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Cancer: on the trail of miR-503

8/17/15

A recent article published by the journal Oncotarget (1), researchers at the Molecular Angiogenesis Laboratory in the GIGA-Cancer Unit at the University of Liege demonstrated the action of endothelial exosomes and the microRNAs they contain in relation to tumor growth. Their study shows that when the miR-503 microRNA is transferred into cancerous human breast cells it can bring about changes resulting in a reduction of the proliferative and invasive ability of these cells.

At the beginning of the 2,000s, biologists realized that the microRNAs, small non-coding RNAs, played a much more important role in mammals and humans that had previously been thought. While scientists were aware that these RNAs played a part in plant metabolism, about which very little is still known for that matter, they were nonetheless unaware that they were strongly expressed in humans, and that they varied greatly in many different pathologies.

Exosomes had already been identified in the 1980s as being small spherical-shaped membranous vesicles with a diameter varying between 40 to 100 nanometers. Initially, it was commonly accepted that they were secreted by the reticulocytes. They were believed to have the function of eliminating proteins that had become useless to the cell after being deposited in the extracellular medium. It later emerged that, not only could they be secreted by different types of cells and that their molecular composition and function depended on the type of cell they came from, but that they also enclosed microRNAs as well as other non-coding forms of RNA.

“Two theories were then put forward. According to the first of these, the cell got rid of an overflow of microRNAs. According to the second theory, these microRNAs played a part in communication between the cells”, explains Ingrid Struman, a research associate at the FNRS and head of the Laboratory of Molecular Biology and Genetic Engineering in the GIGA-Cancer unit of the University of Liege. For the moment, the debate is still open, at least from a semantic point of view, in so far as the notion of "communication" creates the idea of purpose which would imply a voluntary action, something that would not seem to be applicable to a cell.

ExRNAs

Many unknowns

However, the idea came into being that microRNAs transferred by exosomes could have a function. It was, in fact, demonstrated that on contact with a recipient cell, they are capable of modifying the genetic response of these cells and therefore the phenotype.

Currently, the mechanisms by which the export of microRNAs into the exosomes is regulated is still not fully understood even though some work has demonstrated the intervention of a  specific RNA binding protein as being involved in some cases. “At GIGA-Cancer, we are trying to identify the proteins capable of transferring some determined microRNAs but not others to the exosomes”, says Ingrid Struman.

Another unanswered question that remains is: how does the exosome target the cell to which it will transfer its content? Some studies suggest that there is an interaction from protein to protein similar to that of the ligand-receptor type of interaction, between the exosome and the target cell, but this has not been proved to date. In practice, at the moment when it transfers its contents, the exosome is endocytosed by the target cell after fusion of the membranes. However, it remains unknown whether the exosomes produced by a cell are uniform and will always target the same type of cell. “We are not certain, but we think that the content of the exosomes can vary according to the moment they are produced”, explains Ingrid Struman.

The word “contents” must be used reservedly because some authors think that the microRNAs are not necessarily lodged inside the exosomes but can attach themselves to the exosome membrane by means of an electric charge.

The quantity of exosomes secreted depends first and foremost on the type of cell. For example, the endothelial cells produce a lot of exosomes and according to Ingrid Struman, are probably those that produce the most. The cancer cells are not at rest however. Through the work carried out at GIGA-Cancer by the Molecular Angiogenesis Laboratory, it seems that the quantity and content of exosomes are modulated by the conditions of the cells that produce them. The team of researchers from Liege established that endothelial cells placed in conditions of stress (hypoxia, presence of chemotherapeutic agents...) secreted an increased number of exosomes.

MiR-503: antitumor microRNA

At GIGA-Cancer, Ingrid Struman's group focusses on the transfer of the microRNA from endothelial cells into cancer cells during tumor angiogenesis. Before this work, no information was available on the role of endothelial exosomes and the microRNAs they contain for tumor growth. The Molecular Angiogenesis Laboratory was the first to shed light on the existence of a transfer of the microRNAs of the endothelial cells to tumor cells by means of the exosomes (1). The researchers then showed that if endothelial cells were grown in culture in a pro-tumor environment, the content of the exosomes they exported became modified. Among the microRNAs whose presence became reduced, the biologists at GIGA-Cancer identified miR-503 in particular. Functional studies led them to conclude that this microRNA show antitumor properties: when transfected into human breast cancer cells, it caused a phenotypic change that had the consequence of reducing the proliferative and invasive potential of these cells.

Chemotherapy - exosomes“Our theory was that the tumor cells, when implanted in the host, send a signal leading to a reduced export of miR-503 by the endothelial cells thus reducing the antitumor response of the host, explains Ingrid Struman. In order to assess the relevance of this observation in humans, we wanted to determine whether the plasmatic level of microRNAs was modulated in patients affected by breast cancer. The measurement we carried out revealed an increase in the plasmatic RNA level in the blood of patients following treatment by an adjuvant chemotherapy treatment”.

This led to the inevitable conclusion that the chemotherapy prescribed for these patients could have an impact that goes beyond a mere direct action on the cancer cells. In fact, the observed increase in the plasmatic level of Mir-503 could be partly due to an increase in the secretion of antitumor microRNAs by the endothelial cells. At this stage, the researchers did not know if this was indeed the case or whether other cell types were involved.

But this is not the whole story: it also seems that the phenotypic change induced by miR-503 in the tumor cells modifies their production of angiogenic factors. This results in an inhibition of angiogenesis.

