Marissa Williams

Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma

BACKGROUND Malignant pleural mesothelioma (MPM) is recalcitrant to treatment and new approaches to therapy are needed. Reduced expression of miR-15/16 in a range of cancer types has suggested a tumour suppressor function for these microRNAs, and re-expression has been shown to inhibit tumour cell proliferation. The miR-15/16 status in MPM is largely unknown. MATERIALS AND METHODS MicroRNA expression was analysed by TaqMan-based RT-qPCR in MPM tumour specimens and cell lines. MicroRNA expression was restored in vitro using microRNA mimics, and effects on proliferation, drug sensitivity and target gene expression were assessed. Xenograft-bearing mice were treated with miR-16 mimic packaged in minicells targeted with epidermal growth factor receptor (EGFR)-specific antibodies. RESULTS Expression of the miR-15 family was consistently downregulated in MPM tumour specimens and cell lines. A decrease of 4- to 22-fold was found when tumour specimens were compared with normal pleura. When MPM cell lines were compared with the normal mesothelial cell line MeT-5A, the downregulation of miR-15/16 was 2- to 10-fold. Using synthetic mimics to restore miR-15/16 expression led to growth inhibition in MPM cell lines but not in MeT-5A cells. Growth inhibition caused by miR-16 correlated with downregulation of target genes including Bcl-2 and CCND1, and miR-16 re-expression sensitised MPM cells to pemetrexed and gemcitabine. In xenograft-bearing nude mice, intravenous administration of miR-16 mimics packaged in minicells led to consistent and dose-dependent inhibition of MPM tumour growth. CONCLUSIONS The miR-15/16 family is downregulated and has tumour suppressor function in MPM. Restoring miR-16 expression represents a novel therapeutic approach for MPM.

Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma

Introduction: The upregulation of programmed death ligand 1 (PD‐L1) is found in many cancers and contributes to evasion of the hosts immune defense. In malignant pleural mesothelioma (MPM), PD‐L1 expression is associated with the nonepithelioid histological subtype and poor prognosis, but the pathways involved in control of PD‐L1 expression in MPM are poorly understood. To address one possible means of PD‐L1 regulation we investigated the relationship between dysregulated microRNA levels and PD‐L1 expression. Methods: PD‐L1 expression was analyzed by immunohistochemistry in tissue microarrays prepared from samples from patients undergoing an operation (pleurectomy with or without decortication). MicroRNA expression was analyzed by reverse‐transcriptase quantitative polymerase chain reaction. Regulation of PD‐L1 expression in cell lines was assessed after transfection with microRNA mimics and small interfering RNAs. Interaction between microRNAs and PD‐L1 was analyzed by using argonaute‐2 immunoprecipitation and a luciferase reporter assay. Results: In a series of 72 patients with MPM, 18 (25%) had positive PD‐L1 staining, and this was more common in patients with the nonepithelioid subtype (p = 0.01). PD‐L1 expression was associated with poor survival (median overall survival 4.0 versus 9.2 months with positive versus negative PD‐L1 expression [p < 0.001]), and in multivariate analyses, PD‐L1 expression remained a significant adverse prognostic indicator (hazard ratio = 2.2, 95% confidence interval: 1.2–4.1, p < 0.01). In the same patient series, PD‐L1 expression was also associated with downregulation of microRNAs previously shown to have tumor suppressor activity in MPM. The median microRNA expression levels of miR‐15b, miR‐16, miR‐193a‐3p, miR‐195, and miR‐200c were significantly lower in the PD‐L1–positive samples. Transfecting MPM cell lines with mimics corresponding to miR‐15a and miR‐16, both of which are predicted to target PD‐L1, led to downregulation of PD‐L1 mRNA and protein. In addition, miR‐193a‐3p, with an alternative G‐U–containing target site, also caused PD‐L1 downregulation. Conclusions: Together, these data suggest that tumor suppressor microRNAs contribute to the regulation of PD‐L1 expression in MPM.

