HOXB13 promotes proliferation,migration, and invasion of glioblastoma through transcriptional upregulation of lncRNA HOXC-AS3

HOXB13 exerts a close relation in several human cancers. This study explored the role of HOXB13 in glioblastoma (GBM), a brain tissue with the highest aggressive rate and mortality in adults. Through microarray and immunohistochemistry analyses, HOXB13 was highly expressed in GBM tissues. Furthermore, we showed that high-level expression of HOXB13 in GBM was associated with worse survival, suggesting that HOXB13 could be a prognostic marker for patients with GBM. GBM cells U87 and U251 overexpressing HOXB13 showed enhanced proliferation, migration, and invasion relative to the control cells, while knockdown of HOXB13 led to decreased cell proliferation, migration, and invasion abilities. In addition, dual-luciferase report assay, chromatin immunoprecipitation assay, and quantitative real-time polymerase chain reaction data showed that HOXB13 directly bound to HOXC-AS3 promoter. HOXC-AS3 was involved in HOXB13-induced proliferation, migration, and invasion of GBM cells. In summary, this study revealed the prognostic potential of HOXB13 in GBM. We believed that HOXB13/HOXC-AS3 signaling axis can be served as therapeutic targets for this highly aggressive cancer.     

MALT1 is a potential therapeutic target in glioblastoma and plays a crucial role in EGFR‐induced NF‐κB activation

Glioblastoma multiforme (GBM) is the most common malignant tumour in the adult brain and hard to treat. Nuclear factor κB (NF‐κB) signalling has a crucial role in the tumorigenesis of GBM. EGFR signalling is an important driver of NF‐κB activation in GBM; however, the correlation between EGFR and the NF‐κB pathway remains unclear. In this study, we investigated the role of mucosa‐associated lymphoma antigen 1 (MALT1) in glioma progression and evaluated the anti‐tumour activity and effectiveness of MI‐2, a MALT1 inhibitor in a pre‐clinical GBM model. We identified a paracaspase MALT1 that is involved in EGFR‐induced NF‐kB activation in GBM. MALT1 deficiency or inhibition significantly affected the proliferation, survival, migration and invasion of GBM cells both in vitro and in vivo. Moreover, MALT1 inhibition caused G1 cell cycle arrest by regulating multiple cell cycle–associated proteins. Mechanistically, MALTI inhibition blocks the degradation of IκBα and prevents the nuclear accumulation of the NF‐κB p65 subunit in GBM cells. This study found that MALT1, a key signal transduction cascade, can mediate EGFR‐induced NF‐kB activation in GBM and may be potentially used as a novel therapeutic target for GBM.     

CFTR activation suppresses glioblastoma cell proliferation,migration and invasion

The aim of this study was to investigate the function of Cystic fibrosis transmembrane conductance regulator (CFTR) in human glioblastoma (GBM) cells. Data dining results of the Human Protein Atlas showed that low CFTR expression was associated with poor prognosis for GBM patients. We found that CFTR protein expression was lower in U87 and U251 GBM cells than that in normal humane astrocyte cells. CFTR activation significantly reduced GBM cell proliferation. In addition, CFTR activation significantly abrogated migration and invasion of GBM cells. Besides, CFTR activator Forskolin treatment markedly reduced MMP-2 protein expression. These effects of CFTR activation were significantly inhibited by CFTR inhibitor CFTRinh-172 pretreatment. Our findings suggested that JAK2/STAT3 signaling was involved in the anti-glioblastoma effects of CFTR activation. Moreover, CFTR overexpression in combination with Forskolin induced a synergistic anti-proliferative response in U87?cells. Overall, our findings demonstrated that CFTR activation suppressed GBM cell proliferation, migration and invasion likely through the inhibition of JAK2/STAT3 signaling.     

