Thanks to Laurence de Nijs and the European College of Neuropsychopharmacology (ENCP) for allowing us to adapt their press release into a blog.


 

Individuals affected with PTSD (Post-Traumatic Stress Disorder) demonstrate changes in microRNA (miRNA) molecules associated with gene regulation. A controlled study, involving Dutch military personnel on deployment to a combat zone in Afghanistan, provided evidence for the role of blood-based miRNAs as candidate biomarkers for symptoms of PTSD. This finding may offer an approach towards screening for symptoms of PTSD, and it holds promise for understanding other trauma-related psychiatric disorders. Limitations of the study are that this was a small pilot study, and the findings need to be validated, extended, and confirmed. First results were presented at the 30th conference of the European College of Neuropsychopharmacology (ENCP) in Paris in early September.

PTSD is a psychiatric disorder which can manifest following exposure to a traumatic event, such as combat, assault, or natural disaster. Among individuals exposed to traumatic events, only a minority of individuals will develop PTSD, while others will show resiliency. Little is known of the mechanisms behind these different responses. The last few years have seen much attention given to whether the modification and expression of genes – epigenetic modifications – might be involved. But there are several practical and ethical challenges in designing a research study on humans undergoing such experiences, meaning that designing relevant study approaches is difficult.

A research group from the Netherlands worked with just over 1,000 Dutch soldiers and the Dutch Ministry of Defense to study changes in biology in relation to changes in presentations of symptoms of PTSD in soldiers who were deployed to a combat zone in Afghanistan. In a longitudinal study, they collected blood samples before deployment as well as 6 months after deployment. Most of the soldiers had been exposed to trauma, and some of the soldiers had developed symptoms of PTSD.

For this pilot study, from the initial group, 24 subjects were selected in 3 subgroups of 8. Eight of the soldiers had developed symptoms of PTSD; 8 had endorsed traumatic experiences but had not developed symptoms of PTSD; and another 8 had not been in serious traumatic circumstances and served as a control group. Using modern sequencing techniques, several types of miRNAs for which the blood levels differed between the groups were identified.

MiRNAs (Micro RiboNucleic Acids) are small molecules with chemical building blocks similar to DNA. Unlike the more famous DNA, miRNAs are typically very short – comprising only around 20 to 25 base units (the building blocks of nucleic acids), and they do not code, in other words they do not specify the production of a protein or peptide. However, they have very important roles in biology (every miRNA regulates the expression, and thereby also the activity of several other genes), and they are known to regulate the impact of environmental factors on biology. In addition, brain-derived miRNA can circulate throughout the human body and can be detected in the blood.

Differences in miRNA levels have been associated with certain diseases, such as some cancers, kidney disease, and even alcoholism. This regulatory role makes them also a candidate for investigation in PTSD.

“We discovered that these small molecules, called miRNAs, are present in different amounts in the blood of persons suffering from PTSD compared to trauma-exposed and control subjects without PTSD,” said first author Dr Laurence de Nijs of Maastricht University.

“We identified over 900 different types of these small molecules. 40 of them were regulated differently in people who developed PTSD, whereas there were differences in 27 of the miRNAs in trauma-exposed individuals who did not develop PTSD.”

“Interestingly, previous studies have found circulating miRNA levels to be not only correlated with different types of cancer, but also with certain psychiatric disorders including major depressive disorders. These preliminary results of our pilot study suggest that miRNAs might indeed be candidates as predictive blood markers (biomarkers) to distinguish between persons at high and low risk of developing PTSD. However, several steps need to be performed before such results can really have an impact on the larger field and in clinical practice. In addition to working towards biomarkers, the results may also provide novel information about the biological mechanisms underlying the development of PTSD.”

Dr de Nijs explained:
“Most of our stressful experiences don’t leave a long-lasting psychological scar. However, for some people who experience chronic severe stress or really terrible traumatic events, the stress does not go away. They are stuck with it, and the body’s stress response is stuck in ‘on’ mode. This can lead to the development of mental illness such as PTSD.

