Linköping University | Department of Physics, Chemistry and Biology Type of thesis, 16 hp | Educational Program: Physics, Chemistry and Biology Spring term 2020 | LITH-IFM-G-EX—20/3869—SE
The Tumor Promoting Actions of
Exosomal miRNAs
Emil Johansson
Examinator: Jordi Altimiras Tutor: Carlos Guerrero-Bosagna
Datum 2020 05 29 Datum Dae Avdelning, institution
Division, Department
Department of Physics, Chemistry and Biology Linköping University
URL för elektronisk version
ISBN
ISRN: LITH-IFM-G-EX--20/3869--SE
_________________________________________________________________ Serietitel och serienummer ISSN
Title of series, numbering 20/3869
Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel
The Tumor Promoting Actions of miRNAs
Författare
Emil Johansson
Nyckelord
Exosome, exosomal miRNA, cancer, tumor proliferation, gene regulation, miRNA, tumor
Sammanfattning
Exosomes are nanosized vesicles that contain proteins and nucleic acids. They are released and taken up by many different cell types as a way of cell-to-cell communication. It has previously been recognized that exosomes released by cancer cells promote tumor progression. Moreover, recent studies have increasingly found evidence that microRNA contained within cancer derived exosomes plays an important role in tumor progression. In this manuscript, the current knowledge on exosomes, the sorting of microRNA into exosomes as cargo, the gene regulatory mechanisms of microRNA, and recent findings on the tumor promoting actions of exosomal microRNAs on gastric cancer, hepatocellular carcinoma and breast cancer, is reviewed. The information gathered emphasizes the importance of exosomal microRNAs that increase tumor growth in the development of cancer treatment and research.
Table of contents
Abstract ... 1
1. Background ... 2
2. Exosomal biogenesis, release and uptake ... 3
3. Exosomal miRNA sorting ... 4
4. MiRNA-mediated gene regulation ... 5
5. Exosomal miRNA and its role in tumor progression ... 7
6. Conclusion ... 10
7. Ethical considerations ... 10
8. Social relevance ... 11
9. Acknowledgements ... 11
1
Abstract
Exosomes are nanosized vesicles that contain proteins and nucleic acids. They are released and taken up by many different cell types as a way of cell-to-cell communication. It has previously been recognized that exosomes released by cancer cells promote tumor progression. Moreover, recent studies have increasingly found evidence that microRNA contained within cancer derived exosomes plays an important role in tumor progression. In this manuscript, the current knowledge on exosomes, the sorting of microRNA into exosomes as cargo, the gene regulatory mechanisms of microRNA, and recent findings on the tumor promoting actions of exosomal microRNAs on gastric cancer, hepatocellular carcinoma and breast cancer, is reviewed. The information gathered emphasizes the importance of exosomal microRNAs that increase tumor growth in the development of cancer treatment and research.
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1. Background
Exosomes are small vesicles, with a diameter of 30-150 nm, released by a variety of cells to the extracellular space through exocytosis (Hessvik & Llorente, 2018 ; Farooqi et al., 2018). These vesicles function as carriers of cargo and as a delivery system between cells in the body (Simons & Raposo, 2009). Because of the exosomes’ ability to deliver substances from one cell to another, they play an important role in cell-to-cell communication and several physiological and pathological functions (Mathivanan et al., 2010). How exosomes can influence cell functions depends on the content of the small vesicles. This exosomal cargo has previously been identified to consist of proteins, however various nucleic acids such as
mRNAs, non-codingRNAs and most importantly microRNAs (miRNAs), a class of small non-coding RNAs of 17-22 nt in length, have recently been identified as well (Zhang et al., 2015).
