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As part of the Extracellular RNA Communication Consortium (ERCC) seminar series, on 30 June 2016, Dr. Janusz Rak from McGill University presented his work on “Extracellular Oncogenes as Biological Effectors and Biomarkers.”

When focusing on a single cancer cell, we consider how particular mutations alter the cell’s properties, causing it to grow, replicate, evade immune detection, and do all the other things cancer cells can do. But it’s clear that cancer is more than a collection of cells operating autonomously. Perhaps the most interesting and frightening part of cancer is that tumors are complex, heterogeneous entities that operate and interact with each other and with their environment in characteristic ways. Researchers have been studying cancer cell signaling pathways for years. An example is paracrine interactions with neighboring cells that induce irregular vascular growth. What is new and exciting about Dr. Rak’s work is the focus on oncogenes as cargo in extracellular vesicles (EVs), which is a nontraditional means of communication between cancer cells and from cancer to non-cancer cells.

Dr. Rak has followed several interesting lines of research. He addresses fundamental questions; for example:

• With which cell types do cancer-cell-derived EVs (CCEV) interact?
• Are tumor-associated mutant proteins found on or in CCEVs?
• Are these mutant proteins then expressed on recipient cells?

Preliminary work in these areas has led to the question: Can cancer cells transform non-cancer cells (i.e. make them cancer-like) via EV-mediated transfer of oncogenic proteins? And is this process complete and permanent? Numerous lines of inquiry, from in vitro to mouse studies, lead to the conclusion that the answer is probably no. Dr. Rak argues that such a finding is not surprising: if horizontal oncogenic transformation were occurring regularly between cell types, we would see more cancer patients with mixed tumor types. Secondly, phylogenetic approaches have been used to trace tumors back to single cell ancestors, another argument against the model of horizontal transformation. The current leading hypothesis is that CCEVs, rather than inducing oncogenic transformation, play a modulatory role, regulating interactions between cancer cells and between cancer and host cells but without changing their identity.

A better understanding of cancer EV biology has promise for improving cancer therapy and diagnosis. CCEVs and their oncogene cargo may represent interesting and novel pharmaceutical targets. They also constitute a unique reservoir of mutant molecules in blood, far away from an inaccessible tumor. Efforts are underway to advance cancer diagnosis through detection of CCEVs, particularly in tumor classes like glioblastoma multiforme (GBM) where no other methods of early detection are available. Research in humans has indicated that GBM patients can be identified by EVs in blood serum. As research such as Dr. Rak’s progresses, and EV biology is further elucidated, we will continue moving towards a future where we can utilize EVs in clinical practice to improve health outcomes.

Members of the Extracellular RNA Communication Consortium have recently elucidated a mechanism through which hypoxia leads to increased tumor aggressiveness and metastasis. Anil Sood and his group at University of Texas MD Anderson Cancer Center have identified a miRNA that downregulates the very pathway responsible for miRNA biogenesis, a finding that should generate excitement due to the identification of a potential new way of treating cancers: through miRNA or RNA interference-based gene targeting.

The development of tumors is a complex process that involves the cooperation of cancer cells with non-cancer cells, together making up the tumor microenvironment that is vital for tumor survival. As tumors grow and become dense, they begin signaling for the formation of new blood vessels to bring oxygen and nutrients to their core, a process called angiogenesis. Inhibiting angiogenesis is thus a highly attractive therapeutic avenue and is typically accomplished through the targeting of vascular endothelial growth factor (VEGF) and the subsequent induction of hypoxia, or a condition of low oxygen, in the tumor. Hypoxia, however, comes with its own set of problems.

The recent study suggests that hypoxia itself can lead to an increase in the aggressiveness and metastatic potential of a tumor (Rupaimoole et al, 2016). Previous studies have found that hypoxic conditions lead to the downregulation of Drosha and Dicer, two components of the miRNA biogenesis pathway, and that this decrease in expression is associated with poor clinical outcomes through a decrease in the pool of miRNA present in the tumor. In investigating the root cause of Dicer downregulation, Sood and his team identified a miRNA, miR-630, that is upregulated during hypoxia and that targets the 3’ UTR of Dicer. The study validated this targeting by monitoring Dicer mRNA and protein levels both in cells and in vivo in mouse models. Mice that had miR-630 delivered to them via nanoliposomes developed larger tumors and metastases in more places than control mice.