These individuals experience symptoms including re-experiencing of the traumatic event through flashbacks or recurrent nightmares, constant avoidance of reminders of the event, negative mood, and extreme arousal. This can manifest itself through insomnia and or hyper-alertness. Individuals with PTSD are six times more at risk of committing suicide and having marital problems, and the annual loss of productivity is estimated to be approximately $3 billion. Currently, there is no definite cure for patients with PTSD, and available treatments often are not effective.”

Commenting, Professor Josef Zohar (Ex-ECNP Chair, Tel Aviv, Israel) said:
“The relevance of a better understanding of stress-related events is unfortunately becoming clearer and clearer after each terror attack. This work points to an innovative avenue regarding the potential identification of risk factors for susceptibility to developing post-traumatic stress disorder.”


Funding: Dr de Nijs was awarded a Marie Curie fellowship grant by the European Union to perform this study, within a network of other expert scientists in PTSD and epigenetics. The Dutch cohort of soldiers (PRISMO) was funded through the Dutch Ministry of Defence.

Glioblastoma multiforme is the most common type of malignant brain tumor. These tumors actively divide and send invasive cells throughout the brain. This migration complicates patient treatment, because simply removing the tumor does not clear the brain of migrating cells, which can initiate new tumors elsewhere. This problem has nearly halted progress in prognosis or treatment of glioblastoma over the past 20 years, and has afflicted many of our most important political figures, including Ted Kennedy and John McCain. This astonishing lack of improvement in treatment has motivated Dr. Xandra Breakefield and her team to explore an alternative pathway for treatment. Dr. Breakefield discussed her work in a recent Wednesday afternoon lecture at NIH.

Saboteurs

To start their investigation, Dr. Breakefield gathered a “fresh” sample of glioblastoma multiforme that came straight out of the operating room. The idea of studying a fresh sample was novel; previously, samples were a few hours old or from mouse models. Examining fresh samples in a living condition revealed that glioblastomas are extremely physically active, constantly extending protrusions from the surface of the tumor cells. These protrusions turn into extracellular vesicles (EVs) of many sizes, released at the rate of about 10,000 vesicles per cell per day. When profiled, these EVs were found to contain RNAs, enzymes, transcription factors, other proteins, and many other cellular components. The team hypothesized that the EVs released by the tumor must somehow promote tumor progression.

Using a fluorescent tag that labels both cell and vesicle membranes, they tracked the tumor EVs in the living brain of mice and found that many of them are taken up by surrounding healthy myeloid cells — microglia and macrophages. Macrophages are the primary form of defense in the central nervous system. Normally functioning microglia are sentinels, warriors, and nurturers. As sentinels, they actively scan the brain for damage, then rush in to nurture injured areas to repair the damage. In their warrior role they kill and eat invading cells. Ironically, a higher density of microglia and macrophages in a tumor results in a worse prognosis. They are attracted to but then subjugated by the glioblastoma, and are coerced into an abnormal function of supporting tumor growth. Understanding how glioblastoma sabotages these immune cells and co-opts them to support tumor growth is key to ultimately finding a cure for the disease.

miR-21

Glioblastomas and their EVs typically have very high levels of miR-21, a microRNA affiliated with various cancer pathways. Dr. Breakefield and her team found that microglia grown near a glioblastoma and exposed to its EVs had higher levels of miR-21, higher proliferation rates, and lower expression of proteins from pathways involved in sensing and attacking invaders. They hypothesized that miR-21 transferred from the tumor to surrounding benign microglia by EVs plays a key role in recruiting and transforming them. Then they asked whether microglia that have taken up many tumor vesicles have a different phenotype than microglia that haven’t taken up as many. To test this question, the Breakefield lab implanted fluorescently (GFP) labelled gliomas into the brains of mice and developed a flow cytometry method to sort cells extracted from that environment based on their level of GFP. The more tumor vesicles the surrounding normal cells have taken up, the more GFP they should have. The researchers used single-cell RNA sequencing to compare the expression profile of mRNAs in microglia and macrophages with high vs. low levels of GFP, indicating uptake of tumor vesicles. They found significant differences in mRNA expression in brain microglia but not in macrophages.

