Graham Brock

Overexpression of the MicroRNA hsa-miR-200c Leads to Reduced Expression of Transcription Factor 8 and Increased Expression of E-Cadherin

MicroRNAs are approximately 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, deltaEF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-cadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.

Stable expression of miR-200c alone is sufficient to regulate TCF8 (ZEB1) and restore E-cadherin expression.

Regulation of the transcription factor TCF8 (ZEB1) by the microRNA miR-200c and was first identified using a bioinformatic and relative quantitative PCR based approach1. Using stable ectopic expression of miR-200c we then demonstrated loss of TCF8 (ZEB1) mRNA and restoration of its primary regulatory target, E-cadherin2. Recently, other members of the miR-200 ‘family’ and an additional unrelated microRNA, miR-205, have been reported to be essential for the regulation of TCF8 (ZEB1) and restoration of E-cadherin3-5. To investigate, we repeated our initial method(s) and generated individual stable cell-lines, expressing the miR-200 ‘family’ members; miR-200c, miR-200b, miR-141 and the related miR-205. Of these lines, miR-200b produced no mature transcript and miR-205 and miR-141 cells were either morphologically similar to controls or showed some slight changes respectively. However no reduction in TCF8/ZEB1 mRNA or restoration of E-cadherin could be detected in either line. In contrast, cells expressing miR-200c had a significantly altered morphology from mesenchymal to epithelial and restored expression of E-cadherin. These contrasting findings demonstrate that, as transient transfection is in essence an RNAi experiment, results should be treated with caution when investigating microRNAs and that an examination of microRNAs with similar seed regions requires stable ectopic expression to accurately reflect the endogenous mechanism(s).

Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR.

Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated that ~3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against ~600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2’-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.

Liquid biopsy for cancer screening, patient stratification and monitoring

Molecular characterization of a patient’s tumor to guide treatment decisions is increasingly being applied in clinical care and can have a significant impact on disease outcome. These molecular analyses, including mutation characterization, are typically performed on tissue acquired through a biopsy at diagnosis. However, tumors are highly heterogeneous and sampling in its entirety is challenging. Furthermore, tumors evolve over time and can alter their molecular genotype, making clinical decisions based on historical biopsy data suboptimal. Personalized medicine for cancer patients aims to tailor the best treatment options for the individual at diagnosis and during treatment. To fully enable personalized medicine it is desirable to have an easily accessible, minimally invasive way to determine and follow the molecular makeup of a patient’s tumor longitudinally. One such approach is through a liquid biopsy, where the genetic makeup of the tumor can be assessed through a biofluid sample. Liquid biopsies have the potential to help clinicians screen for disease, stratify patients to the best treatment and monitor treatment response and resistance mechanisms in the tumor. A liquid biopsy can be used for molecular characterization of the tumor and its non-invasive nature allows repeat sampling to monitor genetic changes over time without the need for a tissue biopsy. This review will summarize three approaches in the liquid biopsy field: circulating tumor cells (CTCs), cell free DNA (cfDNA) and exosomes. We also outline some of the analytical challenges encountered using liquid biopsy techniques to detect rare mutations in a background of wild-type sequences.

Abstract 4969: Exosomal RNA based liquid biopsy detection of androgen receptor variant 7 in plasma from prostate cancer patients

