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L. Girard
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-073 - Stromal Hedgehog Pathway Activation Suppresses Growth and Metastasis of Lung Adenocarcinoma (ID 9880)
09:30 - 09:30 | Author(s): L. Girard
- Abstract
Background:
Aberrant activation of the Hedgehog (Hh) signaling pathway, a crucial developmental pathway, drives the tumor growth of Gorlin-type cancers. However, recent data suggest that paracrine activation of the pathway is tumor suppressive rather than oncogenic in sporadic epithelial cancers. The role of the pathway in non-small lung cancer is poorly understood. Thus, we explored the role of stromal Hh pathway activation in growth of lung adenocarcinoma.
Method:
Human lung adenocarcinoma cell lines were used to probe SHH mRNA and protein expression. Co-culture of high SHH expressing cell lines with murine embryonic and lung fibroblasts were used to confirm and probe the role of paracrine SHH expression on the growth of lung cancer cells. The in vivo role of paracrine SHH was tested using autochthonous lung cancer models with conditional KRAS[G12D]activation, p53 loss, and SHH loss compared to wild-type SHH.
Result:
In human lung adenocarcinoma patients, higher expression of SHH mRNA in lung adenocarcinoma correlated with poor overall and progression free survival. A scan of 35 human lung adenocarcinoma cell lines revealed heterogeneous expression of SHH and IHH with high expression found predominantly in mutant K-Ras lines. Co-culture of high SHH expressing tumor epithelial cells and Shh-Light2 reporter cell lines demonstrated that SHH activated the fibroblast reporter in a paracrine manner, rather than an autocrine effect on cancer cells. Treatment with the SMO inhibitor, KAAD-cyclopamine, also inhibited the growth of tumor epithelial cells in co-culture with NIH-3T3 fibroblast cells but the effect was decreased when co-cultured with lung fibroblasts. Genetic loss of SHH in an autochthonous mouse model, LSL-Kras[G12D/+];Trp53[fl/fl]; Shh[fl/fl ](KPS) did not affect overall survival compared to LSL-Kras[G12D/+];Trp53[fl/fl](KP) mice However, early inhibition of the Hh pathway by anti-SHH/IHH antibody, 5E1, on KP mice resulted in significantly worse survival rates with increased metastatic burden compared to IgG treatment. Analysis of KP tumors revealed unexpected high levels of IHH mRNA by in situ hybridization and qPCR that may account for the survival differences seen between genetic ablation and pharmaceutical inhibition of the Hh ligands.
Conclusion:
The Hh signaling pathway acts upon lung stromal cells in a paracrine fashion and induces distinct transcriptional programs in murine embryonic and lung fibrtoblasts. Inhibition of paracrine Hh pathway activity in vivo worsened mortality rate due to increase in tumor growth and metastases. Furthermore, mutant Kras lung adenocarcinomas express high levels of IHH that dominates the tumor suppressive effects in our mouse models.
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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): L. Girard
- 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.