The genetic pathways responsible for microglia’s sentinel role have been called the “microglial sensome” (Hickman et al., 2013). The mRNA for most sensome genes were down-regulated in microglia that had taken up more tumor EVs – thus, the tumor EVs “blinded” the microglia. Pathways for immune suppression were up-regulated – showing that uptake of the tumor EVs compromised the microglias’ warrior function. Finally, pathways for tissue repair were up-regulated – indicating that the tumor EVs co-opted the microglias’ nurturing function to support the tumor instead of normal brain cells.

Biomarkers

EVs are a promising biomarker for earlier diagnosis of disease. They are released by all cells and found in all biofluids. They can contain a wide variety of different information, including cell specific proteins and RNAs.

Dr. Breakefield was interested in finding biomarkers for glioblastoma that could be found in the bloodstream or other accessible biofluids. She targeted EGFRvIII, a deletion in the EGFR gene quite common in glioblastomas, and IDH1/2, a single point mutation that results in lower grade tumors with better prognosis. Her team is examining cerebrospinal fluid (CSF) and serum from patients to identify the presence or absence of EGFRvIII with promising success. Examining exRNA and free circulating DNA in plasma, her team was able to differentiate IDH1/2 mutant carriers from healthy volunteers. That work is being developed into a clinical tool for earlier diagnosis of low-grade gliomas which have a better prognosis and different treatment options than non-IDH1/2 gliomas.

Therapy EVs are proven, effective therapies for a variety of diseases. They can be obtained from many cell types and protect their fragile molecular cargo when administered into the body. They can also be efficiently taken up by specific target cells in vivo. EVs have been used therapeutically in immune modulation, tissue repair, and antigen presentation for vaccination.

After working on glioblastomas for an extended period, Dr. Breakefield decided to study schwannomas, a related but benign class of tumor. There are three types of hereditary diseases that stem from schwannomas: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. These diseases cause motor dysfunction, pain, and potential hearing loss.

In NF2, tumors form along nerve fibers, including those at the base of the spine. The current treatment is surgical removal of the tumor, but that can cause irreparable nerve damage. Because these tumors are not malignant, reducing their size could be an effective alternative treatment.

Dr. Breakefield and colleagues made a model system using human Schwann cells from an NF2 patient. They implanted those cells in the sciatic nerve of mice, forming tumors. They then created an adenovirus-associated virus (AAV) vector containing the pro-inflammatory enzyme caspase 1 attached to a promoter (P0) exclusively active in Schwann cells. They then injected this vector into the tumors to try to shrink them.

In this model system, the tumor grew when the AAV-GFP was injected. It regressed when AAV-P0-ICE was injected. P0-ICE creates a “bystander” effect, in which cells surrounding the injected cell are also killed. Surprisingly, Schwann cells appeared completely normal after treatment with P0-ICE, while the tumor (schwannoma) shrunk after injection of the tumor with AAV-P0-ICE. Although the EVs’ role in this finding is not known, Dr. Breakefield theorizes that caspase-1 is incorporated into the EVs and transferred to surrounding schwannoma cells.

There is still much to be discovered about EVs’ roles in tumors, both benign and malignant. We have not yet found glioblastoma’s Achilles’ heel, but further research may yet do so. Dr. Breakefield looks forward to continuing her work and potentially creating better treatment options for those afflicted by these devastating tumors. The previously unrecognized role of the tumor-derived EVs in transferring genetic information and co-opting other cells in their microenviroment may be an important key.