Introduction Androgenic signaling is a critical part in the growth and survival of prostate cancer (PCa), especially in advanced, metastatic or recurrent disease. Consequently, the androgen receptor (AR) is a target for hormone therapies. Full-length androgen receptor mRNA is derived from 8 exons and encodes an N-terminal transactivation domain (exons 1-2), a DNA-binding domain (exons 2-3) and C-terminal ligand binding domain (exons 4-8). However, AR variants have been identified in which the ligand binding domain is removed by splicing between exons 1-3 and intronic, cryptic exons. One of these variants, ARv7, is highly expressed in patients and cultured PCa cells, which demonstrate resistance to hormone therapy treatment. To avoid ineffective procedures, the development of a liquid biopsy test for ARv7 expression would be a useful and non-invasive means of determining treatment strategies for PCa patients. Methods We have developed a diagnostic test to detect the presence of full-length androgen receptor and ARv7 transcripts in plasma samples taken from patients with advanced prostate cancer. The ExoDx™ ARv7 assay comprises a column-based isolation step yielding total exosomal RNA from 1-4 mls of patient plasma, followed by detection of both full-length androgen and the v7 receptor variants, using qPCR. Assay quality is monitored by inclusion of internal and external controls. Following validation, using both synthetic spikes and human samples, we monitored AR expression in plasma samples from treatment-resistant PCa patients. The data was analyzed and aligned with patient9s response data. Results Here, we present proof of concept data for the ExoDX ARv7 assay. Using this novel diagnostic test, we were able to detect ARv7 variant mRNA in exosomal RNA isolated from patient plasma. We determined the profile of ARv7/FL transcripts in a cohort of prostate cancer patients with both high sensitivity and specificity, and observed concordance of the qPCR-based plasma results. Conclusions Liquid biopsies represent a low-risk, non-invasive and viable approach to testing for biomarkers in prostate cancer patients. Here, we demonstrate the capability of our validated diagnostic test to determine the presence of AR alternate transcripts in plasma. Since this analyte is the result of alternative splicing, it would not be detected in cell-free DNA alone. Monitoring plasma levels of ARv7 transcripts could enable effective personalized treatment for patients with advanced prostate cancer and has clear clinical application. Citation Format: James Hurley, Vincent O’Neill, Graham Brock, Javed Siddiqui, Rohit Malik, Arul Chinnaiyan, Johan Skog. Exosomal RNA based liquid biopsy detection of androgen receptor variant 7 in plasma from prostate cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4969.

The 3 prime paradigm of the miR-200 family and other microRNAs

The number of predicted human microRNAs in Sanger miRBase currently stands at over a thousand, with each of these in turn predicted to target numerous mRNAs. However, those microRNAs for which mRNA targets have been evaluated, verified and reported in the literature are still in the minority and the bulk of microRNA/mRNA interactions are yet to be confirmed. Confirmation of microRNA interaction with predicted mRNA targets represents a considerable undertaking, made more complex by potential synergistic effects of multiple microRNAs and the three possible outcomes (translational repression, degradation or a mixture of both). In addition, contrasting results obtained when either stably expressing or transiently transfecting members of the miR-200 family illustrate limitations in the verification methods currently in use. In this article we suggest that instead of allowing computational predictions to drive investigation, it would be desirable, when possible, to systematically evaluate microRNA targets using inducible, stable, ectopic expression. The advantage of stable lines ectopically expressing microRNA(s) is that they allow an analysis of changes to both the proteome and the transcriptome. This would allow verification of targets, improve the design of prediction algorithms and greatly increase our understanding of the outcome of microRNA/mRNA interaction.

Abstract 5686: Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA

Growing research in oncology implicates exosomes in cell-to-cell communication and various cancer patho-physiologies. Vesicle-mediated signaling is thought to occur via both protein and RNA transport. However, there is a fundamental lack of understanding regarding the long RNA cargo within exosomes. Previous RNASeq studies on exosomes have largely focused on the small RNA fraction, although the clinical biomarker space remains dominated by mRNA. These studies have reported a relatively small proportion and poor transcript coverage of long RNA which has led many to conclude that exosomes only carry short fragments of mRNA and ncRNA. To address this, we have developed a novel platform specifically designed to perform RNASeq on long RNA from exosomes to efficiently interrogate these as RNA biomarkers. Paired-end sequencing revealed the presence of a wider diversity of annotated mRNA and ncRNAs than previously recognized in exosomes. Employing our whole transcriptome unique molecular indexing strategy, we were able to accurately quantify the number of unique RNA molecules. We detected a total of over 1.4 million unique RNA molecules in our libraries, with 271,308 mapped to protein-coding regions from as little as 2 ml of plasma. We observed that even at a shallow sequencing depth (15 million read pairs), we could detect more than 10,000 genes, composed of 40,000 mRNA and 37,000 ncRNA annotated transcripts. ERCC spike-in analysis indicates that our RNASeq platform is highly sensitive, detecting as few as 8 copies of RNA. Long non-coding RNAs dominated the ncRNA classes and accounted for over 95% of all uniquely detected ncRNAs. We also observed a bimodal distribution of transcript coverage, similar to cellular and tissue derived RNA. In excess of 15,000 transcripts were detected with ≥80% coverage and of these high-coverage transcripts, more than 25% were over 1kb in length, the longest being 17.6kb. Taken together, these findings suggest exosomes harbor more than just small RNA transcripts and short fragments of mRNAs as was previously thought and is a treasure trove of RNA biomarkers. Current research in the liquid biopsy space has been limited to variant detection in cell-free DNA and circulating tumor cells but transcriptional dysregulation is also an important feature of cancer. Our results shed new light on the biology of exosomes and indicate that, in addition to well-recognized small RNA cargo, plasma exosomes carry an abundance of long RNAs that can be interrogated by our optimized workflow. As exosomes provide a rich and protected source of RNA in biofluids, sequencing both long and small RNA from exosomes has the potential to identify biomarkers without the need for invasive tissue biopsies and allow us to have a more complete picture of how the RNA transcriptome in plasma changes in health and disease. Citation Format: Sudipto K. Chakrabortty, Lisa Bedford, Hidefumi Uchiyama, Vasisht Tadigotla, Michael D. Valentino, Dominik Grimm, Dalin Chan, Sunita Badola, Graham Brock, Johan Skog. Long RNA sequencing of human plasma exosomes reveals full coverage of diverse protein coding and long non coding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5686. doi:10.1158/1538-7445.AM2017-5686