C3G downregulation induces the acquisition of a mesenchymal phenotype that enhances aggressiveness of glioblastoma cells

Glioblastoma (GBM) is the most aggressive tumor from the central nervous system (CNS). The current lack of efficient therapies makes essential to find new treatment strategies. C3G, a guanine nucleotide exchange factor for some Ras proteins, plays a dual role in cancer, but its function in GBM remains unknown. Database analyses revealed a reduced C3G mRNA expression in GBM patient samples. C3G protein levels were also decreased in a panel of human GBM cell lines as compared to astrocytes. Based on this, we characterized C3G function in GBM using in vitro and in vivo human GBM models. We report here that C3G downregulation promoted the acquisition of a more mesenchymal phenotype that enhanced the migratory and invasive capacity of GBM cells. This facilitates foci formation in anchorage-dependent and -independent growth assays and the generation of larger tumors in xenografts and chick chorioallantoic membrane (CAM) assays, but with a lower cell density, as proliferation was reduced. Mechanistically, C3G knock-down impairs EGFR signaling by reducing cell surface EGFR through recycling inhibition, while upregulating the activation of several other receptor tyrosine kinases (RTKs) that might promote invasion. In particular, FGF2, likely acting through FGFR1, promoted invasion of C3G-silenced GBM cells. Moreover, ERKs mediate this invasiveness, both in response to FGF2- and serum-induced chemoattraction. In conclusion, our data show the distinct dependency of GBM tumors on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibition protocols. Hence, patients with a low C3G expression might not respond to EGFR inhibitors.Subject terms: CNS cancer, Metastasis     

Microenvironmental Stiffness Enhances Glioma Cell Proliferation by Stimulating Epidermal Growth Factor Receptor Signaling

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.     

ZC3H13 suppresses colorectal cancer proliferation and invasion via inactivating Ras–ERK signaling

ZC3H13 is a canonical CCCH zinc finger protein, which harbors a somatic frame-shift mutation in colorectal cancer (CRC). However, its expression and biological function were still uncertain. In the current study, we found that ZC3H13 was served as a tumor suppressor in CRC cells, which decreased the expression of Snail, Cyclin D1, and Cyclin E1, and increased the expression of Occludin and Zo-1 through inactivating Ras–ERK signaling pathway. Furthermore, reduction of ZC3H13 associated with advanced TNM stage (p = 0.02), positive regional lymph node metastasis ( p = 0.01). Taken together, the current study indicated that ZC3H13 may be an upstream regulator of Ras–ERK signaling pathway and suppressed invasion and proliferation of CRC.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

CPSF6在胶质母细胞瘤进展中的作用及相关调控机制研究*

目的: 研究mRNA前体切割和多聚腺苷酸化特异性因子6(polyadenylation specific factor 6,CPSF6)对人胶质母细胞瘤(glioblastoma,GBM)细胞系U87和U251的增殖、迁移、侵袭以及ATP水平的影响,进一步探究其相关调控机制。方法: 通过Western blot和免疫组化检测CPSF6在GBM组织中的表达水平,利用在线数据库分析CPSF6在GBM组织和配对的非肿瘤组织中的表达水平,同时分析CPSF6与GBM的组织学级别和患者预后的关系。构建敲低CPSF6的U87和U251稳定表达细胞株,并采用RT-qPCR和Western blot方法分别验证U87和U251细胞中CPSF6的敲低效率;利用CCK8和Transwell实验分别检测CPSF6敲降对细胞增殖、迁移和侵袭能力的影响;ATP实验检测细胞内的ATP水平变化,确定CPSF6在GBM中的致癌作用。通过RNA-seq分析敲低CPSF6后GBM内mRNA 3'UTR变化情况,KEGG富集分析差异靶基因相关的信号通路。在富集出的信号通路指示下,利用透射电镜和Western blot实验进一步验证敲低CPSF6后GBM自噬的发生情况。 结果: CPSF6在GBM组织中呈现出高表达,其表达水平随组织学级别的增加而升高,且与患者不良预后相关。在U87和U251中敲低CPSF6后,细胞的增殖、迁移及侵袭能力均明显降低,细胞内ATP水平下降。对RNA-seq结果分析表明,敲低CPSF6后发生3'UTR缩短事件的基因远多于3'UTR延长事件的基因;KEGG富集到自噬信号通路与肿瘤进展密切相关,透射电镜和Western blot实验验证敲低CPSF6可以促进自噬通路的激活。结论: CPSF6在GBM中高表达,且与GBM的组织学级别和患者不良预后呈正相关,CPSF6可能通过抑制自噬通路的激活来促进U87和U251细胞的增殖、迁移、侵袭以及ATP的生成,进而促进GBM发生、发展。     

The duality of epidermal growth factor receptor (EGFR) signaling and neural stem cell phenotype: cell enhancer or cell transformer?