All the available data reveals for the first time the involvement of endothelium in the modulation of tumor development through the export of the miR-503 microRNA in response to chemotherapy. “This process could be complementary to the direct effects generated by the miR-503 microRNA on the tumor and enhance the response of the host during treatment of the disease”, explains Ingrid Struman.

Therapeutic prospects

Communication (the term is used reservedly) between endothelial cells and tumor cells was established in vitro. But up to the present, the researchers in the Molecular Angiogenesis Laboratory have not been able to show the origin of miR-503 in vivo. They would specifically like to isolate endothelial exosomes in patients suffering from breast cancer and demonstrate an eventual modification of the expression of microRNA. Unfortunately, this objective is faces an obstacle in terms of the absence of techniques in relation to research into specific types of exosomes by means of antibodies.

What therapeutic possibilities can be anticipated based on the work of the GIGA-Cancer team? According to Ingrid Struman, two avenues can be explored. The first would involve the injection of the antitumor microRNA into the blood stream in order to increase the level beyond the increase brought about by chemotherapy. Applied to other microRNAs, this technique is at the heart of clinical trials for several pathologies. Ingrid Struman cites the example of miR-122, in phase two of the treatment for hepatitis C. The second approach would be centred on the increase of the miR-503 load in the secreted exosomes. “For the moment, we do not know how to increase the export of microRNAs in these vesicles”, comments the researcher. “It is an objective of our future studies. If we succeed in identifying the proteins involved, we will be able to act on them.”

Cancer sein exosomes

 

An early function

Although it has concentrated most of its efforts on miR-503, the Molecular Angiogenesis Laboratory has also identified some other microRNAs capable of intervening in the communication between endothelial and tumor cells in breast cancer. The study of some of them has revealed, for example, that the variations in the level of miR-146 exported by the endothelial cells were modelled on those of miR-503. In addition, this interest in the therapeutic action based on miR-146 might be to exclude the fact that, as studied in peripartum cardiomyopathy (2), this microRNA has been shown to be actively involved in immunity (3).

Beyond the research undertaken at the University of Liege (in collaboration with the CHU) in the context of breast cancer, many other studies have shown that the circulating levels of various microRNAs changed in case of tumor development and following chemotherapy treatment(4). “A long-term study (5) monitored smokers”, says Ingrid Struman. “Some of them developed lung cancer. Among those who developed lung cancer, we noticed that the profile of their circulating levels of certain microRNAs were already activated even before we were able to detect the presence of tumor cells in the lungs”.  As the researcher explains, the early modifications of the circulating levels of these microRNAs leads us to the conclusion that they were involved in tumor development at an early stage. In addition, as is the case with miR-503, their presence can be observed in different cancers having higher or lower plasmatic levels according to the type of tumor. It would also seem to be the case that according to their nature but also to the category of cancer, the microRNAs can exert either an antitumor or a protumor action.

Murine models

In the aftermath of work which merited publication in the journal Oncotarget in April 2015(1),Ingrid Struman’s team began a study of patients who had suffered from breast cancer in order to determine whether certain microRNAs, and in particular miR-503, could be predictive of a relapse and, consequently, could serve as biomarkers. Another ambition: during resistance to adjuvant chemotherapy, to determine whether the tumor cells are capable of modifying their ability to respond to miR-503 or other microRNAs or whether in response to the action of cancer cells, the eventual transfer of circulating microRNAs by the endothelial cells dries up.

Another study, on murine models in this case, commenced recently. Its objective is the observation, in vivo, of the effect of miR-503 on tumor progression. One particular test involved the production of exosomes overloaded with this microRNA and their injection into the blood stream. The result has not been conclusive to date. The reason for this is possibly because, in natural conditions, the exchanges which take place between endothelial and cancer cells via the exosomes occur over a short distance, that is to say, in the tumor microenvironment. Also the remote injection of exosomes seems to be problematic. “This is why we are going to try to directly implant into the targeted organ both the tumor and the exosomes”, says Ingrid Struman.

It remains to be established how miR-503 influences tumor progression in the animal.

 

(1) Bovy N., Blomme B., Frères P., Dederen S., Nivelles O., Lion M., Carnet O., Martial J.A., Noël A., Thiry M., Jérusalem G., Josse C., Bours V., Tabruyn S.P., Struman I., Endothelial exosomes contribute to the antitumor response during breast cancer neoadjuvant chemotherapy via microRNA transfer, Oncotarget, 2015 Apr 30;6(12):10253-66.

(2) Halkein, J., Tabruyn, S.P., Ricke-Hoch, M., Haghikia, A., Nguyen, N.Q., Scherr, M., Castermans, K., Malvaux, L., Lambert, V., Thiry, M., Sliwa, K., Noel, A., Martial, J.A., Hilfiker-Kleiner, D., and Struman, I. (2013). MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy. The Journal of clinical investigation 123, 2143-2154.
(3) Boldin, M.P., Taganov, K.D., Rao, D.S., Yang, L., Zhao, J.L., Kalwani, M., Garcia-Flores, Y., Luong, M., Devrekanli, A., Xu, J., Sun, G., Tay, J., Linsley, P.S., and Baltimore, D. (2011). miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. J Exp Med 208, 1189-1201.
(4) Freres, P., Josse, C., Bovy, N., Boukerroucha, M., Struman, I., Bours, V., and Jerusalem, G. (2015). Neoadjuvant chemotherapy in breast cancer patients induces miR-34a and miR-122 expression. Journal of cellular physiology 230, 473-481.
(5) Boeri, M., Verri, C., Conte, D., Roz, L., Modena, P., Facchinetti, F., Calabro, E., Croce, C.M., Pastorino, U., and Sozzi, G. (2011). MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A 108, 3713-3718.


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