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P. Dowling
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-109 - Epithelial-To-Mesenchymal Transition (EMT) and Acquired Resistance to PI3K-mTOR Inhibition in NSCLC (ID 934)
09:30 - 09:30 | Author(s): P. Dowling
- Abstract
Background:
The PI3K-Akt-mTOR pathway regulates cell growth and proliferation and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in Phase II clinical trials for solid tumours. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. The aims of this project are to (i) elucidate the frequency of PIK3CA mutations in an Irish cohort of NSCLC patients and (ii) develop and characterise three cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.
Methods:
DNA was extracted from 250 NSCLC patient tissue samples, and screened for 547 clinically relevant mutations in 46 genes using the Sequenom platform. H460, A549, and H1975 cells were cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Development of resistance was assessed by monthly BrdU proliferation assays. Cell growth patterns were compared across the sensitive and resistant cell lines in real time using the xCELLigence platform. Cell lines were then interrogated for alterations in DNA (Sequenom), mRNA (SABiosciences arrays profiling expression of >150 genes), miRNA (Exiqon expression profiling of 2100 miRNAs) and protein (R&D Phospho Kinase array expression profiling of 43 kinases and 2 associated total proteins, PTMScan[®] Ubiquitin Remnant Motif (K-ε-GG) Kit from CST and Western blot analysis).
Results:
PIK3CA mutations occur in ~5% adenocarcinomas & 12% squamous cell carcinomas. H1975 cells (PIK3CA mutant and activated pAkt (Ser473/Thr308), pmTOR, pS6R) were most sensitive to GDC-0980, however they were the first to develop resistance to the drug. Results obtained from xCELLigence studies identified H1975 resistant (H1975R) cells as having the highest cell index out of all parent and resistant cell lines after 100 hours of cell growth, suggesting that these are the most aggressive cells. Initially a 33 miRNA signature was identified contrasting H1975P and H1975R. qPCR validation of miR-205 (a regulator of EMT) identified expression in H1975P cells but miR-205 was undetectable in H1975R cells. mRNA expression of Zeb1 & Zeb2 (direct targets of miR-205) were increased in H1975R cells compared to H1975P cells. 1,200 proteins were found to be differentially expressed between H1975P and H1975R cells. Increased expression of EMT proteins vimentin, desmin and filamin was detected in H1975R cells (p < 0.05, fold change >2). Vimentin overexpression in H1975R cells was confirmed by western blot analyis. Activation of EMT was identified as one potential mechanism of resistance to GDC-0980 in H1975R cells.
Conclusion:
The PI3K-mTOR pathway is frequently mutated in NSCLC, in particular squamous cell carcinoma, making it an ideal therapeutic target. Acquired resistance to GDC-0980 developed rapidly in NSCLC cell lines, (4-6 months) and correlates to the induction of EMT. Further elucidation of EMT regulation is under investigation and is crucial to the design of improved treatment protocols.
