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Michael Grusch
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EP1.03 - Biology (ID 193)
- Event: WCLC 2019
- Type: E-Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/08/2019, 08:00 - 18:00, Exhibit Hall
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EP1.03-31 - Cell Migration and Epithelial Mesenchymal Transition in Lung Cancer - Different Roads and Common Themes (Now Available) (ID 930)
08:00 - 18:00 | Author(s): Michael Grusch
- Abstract
Background
Cell migration is an indispensable function for many cells in multicellular organisms. When deregulated, however, especially in conjunction with the ability to degrade extracellular matrix and invade surrounding tissues, it is a hallmark of malignancy and forms the basis for cancer metastasis. This is often linked to epithelial mesenchymal transition (EMT) which is widely recognized in cancer cell biology to be intricately connected to metastasis, drug resistance and stemness. Multiple extracellular stimuli that induce EMT and cell migration in diverse cellular contexts have been described, nevertheless, a lot still needs to be learned about pathway-specific mechanisms. We have chosen the Ras-mutated A549 lung adenocarcinoma cell line for investigating how the two growth factors EGF and TGFb, which each play fundamental roles in tumor development but activate clearly distinct signaling cascades both stimulate EMT and migration individually and when acting in cooperation.
Method
A549 cells were treated with EGF, TGFb or a combination of both in serum-free conditions. Also, inhibitors of downstream pathways were used at sub-lethal concentrations. Changes in cell morphology were determined using ImageJ from microscopy images. Cell migration was assessed by live cell videomicroscopy followed by singe cell tracking. The invasive capacity was determined by a 3D sprouting assay. Expression changes were identified by qPCR and immunoblots.
Result
Treatment with TGFb and EGF resulted in cell scattering and distinct changes in cell morphology, which were different for each growth factor. While cells treated with TGFb showed classic EMT-like, elongated morphology, cells exposed to EGF rounded up. Combining both factors resulted in a mixed population. EGF-induced changes could be prevented by Akt but not MAPK inhibitors. Importantly, each growth factor induced a significant increase in cell migration compared to untreated cells and the combination of both factors stimulated migration even further. Interestingly, the increase in migration occurred earlier with EGF than with TGFb, and this was in concordance with increased pERK levels. However, only TGFb was able to induce significantly increased sprouting.
Conclusion
Our data describes two independent signaling pathways which both are able to induce cell scattering and cell migration, albeit along different roads. Especially in cancer cells, a better understanding of signaling pathway-dependency of EMT and migration and potential cross talks could lead to more effective antimetastatic therapies.
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P2.06 - Mesothelioma (ID 170)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Mesothelioma
- Presentations: 1
- Now Available
- Moderators:
- Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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P2.06-06 - Y-box Binding Protein-1, a Potential Target in Malignant Pleural Mesothelioma, Drives Growth Through Distinct Mechanisms (Now Available) (ID 2042)
10:15 - 18:15 | Author(s): Michael Grusch
- Abstract
Background
Malignant pleural mesothelioma (MPM) is an asbestos-related disease with a five‑year survival of five percent. Current therapy provides limited success and finding other targetable molecules remains a top priority. We recently identified Y-box binding protein-1 (YB‑1) as a significantly overexpressed oncogene with prognostic relevance in MPM. YB-1 is a multifunctional transcription and translation factor of the cold-shock protein family. Using siRNA‑mediated knockdown of YB-1 we showed that silencing YB-1 inhibited the proliferation, migration and invasion of four MPM cells by an unknown mechanism. Here we extend this work to examine how YB-1 regulates MPM growth.
Method
Functional activity of YB-1 was investigated by siRNA-mediated knockdown in MPM cells followed by TALI apoptosis assays, multi-dimension flow cytometry or live-cell imaging. Transcript expression was determined using reverse transcription qPCR (RT-qPCR) and RNA sequencing (RNA‑seq) with poly(A) selection.
Result
Following our previous data demonstrating growth inhibition after YB-1 knockdown, we transfected three MPM cell lines with YB-1 siRNA and conducted multi‑dimension flow cytometry and TALI apoptosis assays to begin understanding how this growth inhibition was occurring. We found that cells underwent either apoptosis or a G0/G1 cell cycle arrest. Using live-cell imaging and single cell fate mapping we found that each cell line undertook a distinct mechanism of growth inhibition. MSTO cells displayed apoptosis during interphase, VMC23 cells showed no death but underwent a G0/G1 cell cycle arrest, while REN cells did not delay during interphase but entered prolonged aberrant mitosis resulting in mitotic catastrophe and cell death. To examine the interphase arrest in MSTO and VMC23 further we analysed the expression of cyclin D1 and Myc, known cell cycle targets of YB-1, in knockdown samples using RT-qPCR. Transcripts of cyclin D1 and Myc were downregulated in both cell lines in response to reducing YB‑1, partially explaining the growth inhibition observed. To further understand the effects of YB-1 inhibition we have undertaken a global analysis of downstream targets and pathways after YB‑1 siRNA transfection in all three cell lines using RNA‑seq analysis.
Conclusion
This project delves into the complex mechanism underlying YB-1-driven MPM proliferation and found it plays a broader role than expected due to its influence over multiple cancer-promoting genes and pathways. Our study significantly extends our understanding of this important protein in MPM, a disease in dire need of actionable targets.