Early detection of viral infections is extremely important for control of disease transmission, prompt initiation of treatment, and prevention of infection-related complications. Because of our hypothesis that viral DNA, messenger RNA, and proteins cannot be detected in all infected individuals, we wanted to determine whether detection of exogenous miRNAs encoded by viruses represents a more sensitive assay of the true prevalence of infection by viruses including latent Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV). Therefore, we measured plasma miRNAs using RT-qPCR and compared it to the current standard method for detection of viral infection, an enzyme-linked immunosorbent assay (ELISA) of blood plasma, which detects antibodies generated by the host against the infecting virus. Our study population was 214 Caucasian patients from the United States and Romania, separated into four independent patient cohorts. We examined a total of 300 plasma samples from this population. This study enabled us to develop an approach to detect infection by KSHV using multiplexed RT-qPCR of multiple viral miRNAs.

We found that our method had clear advantages over the current ELISA-based approach. It detected a significantly higher prevalence of KSHV infection than that determined by seropositivity, with the difference most pronounced in immuno-depressed patients. When applied to EBV, our new method based on plasma viral miRNA quantification proved that EBV infection is ubiquitous. This strategy has the potential to become a gold standard method in clinical practice to detect latency of viruses and viremia — viral infection of the bloodstream — in both general and immune-compromised populations.

Detecting viral infection by amplifying viral miRNA has clear advantages over the current ELISA-based approach.

Fuentes-Mattei E, Giza DE, Shimizu M, Ivan C, Manning JT, Tudor S, Ciccone M, Kargin OA, Zhang X, Mur P, do Amaral NS, Chen M, Tarrand JJ, Lupu F, Ferrajoli A, Keating MJ, Vasilescu C, Yeung SJ, Calin GA. Plasma viral miRNAs indicate a high prevalence of occult viral infections. EBioMedicine. (2017) 20:182-192. doi: 10.1016/j.ebiom.2017.04.018. Pubmed: 28465156.

Extracellular vesicles, such as exosomes and microvesicles, are small vesicular particles that are constantly being produced and shed by cells. Due to their natural origin, and their ability to efficiently deliver their cargo to target cells and alter biological functions, exosomes attracted researchers to study their potential use as drug delivery systems. In the past few years, numerous studies have reported the effective use of exosomes to deliver therapeutic cargo ranging from miRNA, siRNA and even small molecule drugs in both in vitro cell models and in vivo animal models. However, since exosomes are cell-derived vesicles, it is unclear how these natural carriers of biomolecules may induce immune responses or induce toxicity either in animal models of disease or eventually in humans as we progress toward clinical evaluation of exosomes in healthy volunteers or in patients. Furthermore, what can we conclude about the presence or lack of immunogenicity or toxicity in our animal models as we work toward delivery of exosomes in humans?

Our lab has been studying the production of therapeutic exosomes using genetically engineered HEK293T cells for treatment of hepatocellular carcinoma (HCC). We developed engineered HEK293T cells that endogenously package miR-199a-3p, a miR commonly downregulated in HCC, into exosomes, and we are evaluating these and also exosomes exogenously loaded with therapeutic miRs in vitro and in vivo. Although demonstrating in vivo efficacy is a major milestone for all drug development efforts, understanding the potential toxicities and immunogenic responses associated with exosome therapy is equally important. The ability to identify and characterize adverse responses in preclinical models is critical to the drug development process and a necessary component of an Investigational New Drug (IND) application. Therefore, approaches to characterizing potential toxicities and immune responses induced by exosomes will be a necessary component of any effort to develop therapeutic exosomes.

In our article, “Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells,” that was just published online in the Journal of Extracellular Vesicles, we provide a general template process for comprehensively evaluating toxicity and immunogenicity of therapeutic exosomes in preclinical animal models. We started by dosing mice with wild type or engineered HEK293T-derived exosomes over a period of three weeks. Mice received 10 doses via intraperitoneal and intravenous routes of injection, and blood samples were collected at various times throughout the 3-week study. Animals were euthanized 24 hours after the last dose, and blood and all organs were collected from each animal for gross necropsy and evaluation of various markers of immune response and potential exosome-induced toxicity markers.

