusion, YAP and TAZ activation via NF2-Hippo pathway inactivation is essential for MM cells to acquire more malignant phenotypes, and therefore, detailed understanding of the biology of this pathway is required to develop new therapeutic modalities against MM based on dysregulation of this pathway.
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P3.03 - Chemotherapy/Targeted Therapy (ID 719)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Chemotherapy/Targeted Therapy
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.03-013 - Identification of Proteasomal Catalytic Subunit PSMA6 as a Therapeutic Target for Lung Cancer through a Pooled shRNA Screen (ID 8867)
09:30 - 09:30 | Author(s): Yoshitaka Sekido
- Abstract
Background:
Recent advances in high-throughput genetic analysis revealed that single lung cancer cells harbour a number of genetic and epigenetic changes. Nevertheless, findings from cancer epidemiology and the experimental models of the multi-step lung carcinogenic process, which were developed by our group and others, suggested that only a handful of changes are ‘drivers’ whereas others are only ‘passengers’. Thus, it is very important to identify those that truly contribute to the oncogenic properties of cancer cells by performing functional screening. To this end, we performed screening with a pooled shRNA library in search for genes that are critical for the survival and/or proliferation of lung cancer cells using a lung cancer cell line.
Method:
NCI-H460 cell line was used for semi-genome-wide dropout viability analysis using a pooled shRNA library that targeted 5,043 genes. Two Cdk4/hTERT-immortalised normal human bronchial epithelial cell lines, HBEC3 and HBEC4 were used as controls. Pathway analysis was done using NIH-DAVID. Microarray gene expression analysis was done using Illumina Human WG-6 v3.0 Expression BeadChip for 163 non-small cell lung cancer (NSCLC) cell lines and 59 normal control cell lines. DNA copy number analysis with array CGH was done for 108 NSCLC cell lines. Proteasome activity was measured using a 20S proteasome activity assay kit. 20 pairs of resected lung cancer and matched normal lung samples were used for immunohistochemistry of PSMA6. Cell growth was evaluated by WST-1 colorimetric proliferation assay. Cell cycle analysis was done using FACS for cells stained with propidium iodide.
Result:
shRNA screening targeting 5,043 genes in NCI-H460 identified 51 genes as candidates for therapeutic targets. Pathway analysis revealed that the 51 genes were enriched for the five pathways, including ribosome, proteasome, RNA polymerase, pyrimidine metabolism and spliceosome pathways. We focused on the proteasome pathway that involved six candidate genes because its activation has been demonstrated in diverse human malignancies, including lung cancer. Microarray expression and array CGH data showed that PSMA6, a proteasomal subunit of a 20S catalytic core complex, was highly expressed in lung cancer cell lines, with recurrent gene amplifications in some cases. Therefore, we further examined the roles of PSMA6 in lung cancer. Silencing of PSMA6 induced apoptosis or G2/M cell cycle arrest in cancer cell lines but not in an immortalised normal lung cell line.
Conclusion:
Our data suggested that PSMA6 serves as an attractive target with a high therapeutic index for lung cancer.