Interestingly enough, studies have frequently shown that exosomes released from various types of cancer cells promote the progression of tumors (Zhang et al., 2015 ; Hessvik & Llorente, 2018 ; Maia et al., 2018). An explanation for this phenomenon proposes that interactions between neighbouring cells in the primary tumor are important for the tumor growth and development. Because of this, and that exosomes can move from cell to cell in the tumor microenvironment, exosomes play an important role in the interaction between cells located far from each other to modify tumor microenvironments by projecting
pro-tumorigenic properties. (Maia et al., 2018). While this is a broad explanation for why exosomes pose as tumor progressing delivery systems, the underlying mechanisms of how exosomes can modify tumor microenvironments is not yet fully understood.
However, it has been shown that certain miRNAs are present in higher concentrations inside exosomes relative to cells and that these miRNAs seem to be sorted into exosomes (Hessvik & Llorente, 2018). Since the identification of miRNAs inside exosomes, researchers have therefore begun to study whether these exosomal miRNAs cause tumor progression through miRNA mediated gene regulation (Sun et al., 2018). In this review, the exosome and its miRNA cargo will be placed under the microscope to investigate the tumor progression actions of exosomal miRNA. The review will focus on the impact of exosomal miRNA on the tumor progression seen in different types of cancers, the mechanisms by which the exosomal miRNA from one cell can promote tumor progression on other cells, and also what
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2. Exosomal biogenesis, release and uptake
The formation of exosomes begins with the generation of multivesicular bodies (MVBs) from late endosomes, which are eventually released by the cell via exocytosis (Farooqi et al., 2018). As the word multivesicular suggests, MVBs are late endosomes that contain several intralumenal vesicles (ILVs) that accumulates during endosomal maturation. These ILVs are formed through invagination of the endosomal membrane when the cargo is sorted into late endosomes for transport towards lysosomes with, for instance, a degradation function (Huotari & Helenius, 2011). The MVB formation and endosomal cargo sorting requires the endosomal sorting complex for transport (ESCRT) (Simpson et al., 2008). It is this protein complex that facilitates the generation of ILVs by causing the endosomal membrane to invaginate away from the cytoplasm into the lumen of the endosome (Henne et al., 2013).
When an MVB has been formed, it can be subjected to two possible fates as shown in figure 1. It may be transported to and merged with lysosomes in the cell where the MVB will be degraded along with its contents, or it may be transported to the cell membrane where it merges with the cell membrane to release all ILVs to the extracellular space. These released ILVs are then referred to as exosomes (Mathivanan et al., 2010). Exosomes in the
extracellular space may then be internalized by other cells via endocytosis or fusion with the plasma membrane of the cell. The specific mechanisms of endocytosis depend on the type of cell internalizing the exosome, the cells’ physiologic state and on whether ligands or receptors on the exosomes surface match with the corresponding type on the cell membrane. Instead, membrane fusion of exosomes with the cell’s plasma membrane requires low pH, a condition that is often found in tumors (Abels & Breakefield, 2016).
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Figure 1: Schematic figure showing the two possible fates of MVBs in the cell. Pathway A shows the MVB moving to the lysosome where it is degraded along with the ILVs and their content. Pathway B shows the MVB moving to and merging with the cell membrane to release the ILVs (now exosomes) into the extracellular space through exocytosis.
3. Exosomal miRNA sorting
Ever since exosomes were found to contain nucleic acids such as miRNAs, studies have shown that certain miRNAs are enriched in exosomes relative to their transmitting cells (Guduric-Fuchs et al., 2012 ; Goldie et al., 2014). This suggests that there is some sort of mechanism regulating selective sorting of miRNAs into exosomes and may be used to target functions of similar cells (Guduric-Fuchs et al., 2012).
Firstly, one described mechanism of miRNA secretion relates to the enzyme neutral sphingomyelinase 2 (nSMase2). Kosaka et al. demonstrated that reduction in the cellular activity of nSMase2 enzymes led to the reduction of miRNA secretion into exosomes, while overexpression of nSMase2 led to increased levels of exosomal miRNA instead (Kosaka et al., 2013). This suggests that nSMase2 plays an important role in miRNA secretion through exosomes.