Abstract LB-78: Pharmacogenomic analysis of a phase III trial of gemcitabine/axitinib versus gemcitabine/placebo in patients with advanced pancreatic cancer

Background: The oral antiangiogenic agent axitinib (AG-013736) is a potent, selective inhibitor of vascular endothelial growth factor (VEGF) receptors 1, 2 and 3. We explored potential associations between germline single nucleotide polymorphisms (SNPs) in genes regulating angiogenesis and response to axitinib in pts with advanced pancreatic cancer. In a previous Phase III study of pts with advanced breast cancer treated with bevacizumab, VEGF-2578 (rs699947) AA genotype was associated with increased overall survival (OS). A VEGFR1 SNP ( rs9582036 ) has also been associated with OS in pts with metastatic pancreatic cancer who received bevacizumab. Methods: This analysis was based on a double-blind Phase III trial of pts with metastatic or locally advanced nonresectable pancreatic adenocarcinoma who were randomized 1:1 to gemcitabine 1000 mg/m 2 + axitinib 5 mg twice daily (n=316) or gemcitabine + placebo (n=316). Twelve angiogenesis-related SNPs were analyzed including VEGFA, VEGFR1, VEGFR2, HIF1A, and WHSC2 using DNA isolated from peripheral blood samples. All 12 SNPs were analyzed for associations with OS. The 5 VEGFA SNPs ( rs699947, rs1570360, rs833061, rs2010963, rs3025039 ) were also examined for possible relationships with OS, hypertension (Grade ≥3), and blood pressure (BP; at least 1 measurement of diastolic BP ≥90 mmHg during treatment). This analysis focused on the Caucasian subpopulation (68% of total). Results: Of the 632 pts, 301 voluntarily donated blood samples for genotyping. Most samples evaluable for association analyses (as-treated population) came from Caucasians (260/297, 88%). Among the subpopulation of pts who consented to genotyping, significantly more had metastatic disease than locally advanced disease compared with those who did not consent (238/59 vs 241/92; P =0.0249). Pts with available genotyping data had a correspondingly shorter OS (median OS 7.15 mo vs 9.48 mo; P =0.0068). The genotyped pts had a significantly lower ratio of Asian to Caucasian pts (22/260 vs 151/171; P =2.2×10- 16 ). No significant correlations were found between any of the SNPs examined and OS. Furthermore, no associations were noted between the VEGFA SNPs and either hypertension or high BP. Conclusions: No statistically significant associations were seen between examined SNPs and clinical end points in pts with advanced pancreatic cancer treated with gemcitabine/axitinib. Owing to differences in baseline demographics and stratification factors between the genotyped and nongenotyped pts, these results cannot be extrapolated beyond this subpopulation. This pharmacogenomic component demonstrated that it is feasible to collect donated samples from nearly 50% of pts in a large international Phase III trial. Further studies will examine possible associations between VEGFA SNPs or other polymorphisms and clinical outcomes in VEGF-driven tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-78.