This study demonstrates one approach to immunogenicity and toxicity evaluations of human-derived exosomes in mice, and it highlights some of the variables that must be considered during these evaluations. For example, what are appropriate animal models in which to study immunogenicity and toxicity? What doses and dose regimens should be evaluated? How might the cell type from which the exosomes were harvested impact immunogenicity and toxicity? Just as with efficacy evaluations in animals, comprehensive study of these other factors will be necessary for safely moving therapeutic exosomes into human trials.

Acute liver failure is a potentially fatal consequence of severe liver injury. Liver transplantation may be necessary for survival if the liver injury exceeds the ability of the liver to regenerate (also called fulminant liver failure). New therapeutic interventions are needed to enhance tissue regeneration and improve the outcome of acute or fulminant liver failure. Previous studies have reported that mesenchymal stem cell (MSC) transfusions can improve function in liver facing acute failure. Stem cells can grow into multiple cell types and may support the replacement of functional liver tissue. In addition, paracrine effects — signalling between nearby cells — resulting from the release of soluble factors and extracellular vesicles (EV) may contribute to some of the beneficial effects observed with stem cell therapies.

A new study by Haga et al. reports on the beneficial effects of EV derived from stem cells. EV were isolated using classical ultracentrifugation methods. To mimic liver injury, D-galactosamine and recombinant tumor necrosis factor-α were injected into male mice. Subsequently, systemic administration of EV was shown to result in a dramatic improvement in survival. Whereas control animals receiving placebo showed complete lethality within 12 hours of D-galactosamine/TNF-α injection, the mice injected with mouse stem-cell-derived EV had a 57% survival at 24 hrs. When human MSC-EV were administered, a 37.5% survival was noted. Also noteworthy, survival was observed even with EV that had been cryopreserved. The EV reduced hepatic inflammation, likely by protecting the hepatocytes from apoptosis and recruiting Kupffer cells that protect from liver injury. The figure shows an overview of this process. Some of the beneficial effects were shown to be mediated by Y-RNA-1, a long non-coding RNA that is enriched within MSC-EV.

Stem-cell-derived extracellular vesicles repair tissue after acute liver failure

The beneficial effects of stem cells, mediated through EV and their RNA content, in severe injury models provides new avenues for investigation of the pathophysiology of liver injury and inflammation. These observations provide a very compelling justification for the future use of MSC-EV as therapeutics for severe liver injury.

Reference:
Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Stem Cells Transl Med. (2017) 6:1262-1272. doi: 10.1002/sctm.16-0226.

The role of extracellular vesicles (EVs) in cancer has recently become a promising area of research. A primary function of EVs is to deliver molecules from a donor cell to regulate cellular processes in a target cell. Little research has investigated what effect departing EVs may have on the donor cell.

In cancer, EVs from tumor cells can deviate from their original purpose. Altering vesicular content could benefit tumors, for example by increasing tumor proliferation or strengthening drug resistance.
 

Exosomal Packaging
Could donor tumor cells selectively shunt cancer-fighting molecules into secreted vesicles to escape their effects? Teng et al. thought this might be the case. They hypothesized that tumor cells specifically secrete tumor suppressor miRNA, namely miR-193a, into exosomes, a class of EV secreted via the endocytic membrane transport pathway, while oncogenic miRNAs are kept.

To test this theory, they validated miR-193a’s function in a mouse model. Specifically, they found that miR-193a targets Caprin1, a cell-cycle-associated protein, and arrests the cell cycle in phase G1. Thus, secreting miR-193a from the cell in exosomes would restart the cell cycle and enable a tumor cell to proliferate.

After studying miR-193a’s primary function, the authors explored what might facilitate its secretion. They showed that MVP (major vault protein) complexes with miR-193a and that knock-down of MVP leads to higher levels of miR-193a in the cell and less miR-193a in exosomes. They concluded that MVP mediates the sorting of miR-193a into exosomes. They also found that a higher level of MVP in the cell correlates with lower levels of miR-193a and higher levels of Caprin1, indicating that MVP aids in cell proliferation. Lastly, the researchers determined in a mouse model that export of miR-193a by MVP promotes metastasis of colon cancer to the liver.