A mechanism of sorting was then suggested by Villarroya-Beltri et al. in 2013. They found that the loading of certain miRNAs into exosomes was directed by the protein hnRNPA2B1 through the recognition of specific sequence motifs present in these miRNAs. They also found that the hnRNPA2B1 protein inside exosomes were sumoylated (Villarroya-Beltri et al., 2013), a post transcriptional modification where a member of the small ubiquitin-like modifier protein family is attached to lysine residues in a target protein (Yang et al., 2017). Moreover, the researchers found that this sumoylation controls the binding of the protein to the miRNA motifs and consequently miRNA sorting (Villarroya-Beltri et al., 2013). Another study by Koppers-Lalic et al. suggests that the 3′-end of the miRNA may contribute to the miRNAs sorting into exosomes. The researchers found that miRNAs in B cells possess different 3′-ends compared to the same miRNA species found in exosomes. While miRNA enriched in B cells had adenylated 3′-ends, miRNA enriched in exosomes were uridylated instead, suggesting that these posttranscriptional modifications may affect miRNA sorting into exosomes (Koppers-Lalic et al., 2014).
It has also been suggested that the miRNA induced silencing complex (miRISC) also plays a role in the sorting of miRNA into exosomes. The evidence supporting this includes the
5 identification of the well-known miRISC component AGO2 inside exosomes (Gibbings et al., 2009). Additionally, AGO2 knockout is suggested to decrease the types or abundance of certain miRNAs that are preferentially exported by exosomes (Guduric-Fuchs et al., 2012). Furthermore, the main components of the miRISC complex have been discovered to be located with MVBs (Gibbings et al., 2009). Interestingly, the blocked movement of MVBs to lysosomes leads to over-accumulation of miRISCs in cells, while blockage of MVB formation led to the loss of miRISCs (Lee et al., 2009).
4. miRNA-mediated gene regulation
After the exosomal miRNA has been delivered to the target cell, the miRNA can influence the cellular functions in two main ways. The first one is the well understood function of
regulating and changing the expression levels of target genes. However, it can in some cases also function as a ligand that binds to toll-like-receptors, thereby activating immune cells (Zhang et al., 2015). While the latter function is interesting and needs further investigation as well, this review will not discuss this function.
miRNAs regulate gene expression through interference with a target gene’s mRNA product (Krebs et al., 2018). It is important to note that the miRNA-mediated gene regulation will not function with the miRNA in isolation. The miRNA must first be incorporated into a complex known as the RNA-induced silencing complex (RISC). RISC consists of a single stranded miRNA and several proteins, most notably the Argonaute RISC Catalytic Component 2 (Ago2) (Santhekadur & Kumar, 2020). RISC may control mRNA expression in two main ways, either by degradation of the mRNA or by inhibition of the mRNA’s translation. While most mechanisms of miRNA are inhibitory, miRNA may in some cases be required for translation activation as well (Krebs et al., 2018). Although originally it was thought that animal miRNA represses gene expression only by translation inhibition and not degradation, while in plants it was thought to be the other way around, we now know that miRNAs can regulate gene expression through both mechanisms in both kingdoms (Huntzinger &
Izaurralde, 2011). These two miRNA-mediated downregulation types can be further divided into four mechanisms that act directly and indirectly on the mRNA. These include translation initiation, post-initiation, co-translation, as well as miRNA-mediated mRNA degradation (Eulalio et al., 2008).
