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Glen Reid
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ES03 - BAP-1 and Other Novel Molecular and Metabolic Targets in Mesothelioma (ID 6)
- Event: WCLC 2019
- Type: Educational Session
- Track: Mesothelioma
- Presentations: 1
- Now Available
- Moderators:Michele Carbone, Miyako Satouchi
- Coordinates: 9/08/2019, 10:30 - 12:00, Tokyo (1982)
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ES03.01 - Targeting Altered microRNA Expression in Mesothelioma (Now Available) (ID 3161)
10:30 - 12:00 | Presenting Author(s): Glen Reid
- Abstract
- Presentation
Abstract
MicroRNAs (miRNAs) are an important class of noncoding RNA that post-transcriptionally regulate the expression of most protein-coding genes. In addition to central roles in normal biology, their aberrant expression in tumours contributes to all of the hallmarks of cancer. In common with other tumour types, changes in miRNA expression in malignant pleural mesothelioma (MPM) are characterised by a global downregulation, although elevated levels of some miRNAs are also found. While altered expression has been demonstrated for many miRNAs in MPM, relatively few have been functionally characterised. Early studies reported modest tumour suppressive activities of, among others, miR-29c, miR-31 and miR-145. More recently, miR-16, miR-193a and the miR-34 family were shown to have clear tumour suppressor activity; restoring the levels of these miRNAs leads to a range of effects including inhibition of cell growth, induction of apoptosis, reversal of drug resistance and reduced migration and invasion. In the case of miR-16 and miR-193a, targeted delivery of mimics to tumours in a xenograft model led to significant inhibitory effects on tumour growth. These results laid the foundation for the phase I MesomiR-1 trial, investigating the safety and optimal dose of a miR-16-based mimic delivered in anti-EGFR antibody-targeted bacterial minicells. This trial of 27 patients demonstrated safety of the treatment as well as initial signs of activity, with one objective response and stable disease in a further 15 patients. With miR-16 also impacting response to pemetrexed and contributing to PD-L1 regulation in vitro, restoration of miR-16 levels in combination with chemo or immunotherapy are potential future applications of this approach. Other miRNAs with pronounced tumour suppressor activity, including miR-137 and miR-193a, are further candidates for clinical development. The lower levels of miR-193a recently found to be associated with shorter overall survival in the TCGA study lend support to this notion.
In contrast to the use of mimics to restore levels of miRNAs downregulated in MPM, inhibition of overexpressed miRNAs with antisense oligonucleotides is an alternative strategy for modulating miRNA levels. This approach is attractive as it may be amenable to local delivery, avoiding the problems associated with tumour targeting via systemic administration. While the number of miRNAs found to be consistently overexpressed in MPM is relatively small, recent studies suggest that their inhibition can have profound effects on MPM growth. One such example was the report of the effects of inhibiting the overexpressed miR-182 and miR-183. They are upregulated in MPM cell lines where they promote proliferation and invasion, at least in part due to suppression of FOXO1. Reducing their levels with miRNA inhibitors reversed these effects, with dual inhibition showing additive effects. An oncogenic role for miR-182 was first demonstrated in melanoma, in which this miRNA enhances migration, invasion and metastasis via inhibition of FOXO3 and MITF. Upregulation of miR-182 in melanoma is due to amplification (at 7q31) of a miRNA cluster which also contains the related miR-183 and miR-96. As this region appears to be more frequently lost in MPM, the mechanism for overexpression remains to be determined. Another miRNA with oncogenic activity in MPM is miR-24, which was identified via a screen of polysome-associated miRNAs and is upregulated in cell lines and tumour samples. This miRNA regulates a range of genes involved in cell adhesion and communication, many of which are associated with good prognosis, and miR-24 knockdown reduced migration and invasion in vitro and in vivo. Although the targets of miR-24 identified in this study had no obvious link to MPM biology, it is intriguing that in other cancers miR-24 regulates both transcripts produced by the CDKN2A locus. Whether other well known oncogenic miRNAs such as miR-155, miR-10b and miR-21 promote MPM tumour progression remains to be seen, but the initial results with miR-182, miR-183 and miR-24 warrant further pre-clinical development.
Despite progress in the development of miRNAs as therapeutic targets for MPM, outstanding questions remain. Of particular relevance is the question of the cell of origin of dysregulated miRNAs. As tumour samples consist of a mix of cancer, stroma and immune cells, the source of miRNA contributing to changes in expression is unknown and thus impacts the interpretation of results. For example, altered expression of miR-223 was linked to changes in migratory behaviour of MPM, but in xenograft studies this miRNA is clearly derived from the stroma. Another confounding observation concerns the apparent contradictory findings from studies of the same miRNA. For example, miR-137 has clear tumour suppressor function in MPM cells in vitro, but its expression varies widely across MPM lines and is found at higher levels in tumours from patients with shorter survival. Similarly, miR-31 has growth inhibitory activity but higher expression is associated with poor prognosis in sarcomatoid tumours. There are also cases of different miRNAs showing apparent differences in MPM compared with other cancers. A prime example of this is miR-21; an oncomiR in most cancer types, but growth inhibitory when overexpressed MPM cell lines. Finally, because the activity of miRNAs typically leads to modest reductions in target genes, demonstrating in vivo effects is not trivial. In addition, because the miRNA being restored (or inhibited) is usually identical to its endogenous miRNA counterpart, measuring delivery can be difficult. Due to their short size, even novel sequences such as that used in the TargomiR study are difficult to definitively measure by RT-qPCR or RNA-seq.
In summary, the changes in miRNA expression in MPM provide avenues to develop new therapetuic approaches. The successful phase I trial of TargomiRs demonstrated that miRNA targeting is feasible in MPM and while the majority of miRNA studies in MPM have focused on miRNA mimics, recent studies suggest that antisense inhibitors have similar potential. Notwithstanding the ongoing difficulties in delivering nucleic acid-based drugs, the recent FDA approval of the first siRNA therapy – together with ongoing clinical trials of a number of miRNA mimic drugs – means that gene silencing drugs have moved from concept to reality. Continued preclinical studies and early phase clinical trials are needed to determine the true potential of miRNA targeting in MPM treatment.
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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): Glen Reid
- 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.