Figure 1 shows a model of these interactions. In the pre-metastatic cell, miR-193a is freely expressed. In the tumor metastatic cell, MVP has complexed with miR-193a, driving it into exosomes to be secreted.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.

Figure 1: Model for the mechanism of colon cancer metastasis to the liver. Tumor suppressor mir-193a is sorted into exosomes by Major Vault Protein (MVP) and then secreted from the cell.


 

Cancer Biomarker
This finding implicates exosomal miR-193a as a potential biomarker for colon cancer. Could higher levels of exosomal miR-193a indicate a more aggressive disease? To answer these questions, the authors applied their findings in the clinical setting. Teng et al. examined the livers of mice with metastatic colon cancer, performing an extensive characterization of the levels of tumor suppressive and oncogenic miRNAs in normal and tumor cells and in the exosomes secreted by both. From that extensive analysis, they chose three miRNAs upregulated (miR-193a, miR-126 and miR-148a) and one miRNA downregulated (miR-196b) in exosomes from tumor cells and examined the levels of those miRNAs in exosomes purified from the blood (plasma) of 40 colon cancer patients, 15 with metastasis to the liver and 25 without. They found the same upregulation and downregulation of the 4 miRNAs in the patient population, with higher levels in the population with liver metastasis.

This exciting study identifies potential biomarkers for colon cancer. The two main findings highlight the importance of exosomes in cancer proliferation. First, the authors found that tumor suppressing miRNAs are packed into exosomes, while oncogenic miRNAs remain in the cell. Next, they found that there are higher amounts of tumor suppressing miRNAs in tumor-derived exosomes relative to exosomes from healthy cells. They also found that MVP acts as a mediator of these differences between metastatic and healthy cells. Teng et al. believe that once we develop a dependable method to purify exosomes, scientists may uncover further roles for exosomes in cancer progression.

Recent research from members of the ExRNA Communication Consortium (ERCC) suggests that extracellular RNAs (exRNAs) circulating in plasma play an active role in insulin resistance (IR). Insulin resistance is an incurable but manageable syndrome where the body stops reacting efficiently to the insulin hormone, which stimulates the uptake of glucose in the blood into cells and inhibits the body from using fat for energy, resulting in high blood sugar levels. The study, by Shav et al., points to certain exRNAs, particularly miR-122 and miR-192, as indicators and active players in IR regardless of the age, sex, or BMI of a person, which suggests that they may serve as more than metabolic markers and that they perhaps have functional, trans-organ roles in mediating IR.

Previous research has demonstrated that exRNAs have different functions in pathways relating to metabolic syndrome, which is a series of conditions that increase the risk of heart disease, stroke, and diabetes. For example, there is measurable miRNA dysregulation in obesity and in the progression of cardiometabolic disease. Other miRNAs are involved in brown/white fat specification, adipose tissue inflammation (Karbiener and Scheideler, 2014), and hepatic steatosis (fatty liver disease, Becker et al., 2015). Although these studies have identified specific exRNAs and miRNA networks that also have roles in IR, they have had either small sample sizes and lack validation in large populations or have been carried out in non-human models. In order to validate these data, the authors carried out a large-scale human translational study in which they analyzed detailed obesity-related phenotypic data from over 2,500 participants (most of whom were non-diabetic) from the Framingham Heart Study (FHS), an unrelated cardiovascular disease study (Feinleib et al., 1975).