Recruitment of neural stem cells (NSCs) represents an elegant strategy for replacing adult central nervous system (CNS) cells lost to injury or disease. However, except in the rostral migratory stream to the olfactory bulb, the adult CNS harbors a relatively non permissive environment for motility of neural stem cells. This opens the possibility of therapeutic enhancement of NSC motility towards sites of CNS injury or disease. The Epidermal Growth Factor Receptor (EGFR) is involved in the activation of a number of downstream pathways that regulate the phenotype of progenitor cells. Activated EGFR tyrosine kinase activity enhances NSC migration, proliferation, and survival. However, EGFR signaling is also known to play a role in the most malignant and highly invasive of human tumors, glioblastoma multiforme (GBM). Recent evidence supports the theory that GBM derives from a 'cancer stem cell' and that EGFR signals are commonly altered in these precursor cells. This article will review the role of EGFR signaling as it relates to neural stem cell motility and invasion. The duality of altered EGFR signaling in neural progenitor cells is discussed and opportunities for enhancing the recruitment of adult progenitors, and consequences of altering EGFR signaling in progenitor cells will be highlighted.     

Ganoderic acid A holds promising cytotoxicity on human glioblastoma mediated by incurring apoptosis and autophagy and inactivating PI3K/AKT signaling pathway

Ganoderic acid A (GA‐A), recognized as a lanostanetriterpene isolated from Ganoderma lucidum, demonstrates an efficient antitumor activity in multiple cancers. To date, it is unclear whether and how GA‐A functions on human glioblastoma (GBM). To unravel the functional significance of GA‐A on human glioblastoma (GBM), the cell‐counting kit‐8 and transwell assays were used to detect proliferation, migration, and invasion of human GBM cell after GA‐A treatment. Then, we utilized the flow cytometry and western blot to further evaluate the effect of GA‐A on GBM cell. Further, activities of autophagy and PI3K/AKT signaling were assessed by Western blot assay. We found that GA‐A significantly inhibited proliferation, migration, and invasion of GBM cell. Additionally, GA‐A markedly triggered cell apoptosis, which incarnated an elevation trend in apoptotic percentage, simultaneously, an increased level of proapoptosis protein (Bax and active caspase‐3) and a decreased level of antiapoptosis protein (Bcl‐2), induced by GA‐A treatment. Meanwhile, levels of two well‐known autophagy markers (beclin 1 and LC3 II) increased while another autophagic substrate (P‐62) was reduced. Moreover, the expressions levels of phosphorylated AKT, mTOR, p‐P70S6K, and cyclin D1 in the PI3K/AKT pathway were significantly reduced, which revealed GA‐A repressed the activation of PI3K/AKT signaling pathway. Collectively, these results indicate that GA‐A may encourage U251 cell growth and invasion/migration inhibition, apoptosis, and autophagy through the inactivation of PI3K/AKT signaling pathway in human GBM. Hence, GA‐A may be a potent antitumorigenic agent for human GBM in future clinical practice.     

