Please notice that this presentation aims at summarizing currently available data only without the claim of representing established knowledge. Thus, information presented in this course may be subject to change in the future due to the rapid development of the field of extracellular vesicles. Okay. So this presentation has been prepared by these lovely human beings here. So you've Carolina Soekmadji, David Greening, and Mary Bebawy all based in Australia. So my name is Dave Carter, I'm just narrating this, that these guys put together this beautiful presentation for me to describe for you. So let's get started then. So the cancer progression requires the ability of cancer cells to evade any onslaughts that threaten their ability to grow at the primary site, but also at the secondary metastatic site. In the past few decades, study on cancer progression has focused on its ability to drive some key pathways known as the cancer hallmarks. These hallmark capabilities, they are multi-step process of human tumor pathogenesis that allows cells to become more aggressive, more tumorigenic, and ultimately malignant. We'll talk more about some of these cancer hallmarks in one of the other MOOC presentations that we'll be doing. So the hallmark capabilities that can be driven by changes in the cells themselves are shown for example by alterations that cause genomic instability, cause mutations but also changes in the epigenetics. But they're also mediated by surrounding tissue microenvironments, so stromal remodeling. This interaction with the tumor microenvironment, the tumor cells with the environment has emerged as a really important factor in regulating tumor behavior. So the complex tissue microenvironments that the interaction there, it can support cancer sustained growth, invasion, and the ability to metastasize. Now, this communication is bidirectional. So it occurs between cells and the environment, and it's traditionally known to occur via various methods, clothing direct cell to cell contact, or the secretion of soluble factors for example, cytokines, chemokines, and growth factors. However, in the last few decades, it's become really clear that extracellular vesicle trafficking is an important mechanism of cell-to-cell communication, that can support cancer growth and cancer development. We now know that EVs have an important functional role in long-range communication between tumor cells and distant sites of metastasis. So we know that all cells ubiquitously release extracellular vesicles, and we know that they can contribute to the various hallmarks of cancer, and they can do that by transferring cargo between cells, and between cancer cells, and between cells in the tumor microenvironment. There are many different ways in which they can do this. So for example, they can contribute towards antigenesis. So communication between cancer cells and endothelial cells can promote and sustain antigenesis. We know that for example in the bottom left here, we know that EVs released by cancer cells can modulate the behavior of for example, fibroblasts or other cells in the tumor microenvironment through stromal remodeling. We know that EVs are involved in immune regulation, so [inaudible] cancer cells and the EVs that they release is involved in immune evasion for example. We also know that cancer cell can help to determine organ tropism in metastasis, but it can also be involved in epithelial-to-mesenchymal transition of various cell types, and so we know that they're involved in metastasis. Now, in this particular lecture, we're going to be focusing on the role of EVs in cancer proliferation and survival, particularly. So we'll be discussing some of the various mechanisms by which cancer cells can alter proliferation and survival of tumor cells which obviously has impacts on tumor progression, and drug resistance, etc. The potential freebies to serve as vehicle to transfer information between cells, has really stimulated a lot of interest in the past decade. So this mode of communication allows the ability to expand the repertoire of communication molecules from just secreted proteins and small molecules to include nucleic acids, lipids, membranes, and cytoplasmic constituents. So communication via information transfer might occur by the fusion of EVs with the recipient cell for example, either at the plasma membrane or at an endosomal membrane following uptake, resulting ultimately in the functional delivery of EV cargo. EVs can deliver cargoes which drive cancer proliferation, and we know this. But these include but are not limited to molecules which can activate pathways by growth factors inhibiting pathways regulated by tumor suppressor genes, can increase expression or mutations of oncogenes, and activate intracellular signaling pathways, or potentially disrupt negative feedback mechanisms. So all of these