MicroRNA Interrelated Epithelial Mesenchymal Transition (EMT) in Glioblastoma

MicroRNA Interrelated Epithelial Mesenchymal Transition (EMT) in Glioblastoma: Comparison

Please note this is a comparison between Version 1 by Rodney Hull and Version 3 by Lindsay Dong.

MicroRNAs (miRNA) are small non-coding RNAs that are 20–23 nucleotides in length, functioning as regulators of oncogenes or tumor suppressor genes. They are molecular modulators that regulate gene expression by suppressing gene translation through gene silencing/degradation, or by promoting translation of messenger RNA (mRNA) into proteins. Circulating miRNAs have attracted attention as possible prognostic markers of cancer, which could aid in the early detection of the disease. Epithelial to mesenchymal transition (EMT) has been implicated in tumorigenic processes, primarily by promoting tumor invasiveness and metastatic activity; this is a process that could be manipulated to halt or prevent brain metastasis. Studies show that miRNAs influence the function of EMT in glioblastomas. Thus, miRNA-related EMT can be exploited as a potential therapeutic target in glioblastomas.

microRNA
EMT

1. Introduction

Gliomas are the most common primary carcinomas of the central nervous system (CNS), arising from structural supporting brain cells known as glial cells. Glial cells have been shown to play an important role in the development and function of the CNS. Glioblastomas occasionally arise from genetic syndromes or from exposure to ionizing radiation ^[1][3]. The regulatory mechanisms of miRNAs in glioblastoma processes are still not fully elucidated. Several miRNA signatures have been associated with either tumor suppression or promotion. Glioblastomas express several miRNAs, which have been shown to prevent progression of the disease whilst facilitating apoptosis. An occurrence was observed following treatment with chemotherapeutic agents. In this case, some of the miRNAs acted as apoptotic genes, suppressing the proliferation and survival of glioblastomas or silencers of anti-apoptotic genes, thus favoring tumor growth. This potentiates the use of miRNA signatures as diagnostic biomarkers for glioblastoma, primarily with the use of liquid biopsies, which are easily obtainable ^[2][4]. miRNAs have been shown to regulate epithelial to mesenchymal transition (EMT) ^[3][5], a process whereby epithelial cells undergo biological processes that allow them to take on the phenotype of mesenchymal stem cells and adopt their enhanced migratory ability, invasive capacity and increased ability to resist apoptosis ^[4][6]. EMT is closely associated with malignant progression and clinical outcome in gliomas ^[5][7]. Manipulation of miRNA-EMT regulatory mechanisms for treatment purposes includes targeting EMT transcription factors and structural components of the epithelial cells in an attempt to reverse the EMT processes, as well as targeting the miRNA signaling pathways involved in these processes ^[6][8].

2. Epithelial Mesenchymal Transition in Glioblastoma

During EMT, epithelial cells develop a mesenchymal phenotype and properties including migration. Cancer cells acquire these abilities and migrate to distant sites away from the primary tumor to establish metastatic foci. The process of EMT and the resultant migratory abilities are also associated with drug resistance. A number of processes contributing to glioma EMT have been described. The induction of these processes in glioblastomas differ from those observed in epithelial cancers because of the absence of the basement membrane ^[7][8][9,10]. The basement membrane is a specialized extracellular matrix that forms the base for endothelial and epithelial cells. It plays a pivotal role in maintaining the structural integrity, exchanging biochemical signals, and regulating cellular function ^[9][10][11,12]. Typically, for tumor cells to migrate to other parts of the body, they need to detach from the basement membrane. In the case of gliomas, glial cells are the most abundant cells in the brain, and provide protective and structural support ^[11][13]. A type of glial cell, referred to as astrocyte, becomes activated and surrounds the tumor stroma, thus keeping cancer cells and the cluster intact. These cells can undergo EMT processes induced by cancer cells and EMT-associated transcription factors ^[12][14] (Figure 1).

