The Promotive Effect Of Histone Deacetylase 3 On Vasculogenic Mimicry Of Gliomas

Glioma is the one of the most common primary brain tumor, accounting for 40-60% of all intracranial tumors, with high incidence rate, poor prognosis, high recurrence rate or other characteristics. At present, the main treatment is surgical operation with postoperative radiotherapy and chemotherapy. However the patient is still difficult to obtain a longer survival. The accepted consensus underlying tumor survival has been that a blood supply is required to sustain tumor genesis, growth and metastasize. This important premise ignited the field of neoplastic angiogenesis research and served as the major organizing principle for drug discovery and development and clinical trials. However, recent studies have found that anti-antigenic therapy only to the leave the tumor cells surfing hypoxia and ischemia, which not only cannot effectively control the tumor but may even increase the degree of malignancy and accelerate tumor metastasis. These studies suggest that the development of malignant tumors also lurks other mechanisms.In 1999, vasculogenic mimicry (VM) was first introduced by Maniotis et al. Vascular mimicry is a new blood supply system and tumor microenvironment independent of endothelial vessels, which refer that in order to meet their blood supply, the tumor cells could simulate the vascular endothelial cells by deformation and extracellular matrix remodeling to form a channel that was similar to the normal vessels in exhibiting the functions. At present, it has been found between angiogenesis melanoma, liver cancer, invasive ovarian cancer, gastrointestinal stromal tumors, breast cancer, glioma, colorectal cancer, prostate cancer, throat, squamous cell carcinoma, etc. Both our previous study and many other reports demonstrated that VM exists in gliomas and is a significant prognostic factor for patient survival. Since, apart from traditional anti-antigenic therapy, anti-VM therapy should also be taken into account in treatment strategies targeting tumor microcirculation.At present, the VM formation mechanism is still not very clear, but since the concept was put forward, a number of molecules involved in the formation of vasculogenic mimicry have been confirmed in different tumors by Maniotis and many other researchers, including endothelial cells cadherin (vascular endothelial-cadherin, VE-cadherin), Eph receptor tyrosine kinases A2 (Erythropoietin-producing hepatocellular carcinoma-A2, EphA2), matrix metalloproteinase (Matrix metalloproteinase, MMPS), phosphatidylinositol 3-kinase (Phosphoinositide 3-Kinase, PI3K), laminin (Laminin-5 y2), vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), hypoxia-inducible factor (Hypoxia-inducible factor, HIF) and tissue factor pathway inhibitory factor -2 (tissue factor pathway inhibitor-2, TFPI-2). Depth study of these molecules involved in vasculogenic mimicry, a classical model of vasculogenic mimicry signal pathway formed gradually. In 2001, Seftor, RE et al. Demonstrated that the expression of MMP-2 and MT1-MMP (MMP-14) are capable of lysing laminin5 γ2 chain, resulting in splitting fragments γ2’ and γ2x, then facilitating the generation of VM. Hess, AR in 2003 had verified that PI3K could regulates MT1-MMP, thus affect the VM. Therefore, PI3K, MMPs and LAMC2 are important molecules for VM on signaling pathways.Histone deacetylase (HDAC) are a class of enzymes which plays an important role in regulating the gene expression and structure modification of chromosome structure, which, regulating the de-acetylation, is closely related to the inhibition of gene transcription and gene silencing, and is a hot target for the design of anticancer drugs. Different HDACs types have different acetylation on nuclear transcription factors and protein, inhibiting the expression of a variety of tumor suppressor protein, which is closely associated with a variety of oncogenes, leading to cells excessive proliferation and then tumorigenesis. Importantly, there are reports verified that it is also closely related to angiogenesis; Liby, etc. found that in glioblastoma the expression of HDAC3 increased in the more malignancy tumor cells; HDAC3 perhaps is a necessary molecule for gliomas cell proliferation, which in the process of malignant transformation and growth of glioma plays an important role; Some study also reported that histone deacetylase inhibitor TSA, SAHA and other drugs blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. What’s more, PU3K (AKT) was the last cascade for VM related signaling pathways. Another report showed that HDAC3 is specific reactant of HIF-la which is one of the most important factors that promote to VM.Thus, HDAC3 are closely related to the formation of vasculogenic mimicry, and there is a crosstalk between signal pathways in a variety of molecules involved in vasculogenic mimicry formation. However, there were no reports about the relationship between HDAC3 and the vascular mimicry. If we can further clarify whether HDAC3 associated with glioma VM and define HDAC3 molecular signaling pathways associated with VM, it will be beneficial for identifying new mechanisms of vasculogenic mimicry formation and new therapeutic targets and methods, which has a very important significance in improving the prognosis of patients with glioma. Chapter 1 Relationship between VM and clinicopathological data in glioma tissuesObjective To analysis the correlation between glioma VM or HDAC3 expression and clinical data for exploring the positive relationship between VM and clinicopathological data in glioma tissues.Method Tissues collection and analysis in this study were approved by the Research Ethics Committee of Southern Medical University, China. Specimens (n= 102) consisting of formalin-fixed paraffin-embedded (FFPE) Glioma tissues were obtained from the Department of Pathology in Zhujiang Hospital at Southern Medical University between 2010 to 2013. All tissues were randomly collected from patients who did not undergo any therapy before undergoing a surgical operation to remove