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets.MethodsUtilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy.ResultsWe identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration.ConclusionThese functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

DNA Methylation as a Noninvasive Epigenetic Biomarker for the Detection of Cancer

In light of the high incidence and mortality rates of cancer, early and accurate diagnosis is an important priority for assigning optimal treatment for each individual with suspected illness. Biomarkers are crucial in the screening of patients with a high risk of developing cancer, diagnosing patients with suspicious tumours at the earliest possible stage, establishing an accurate prognosis, and predicting and monitoring the response to specific therapies. Epigenetic alterations are innovative biomarkers for cancer, due to their stability, frequency, and noninvasive accessibility in bodily fluids. Epigenetic modifications are also reversible and potentially useful as therapeutic targets. Despite this, there is still a lack of accurate biomarkers for the conclusive diagnosis of most cancer types; thus, there is a strong need for continued investigation to expand this area of research. In this review, we summarise current knowledge on methylated DNA and its implications in cancer to explore its potential as an epigenetic biomarker to be translated for clinical application. We propose that the identification of biomarkers with higher accuracy and more effective detection methods will enable improved clinical management of patients and the intervention at early-stage disease.

Abstract 3976: Targeted delivery of a synthetic microRNA-based mimic as an approach to cancer therapy

MicroRNA expression is commonly suppressed in cancer, contributing to tumor cell biology. Recently we demonstrated that multiple members of the miR-15/16 family are downregulated and have tumor suppressor functions in malignant pleural mesothelioma (MPM), an asbestos-related cancer for which few treatments are available. These results are similar to previous findings in prostate and non-small cell lung cancer (NSCLC). Using mimics to restore levels of miR 15a, miR-15b or miR-16 led to growth inhibition and induction of apoptosis of MPM cells in vitro. The miR-16 mimic, packaged in bacterially-derived, EGFR antibody-targeted, EDV(TM)nanocells, inhibited xenograft tumor growth in vivo. As multiple microRNAs from the same family are downregulated in MPM, we investigated whether a single synthetic mimic based on the consensus sequence of all family members could restore activity of the entire family. To this end we generated four novel mimics derived from the consensus sequence of the miR-15/16 family and tested them in a range of tumor cell lines. Compared with a mimic corresponding in sequence to native miR-16, the consensus mimics had enhanced growth inhibitory activity in MPM, NSCLC and prostate cancer lines, three tumor types in which miR-15/16 expression is suppressed. They were also active in cell lines derived from breast and colon cancer. When packaged in minicells, the synthetic mimics inhibited growth of MPM xenograft tumors in vivo. Based on these preclinical studies a Phase I clinical trial has been initiated for patients with MPM or NSCLC failing standard therapy. This represents only the second trial of microRNA replacement as a cancer therapy, and the first for thoracic cancer. Citation Format: Glen Reid, Marissa Williams, Michaela B. Kirschner, Nancy Mugridge, Jocelyn Weiss, Himanshu Brahmbhatt, Jennifer MacDiarmid, Nico van Zandwijk. Targeted delivery of a synthetic microRNA-based mimic as an approach to cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3976. doi:10.1158/1538-7445.AM2015-3976

Exploring Mechanisms of MicroRNA Downregulation in Cancer

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at a posttranscriptional level. Each miRNA controls the expression of multiple messenger RNAs (mRNAs) and their dysregulation has been implicated in multiple cancer phenotypes. While some miRNAs are upregulated, global downregulation of miRNA expression is often the rule in cancer. A multitude of potential mechanisms drive aberrant miRNA expression in cancer; miRNA coding regions can harbour genomic defects including mutations, amplifications or deletions, and some miRNAs are broadly repressed by transcription factors such as Myc or have epigenetic modifications to their promoter regions such as hypermethylation of CpG islands. Additionally, the suppression of components of the miRNA processing machinery has been shown to reduce mature miRNA expression and contribute to the malignant phenotype. Understanding the mechanisms driving miRNA downregulation is important in uncovering the critical and complex role of miRNAs in cancer biology. This review will outline the multiple mechanisms by which cancer cells suppress miRNA expression.

Sequestration of AS-DACA into acidic compartments of the membrane trafficking system as a mechanism of drug resistance in rhabdomyosarcoma.