different pathways can occur as a means to drive in proliferation. So EVs can also drive cellular proliferation by delivering cargo which enables the target cells to acquire drug resistance phenotype, or a drug resistance phenotype. That also contributes ultimately to cancer proliferation. Let's start looking at some examples of how these EVs can stimulate the growth and proliferation of cancer cells. So first, we're going to give some examples of EVs associated growth factors and their receptors. So growth factors are a broad terms, a broad term which covers molecules that can induce cell proliferation, and they can act as signaling molecules between cells, and have been shown in the past that they are abundant in condition medium. Growth factors include homologues of the fibroblast growth factor, FGF epidermal growth factor, EGF, and transforming growth factor beta TGF beta families. So there's now accumulating evidence that shows that EVs can transfer growth factors or their receptors, and activate multiple signaling pathways in target cells including BMPs, FGFs, SHH, and the wind signaling pathways, amongst others. So let's start having a look at some of these examples. So in their role in mediating cell proliferation and survival, Al Nedawi et al have shown that EVs can transfer active oncoprotein, EGFR, epidermal growth factor receptor variance, and this particular example here's EGFR of variant three, or the EGFRvIII from cancer cells. Now, these EVs were shown to cause a horizontal propagation of proliferative survival and mitogenic and antigenic capacity. So these vesicles can transfer EGFRvlll from the expressing cells. The EGFRvlll expressing glioma cells to glioma cells that lack this variant, this EGFRvlll varying, and these EVs can then also cancer cell morphology, and increase anchorage independent growth capacity in the recipient cells in vitro. So upon EV-mediated uptake by these recipient's cells, the EGFRvlll can then activate MAP-Kinase signaling pathways and Protein Kinase B and Akt signaling pathways thereby inducing morphological transformation and accelerating cancer growth. Then cellular transformation downstream of EGFRvlll is mediated by changes in cellular proliferation and survival reflected by an increase in expression of the anti-apoptotic protein Bcl-xL of cells treated with EGFRvlll containing vesicles. So they have a number of different effects on the recipient's cells. Now, EVs derived from cancer cells overexpressing wild-type EGFR can also induce changes. They can induce angiogenesis for example by transferring the receptor, the EGF receptor to nearby endothelial cells. This then triggers the release of vascular endothelial growth factor VEGF and subsequent autocrine signaling activation by its receptor VEGFR2. So there's an example of how these vesicles when they're received by the endothelial cell can induce angiogenesis. In brain metastasis, astrocytes may epigenetically down-regulate the expression of tumor suppressor PTEN in metastatic cancer cells, by transferring a set of microRNAs via vesicles. So these microRNAs can then down-regulate the tumor suppressors in recipient cells. This leads to increased secretion of chemokines like CCL2 which then recruits Iba-1 expressing myeloid cells that will then reciprocally enhance the outgrowth of brain metastatic cells. So this then contributes to that proliferation and the expansion and progression of the cancer. Another growth factor like TGF Beta 1 has also been reported to be transferred via vesicles via EVs, which is able to then cause transformation of fibroblasts into myofibroblasts. So these myofibroblast cells involved in wound healing and inflammation and in cancer, we know that they can potentially play a role in cancer progression, and they may be indicative of cancer progression. In this particular instance for example, it's not necessarily directly linked to proliferation, but it shows that these EVs have multiple different effects. So the role of growth factors in the receptors in EVs are really intensely studied, and they've been continuously investigated as demonstrated by increasing number of publications that have been reported, that have been showing to involve looking at growth factors in vesicles for example, like FGF, or interleukins. EVs are also associated with oncogenes and mutants, and EVs may transfer tumor DNA. A study by Bergsmedh et al showed that apoptotic vesicles can transfer mutant H-ras_V12 the DNA encoding this H-ras_V12 and human c-myc from transfected fibroblast to wild-type fibroblasts. These then induce a tumorigenic phenotype in liver. So specifically, what they showed is that these EV transfer induced loss of contact inhibition and I encourage independence and tumorgenicity in mice after the transfer to recipient p53 knockout cells. So more details on the role of EVs remediating