Of particular importance, the researchers treated mice with a combination of anti-miR-630 or anti-VEGF therapy (bevacizumab). Mice that were treated with both bevacizumab and anti-miR-630 developed smaller tumors and fewer metastatic nodules compared with mice treated with bevacizumab alone; Dicer expression was rescued upon treatment with anti-miR-630.

Apart from furthering our understanding of Dicer regulation during cancer, this research demonstrates the potential of treating cancers with anti-miRNA therapies, which would be particularly useful in situations where antibodies or chemical agents are not able to reach a target.

In previous studies in humans, measurement of extracellular RNAs (exRNAs) have primarily focused on microRNAs (miRNAs) or studied a small handful of subjects. The specific question of how large numbers of exRNAs are expressed in broader, non-diseased populations has remained. To examine this question, several groups from multiple institutions collaborated to measure exRNAs in the blood plasma of participants from the Framingham Heart Study, an observational cohort study based in Framingham, MA. In their recent publication (1), they first analyzed RNA sequencing data from the plasma of 40 individuals and identified over a thousand human exRNAs including miRNAs, piwi-interacting RNA (piRNAs), and small nucleolar RNAs (snoRNAs).

Study Design

Although miRNAs have been commonly observed in the circulation and plasma, little is known about the presence of other common varieties of small human RNAs such as piRNAs and snoRNAs, known to be key components of molecular interactions and gene regulation in eukaryotes. Using a targeted RT-qPCR approach in an additional 2,763 individuals, the groups then characterized almost 500 of the most abundant extracellular RNA transcripts. The presence in plasma of many non-microRNA small RNAs was confirmed in this independent cohort. The findings show that diverse classes of circulating non-cellular small RNAs, beyond miRNAs, are consistently present in plasma from multiple human populations. Further work will determine how the presence of these exRNAs in the circulation correlates with the presence and progression of a broad number of human traits and diseases.

1. Freedman JE, Gerstein M, Mick E, Rozowsky J, Levy D, Kitchen R, Das S, Shah R, Danielson K, Beaulieu L, Navarro FCP, Wang Y, Galeev TR, Holman A,, Kwong RY, Murthy V, Tanriverdi SE, Koupenova-Zamor M, Mikalev E, Tanriverdi K. Diverse Human Extracellular RNAs are Widely Detected in Plasma. Nature Communications. Published online 26 April 2016.

Overly active KRAS leads to increased serine phosphorylation of Ago2 downstream of MEK and ERK. Phosphorylated Ago2 associates more with P-bodies than with multivesicular endosomes, which reduces the sorting of Ago2 and miRNAs into exosomes bound for export from the cell.

Source: Cell Reports

miRNA release into extracellular vesicles (EVs) is a mechanism to control the gene expression and cellular phenotypes of neighboring cells. A key question is how specific miRNAs are sorted into EVs. Active sorting of RNAs to extracellular carriers such as EVs likely depends on binding to specific RNA binding proteins. As a key member of the RNA-induced silencing complex (RISC) machinery that directly binds miRNA, Argonaute 2 (Ago2) has been a strong candidate as a miRNA carrier in EVs. However, the presence of Ago2 in EVs has been controversial.

In a new paper, we show that Ago2 is carried in both microvesicles and exosomes. Using isogenic cell lines for mutant oncogenic KRAS, we show that Ago2 sorting to exosomes is specifically down-regulated by KRAS-MEK-ERK signaling at late endosomes. Tests of three candidate miRNAs showed that this mechanism can regulate sorting of miRNAs to exosomes. Overall, these data indicate that Ago2 sorting to exosomes is a regulated event and may control miRNA sorting. Furthermore, previous studies that were performed in the presence of serum or growth factors in the media may have detected little Ago2 in exosomes due to growth factor activation of KRAS-MEK-ERK signaling. We hypothesize that this may be a mechanism for cells to sense the growth factor milieu and send that information to other cells via alterations in Ago2 and miRNA secretion.