tumors. Tumor sections were reviewed by two neuropathologists to verify the diagnosis of glioma according to the 2007 World Health Organization classification of tumors of the central nervous system.Results Twenty-six specimens (25.49%) with VM structures were identified out of 102 glioma specimens by CD34-PAS dual staining. The results showed that the pathological grade (based on WHO standards) of glioma differed significantly between groups with VM and without VM (P= 0.048). No significant differences were found in other clinicopathological characteristics such as gender, age, tumor size, and preoperative Karnofsky performance scores (KPS). VM was detected preferentially in high-grade gliomas:13 of 33 WHO grade IV (39.39%), seven of 23 WHO grade Ⅲ (30.43%), and six of 40 WHO grade Ⅱ (15%). VM was not detected in any WHO grade I gliomas. The expression of HDAC3 significantly correlated with the WHO pathological grade of gliomas (P< 0.001). The high HDAC3 expressers (HDAC3++ and HDAC3+++) were 16.67%(1 of 6) of WHO I,27.50%(11 of 40) of WHO Ⅱ,73.91% (17 of 23) of WHO Ⅲ, and 87.88%(29 of 33) of WHO IV. More importantly, it is observed that the percent of high expression of HDAC3 in gliomas with VM was 76.92% (20 of 26) compared with 51.31% (39 of 76) that without VM. This difference was statistically significant (P= 0.045). mRNA levels of HDAC3 in 102 glioma tissues were also analyzed by qPCR. Prior to qPCR, samples were divided into two groups based on the presence or absence of VM. The results show that the mRNA levels of HDAC3 in VM-positive gliomas were significantly higher than those in VM-negative gliomas (P< 0.001). The highest median value of the VM channels was detected in HDAC3+++ cases. Moreover, a significant difference in the VM numbers was observed when HDAC3+++ was compared to HDAC3++ or HDAC3++ combined with HDAC3-/+(P= 0.024 and P= 0.007, respectively).Conclusions The VM and expression of HDAC3 releate to pathological grade (based on WHO standards) of glioma; What’s more, HDAC3 have a positive correlation with VM.Chapter Ⅱ HDAC3 had influence on VM and VM-related molecules on U87MG cells.Objective To establish three-dimensional culture (vasculogenic mimicry formation) model of U87MG cells, and observe the influence of HDAC3 on VM and VM-related molecules in U87MG cells.Method 1, the ability of vasculogenic mimicry formation by U87-MG cells was studied after U87MG cells were treated by different doses of HDAC3 inhibitor or HDAC3-siRNAs.2, the migration and invasion of U87MG were detected by MTT and Transwell invasive experimental, using U87MG cells that were treated by different doses of HDAC3 inhibitors and HDAC3 siRNAs.3, to verify the involvement of LAMC2 and MMP-14 in VM, vascular mimicry model were established after U87MG cells were treated by different doses of HDAC3 inhibitors and HDAC3 siRNAs.4, to analyze the relationship between HDAC3 and vascular mimicry, the expression of VM-related molecular in U87MG cells, which were treated by different doses of HDAC3 inhibitors and HDAC3 siRNAs, were tested useing qPCR and Western blot.5, to verify the involvement of HDAC3 in VM in vivo, tumor formation in nude mice was taken.Results 1, U87MG cells three-dimensional culture models were successfully established; 2, the number of intersections and the total length of tubular structure in treated group, without siRNA control group, decreased apparently when compared with that of the normal U87MG group; 3, the treated cells all can not formed tubular structures successfully; 4, the detection of VM-related molecules by qPCR:HDAC3 mRNA, MMP-2 mRNA, MMP-14 mRNA, LAMC2 mRNA expression levels were significantly decresed in treated group; the detection of VM-related molecules by Western blot:the HDAC3, MMP-2, MMP-14 and LAMC2 protein level were significantly reduced when cells were treated by siRNAs and HDAC3 inhibitors.5, the protein and mRNA expression levels of LAMC2 and MMP2/14 tested by Western blot and qPCR were decreased significantly when cells were treated by HDAC3 inhibitors and siRNAs.6, tumor-bearing model were established successfully in vivo; the isolated tumor from mice had lower levels in terms of volume or weight in group that was stablely low expression in HDAC3 than that of normal U87MG group; PAS-CD34 tests showed that VM positive rate were lowere in group that was stablely low expression in HDAC3; Western blot and qPCR detection showd that VM related molecules had lowere expression in group that was stablely low expression in HDAC3.Conclusions The VM and HDAC3 were releated to pathological grade (based on WHO standards) of glioma; What’s more, HDAC3 have a positive correlation with VM.Chapter Ⅲ HDAC3 expression correlates with vasculogenic mimicry via the PI3K/ERK-MMPs-Ln5y2 signaling pathwayObjective To further investigate the mechanism that HDAC3 regulated VM.Method The ability of vasculogenic mimicry formation by U87-MG cells was studied after U87MG cells were treated by HDAC3 inhibitor or HDAC3-siRNAs. We evaluated the expression of HDAC3, ERK, ρ-ERK, PI3K (AKT) and p-PI3K (p-AKT) in HDAC3-depleted U87MG cells by western blot.Results A significant decrease in levels of both ρ-AKT and ρ-ERK were found when we altered HDAC3 level by siRNA, which indicating that AKT and/or ERK signaling pathways may indeed be involved in VM formation regulated by HDAC3; p-AKT exhibited a decrease, with no change being observed in ρ-ERK, when AKT expression was inhibited by LY294002; similarly, ρ-AKT did not decrease with the inhibition of ERK by U0126, which indicating that ERK or AKT signaling pathways did not act upstream or downstream from each other; All molecules except ERK and AKT showed a significant reduction in expression when U0126 and LY294002 were both used and expression of MMP-14 and LAMC2 showed a relatively higher reduction than that treated with only one inhibitor (LY294002 or U0126).Conclusions These interesting results clearly indicated that the PI3K and ERK signaling pathways play key roles in VM. More importantly, ERK or AKT signaling pathways did not act upstream or downstream from each other.