The initiation inhibitory mechanisms include the competition between RISC and a variety of initiation factors. These involve binding to the mRNA cap instead of the factor eIF4E (figure 2.A) and recruiting the eIF6 factor, which prevents the large 60s ribosomal subunit from
6 joining the small subunit (figure 2.B). RISC may also prevent a closed-loop formation
required for translation through deadenylation (figure 2.C), effectively inhibiting the
translation initiation phase (Eulalio et al., 2008 ; Orang et al., 2014 ; Krebs et al., 2018). Post-initiation mechanisms include RISC interfering with ongoing translation by simply blocking elongation or by prematurely promoting dissociation of the ribosome (figure 2.D) (Eulalio et al., 2008 ; Orang et al., 2014 ; Krebs et al., 2018). The co-translational mechanism involves RISC inducing degradation of the translated protein product through proteolysis by an
unknown protease (figure 2.E). This occurs during the process of elongation in translation and leads to degradation of the peptide product (Eulalio et al., 2008 ; Krebs et al., 2018). Finally, RISC may also recruit a variety of proteins and enzymes that facilitate mRNA cleavage and degradation through decapping and deadenylation (figure 2.F) (Eulalio et al., 2008 ; Orang et al., 2014 ; Krebs et al., 2018).
Figure 2: The possible mechanisms of downregulation, mediated by miRNA. A) RISC
competes with eIF4E by binding to the mRNA cap. B) RISC recruits eIF6, hindering the large ribosomal subunit from joining to the small subunit. C) RISC initiates deadenylation,
hindering the formation of a closed-loop formation. D) RISC blocks translation elongation and promoting premature dissociation of ribosomes. E) RISC induces proteolysis of the translational product. F) RISC induces mRNA degradation by recruiting decapping and deadenylation enzymes. Figure modified from Krebs et al. 2018
In previous studies, miRNAs have proven to be important to understanding cancer
7 overexpressed in Hepatocellular carcinoma tissue, where the miRNA acts as an oncogene by targeting and silencing the tumor suppressor gene CSMD1 (Zhu et al., 2016). Moreover, miR-21 has been shown to act as a oncogene that promotes apoptosis resistance and metastasis in prostate cancer by targeting MARCKS (Li et al., 2009). The same miRNA has also been shown to target two other tumor suppressor genes, providing further support for its role as an oncogene (Schramedei et al., 2011).
5. Exosomal miRNAs and their role in tumor progression
Tumors do not only consist of malignant cells of one type, but also of other non-malignant cell types such as lymphocytes, cells associated with vasculature, fibroblasts and many more. These cell types form the tumor microenvironment (TME) together with the malignant cells, , with the non-malignant cells often having tumor promoting functions. Because of this, it may be of importance to not only target malignant cells during cancer treatment, but also the non-malignant cell types within the TME (Balkwill et al., 2012). Evidence suggests that cancer-derived exosomes may influence the TME to form a pro-tumorogenic soil and, in that way, manipulate the complex environment to promote cancer growth and spread (Kahlert & Kalluri, 2013). Since miRNAs have been shown to promote cancer already (Li et al., 2009 ; Schramedei et al., 2011 ; Zhu et al., 2016), the idea that exosomal miRNAs promotes tumor progression is not farfetched. Therefore, many studies have been conducted to test the hypothesis that exosomal miRNAs promote tumor progression through miRNA mediated gene regulation.
It has been previously shown that exosomes from gastric cancer (GC) cells promote tumor proliferation, in part by activating the Phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/extracellular-regulated protein kinase pathways (Qu et al., 2009). Since then, research on the exosomal actions on GC proliferation have expanded towards exosomal miRNAs as well. In 2017, Ren et al. showed that exosomes derived from GC cells contained more types of miRNAs than exosomes from normal cells. They also showed that hundreds of particular miRNAs were upregulated in exosomes from cancer cells compared to normal cells (Ren et al., 2017).
