RETRACTED: MMP-9 induces CD44 cleavage and CD44 mediated cell migration in glioblastoma xenograft cells.
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ABSTRACT:
SUBMITTER: Chetty C
PROVIDER: S-EPMC3481542 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Glioblastoma (GBM) is an aggressive malignant brain tumor with 2-year survival rates of 6.7% [1], [2]. One key characteristic of the disease is the ability of glioblastoma cells to migrate rapidly and spread throughout healthy brain tissue[3], [4]. To develop treatments that effectively target cell migration, it is important to understand the fundamental mechanism driving cell migration in brain tissue. Here we utilized confocal imaging to measure traction dynamics and migration speeds of glioblastoma cells in mouse organotypic brain slices to identify the mode of cell migration. Through imaging cell-vasculature interactions and utilizing drugs, antibodies, and genetic modifications to target motors and clutches, we find that glioblastoma cell migration is most consistent with a motor-clutch mechanism to migrate through brain tissue ex vivo, and that both integrins and CD44, as well as myosin motors, play an important role in constituting the adhesive clutch.
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Project description:In more than 90% of cancers including glioma, telomere elongation reverse transcriptase (hTERT) is overexpressed. In the present study, we sought to explore whether matrix metalloproteinase-9 (MMP-9) shRNA could alter hTERT-mediated proliferation in glioma cells. MMP-9 shRNA induced senescence and apoptosis in glioma cells by inhibiting hTERT expression and telomere activity. MMP-9 silencing decreased oncogenic c-Myc expression (hTERT activator), whereas the expression of the c-Myc antagonist MAD increased drastically (hTERT repressor); both c-Myc and MAD are transcription factors for hTERT. In addition, MMP-9 suppression turns the switch from c-Myc/MAX to MAD/MAX heterodimer binding to the hTERT promoter as determined by chromatin immunoprecipitation assay. We also show that silencing MAD via siRNA restored hTERT expression and inhibited senescence in glioma cells. MMP-9 transcriptional suppression decreased the expression of FAK, phospho FAK and ?1 integrin in glioma xenograft cells. Further, MMP-9 suppression decreased the interaction of ?1 integrin/FAK and also MMP-9/?1 integrin as confirmed by immunoprecipitation analysis. Studies with either function blocking ?1 integrin or FAK shRNA indicate that suppression of MMP-9 decreased ?1 integrin-mediated induction of FAK, which led to decreased hTERT expression. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of FAK/c-Myc and elevated MAD levels. Decreased co-localization of ?1 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, immunoprecipitation analysis showed decreased association of proteins involved in telomere end repair in MMP-9 shRNA-treated glioma cells. Elevated levels of p73 and TRAIL and the results of the FACS analysis show induction of apoptosis in MMP-9-silenced glioma cells. Taken together, these data provide new insights into the mechanisms underlying MMP-9-mediated hTERT expression in glioma proliferation.
| S-EPMC3184383 | biostudies-literature
Project description:Glioblastoma multiforme (GBM) is a lethal brain tumor, characterized by enhanced proliferation and invasion, as well as increased vascularization and chemoresistance. The expression of the hyaluronan receptor CD44 has been shown to correlate with GBM progression and poor prognosis. Here, we sought to elucidate the molecular mechanisms by which CD44 promotes GBM progression by knocking out (KO) CD44, employing CRISPR/Cas9 gene editing in U251MG cells. CD44-depleted cells exhibited an impaired proliferation rate, as shown by the decreased cell numbers, decreased Ki67-positive cell nuclei, diminished phosphorylation of CREB, and increased levels of the cell cycle inhibitor p16 compared to control cells. Furthermore, the CD44 KO cells showed decreased stemness and increased senescence, which was manifested upon serum deprivation. In stem cell-like enriched spheres, RNA-sequencing analysis of U251MG cells revealed a CD44 dependence for gene signatures related to hypoxia, the glycolytic pathway, and G2 to M phase transition. Partially similar results were obtained when cells were treated with the γ-secretase inhibitor DAPT, which inhibits CD44 cleavage and therefore inhibits the release of the intracellular domain (ICD) of CD44, suggesting that certain transcriptional responses are dependent on CD44-ICD. Interestingly, the expression of molecules involved in hyaluronan synthesis, degradation, and interacting matrix proteins, as well as of platelet-derived growth factor (PDGF) isoforms and PDGF receptors, were also deregulated in CD44 KO cells. These results were confirmed by the knockdown of CD44 in another GBM cell line, U2990. Notably, downregulation of hyaluronan synthase 2 (HAS2) impaired the hypoxia-related genes and decreased the CD44 protein levels, suggesting a CD44/hyaluronan feedback circuit contributing to GBM progression.
| S-EPMC9367353 | biostudies-literature
Project description:The molecular mechanisms that control the ability of adipose-derived mesenchymal stem cells (AMSCs) to remodel three-dimensional extracellular matrix barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of AMSCs to endothelial cells (ECs) in vitro. MSCs were isolated from porcine abdominal adipose tissue, and characterized by immunopositivity to CD44, CD90, CD105, and immunonegativity to CD14 and CD45. Plasticity of AMSCs was confirmed by multilineage differentiation. The mRNA transcripts for MMPs and Tissue Inhibitor of Metalloproteinases (TIMPs), and protein expression of EC markers were analyzed. The enzyme activity and protein expression were analyzed by gelatin zymography, enzyme-linked immunosorbent assay (ELISA), and Western blot. The differentiation of AMSCs to ECs was confirmed by mRNA and protein expressions of the endothelial markers. The mRNA transcripts for MMP-2 and MMP-14 were significantly increased during the differentiation of MSCs into ECs. Findings revealed an elevated MMP-14 and MMP-2 expression, and MMP2 enzyme activity. Silencing of MMP-2 and MMP-14 significantly increased the expression of EC markers, formation of capillary tubes, and acetylated-low-density lipoprotein uptake, and decreased the cleavage of vascular endothelial growth factor receptor type 2 (VEGFR2). Inhibition of VEGFR2 significantly decreased the expression of EC markers. These novel findings demonstrate that the upregulation of MMP2 and MMP14 has an inhibitory effect on the differentiation of AMSCs to ECs, and silencing these MMPs inhibit the cleavage of VEGFR2 and stimulate the differentiation of AMSCs to ECs. These findings provide a potential mechanism for the regulatory role of MMP-2 and MMP-14 in the re-endothelialization of coronary arteries following intervention. Stem Cells Translational Medicine 2017;6:1385-1398.