Prostaglandin E2 regulates cell migration via the intracellular activation of the epidermal growth factor receptor

Over the past decade cyclooxygenase-2-derived prostaglandins have been implicated in the development and progression of many types of cancer. Recently our laboratory has shown that treatment with prostaglandin E2 (PGE2) induces increased proliferation, migration, and invasiveness of colorectal carcinoma cells (Sheng, H., Shao, J., Washington, M. K., and DuBois, R. N. (2001) J. Biol. Chem. 276, 18075-18081). The stimulatory effects of PGE2 were dependent upon the activation of the phosphatidylinositol 3-kinase/Akt pathway. However, the exact signaling cascade responsible for phosphatidylinositol 3-kinase/Akt activation by PGE2 remains poorly defined. In the present study, we demonstrate that the PGE2-induced migration and invasion occurs via rapid transactivation and phosphorylation of the epidermal growth factor receptor (EGFR). Within minutes following treatment, PGE2 induces the activation of Akt. This effect was completely abolished by EGFR-specific tyrosine kinase inhibitors providing evidence for the role of the EGFR in this response. The rapid transactivation of the EGFR occurs via an intracellular Src-mediated event but not through the release of an extracellular epidermal growth factor-like ligand. EGFR transactivation was also observed in vivo by the direct comparison of normal and malignant human colorectal samples. These results suggest that in developing colonic carcinomas, the early effects of cyclooxygenase-2-derived PGE2 are in part mediated by the EGFR, and this transactivation is responsible for subsequent down-stream effects including the stimulation of cell migration and invasion.     

Ras-PI3K信号负性调节的H3K56乙酰化促进乳腺癌细胞MCF-7的增殖和迁移能力

Ras蛋白常见的第12、13氨基酸残基突变引起的Ras信号通路异常与人类恶性肿瘤发生相关。然而,Ras致瘤信号通路是否涉及表观遗传学因素尚不明了。本研究旨在阐明人乳腺癌MCF-7细胞中组蛋白H3第56位赖氨酸残基乙酰化修饰（H3K56ac）水平是否受Ras信号通路调控,以及H3K56ac水平对MCF-7细胞增殖和迁移能力的影响。点突变结合基因转染揭示,与野生型比较,第12位氨基酸突变的Ras质粒（pEGFP-H-RasG12V）转染导致MCF-7细胞内H3K56ac水平明显降低。采用可特异激活Ras下游3条通路（Ras-Raf、Ras-RalGEF和Ras-PI3K）的3种质粒（pEGFP-H-RasG12V T35S,pEGFP-H-RasG12V E37G和pEGFP-H-RasG12V Y40C）转染证明,只有转染pEGFP-H-RasG12V Y40C的MCF-7细胞内不仅有Ras-PI3K-AKT通路被激活,且与H3K56ac水平下调相伴;而其他两条通路的激活不影响H3K56ac水平。MTT法结合Transwell、软琼脂克隆形成能力实验证明,RasG12V Y40C转染增强细胞增殖、迁移和克隆形成能力。上述结果表明,MCF-7细胞中H3K56ac水平受Ras-PI3K通路的负性调控,但不受Raf和RalGEF通路影响。Ras-PI3K激活导致的H3K56ac水平降低可增强乳腺癌MCF-7细胞的增殖和迁移能力。总之,这些结果提示,组蛋白H3K56ac是Ras-PI3K致瘤信号通路中的重要成员。Ras信号通路与组蛋白修饰相结合研究将会加深对乳腺癌细胞增殖和迁移调控机制的认识。     

PTBP3 promotes tumorigenesis of glioblastoma by stabilizing Twist1

ObjectiveGlioblastoma (GBM) is the most common malignancy tumor of central nervous system. PTBP3 was closely associated with the development of tumor. However, the function and molecular mechanism of PTBP3 in GBM is little known.MethodsqPCR and immunoblotting were used to detect PTBP3 expression levels in glioma tissues and cells. CCK8, Edu, flow cytometry, wound healing, and transwell assays were used to examined the function of PTBP3 in GBM. qPCR, Immunoblotting, and ubiquitination assays were performed to identify the mechanism of PTBP3.ResultsWe found that PTBP3 was upregulated in GBM, and high expression of PTBP3 correlated with the poor survival of GBM patients. PTBP3 knockdown reduced proliferation, invasion, and migration of GBM. Conversely, overexpressing PTBP3 has an opposite effect. Moreover, PTBP3 had an effect on the EMT of GBM. More importantly, we found that PTBP3 stabilized Twist1 by decreasing its ubiquitination and degradation. Furthermore, orthotopic xenograft models were used to demonstrate the PTBP3 on the development of GBM in vivo.ConclusionThis study proved that PTBP3 promoted tumorigenesis of GBM by stabilizing Twist1, which provided a new therapeutic target for GBM.     