Yang et al. later showed that exosomal miRNAs in GC cells had elevated levels of miR-423-5p compared to normal cells. They also found that the expression level of miR-423-miR-423-5p was correlated with lymph node metastasis, and that high levels of miR-423-5p were associated with poor clinical outcome. Moreover, this miRNA could be internalized into GC cells to enhance cell proliferation and migration by inhibiting the expression of the tumor suppressor,
8 suppressor of fused protein (SUFU) (Huan Yang et al., 2018). In another study it was further discovered that miR-130a was significantly upregulated in GC cells and their secreted exosomes. This miRNA was then shown to be internalized by vascular endothelial cells, where it promoted angiogenesis and tumor growth by targeting and downregulating the translation of the transcription factor c-MYB, shown to be correlated with angiogenesis (Haiou Yang et al., 2018).
Similar to other cancers, such as pancreatic cancer (Li et al., 2009 ; Schramedei et al., 2011), Meng et al. (2007) showed that miR-21 was overexpressed in Hepatocellular carcinoma (HCC) cells. Furthermore, they found that the miRNA could promote cell invasion, migration and growth via repression of the tumor suppressor phosphate and tensin homolog gene
(PTEN) (Meng et al., 2007). Later, in 2018, Zhou et al. found elevated expression of miR-21 in exosomes derived from HCC cells, suggesting that this tumor progression related miRNA is secreted by exosomes (Zhou et al., 2018). Moreover, when HCC derived exosomes
containing miR-21 were transferred to hepatic stellate cells (HSCs) in the tumor parenchyma, miR-21 could convert the HSCs to cancer-associated fibroblasts, again, by targeting PTEN and promoting HCC proliferation (Zhou et al., 2018).
In 2019 Tian et al. suggested that dysregulated pH, a hallmark of early-stage HCC, correlated with poor prognosis and promoted the upregulation of exosomal miR-21 and miR-10b, which in turn also promotes tumor proliferation in HCC (Tian et al., 2019). They suggested that increased activity of hypoxia inducible factors (HIFs), specifically HIF-1α and HIF-2α promoted this upregulation. It was shown that HIF-1α and HIF-2α could directly bind to the promoter regions of miR-21 and miR-10b, thereby upregulating their cellular expression (Tian et al., 2019). This was suggested to result in increased miR-21 and miR-10b levels in HCC derived exosomes (Tian et al., 2019).
Other studies have shown that the expression levels of exosomal miR-224 and miR-155 is also increased in exosomes derived from HCC cells (Cui et al., 2019 ; Sun et al., 2019). miR-224 was shown to promote proliferation and invasion of HCC cells by directly targeting the 3′-UTR region of glycine N-methyltransferase (GNMT) mRNA (Cui et al., 2019), a tumor suppressor of HCC (Hung et al., 2018). The exosomal miR-155 was shown to target and downregulate PTEN instead, thereby triggering the PI3K-Akt pathway activity in target cells and mediating HCC development (Sun et al., 2019).
In breast cancer (BC), tumor associated macrophages (TAMs) have been reported to promote BC progression and metastasis. Furthermore, macrophages have been shown to produce
9 exosomes containing miRNA that is taken up by adjacent cells in the microenvironment (Yang et al., 2011). Because of this, Yang et al. investigated whether exosomal miRNA could explain the TAMs ability to promote BC progression and metastasis. They found that
exosomes secreted from IL-4-activated macrophages delivered miR-223 to BC cells, promoting BC cell invasion (Yang et al., 2011).
In another study on exosome-mediated promotion of BC invasion, Singh et al. found that metastatic BC MDA-MB-231 cells contained high levels of miR-10b. In contrast,
non-metastatic BC cells and non-malignant breast cells did not contain similar levels. They further found that released exosomes containing miR-10b could be taken up by non-malignant
epithelial breast cells to promote cell invasion through the translational repression of HOXD10 (Singh et al., 2014), an angiogenesis suppressor gene (Myers et al., 2002).