cancer proliferation and survival by specifically by inhibiting senescence mediated processes and apoptosis are going to be discussed in more detail in this MOC 2 lecture series in the apoptosis and senescence lecture. Another example, in prostate cancer, the transcript variant androgen receptor splice variants 7 was also detected in EVs isolated from plasma. This variant has been associated with prostate cancer growth and survival. Implicating the potential of EV derived ARv7 in prostate cancer. In pancreatic cancer, EVs have been reported to associate with a K-ras mutation at codon 12. So the K-ras_G12D mutation and the DNA encoding this mutant variant has been identified in serum derived extracellular vesicles. So these findings indicative of potential transfer of oncogenes via extracellular vesicles. Expression of tissue factor; primary cellular initiator of blood coagulation and the modulator of angiogenesis and metastasis in cancer like colorectal cancer, directly links the genetic status of the cells such as an activated K-ras gene or loss of function mutation in p53 to there in the lower angiogenesis and growth capacity. EVs have also been shown to carry DNA encoding tumor suppressors often with mutations related to cancer progression. So for example, Yang et al reported the presence of TP53 mutant DNA, specifically the TP53_R273H mutation in serum from pancreatic cancer patients. It was reported that the Gamma radiation can also EV cargo in a P53 dependent manner suggesting in direct regulation mediated by p53 on EV secretion. PTEN protein was also identified in EVs, and the secretion of PTEN is dependent on NDFIP1, a member of the nedd4 family of E3 ubiquitin ligases. In cancer therapy, transfer C-terminus regulatory domain of PTEN has been proposed as a strategy to target breast cancer cells. The C-terminus of PTEN when expressed in stable cell lines were shown to inhibit tumorigenesis in syngeneic breast tumor models. While EVs can transfer cargo which are well 9's mediated cancer proliferation and survival such as the growth factors oncogene and the mutants, other reports have shown that normal targets can also mediates some proliferation via EV's. So one example of this is via transfer of the endogenous RNA RN7SL1. So triggering the stromal cells in breast cancer and breast cancer model generates unshielded RNA7SL1, allowing activation of the pattern-recognition receptor RIG-1 which then enhances tumor growth and metastasis and therapy resistance. EVs can also transfer non-coding transcripts and transposable elements from stromal to breast cancer cells which can then stimulate the pattern recognition receptors, RIG-1 to activate STAT1 dependent antiviral signaling and activate NOTCH3 on breast cancers cell. This leads the expansion of sub-populations of breast cancer cells that are resistant to therapy and further re-initiating the tumor growth. In prostate cancers that Madji and team have reported that knockdown of the well-known EV marker CD19 reduce our proliferation of androgen receptor positive cells. Treatment of cells with CD9 enriched EVs then increased cell proliferation in androgen deprive cells. So this implicates that CD9 positive EVs are important factor for the growth of prostate cancers even when cells are undergoing androgen deprivation. These studies strongly suggests that there's a strong connection between EV biogenesis and secretion and cell proliferation and survival. So in the next few slides, we'll discuss in more detail how EVs can also transfer drug resistance phenotype to non-resistant cancer cells. The content of EVs have also reprogrammed and regulated as a consequence of energy metabolism and activity. Under hypoxic conditions, multiple enzymes and glycolytic associated pathways are upregulated, resulting in acidification of the tumor micro-environment. Hypoxia, which is reduction in the normal level of tissue oxygen tension is a common characteristic of solid tumors and downwind with malignant aggressive and treatment refractory property. So cells regulate such processes by employing proton pumps that correct their intracellular pH and further render them resistant to changes in extracellular pH, as such this can increase the secretion and uptake of EVs, facilitating this enhanced communication in the tumor micro-environment. Hypoxia we shown to increase EV release from cancer cells by the hypoxia-inducible heterodimeric transcription factor, HIF dependent RAB22A expression, resulting in functional changes in cell invasion and enhanced extravasation in vivo. Further, such conditions facilitate spontaneous breast cancer metastasis to the lungs. So these studies highlight that metabolic changes in cancer cells within hypoxic and acidic environments promote active EV release to promote cancer evasiveness and progression. Drug resistance is a significant obstacle