Micromanaging microRNAs to treat malignant pleural mesothelioma.
MicroRNAs are a class of short gene regulators that are frequently dysregulated in cancer including mesothelioma. We have discovered that the majority of microRNAs are suppressed in malignant pleural mesothelioma (MPM) and potentially act as tumour suppressors. In this project we are systematically testing the functional significance of all downregulated microRNAs. This allows the characterisation of the full repertoire of tumour suppressor microRNAs and their roles in MPM. We have designed experiments to test the effects of individual microRNA mimics using in vitro and in vivo models. The combinations of mimics will also be investigated for their effect on MPM cell and tumour growth. The synergistic effects of microRNA combinations and their subsequent cellular pathway involvement will also be tested. At the final stage of this project, we will test how restoration of these microRNAs individually or in combination affect tumour growth using a newly established inter-pleural MPM model. In this model we will use an in vivo-jetPEI from Polyplus to deliver the microRNAs, this method has already been applied in a current clinical trial. As such, the project has the potential to be rapidly tested in the clinic as a treatment for MPM patients. This project is funded by iCare Dust Diseases Authority
YB-1 plays a dual role in MPM growth, which has implications on its role in drug resistance
Following the findings in our recent publication in the Journal of Thoracic Oncology, we were interested in the mechanisms behind the growth-promoting role YB-1 plays in MPM cells. YB-1 was inhibited in MPM cell lines via siRNA transfection and TALI apoptosis assays and cell cycle analysis using cell-flow cytometry was conducted. We found that some MPM cell lines displayed an increase in apoptosis while others displayed G1-G0 cell cycle arrest in response to YB-1 silencing. This led us to investigate the potential role YB-1 might play in MPM chemotherapy resistance, firstly because target-induced apoptosis can act synergistically with traditional chemotherapy and secondly, because YB-1 is known to be involved in the drug resistance of many other cancers. MPM cells were transfected with low doses of YB-1 or control siRNA and treated with different concentrations of cisplatin or vinorelbine chemotherapy. We found that cell lines undergoing apoptosis in response to YB-1 siRNA were also sensitised to these chemotherapies, while those undergoing cell-cycle arrest displayed increased resistance. These findings have implications on the therapeutic potential of targeting YB-1 in patients suffering MPM, and we have ethics approval to conduct in vivo studies on the effect of YB-1 on growth and drug resistance using our novel intraperitoneal mouse model. This exciting new data has been presented at the World Conference on Lung Cancer in Yokohama, Japan, and at the ECR Cancer Research Symposium, Sydney.
Using artificial miRs to control the growth of MPM
Most cancer chemotherapy is based on ‘one therapy, one target’ model. Through this principle single gene or secondary pathway mutations can lead to loss of drug efficacy and resistance to therapy. Therefore, a therapy that has more than one target pathway has potential advantages. MicroRNAs have this property as they control expression of multiple genes across different pathways. Given the sequence-specific interaction of natural microRNAs with the 3’ untranslated region of their target mRNAs, it is possible to use this principle to design artificial microRNAs that control expression of pre-selected target genes. In theory, controlling multiple genes across different pathways has the potential to avoid resistance associated in a single gene therapy. We have successfully applied an artificial microRNA concept to target genes in multiple pathways in malignant pleural mesothelioma (MPM), and continue to develop this concept in preclinical models.
Regulation of PD-L1 expression by microRNAs in MPM
Cancer immunotherapy, particularly using immune checkpoint blocking antibodies, is leading to long-term responses in some cancer patients. The interaction between programmed death 1 (PD-1) and its ligand PD-L1 are one such target, as they play a significant role in suppressing host immune response in many cancer types. Our study confirms that PD-L1 is an adverse prognostic indicator in malignant pleural mesothelioma (MPM). Although PD-L1 expression is upregulated and associated with poor prognosis in MPM the mechanisms causing its dysregulation are poorly understood. Elevated PD-L1 expression in MPM patient samples correlates with downregulation of tumour suppressor microRNAs that were in turn shown to directly regulate PD-L1 expression in vitro. The relationship between miR-193a-3p and PD-L1 was shown to be due to an unusual interaction between the microRNA and non-canonical a site in the PD-L1 mRNA. This work was published in the Journal of Thoracic Oncology.
Identifying microRNAs with therapeutic potential in MPM
We and others have shown that multiple microRNAs have tumour suppressor activity in malignant pleural mesothelioma (MPM) cell lines when the levels are restored using mimics. Results from our lab led to the phase I MesomiR 1 clinical trial, which was the world’s first microRNA replacement strategy (TargomiRs) in mesothelioma patients. In this project we are carrying out a head-to-head comparison of microRNA mimics, both singly and in combination, to identify the most promising microRNAs for future development as therapeutic agents. The initial experiments in this project were carried out by two students from the 2015/16 Summer Studentship program at The University of Sydney. This work is supported by a grant from the DDB.