Since specific exosomal miRNAs have been shown to contribute to tumor proliferation in certain cancer types, they may serve as novel biomarkers for cancer diagnosis and prognosis (Huan Yang et al., 2018 ; Meng et al., 2007 ; Sun et al., 2019). Moreover, the knowledge of certain exosomal miRNAs that promote tumor progression may serve as potential targets to regulate in cancer treatment (Singh et al., 2014 ; Hung et al., 2018). It has also been suggested that some exosomal miRNAs may directly work as therapeutic treatments in cancers. MiR-122 have, for example, been shown to promote chemosensitivity in HCC and could be delivered to HCC cells via exosomes (Lou et al., 2015). Further research on the cancer promoting actions of exosomal miRNAs is needed to unravel the variety of miRNAs contributing to tumor proliferation in order to develop new ways of cancer diagnosis, prognosis and treatment in the future.
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6. Conclusion
Ever since the cancer promoting properties of exosomes derived from cancer cells were recognized, many studies have attempted to seek out how exosomes induce cancer proliferation. Existing studies suggests that exosomal miRNAs and their gene regulatory actions can partly explain this question. Most notably, exosomal miRNAs may act as oncogenes that are upregulated by cancer cells and secreted in higher amount in exosomes. These exosomes can then be internalized into cells within the tumor microenvironment that may or may not be malignant. The most prominent mechanism of cancer promotion related to exosomal miRNAs seems to be downregulation of tumor suppressor genes. However, the exact mechanisms of action have been analyzed for only a few specific exosomal miRNAs. Moreover, it seems like certain exosomal miRNAs are cancer-type specific, which entails that certain miRNAs may be used as novel biomarkers for diagnosis and prognosis of specific cancer types. In some cases, it may also be possible to target or use exosomal miRNAs to treat certain types of cancer. By discovering more miRNAs that promote tumor progression, and by understanding the mechanisms of exosomal delivery and miRNA-mediated gene regulation, we can exploit our knowledge about exosomal miRNAs to further improve cancer treatment and research. Therefore, further research on the actions of exosomal miRNAs on tumor progression is needed. Especially, research is needed on the mechanisms involved in sorting miRNAs into exosomes and their means of gene regulation, since present-day studies lack this significant information.
7. Ethical considerations
While conducting any type of research, it is important to consider the ethics of carrying out a study. In this review, none of the included studies have discussed any ethical considerations about their research. Because of this it can only be assumed that the researchers had
considered the ethical aspects of their studies as well as followed routines for Good Laboratory Practice.
Since reviews are comprised of many different sources which together make up all the important information in the paper, there are a lot of ethical aspects related to the process of writing to consider as well. These include plagiarism, copyright and conflict of interest. Plagiarism can, and should, be avoided by clearly specifying from which source a statement is coming from. If a statement is directly cited, that is without rewriting it in your own words, it is important to write the statement in quotation marks as well. Similarly, copyright issues from using figures or tables based on other sources can be avoided by stating from where the figure or table comes from, or from where it is based.
11 Conflict of interest related to the topic can be a problem while writing since a scientific paper should highlight the true information about the topic even if the authors views about the topic might be contrary to the truth. Because of this it is important to check the studies sources of funding and the authors affiliation to these sources on every reference before including it into a paper. As the author, it is also important to think of your own potential conflicting interests while writing, so that you do not leave out or over emphasize specific sources that contradicts or correspond to your views.
8. Social relevance
From a societal point of view, the understanding of the tumor promoting actions of exosomal miRNAs are of great importance and interest. Since cancer is still one of the leading causes of death worldwide, new understanding of cancers and new modes of diagnosis and prognosis is still needed to further improve cancer treatment. By further understanding exosomal miRNAs cancer promoting actions, new cancer treatment developed by this knowledge may therefore be used to treat cancer patients with cancers that have previously been difficult to treat or diagnose and prognose without this knowledge.
9. Acknowledgements
I would like to thank my supervisor Carlos Guerrero-Bosagna for his guidance and support throughout the project. I would also like to thank my course mates Isabella Blomlöf, Magnus Paulsson and Linnéa Farajzadeh Lindroth for their feedback, as well as my examinator Jordi Altimiras.
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