Role And Mechanism Of HDAC1 And HDAC6 In The Tumorigenesis And Progression Of Glioma

Malignant gliomas, the most prevalent primary central nervous system tumor, account for approximately 45%　of all cases of malignant primary brain tumors that are diagnosed in adults each year. At present, the primary therapy for glioblastoma is surgical resection followed by radiotherapy and chemotherapy with temozolomide. Although this treatment can result in considerable progress in the outcome of patients with glioblastoma, this aggressive tumor almost always relapses soon after treatment and the prognosis remains dismal. Despite positive surgery and conventional radio-chemotherapy, The prognosis for patients with glioma remains dismal. Therefore, it is necessary to further explore the mechanisms involved in the progression of malignant glioma and find novel effective therapeutic approaches for malignant glioma.Tumorigenesis and development of malignant gliomas is an extremely complex process, influenced by multiple factors, such as environment, genetics, molecular biology and epigenetic modifications. Its pathogenesis is not yet entirely clear. Histone acetylation and deacetylation are epigenetic events that govern many physiological and pathological cellular processes. Histone acetylation is mediated by histone acetyltransferases (HATs), which generally activates gene transcription. Conversely, histone deacetylation is catalyzed by histone deacetylases (HDACs), which more likely induces gene repression. Histone acetylation is a reversible, dynamic and highly regulated process for gene regulation. Recently, increasing evidence has suggested regulation of many non-histone proteins through reversible acetylation by HATs and HDACs. Alterations in this dynamic equilibrium, such as those caused by the aberrant expression or functional activation of HATs and HDACs, can disturb cell homeostasis and result in pathological states, including oncogenesis and tumor progression.HDACs, including eighteen different mammalian isoenzymes, are divided into four subcategories. Of these, HDAC1 may be the most important and is associated with oncogenesis and progression of tumors. Numerous studies have indicated that HDAC1 is over-expressed in diverse human malignancies, such as prostate cancer, breast carcinoma, liver cancer, and lung cancer. Aberrant expression of HDAC1 is associated with tumor cell proliferation, migration, invasion, angiogenesis and poor prognosis. Previous studies have indicated that knockdown of HDAC1 by siRNA (small interfering RNA) in some tumor cells suppressed cell growth and proliferation, increased apoptosis, and inhibited the invasion and migration capacity. In view of these observations, HDAC1 is regarded as a promising therapeutic target in the treatment of malignancies.The excessive proliferation, invasion of malignant glioma are correlated with various signaling pathway disorders. Of these, the PI3K/AKT and MEK/ERK signaling pathways are the most frequent inappropriately activated pathways in malignant glioma, and are extremely important in the tumorigenesis and progression of glioma. Studies have indicated that the PI3K/AKT is associated with cell growth, proliferation, invasion, angiogenesis and response to radiotherapy and chemotherapy. Previous research has indicated that abnormal activation of the PI3K/AKT signaling pathway contributes to drug resistance in glioma, thus the PI3K/AKT signaling pathway is considered to be the ideal therapeutic target in glioma. In addition, the MEK/ERK signaling pathway is also involved in cell proliferation, spread, survival, cell cycle progression and other malignant phenotypes of glioma. However, so far, the study on HDAC1 protein in glioma and mRNA expression in glioma rarely reported, and the potential role of HDAC1 in glioma cell proliferation and invasion, and the molecular mechanisms involved is unclear. Whether HDAC1-mediated cell proliferation and invasion of glioma is also associated with aberrant activation of the PI3K/AKT and MEK/ERK signaling pathways requires further investigation.HDAC6 is a relatively unique enzyme of HDACs family. It can affect the function of the cytoplasmic non-histone proteins and plays a key regulatory role in all aspects of tumor biology such as cell cycle, cell proliferation, migration, drug resistance. Therefore, HDAC6 is becoming a very attractive target in cancer treatment. HDAC6 is over-expressed in a variety of malignant tumors, such as liver cancer, bladder cancer,but its expression in glioma tissue is rarely reported, the mechanisms involved in glioma cell proliferation is unclear. Transforming