McKenzie et al. “KRAS-MEK signaling controls Ago2 sorting into exosomes.” Cell Reports AOP 21 April 2016.

This blog post is adapted from a TGen press release, found here.

Uncovering the genetic makeup of patients using DNA sequencing has in recent years provided physicians and their patients with a greater understanding of how best to diagnose and treat the diseases that plague humanity. This is the essence of precision medicine.

Now, researchers at the Translational Genomics Research Institute (TGen) are showing how an even more detailed genetic analysis using RNA sequencing can vastly enhance that understanding, providing doctors and their patients with more precise tools to target the underlying causes of disease and help recommend the best course of action.

In their review, published recently in the journal Nature Reviews Genetics, TGen scientists highlight the many advantages of using RNA-sequencing in the detection and management of everything from cancer to infectious diseases such as Ebola and the rapidly spreading Zika virus.

RNA’s principal role is to act as a messenger carrying instructions from DNA for the synthesis of proteins. Building on the insights provided by DNA profiling, the analysis of RNA provides an even more precise look at how cells behave and how medicine can intervene when things go wrong.

Dr. Sara Byron

“RNA is a dynamic and diverse biomolecule with an essential role in numerous biological processes,” said Dr. Sara Byron, Research Assistant Professor in TGen’s Center for Translational Innovation and the review’s lead author. “From a molecular diagnostic standpoint, RNA-based measurements have the potential for broad application across diverse areas of human health, including disease diagnosis, prognosis, and therapeutic selection.”

DNA (deoxyribonucleic acid) sequencing spells out — in order — the billions of chemical letters that make up the genes that drive all of our biologic make-up and functions, from hair and eye color to whether an individual may be predisposed to cancer or other diseases.

RNA (ribonucleic acid) sequencing provides information on the genes that are actively being made into RNA in a cell and are important for cell function. While more complex, RNA holds the promise of more precise measurement of the human physical condition.

There are more forms of RNA than of DNA present in the body, explains Dr. Byron. “RNA sequencing provides a deeper view of a patient’s genome, revealing detailed information on the diverse spectrum of RNAs being expressed.”

One of the most promising aspects of RNA-based measurements is the potential of using extracellular RNA (exRNAs) as a non-invasive diagnostic indicator of disease. Monitoring exRNA simply takes a blood sample, as opposed to doing a tumor biopsy, which is essentially a minor surgery with greater risks and costs.

“The investigation of exRNAs in biofluids to monitor disease is an area of diagnostic research that is growing rapidly,” said Dr. Kendall Van Keuren-Jensen, TGen Associate Professor of Neurogenomics, Co-Director of TGen’s Center for Noninvasive Diagnostics, and one of the review’s authors. “Measurement of exRNA is appealing as a non-invasive method for monitoring disease. With increased access to biofluids, more frequent sampling can occur over time.”

The first clinical test to measure exRNA was released earlier this year, the review said. The test is for use in evaluating lung cancer progression, and the potential for using RNA-seq in other cancers is expanding rapidly. Commercial RNA-seq tests are now available, providing the opportunity for clinicians to more comprehensively profile cancer and use this information to guide treatment selection for their patients, the review said.

In addition, the authors reported on several recent applications for RNA-seq in the diagnosis and management of infectious diseases, such as monitoring for drug-resistant populations during therapy and tracking the origin and spread of the Ebola virus.

Using examples from discovery and clinical research, the authors also describe how RNA-seq can guide interpretation of genomic DNA sequencing results. The use of integrative sequencing strategies in research studies is growing across a broad range of health applications, which promises to drive the incorporation of RNA-seq into clinical medicine as well, the review said.

The paper, Translating RNA-sequencing into Clinical Diagnostics: Opportunities and Challenges, was published online recently in the journal Nature Reviews Genetics. Authors Kendall Van Keuren-Jensen and David W. Craig are participants in the Extracellular RNA Communication consortium.

Source: Translational Genomics Research Institute

The exRNA pathway portal at WikiPathways was created in 2014 and now includes 50 exRNA-related pathways, including 19 from publications by the Extracellular RNA Communication consortium. We are committed to capturing every published pathway figure from the consortium as a properly modeled pathway at WikiPathways. If your work involves pathways, especially if you are publishing it, look into contributing to WikiPathways.