To begin, the investigators analyzed blood samples from 2,317 non-diabetic study participants and quantified the plasma extracellular circulating exRNAs. They looked at RNAs [including piwi-interacting RNA (piRNA) and small nucleolar RNA (snoRNA)] expressed above a threshold level and excluded RNAs that were not found in at least 100 participants. From the resulting panel of 391 exRNAs, the investigators identified 16 microRNAs (miRNA), 1 piRNA, and 1 snoRNA that were associated with insulin after controlling for age, sex, and BMI. Of note, the abundance of miR-122 was shown to increase in a stepwise fashion as levels of insulin increased across the population. Higher levels of both miR-122 and miR-192 in the plasma were also consistently associated with a series of metabolic phenotypes, such as greater BMI and waist circumference, visceral fat quantity and quality, and liver attenuation. On the other hand, neither miRNA was associated with subcutaneous fat. These results were consistent whether the analysis included only the non-diabetic participants or the entire FHS population.

miRNAs function to regulate gene expression, so the authors conducted a pathway analysis to determine the targets of the 16 identified miRNAs. Almost unsurprisingly, all 16 miRNAs target insulin signaling pathways such that there is ample crosstalk and targeting of multiple IR-related genes by multiple miRNAs. This analysis validated findings from previous studies that implicated several miRNA target genes in the pathogenesis of IR, notably protein tyrosine phosphatase, nonreceptor type 1 (PTP1B) (Stull et al., 2012), mitogen-activated protein kinases (MAPKs) (Wang, Goalstone & Draznin, 2004), and 5′ adenosine monophosphate-activated protein kinase (AMPK) (Ruderman et al., 2013).

For the second part of the study, the investigators determined whether the miR-122 and miR-192 associations to age, sex, and BMI held true in a separate study population, a cohort of 90 overweight or obese young adults involved in the POOL study. Analyses of the youths’ plasma samples indicated that miR-122 (but not miR-192) was associated with greater IR after adjusting for age, sex, and BMI, and that this association remained even after the miRNA was analyzed independently of age, sex, BMI, or metabolite profile.

This study provides additional evidence and translational support for the role of exRNAs in IR. The findings indicate not only an association of exRNAs with insulin levels, but that the exRNAs may be playing an active role in the development or sustainment of IR. It is therefore critical to conduct further mechanistic investigations into the role of exRNAs in the metabolic architecture of IR.

Dr. Alissa Weaver, Vanderbilt University professor and Extracellular RNA Communication consortium (ERCC) member, will be inducted as an AAAS Fellow this Saturday, February 18, 2016. Dr. Weaver joins Dr. James Patton, also of Vanderbilt, and Dr. David Wong of UCLA as consortium members who are also current AAAS Fellows. This honor is bestowed upon her for her contributions to the field of cancer biology and studies of extracellular vesicles (EVs) in cell motility and cancer metastasis.

Alissa Weaver

Dr. Weaver’s academic career began at Stanford University where she double majored in Biology and Political Science. Always aspiring to be a physician, she then attended medical school at the University of Virginia, Charlottesville. However, along the way, she realized that she missed the academics of a PhD. “When I was in medical school, I realized that I really missed thinking about scientific discovery and was not being taught to do research,” she explained. “I really wanted to have the formal training of getting a PhD so I applied for the program from medical school.” After completing her MD/PhD at UVA, she traveled to Washington University, Saint Louis for 5 years where she did a Laboratory Medicine residency and a postdoctoral fellowship in the Department of Cell Biology and Physiology with Dr. John Cooper.

Finally in 2003, she accepted a faculty position at Vanderbilt University where she now remains as a full time researcher. Her lab focuses on all aspects of extracellular vesicles. The interest originally stemmed from her investigations of cell invasion, migration and cancer metastasis. The lab’s focus shifted as they learned that many of the secreted molecules that facilitated invasion were transported by EVs.

Part of the invasive nature of cancer cells in metastasis involves structures called invadopodia, actin-based protrusions of the plasma membrane that facilitate degradation of the extracellular matrix. For cells to invade, they secrete matrix-degrading proteinases. Work in Weaver’s lab demonstrated that not only were these proteinases carried by EVs but that hot spots for their secretion actually aligned with invadopodia.