growth factor β (TGFβ) receptor signaling pathway plays complicated roles in various biological events, and specifically affects the tumorigenesis of glioma. When a ligand binds to a type ⅡTGFβ receptor, it catalyzes the phosphorylation of a type Ⅰ TGFβ receptor, which triggers phosphorylation of two intracellular proteins SMAD2 and SMAD3 to form heteromeric complexes with SMAD4. The activated SMAD complexes then translocate to the nucleus, where they regulate the transcription of target genes. SMAD7 have an inhibitory role on SMAD2/3 signaling. SMAD7 has been shown to be a direct substrate for HDACs, suggesting that HDACs may affect TGFβ receptor signaling. Nevertheless, the relationship between HDACs and TGFβ receptor signaling pathway, especially in glioma, has not been previously documented. So, we hypothesize that TGFβ/SMAD signaling pathway was also involved in HDAC6-mediated glioma cell proliferation.In the current study, we investigated the expression of HDAC1 and HDAC6 in glioma and non-neoplastic brain tissues. We found that HDAC1 and HDAC6 are over-expression in glioma compared with normal brain tissues. Therefore, HDAC1 and HDAC6 may be potential therapeutic targets for novel treatment strategies in patients with glioma. Next, we explored the role and mechanism of HDACland HDAC6 in glioma cell proliferation and invasion. And then,we investigated whether PI3K/AKT and MEK/ERK signaling pathways were involved in HDAC1-mediated glioma cell proliferation and invasion. Moreover, we explored whether TGFβ/SMAD signaling pathway was involved in HDAC6-mediated glioma cell proliferation.Part One Role and mechanism of HDAC1 in the proliferation and invasion of gliomaChapter I　HDAC1 expression in glioma and non-neoplastic brain tissues.Objective:To investigate the expression of HDAC1 in glioma of different malignancy and non-neoplastic brain tissues.Methods:The expression levels of HDAC1 in 71 gliomas of different pathological grades and 12 temporal lobectomy of epilepsy as negative control was detected by RT-qPCR and immunohistochemical staining.Results:HDAC1 mRNA expressions were higher in glioma than that non-neoplastic brain tissues (P=0.03); HDAC1 protein expressions were higher in glioma than that non-neoplastic brain tissues(p=0.000). Moreover, HDAC1 expressions were no significantly different between different pathological grades(P=0.837)Conclusion:Detecting the expression levels of HDAC1 will be helpful to evaluate the biological behaviors of gliomas and prognosis in the patients.Chapter Ⅱ Role of HDAC1 in the proliferation and invasion of malignant gliomaObjective:To investigate whether knockdown and overexpression of HDAC1 can impact U87MG cell proliferation and invasion.Methods:we examined the effect of targeted silencing of HDAC1 by siRNA and overexpression of HDAC1 by plasmid transfection on cell proliferation and invasion in vitro. We detect the HDAC1 protein expression of U87MG cells in each group by using Western Blot. We detect the U87MG cells proliferation and invasive in each group by using MTS assay transwell. Statistical analyses were performed using the SPSS 16.0. Data were presented as mean ± SD. Comparisons between two groups were performed using one-way ANOVA. P values less than 0.05 were considered statistically significant.Results:The results revealed that HDAC1 was expressed in all samples from each group, but lower HDAC1 protein expression level was discovered in the U87MG-siHDAC1 group compared with the normal U87MG cell group or the negative control group. The results of MTS and invasion assays revealed that there was no significant differences of the proliferation and invasion capability between the normal U87MG cell group and the negative control group(p>0.05). But silencing of HDAC1 in the U87MG-siHDAC1 group leads to the proliferation and invasion capability reduction compared with the normal U87MG cell group(P<0.05), whereas over-expression of HDAC1 in the group which transfected with pCMV-3 *　Flag-HDAC1 significantly increased the proliferation and invasion capability compared with the normal U87MG cell group.Conclusion:Our result showed knockdown of HDAC1 by siRNA lead to the glioma cell proliferation and invasion capability reduction and overexpression of HDACl significantly increased the glioma cells proliferation and invasion capability in vitro.These results provide evidence that HDACl play a crucial role in glioma cells proliferation and invasion.Chapter Ⅲ HDACl promotes U87MG cell proliferation and invasion via activation of PI3K/AKT and MEK/ERK signaling pathwayObjective:To investigated the mechanism of PI3K/AKT and