Inhibition of TRPM7 with waixenicin A reduces glioblastoma cellular functions

Glioblastoma (GBM) is the most common malignant primary brain tumour originating in the CNS. Median patient survival is <15 months with standard treatment which consists of surgery alongside radiation therapy and temozolomide chemotherapy. However, because of the aggressive nature of GBM, and the significant toxicity of these adjuvant therapies, long-term therapeutic effects are unsatisfactory. Thus, there is urgency to identify new drug targets for GBM. Recent evidence shows that the transient receptor potential melastatin 7 (TRPM7) cation channel is aberrantly upregulated in GBM and its inhibition leads to reduction of GBM cellular functions. This suggests that TRPM7 may be a potential drug target for GBM treatment. In this study, we assessed the effects of the specific TRPM7 antagonist waixenicin A on human GBM cell lines U87 or U251 both in vitro and in vivo. First, we demonstrated in vitro that application of waixenicin A reduced TRPM7 protein expression and inhibited the TRPM7-like currents in GBM cells. We also observed reduction of GBM cell viability, migration, and invasion. Using an intracranial xenograft GBM mouse model, we showed that with treatment of waixenicin A, there was increased cleaved caspase 3 activity, alongside reduction in Ki-67, cofilin, and Akt activity in vivo. Together, these data demonstrate higher GBM cell apoptosis, and lower proliferation, migration, invasion and survivability following treatment with waixenicin A.     

LncRNA-H19 activates CDC42/PAK1 pathway to promote cell proliferation,migration and invasion by targeting miR-15b in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the main causes of cancer-related death. This study aims to explore the role and underlying mechanism of H19 in HCC.MethodsqRT-PCR detected miR-15b-5p and H19 expression, as well as the mRNA level of EMT-associated genes. Western blotting detected protein level of EMT-associated genes. Immunohistochemistry (IHC) examined CDC42 in HCC tissues. Dual luciferase reporter assay verified the regulatory mechanism among H19, miR-15b and CDC42. Colony formation, wound healing assay, transwell, flow cytometry measured proliferation, migration, invasion and apoptosis, respectively.ResultsH19 and CDC42 were up-regulated while miR-15b was down-regulated in HCC cells and tissues. miR-15b interacted with H19 and CDC42 3′-UTR. H19 knockdown inhibited proliferation, migration and invasion, and increased apoptosis, which was rescued by miR-15b inhibitor. H19 knockdown suppressed CDC42/PAK1 pathway and EMT progress.ConclusionH19 knockdown inhibited proliferation, migration and invasion, and promoted apoptosis of HCC cells via targeting miR-15b/CDC42/PAK1 axis.     

Flotillin-1 is a prognostic biomarker for glioblastoma and promotes cancer development through enhancing invasion and altering tumour microenvironment

Flotillin-1(FLOT1) has long been recognized as a tumour-promoting gene in several types of cancer. However, the expression and function of FLOT1 in glioblastomas (GBM) has not been elucidated. Here, in this study, we find that the expression level of FLOT1 in GBM tissue was much higher than that in normal brain, and the expression was even higher in the more aggressive subtypes and IDH status of glioma. Kaplan–Meier survival revealed that high FLOT1 expression is closely associated with poor outcome in GBM patients. FLOT1 knockdown markedly reduced the proliferation, migration and invasiveness of GBM cells, while FLOT1 overexpression significantly increases GBM cell proliferation, migration and invasiveness. Mechanistically, FLOT1 expression may play a potential role in the microenvironment of GBM. Therefore, FLOT1 promotes GBM proliferation and invasion in vitro and in vivo and may serve as a biomarker of prognosis and therapeutic potential in the fight against GBM.