Specifically, Weaver’s lab established that invadopodia are important sites for the docking and secretion of exosomes. Exosomes are extracellular vesicles secreted from many different cell types. They originate from multivesicular bodies (MVB), which are mature endosomes that contain many smaller vesicles. Secretion of exosomes occurs when these MVBs fuse with the cell membrane, releasing the molecules contained inside. Though normal cells may use environmental cues to regulate exosome secretion, cancerous cells constitutively turn it on.

Exosome cargoes mediate invadopodia biogenesis, stability, and activity

Exosome cargoes mediate invadopodia biogenesis, stability, and activity.
Source: Hoshino, et al. Cell Rep 2013

“One of the big questions we are working on is the cell biological aspects of these vesicles,” Weaver explained. “How they are made, how cargo gets sorted there, and what does that mean for their biological function after they are secreted? So that is where our work with the ERCC comes in.”

She hopes that working with the scientists of the consortium, they can understand how RNA and RNA binding proteins are trafficked into vesicles. Last year, in a paper published in Cell Reports, her group demonstrated one possible mechanism for the sorting of microRNAs into EVs. They demonstrated that Argonaute 2 (Ago2), part of the RISC machinery that binds to miRNAs, is transported in microvesicles and exosomes. Organization of Ago2 into exosomes is regulated by KRAS-MEK signaling. Dr. Weaver highlighted the study in a blog here mid-last year.

Despite these initial findings, Dr. Weaver admits it is difficult to determine how important extracellular RNA and miRNAs are in regulating cancer metastasis. “I honestly don’t think we know yet, and I think that the field is just now really trying to figure out what are the cargo components that are driving all of these phenotypes we have been trying to characterize so well.” She elaborated, “I think for both the protein and the RNA, the next big step for the field is trying to pin individual EV functions back to specific cargo molecules.”

Asked to reflect on her AAAS fellowship, Dr. Weaver turned the focus on her colleagues in the consortium. “I continue to be very impressed by the quality of investigators and the research being done by the ERCC. I mean really top people who are driving forward what I think is a tough problem.” She and fellow AAAS fellows Dr. Patton and Dr. Wong are, as Dr. Weaver pointed out, “just a small snapshot of fabulous investigators that are part of the consortium.”

The microRNA miR-155 plays a significant role in physiological and pathological processes in humans by blocking the functions of functionally important messenger RNAs of protein coding genes. We found that miR-155 was present in higher levels in cancers resistant to chemotherapy. By studying the association of miR-155 and tumor suppressor TP53 with cancer survival in 956 patients with lung cancer, chronic lymphocytic leukemia and acute lymphoblastic leukemia, we demonstrated that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that blocking or down-regulating miR-155 successfully resensitizes tumors to chemotherapy in vivo. We found that high levels of miR-155 and low levels of TP53 characterize the tumors from lung cancer patients with shorter survival time. Our findings support the existence of a miR-155/TP53 feedback loop involved in resistance to chemotherapy. To target this feedback loop and effectively alter resistance to therapy, we have developed a therapeutic nanoformulation of anti-miR-155 in a lipid nanoparticle (DOPC) and have shown it to be non-toxic in vivo for further pre-clinical work.

We thank our co-authors for their work and discussions that led to this blog.

miR-155 TP53 and resistance to chemotherapy

The first version of the miRandola database has been published in 2012. It contained 89 papers and miRNA data. Now, we have updated the database with 272 papers and we redesigned the website!

We are starting to add more RNA molecules such as lncRNAs and circRNAs.

The miRandola database 2017 includes:

  • 272 articles
  • 2704 entries
  • 6 extracellular RNA forms
  • 673 microRNAs
  • 12 long non-coding RNAs
  • 8 circular RNAs
  • 21 drugs
  • 9 organisms and animal models
  • 173 diseases and cell lines
  • More features are coming soon!

The exrna.org Research Portal is linked in our web page.

Website: http://mirandola.iit.cnr.it/