Figure 1. Stimulus from miRNA signaling molecules, EMT transcription factors (N-cadherin, Snail, Snag and Twist) and reduced levels of E-cadherin (needed for maintaining the cell-to-cell adhesion of the epithelial cells) induce the EMT processes. Epithelial cells and astrocytes, which form part of the tumor stroma cells, will then lose their adhesion capacity and acquire a mesenchymal phenotype, leading to cancer cells migrating into and invading the surrounding tissue. Astrocytes can be activated by the tumor. These reactive astrocytes become cancerous and serve as a supporting structure for the cancer cells. At this point, astrocytes can activate the aberrant signaling pathways involved in the induction of EMT, resulting in metastasis.

3. Drivers of EMT in Glioblastoma

3.1. Extracellular Vesicles (EVs)

Extracellular vesicles secreted by tumors facilitate the intercellular signaling and communication within the components of the tumor microenvironment and the tumor stroma ^[13][16]. EVs have been shown to carry a number of biological molecules, including miRNAs ^[14][17], hence their involvement in the transfer of mutations. Furthermore, EVs are involved in cell proliferation, migration and homing, reprogramming energy metabolism, angiogenesis and drug resistance ^[13][16]. Glioblastomas have been classified into proneural (PN), mesenchymal (MES) and the classical tumor-intrinsic transcriptional subtypes implicated in aiding drug resistance ^[15][18]. MES are known to play an important role in EMT induction ^[16][19]. Thus, EVs originating from MES drive PN glioblastoma progression and, ultimately, drug resistance through an NF-κB signal transducer and STAT3 signaling ^[17][20].

3.2. Transforming Growth Factor β Signaling Pathway

Transforming growth factor-β (TGF-β) is a family of multi-functional growth factors involved in cell proliferation, differentiation and apoptosis. During cancer development, TGF-β suppresses cancer growth via cell cycle arrest and apoptosis, but supports cancer progression at a later stage of the disease by promoting invasion, increasing migration capabilities and drug resistance ^[18][23]. The TGF-β complex consists of surface receptor type I (TβRI) and type II (TβRII) transmembrane serine/threonine kinases. Signaling is initiated by the binding of TGF-β to TβRII and TβRI receptors on the cell surface, resulting in a heterocomplex. Phosphorylation by TβRII activates TβRI, resulting in the recruitment and phosphorylation of Smad proteins 2 and 3, collectively known as R-Smads. Translocation to the nucleus requires the binding of R-Smads to Smad 4. The resultant R-Smads/Smad 4 complex will then induce the transcription of target proteins ^[19][24] (Figure 2). Aberrant TGF-β signaling pathways contribute to cancer development ^[20][25], making TGF-β an important driver of EMT in cancers.

Figure 2. Transforming growth factor-β (TGF-β) signaling pathways; the signaling pathway is initiated by the binding of TGF-β to TβRII and, in the process, recruiting TβRI, forming a heterodimer complex. TβRII will activate TβRI by phosphorylation, resulting in recruitment of R-Smads, which will be anchored onto the complex by SARA. Phosphorylation of R-Smad or receptor-activated Smads will take place. Phosphorylated R-Smads will bind to Smad4, creating a complex that then signals translocation into the nucleus, leading to transcription of target proteins. TGF-β signaling can also result in the activation of non-Smad signaling, which also leads to target gene transcription, while I-Smads act as an inhibitor of the TGF-β signaling pathway.

3.3. Autophagy

Autophagy is a homeostatic process that clears out non-essential cellular components through lysosome-mediated intracellular degradation activity ^[21][32]. Cancer cells use autophagy as a gateway to evade oxygen/nutrient deprivation and pharmacotherapy. Autophagy also serves as a source of the energy they require for rapid proliferation and survival.