in the successful treatment of cancers and contributes to treat and failure in over 90 percent of patients with metastatic disease. It manifests clinically is either a lack of reduction of tumor size following drug exposure or by a recurrence or relapse disease following an initial positive response. The overall resistance profile of any tumor is typically heterogeneous and dynamic, and involves a combination of many different mechanisms and all the evolution of phenotypes over time, following drug exposure. The main mechanisms contributing to drug resistance are shown in the slide here, and resistance can arise through any one of these different mechanisms including alterations and insufficiencies in drug uptake carriers, increase detoxification mechanisms, alterations in drug targets, and changes in the cellular death operators, following preventing programmed cell death. So these are just a few of the numerous mechanisms which in simple terms, program the cancer cell for survival. The unique mechanism however contributes to the simultaneous resistant to numerous structurally and functionally unrelated drugs. This phenotype is often referred to as multi-drug resistance or MDR, and it's the deficit of intracellular drug accumulation arising from an enhanced stability capacity for ATP dependent drug efflux at the level of the plasma membrane of cancer cells. So the over-expression of members of this ATP binding cassette superfamily of membrane transporters is synonymous with conferring this phenotype. So among these expression of the multi-drug flux proteins p-glycoprotein and Multidrug Resistance Protein 1 had been correlated with poor survival and treatment outcome across most cancers. These highly promiscuous drug transporters recognize most chemotherapeutic agents or substrates and act to expel them from the cell. So that renders the cancer cell multidrug resistant and treatment unresponsive. MDR can be acquired following the exposure of a single agent or it can be already an inherent ability within the tumor cells. The latter of which this inherent resistance is observed and those cancers rising particularly from pharmacological barriers sites from which these proteins serve as a normal physiological role in xenobiotic detoxification. The context that acquired multidrug resistance. This typically arises following exposure to a toxic insult, usually in the form of exposure to chemotherapeutic agents. Typically MDR is understood to occur through alterations in the cellular apparatus so as to evade the chemotherapeutic insult. In the context of over-expression of proteins such as P-glycoprotein, this has been shown to occur through a number of different mechanisms including gene amplification, increased transcription and also increased protein trafficking and protein half-life. So the acquisition of P-glycoprotein through non-genetic means was first reported in 2005 by Luchenko and colleagues. They showed that P-glycoprotein could be transferred through direct cell to cell contact, though the possibility of mechanisms beyond the direct cellular interaction were excluded at that time. However, the transfer of P-glycoprotein and hence multi-drug resistance by EVs was first reported by the Bowery and her team in 2009. This is depicted schematically here in the picture and slide. If note was the unique approach the team used to study this trait transfer. So specifically, two cell line pairs were utilized, a drug sensitive line and then MDR derivative sublime. The latter of which was established following step-wise increments in drug exposure. So the MDR, the multidrug resistant derivative line, we're shown to spontaneously shed vesicles, harboring the peak like protein which could be used in the study of fascicular transfer resistance to the drug sensitive cell lines. The recipient cells. So this is also important in demonstrating the EVs can transfer very large functional trans-membrane proteins in the vicinity of about a 170 kilodaltons and a fully functional MDR phenotype could be acquired in as little as four hours following EV exposure. So since this study, subsequent studies have also reported on the transfer of P-glycoprotein across different cell types and by different EVs and through the involvement of tumbling [inaudible] for example. In 2013, the vesicular transfer of another membrane, another member of the ABC super family of drug transporters, this time MRP one was also demonstrated. Unlike P-glycoprotein, the kinetics of transfer defer taking up to 12 hours for the transfer and acquisition of functional MRP mediated drug resistance in recipient's cells to occur. So this difference in the kinetic has been attributed to many different things including differences in structure, whereby MRP comprises of larger protein with an additional internal transmembrane region. This transfer of P-glycoprotein