MEK/ERK signaling pathway in HDACl-mediated U87MG cell proliferation and invasion.Methods:The proliferation and invasion of U87MG cells were tested by MTS and transwell assays after knockdown HDACl with siRNA and overexpression HDACl by transfecting with pCMV-3　*Flag-HDACl. We detect the HDACl, phosphorylation of AKT and ERK protein expression of U87MG cells in each group by using Western Blot. Statistical analyses were performed using the SPSS 16.0. Data were presented as mean ± SD. Comparisons between two groups were performed using one-way ANOVA. P values less than 0.05 were considered statistically significant.Results:Our results suggested that knockdown of HDACl suppresses expression of activated AKT and ERK protein, the phosphorylated AKT and ERK significantly reduced in U87MG-siHDACl group compared with normal U87MG cell group. However, compared with normal U87MG cell group, over-expression of HDACl in U87MG-HDAC1 group can significantly increased phosphorylated AKT and ERK. Moreover, knockdown and over-expression of HDACl did not bring about alteration of total protein levels of AKT and ERK. The PI3K inhibitor (LY294002) lowered phosphorylated AKT without impacting phosphorylated ERK and significantly reduced the proliferation and invasion capability in U87MG-HDAC1+LY group compared with U87MG-HDAC1 group (p＜0.01). Meanwhile, compared with U87MG-HDAC1 group, the MEK inhibitor (PD98059) effectively decreased phosphorylated ERK without without leading to a reduction of phosphorylated AKT in U87MG-HDAC1+PD group, and it also cut down the proliferation and invasion capability in U87MG-HDAC1+PD group compared with U87MG-HDAC1 group. In addition, we can observe that the phosphorylated AKT and phosphorylated ERK reduced and the proliferation and invasion capability of U87MG cells Obviously declined in U87MG-HDAC1+LY+PD group compared with U87MG-HDAC1 group (p<0.01).Conclusion:HDAC1 could significantly enhance the proliferation and invasive capability of glioma cells through activation of the PI3K/AKT and MEK/ERK signaling pathway, and may provide a potential therapeutic target for glioma treatment.Part Two Role and mechanism of HDAC6 in the proliferation of gliomaChapter Ⅰ HDAC6 expression in glioma and non-neoplastic brain tissuesObjective:To investigate the mRNA expression of HDAC6 in glioma and non-neoplastic brain tissues.Methods:The mRNA expression levels of HDAC6 in 12 gliomas and 12 temporal lobectomy of epilepsy as negative control was detected by qPCR.Results:HDAC6 mRNA expressions were higher in glioma than that non-neoplastic brain tissues (P<0.05).Conclusion:Detecting the expression levels of HDAC6 will be helpful to evaluate the biological behaviors of gliomas and prognosis in the patients.Chapter Ⅱ HDAC6 promotes glioma cells proliferation through attenuating TGFβ/SMAD signaling pathwayObjective:To investigated the mechanism of TGFβ/SMAD signaling pathway signaling in HDAC6-mediated glioma cells proliferation.Methods:　The proliferation of A-172 and U87MG cells were tested by MTT assay. Recombinant TGFβ1 and ACY-1215 were used to interfere with A-172 and U87MG cells in each group. We detect the HDAC6, SMAD2, phosphorylation of SMAD2 and p21 protein expression of A-172 and U87MG cells in each group by using Western Blot. Statistical analyses were performed using the SPSS 16.0. Data were presented as mean ± SD. Comparisons between two groups were performed using one-way ANOVA. P values less than 0.05 were considered statistically significant.Results:We used two human glioblastoma cell lines A-172 and U87MG for our study. Both lines expressed significant levels of HDAC6(P<0.05, one-way ANOVA). We thus used ACY-1215 to inhibit HDAC6 and then examined its effect on the growth of GBM cells in a MTT assay. We found that HDAC6 inhibition significantly decreased GBM cell growth(P<0.05, one-way ANOVA), suggesting that HDAC6 promotes GBM cell growth. The inhibition of HDAC6 by ACY-1216 was assured by Western blot in both lines. We found that ACY-1215 significantly increased phosphorylation of SMAD2, and subsequently its nuclear target p21, a well-known cell-cycle inhibitor. To examine whether the modulation of SMAD2 phosphorylation is responsible for the effect of HDAC6 on GBM cell growth, we gave both lines recombinant TGFβ1, a strong inducer of SMAD2 phosphorylation. We found that TGFβ1 significantly induced SMAD2 phosphorylation and increased the p21, without affecting HDAC6 levels.Conclusion:HDAC6 could significantly increase the proliferation capability through attenuating TGFβ/SMAD signaling pathway. It may provide a potential therapeutic target for glioma treatment.