The NADPH oxidases (NOXs) of genes are the main suppliers of reactive oxygen species (ROS). The transcription NOX4 subfamily mRNA has been implicated in the proliferation and survival of glioblastoma cells. Together with TGF-β, NOX4 enhanced glioblastoma growth. Patients with higher levels of TGF-β and NOX4 had a poor prognosis, suggesting that the two molecules could serve as useful biomarkers of the disease ^[22][36]. Wnt signaling pathways are involved in embryonic developmental processes and stem cell proliferation in metazoan animals ^[23][37]. The pathway has a dual role in cancer, being composed of a myriad of oncogenes and tumor suppressors, with genes coding for the components of the anaphase-promoting complex (APC) being the most frequently mutated ^[24][38].

3.4. MiRNAs

The expression signatures of a number of miRNAs have been identified as either instigators or inhibitors of EMT in glioblastomas. Zhang et al. assessed the clinical importance of EMT in glioma using independent glioma datasets, GSE16011, Rembrandt and The Cancer Genome Atlas (TCGA) program. The study found 19 miRNA expression signatures that are positively correlated with EMT-driven gliomas, with the most prominent being miR-223 ^[5][7]. Blocking the miR-223/PAX6 pathway improved sensitivity to chemotherapeutic treatment with temozolomide in glioblastomas, indicating the role of miR-223 in drug resistance ^[25][43]. The PI3K/Akt signaling pathway is associated with EMT-related disease severity in glioblastomas. This pathway has been shown to act in collaboration with other EMT-related signaling pathways, including mTOR ^[26][44], CXCR4 signaling ^[27][45] and LASP1 ^[28][46].

4. miRNA-EMT-Related Cancer Cell Invasion and Metastasis

The link between miRNA expression signatures, EMT and, ultimately, cancer metastasis has long been established ^[29][60]. Several miRNA signatures are involved in EMT-associated mechanisms of invasion and metastasis in glioblastomas. Studies on miR-125a-5p found its expression to suppress the mesenchymal capabilities of glioblastoma cells, thus inhibiting EMT ^[30][31][61,62]. Recently, Nan et al. showed that miR-451 expression is able to suppress EMT and metastasis by blocking the PI3K/Akt/Snail signaling pathway through the activation of calcium-binding protein 39 (CAB39) in gliomas ^[32][63]. The expression of miR-451 was associated with the inhibition or reversal of the EMT processes in cancers ^[33][64]. Contrary to this finding, miR-451 expression plays different roles in gliomas, as it was associated with the proliferation of cancer cells by suppressing the CAB39/AMPK/mTOR pathway, and increased metastatic potential, which takes place via the activation of the Rac1/cofilin pathway ^[34][65]. The increased expression of miR-200b-3p prompted E-cadherin (essential for maintaining the structural integrity of epithelial cells) by the deactivation of extracellular signal-regulated kinase 5 (ERK5), resulting in reduced glioma cell proliferation and mesenchymal capabilities ^[35][41]. mR-424 showed promising results as a potential prognostic molecular marker and therapeutic target by blocking EMT processes and the resultant metastatic capabilities by targeting the kinesin-like protein KIF23 in human glioma ^[36][66]. The same effect was observed with miR-378, which inhibited EMT by targeting cis-aconitate decarboxylase (IRG1) in gliomas ^[37][67], miR-139-5p by targeting the notch1 oncogene in gliomas ^[38][68], miR-181a by targeting ZBTB33 expression in glioma cells ^[39][69], miR-623 by targeting TRIM-44 ^[40][70], miR-940 by targeting ZEB2 ^[41][71], and miR-7, which targeted T-Box 2 in glioblastoma ^[42][72]. In addition to these miRNAs, miR-182-targeted MTSS1 enhanced TGF-β1-related EMT in gliomas ^[43][73], and miR-504 inhibited EMT in glioblastomas by targeting the Wnt receptor FZD7/β-catenin pathway. The phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase (PTEN) tumor suppressor inhibits PI3K by dephosphorylating it. The expression of PTEN is inhibited through the action of three separate miRNAs. These are miR-17-5p ^[44][74], miR-23a-3p ^[45][75] and miR-26a-5p ^[46][76]. The p53 tumor suppressor is, itself, downregulated by miR-10b-5p. This miRNA also downregulates the tumor suppressor and cell cycle regulator p16 ^[47][77]. At the same time, the negative regulator of p53, MDM2, is upregulated in glioblastoma by miRNAs such as miR-32-5p, miR-25-3p and miR-17-3p ^[48][78]. The RB1 tumor suppressor is downregulated by the miRNA miR-28-5p. At the same time, the expression and activity of oncogenes that promote cell cycle progression are downregulated by various miRNAs in glioblastoma; these include miR-124-3p suppressing cyclin-dependent kinase (CKD) 4 ^[49][79], and miR-491-5p, miR-491-3p and miR-138-5p ^[50][80] suppressing CKD6. Finally, the proliferative activity of cyclin D is downregulated by miR-195-5p ^[51][81]. A decreased expression ratio of the miRNA miR-504/FZD7 was shown to be a potential molecular marker for identifying the mesenchymal subtype in glioblastoma ^[52][82]. The actions of the miRNAs discussed above are summarized in Figure 34.