| S-EPMC5442711 | biostudies-literature
Project description:Glioblastoma (GBM) is the most aggressive and deadly adult brain tumor, primarily because of its high infiltrative capacity and development of resistance to therapy. Although GBM cells are typically believed to migrate via mesenchymal (e.g., fibroblast-like) migration modes, amoeboid (e.g., leucocyte-like) migration modes have been identified and may constitute a salvage pathway. However, the mesenchymal to amoeboid transition (MAT) process in GB is not well characterized, most likely because most culture models induce MAT via pharmacological or genetic inhibition conditions that are far from physiological. In this study, we examined the ability of hyaluronic acid (HA) content in three-dimensional collagen (Col) hydrogels to induce MAT in U87 GBM cells. HA and Col are naturally-occurring components of the brain extracellular matrix (ECM). In pure Col gels, U87 cells displayed primarily mesenchymal behaviors, including elongated cell morphology, clustered actin and integrin expression, and crawling migration behaviors. Whereas an increasing population of cells displaying amoeboid behaviors, including rounded morphology, cortical actin expression, low/no integrin expression, and squeezing or gliding motility, were observed with increasing HA content (0.1-0.2 wt% in Col). Consistent with amoeboid migration, these behaviors were abrogated by ROCK inhibition with the non-specific small molecule inhibitor Y27632. Toward identification of histological MAT classification criteria, we also examined the correlation between cell and nuclear aspect ratio (AR) in Col and Col-HA gels, finding that nuclear AR has a small variance and is not correlated to cell AR in HA-rich gels. These results suggest that HA may regulate GBM cell motility in a ROCK-dependent manner.
| S-EPMC7473492 | biostudies-literature
Project description:α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.
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Project description:The hyaluronan receptor CD44 can undergo proteolytic cleavage in two steps, leading to the release of its intracellular domain; this domain is translocated to the nucleus, where it affects the transcription of target genes. We report that CD44 cleavage in A549 lung cancer cells and other cells is promoted by transforming growth factor-beta (TGFβ) in a manner that is dependent on ubiquitin ligase tumor necrosis factor receptor-associated factor 4 or 6 (TRAF4 or TRAF6, respectively). Stem-like A549 cells grown in spheres displayed increased TRAF4-dependent expression of CD44 variant isoforms, CD44 cleavage, and hyaluronan synthesis. Mechanistically, TRAF4 activated the small GTPase RAC1. CD44-dependent migration of A549 cells was inhibited by siRNA-mediated knockdown of TRAF4, which was rescued by the transfection of a constitutively active RAC1 mutant. Our findings support the notion that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.
| S-EPMC7957764 | biostudies-literature
Project description:Glioblastomas are the most common and devastating primary tumors of the central nervous system, with high proliferative capacity, aggressive invasion, and resistance to conventional therapies. Differentiation therapy has emerged as a promising candidate modality. Here we show that the traditional phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 is capable of inducing differentiation of C6 glioblastoma cells characterized by morphological changes to astrocytic phenotype, increase in differentiation marker protein glial fibrillary acidic protein and inhibition of proliferation. Small interfering RNA against glycogen synthase kinase-3? (GSK-3?) suppresses the induced-differentiation and invasiveness in C6 cells. LY294002 also inhibits MMP-9 expression and invasion of C6 cells, assembling the role of metalloprotease (MMP) inhibitor AG3340. Taken together, these findings suggest differentiation-inducing and invasion-inhibitory effectiveness of LY294002 in glioblastomas, most likely involving inhibition of GSK-3? and MMP respectively.
| S-EPMC4920038 | biostudies-literature
Project description:Cancer cells utilise the glycolytic pathway to support their rapid growth and proliferation. Since cells in most solid tumours are subjected to severe microenvironmental stresses including low nutrient and oxygen availability, such cancer cells must develop mechanisms to overcome these unfavourable growth conditions by metabolic adaptation. Although the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signalling pathway plays a pivotal role in maintaining energy homeostasis under conditions of metabolic stress, the role of LKB1-AMPK signalling in aiding cancer cell survival and in malignant tumours has not yet been fully elucidated. We show that glucose starvation promotes cancer cell invasiveness and migration through LKB1-AMPK-regulated MMP-9 expression. Most intriguingly, triggering the LKB1-AMPK signalling pathway by glucose starvation-induced oxidative stress facilitates selective autophagy, which in turn enhances Keap1 degradation and the subsequent activation of Nrf2. Following this, Nrf2 regulates the transactivation of MMP-9 via Nrf2 binding sites in the promoter region of the MMP-9 gene. These mechanisms also contribute to the suppression of excessive oxidative stress under glucose starvation, and protect against cell death. Our data clearly shows that LKB1-AMPK signalling not only maintains energy and oxidative stress homeostasis, but could also promote cancer progression during metabolic stress conditions by MMP-9 induction.
| S-EPMC6031623 | biostudies-literature
Project description:CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.
| S-EPMC4963023 | biostudies-literature