by EVs has also been validated in vivo using MCF seven xenograft model where a single subcutaneous injection of P-glycoprotein expressing EVs resulted in the transfer of P-glycoprotein within the tumor, within the tumor core of recipients in a grass in as little as 12 hours following exposure, with expression being stable for at least two weeks. EV mediated transfer of functional resistance proteins and later functional nucleic acids coding these proteins. This provides a novel mechanism for the dissemination and acquisition of MDR in cancer cell populations. In 2013, the first report that EV proteins could reside in two topographical orientations was first reported. P-glycoprotein on the EV surface was shown to reside in both an inside out and right-side out orientation. Other labs have recently demonstrated that a number of other EV membrane proteins are also present in topologically reversed orientations. In the context of P-glycoprotein, this meant that the protein's, cytosolic nucleotide binding regions were now exposed on the surface of the vesicle. In the presence of an ATP regenerating system, P-glycoprotein mediated efflux hence resulted in the intra-vesicular accumulation and sequestration of drug substrates. So this identified a parallel mechanism of resistance through active sequestration and again mediated by the peak like protein harboring EVs. So both passive, through binding to the phospholipid and nucleic acid cargo on EVs and active drug sequestration mechanisms were shown to have a role in drug resistance. Those are shown in the slide in A and B. So this introduced potential strategies which could be exploited for drug loading and in drug delivery. EV shed from MDR cells have since been shown to promote other mechanisms to ensure the survival of the cancer cell population. Mechanisms facilitated by the EV transfer of microRNAs and protein cargo have been shown to promote metastatic capacity. Likewise, the transfer of P-glycoprotein was shown to increase cell stiffness and mediate changes in tissue bio-mechanics. More recently, EV shed from MDR cells were shown to functionally incapacitate macrophage cells and facilitate their engulfment by MDR cells in a process which has been proposed to support priming of the pre-metastatic niche, through immune evasion. In these fluorescent images here, the MDR cells are labeled in red while the macrophages are labeled in green. MDR cells possess a remarkable ability to engulf macrophages, which is not evident in drug sensitive cells or non-malignant HBCD3 cells. Only when sensitive cells acquire multi-drug resistance following EV transfer of P-glycoprotein, can the cells also engulf macrophages. This research is also summarized in the nice YouTube video link provided there on the screen to recommend you go have a look up. Given the detrimental effects that multi-drug resistance has in cancer cell treatment and the roles of EVs in supporting this deleterious phenotype, strategies to target this are a clinical relevance and importance. Targeting EV bio-genesis is indeed really a viable strategy. However, ensuring selected targeting of malignant cell for circulation, walls having no detrimental impact on normal cell fasciculation, is a priority in achieving this goal. The approach so far really is quite promising. Indeed there's evidence to suggest that at rest, malignant cells fasciculate through a calpain driven pathway, which appears to not to dominate in terms of basal levels of the fasciculation in non-malignant cells. So the mechanism appears to be associated with differences in resting calcium levels between the two cell types and future research aims should be to elucidate the unique pathways regulating bio-genesis in malignant compared to non-malignant cells. Okay. So some final remarks and conclusions on this presentation. So the hallmark capabilities that drive tumor progression are reinforced by the communication between cancer cells and the tumor micro-environment, and EVs can play an important part in this communication. It's been demonstrated that EVs have a role in the establishment and maintenance of cancer hallmarks, including sustaining proliferative signaling and their interaction between tumor and stromal cell via transfer of cargo through EVs. So EVs have been shown to transfer and anti-apoptotic phenotype to neighboring cells, the pro-survival phenotype. For example, through the transfer of P-glycoprotein as we've discussed. Also EVs have been shown to re-program energy metabolism. So all of these different roles that EVs have can contribute towards that tumor progression and that's why it's important to try and understand it. So we can potentially intervene with this communication in a targeted and specific way which will hamper tumor communication but keep normal cells perfectly healthy. Here's a nice list of references.