The accumulation of weakly basic drugs into acidic organelles has recently been described as a contributor to resistance in childhood cancer rhabdomyosarcoma (RMS) cell lines with differential sensitivity to a novel topoisomerase II inhibitor, AS-DACA. The current study aims to explore the contribution of the endocytic pathway to AS-DACA sequestration in RMS cell lines. A 24-fold differential in AS-DACA cytotoxicity was detected between the RMS lines RD and Rh30. The effect of inhibitors of the endocytic pathway on AS-DACA sensitivity in RMS cell lines, coupled with the variations of endosomal marker expression, indicated the late endosomal/lysosomal compartment was implicated by confounding lines of evidence. Higher expression levels of Lysosomal-Associated Membrane Protein-1 (LAMP1) in the resistant RMS cell line, RD, provided correlations between the increased amount and activity of these compartments to AS-DACA resistance. The late endosomal inhibitor 3-methyladenine increased AS-DACA sensitivity solely in RD leading to the reduction of AS-DACA in membrane trafficking organelles. Acidification inhibitors did not produce an increase in AS-DACA sensitivity nor reduce its sequestration, indicating that the pH partitioning of weakly basic drugs into acidic compartments does not likely contribute to the AS-DACA sequestering resistance mechanism evident in RMS cells.

A link between the fibroblast growth factor axis and the miR-16 family reveals potential new treatment combinations in Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with very limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth. Several FGFs and FGF receptors (FGFRs) are predicted targets of the miR‐15/16 family, which is downregulated in MPM. The aim of this study was to explore the link between the miR‐15/16 family and the FGF axis in MPM. Expression analyses via RT‐qPCR showed downregulation of the FGF axis after transfection with miR‐15/16 mimics. Direct interaction was confirmed by luciferase reporter assays. Restoration of miR‐15/16 led to dose‐dependent growth inhibition in MPM cell lines, which significantly correlated with their sensitivity to FGFR inhibition. Treatment with recombinant FGF2 prevented growth inhibition and further reduced the levels of FGF/R‐targeting microRNAs, indicating a vicious cycle between miR‐15/16 down‐ and FGF/FGFR signaling upregulation. Combined inhibition of two independent miR‐15/16 targets, the FGF axis and Bcl‐2, resulted in additive or synergistic activity. Our data indicate that post‐transcriptional repression of FGF‐mediated signals contributes to the tumor suppressor function of the microRNA‐15/16 family. Inhibiting hyperactivated FGF signals and Bcl‐2 might serve as a novel therapeutic combination strategy in MPM.

Dysregulated Expression of the MicroRNA miR-137 and Its Target YBX1 Contribute to the Invasive Characteristics of Malignant Pleural Mesothelioma

Introduction: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure. On a genomic level, MPM is characterized by frequent chromosomal deletions of tumor suppressors, including microRNAs. MiR‐137 plays a tumor suppressor role in other cancers, so the aim of this study was to characterize it and its target Y‐box binding protein 1 (YBX1) in MPM. Methods: Expression, methylation, and copy number status of miR‐137 and its host gene MIR137HG were assessed by polymerase chain reaction. Luciferase reporter assays confirmed a direct interaction between miR‐137 and Y‐box binding protein 1 gene (YBX1). Cells were transfected with a miR‐137 inhibitor, miR‐137 mimic, and/or YBX1 small interfering RNA, and growth, colony formation, migration and invasion assays were conducted. Results: MiR‐137 expression varied among MPM cell lines and tissue specimens, which was associated with copy number variation and promoter hypermethylation. High miR‐137 expression was linked to poor patient survival. The miR‐137 inhibitor did not affect target levels or growth, but interestingly, it increased miR‐137 levels by means of mimic transfection suppressed growth, migration, and invasion, which was linked to direct YBX1 downregulation. YBX1 was overexpressed in MPM cell lines and inversely correlated with miR‐137. RNA interference–mediated YBX1 knockdown significantly reduced cell growth, migration, and invasion. Conclusions: MiR‐137 can exhibit a tumor‐suppressive function in MPM by targeting YBX1. YBX1 knockdown significantly reduces tumor growth, migration, and invasion of MPM cells. Therefore, YBX1 represents a potential target for novel MPM treatment strategies.

Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.