Figure 34. miRNA involved in the invasion and metastasis pathways in glioblastoma; miR-451 blocks the PI3K/Akt/Snail signaling pathway to suppress EMT and metastasis. The expression of E-cadherin maintains the structural integrity of epithelial cells. E-cadherin is repressed by ERK5, while the ERK5 pathway is inhibited by miR-200b-3p, reducing EMT and glioma cell proliferation. The Wnt pathway, which promotes EMT and invasion, can be blocked by miR-504, which blocks the Wnt receptor FZD7. The expression of various matrix metalloproteinases and the metalloproteinase inhibitor 3 (TIM3) can also be controlled by miRNAs.

5. miRNA-EMT-Related Angiogenesis

MicroRNAs regulating angiogenesis in cancers have been dubbed angiomiRNAs ^[53][54][85,86]. Several angiomiRNAs have been identified in glioblastomas ^[55][87], with some originating from glioblastoma-derived EVs ^[56][88]. However, studies indicating the relationship between angiomiRNAs and the EMT process in glioblastomas are limited. Dai et al. found that upregulated miR-24 expression induced the expression of the angiogenic markers VEGF and, TGF-β, and matrix metalloproteinases (MMP)-2 and -9, resulting in increased glioblastoma cell proliferation and development ^[57][89] (Figure 45). Increased expression of miR-16 in glioblastoma inhibited EMT processes by targeting polycomb complex protein BMI-1, and reducing the expression levels of the angiogenic markers VEGF-A and VEGF-C ^[58][90]. miR-576-3p was shown to inhibit EMT and the angiogenic properties of hypoxia-treated glioma cells by targeting HIF-1α ^[59][91] (Figure 45).

Figure 45. The role played by miRNAs in regulating angiogenesis in glioblastoma. Upregulated miR-24 induced the expression of VEGF, TGF-β, and MMP-2 and -9, leading to increased angiogenesis. VEGF expression is also downregulated by miR-16. Angiogenesis is stimulated by hypoxia, and this process is inhibited by miR-576-3p.

AngiomiRNA-EMT-Induced Drug Resistance

The potential of Raddeanin A (RA) as treatment in cancers ^[60][61][105,106], including glioblastomas ^[62][107] has been promising thus far. One of the mechanisms employed by RA is the inhibition of EMT and angiogenic processes by the deactivation of β-catenin and EMT pathways, which then targets specific molecules (N-cadherin, vimentin and Snail). This process results in reduced glioblastoma cell proliferation, invasion and metastasis ^[63][52]. EphrinB2 is a subfamily of receptor protein tyrosine kinases implicated in tumorigenesis. Its downregulation in gliomas is controlled by HIF1α via the activation of ZEB2, resulting in cancer cell invasion and anti-angiogenic resistance. Thus, the study suggests that a combinatorial therapeutic strategy with anti-angiogenic treatment aimed at inhibiting hypoxia signaling pathways could improve clinical outcomes.