Co-stimulation of the immune system to improve chemotherapy for MPM
In collaboration with New Zealand biotech company, Innate Immunotherapeutics, we are investigating the ability of a microparticulate immune response modulator, currently in clinical trial for other indications, for its ability to stimulate the immune system and improve response to chemotherapy. Only 40% of malignant pleural mesothelioma (MPM) patients receiving the standard of care (palliative platinum-pemetrexed chemotherapy) respond to treatment and combined-modality treatment (with curative intent) is followed by prolonged survival in selected cases only. Currently, there is no defined second-line treatment and the need for efficient therapies for MPM remains unfulfilled. Recent research has focused on immunotherapy, to harness the immune system as a novel therapeutic approach. Minor impacts on tumour growth have been demonstrated in animal studies following immunotherapy alone, however, when combined with chemotherapy, clear shrinking of tumours was visible. Moreover, following treatment with immunotherapy, mice implanted with a secondary challenge of MPM exhibited tumour inhibition compared to mice who were not treated with immunotherapy. Clinical studies, with antibodies blocking immune checkpoints have shown dramatic responses in melanoma and non-small cell lung cancer. Efficacy of immune checkpoint blockade has also been noted in a relatively small series of MPM patients but so far the responses observed were less impressive than in patients with melanoma and non-small cell lung cancer. Thus the development of new treatment approaches for MPM remains vitally important and immune stimulation in combination with existing (chemotherapy) treatment warrants further exploration.
The contribution of stromal cells to microRNA expression in mesothelioma
In this study, being carried out by Kadir Sarun as an MSc project, we have identified several microRNAs that are significantly upregulated in experimental tumours derived from human malignant pleural mesothelioma (MPM) cell lines when compared to the levels found in the MPM cell lines grown in the laboratory. Amongst these microRNAs species specific pri-miRNA transcripts depicted miR-143-3p, miR-214-3p and miR-223 to be dominantly contributed by mouse stromal cells. Overexpressing these microRNAs in cultured human tumour cells had no significant effect on growth. Hence, indicating that these microRNAs are likely to possess a more biologically relevant role in the stroma. Furthermore, this data also provides a cautionary tale for interpreting microRNA profiles where the results from a MPM biopsy may include microRNAs contributed by stromal cells.
A 3D spheroid culture represents an improved in vitro model of MPM
Most biologists rely on cell culture in the two-dimensional (2D) format for studying tumour context which does not accurately reflect the in vivo state. 3D cell culture techniques provide better cell-to-cell interactions that mimics pathological conditions such as cancer. Malignant pleural mesothelioma (MPM) is a deadly cancer with no effective treatment and is highly drug resistant. This study addressed this problem by growing cells in 3D and therefore creating an environment that more closely mimics the realistic tumour state for molecular and cellular responses. Cells grown in our 3D model displayed greater drug resistance when compared with 2D cells. Most tumour suppressor biomarkers we analysed show downregulation of mRNA expression levels compared with cells in 2D. The data from this study were presented at the IASLC 18th World Conference on Lung Cancer 2017 in Yokohama, Japan.
Establishing malignant pleural mesothelioma cell lines using the spheroid method produces a model with better 3D architecture
Malignant pleural mesothelioma (MPM) is an aggressive malignancy with no effective treatment options. Poor prognosis and drug resistance are the main challenges of this deadly disease. There is also no simple distinctive diagnosis tool for the identification of MPM. Better diagnostic markers may also provide better biological information for treatment development. In this study we established primary MPM cell lines and characterised them with current biomarkers. The ultimate goal was to use these cell lines for the identification of diagnostic biomarkers. Cells grown in 3D provide better tumour architecture when compared with 2D cells. 3D cells also provide more intensity and greater percentage of positive MPM biomarkers. The data from this study were presented at the IASLC 18th World Conference on Lung Cancer 2017 in Yokohama, Japan.
Correcting aberrant microRNA expression as a therapeutic approach in MPM.
The mechanisms causing downregulation of the tumour suppressor microRNAs miR-15a/16-1, miR-15b/16-2 and miR-193a-3p in malignant pleural mesothelioma (MPM) as the specific modes of their downregulation is unknown. To understand the processes involved, the different stages of microRNA biogenesis are being investigated in this PhD project. Marissa Williams started her PhD in 2015 to study aberrant microRNA expression in MPM. This project was funded by Sydney Catalyst