The latest pathways to be curated from consortium publications are:

1. ApoE and miR-146 in inflammation and atherosclerosis
2. miR-148a/miR-31/FIH1/HIF1α-Notch signaling in glioblastoma
3. mir-124 predicted interactions with cell cycle and differentiation
4. miR-517 relationship with ARCN1 and USP1

The goal of the Wikipathways exRNA portal is to build a collection of pathway models for exRNA researchers to use for illustration, data visualization, and analysis. Each pathway is a self-contained data model that connects to identifier and annotation databases. In addition to providing static images for figures and presentations, these pathways can also be used by bioinformatics and network analysis packages such as Cytoscape and PathVisio. Furthermore, as a wiki, anyone can sign up to improve and grow the content. We invite you all to edit, fix, and add to the pathway models in the exRNA pathway portal at WikiPathways.

This blog post is adapted from an article at AAAS.org by Earl Lane. The original can be found here.

Over the past decade, David Wong of UCLA and his colleagues have been developing a method for detecting circulating tumor DNA in bodily fluids such as blood and saliva. The approach, known as a liquid biopsy, holds the promise of quicker, less invasive identification of cancers and easier tracking of disease status during the course of treatment.

In a news briefing at the 2016 annual meeting of the American Association for the Advancement of Science (AAAS), Wong described a prototype device — called electric field-induced release and measurement (EFIRM) — that can detect biomarkers in saliva for a malignancy called non-small cell lung cancer (Wei et al., 2014). The device has high accuracy compared to current sequencing technology and is entering clinical testing in lung cancer patients in Asia this year.

The test requires just one drop of saliva and can be completed in 10 minutes in a physician’s office. Dr. Wong envisions using it in conjunction with other diagnostic tools. If a lung X-ray were to show a suspicious nodule, for example, a doctor could do the saliva-based test to help quickly determine whether a cancer is likely.

The test can reliably find genetic mutations involving epidermal growth factor receptor (EGFR), a protein on the surface of cells. It normally helps the cells grow and divide, but some cells in non-small cell lung cancer have too much EGFR, which makes them grow faster. Several drugs can block the growth signal from EGFR, and their use could be ordered promptly by a clinician.

Saliva-based tests might someday allow screening for a variety of cancers in a doctor’s office or laboratory, says Wong, director of UCLA’s Center for Oral/Head and Neck Oncology Research. He and his colleagues have also been exploring the use of saliva-based liquid biopsy technology to detect mutations linked to cancers of the mouth and the back of the throat (called oropharyngeal cancers). Dr. Wong’s work with the ERC consortium explores the use of liquid biopsy of extracellular RNA to detect gastric cancer.

Source: AAAS

Exosome Diagnostics, Inc. has announced the launch of ExoDx Lung(ALK), the first ever CLIA-validated exosome based blood test. This test detects EML4-ALK fusion transcripts in the plasma of lung cancer patients whose primary tumors carry this mutation. Although these patients make up a small minority of cases, identifying them is important because their tumors are particularly sensitive to ALK inhibitors. Current clinical tests are performed on biopsied tumor tissue. Major drawbacks include not only the risks associated with the invasive biopsy procedure but also low test performance for some of the commonly used methods (especially immunocytochemistry-based tests). In contrast, ExoDx Lung(ALK) requires only a standard blood draw and has 88% sensitivity and 100% specificity (as reported by the manufacturer). This is a major milestone in the application of exosome-based biomarkers to precision medicine, especially in the areas of companion diagnostics and targeted therapies.

The key discovery that let to this test was made eight years ago when Dr. Johan Skog, the Chief Scientific Officer at Exosome Diagnostics, demonstrated that a mutation present in the tumors of patients with glioblastoma could be detected in their blood (Skog et al., 2008). The publication reporting this finding has been cited over 1600 times, indicating its significance and potential for broad application. Now lung cancer patients can directly benefit from the first clinical test based on this discovery, in the form of a non-invasive test to determine the EML4/ALK mutation status of their tumors. We are sure that many more biofluid-based tests, targeting not only cancers but also other conditions where non-invasive testing is desired, will soon follow.