VEGFA upregulates FLJ10540 and modulates migration and invasion of lung cancer via PI3K/AKT pathway.
ABSTRACT: BACKGROUND: Lung adenocarcinoma is the leading cause of cancer-related deaths among both men and women in the world. Despite recent advances in diagnosis and treatment, the mortality rates with an overall 5-year survival of only 15%. This high mortality is probably attributable to early metastasis. Although several well-known markers correlated with poor/metastasis prognosis in lung adenocarcinoma patients by immunohistochemistry was reported, the molecular mechanisms of lung adenocarcinoma development are still not clear. To explore novel molecular markers and their signaling pathways will be crucial for aiding in treatment of lung adenocarcinoma patients. METHODOLOGY/PRINCIPAL FINDINGS: To identify novel lung adenocarcinoma-associated /metastasis genes and to clarify the underlying molecular mechanisms of these targets in lung cancer progression, we created a bioinformatics scheme consisting of integrating three gene expression profile datasets, including pairwise lung adenocarcinoma, secondary metastatic tumors vs. benign tumors, and a series of invasive cell lines. Among the novel targets identified, FLJ10540 was overexpressed in lung cancer tissues and is associated with cell migration and invasion. Furthermore, we employed two co-expression strategies to identify in which pathway FLJ10540 was involved. Lung adenocarcinoma array profiles and tissue microarray IHC staining data showed that FLJ10540 and VEGF-A, as well as FLJ10540 and phospho-AKT exhibit positive correlations, respectively. Stimulation of lung cancer cells with VEGF-A results in an increase in FLJ10540 protein expression and enhances complex formation with PI3K. Treatment with VEGFR2 and PI3K inhibitors affects cell migration and invasion by activating the PI3K/AKT pathway. Moreover, knockdown of FLJ10540 destabilizes formation of the P110-alpha/P85-alpha-(PI3K) complex, further supporting the participation of FLJ10540 in the VEGF-A/PI3K/AKT pathway. CONCLUSIONS/SIGNIFICANCE: This finding set the stage for further testing of FLJ10540 as a new therapeutic target for treating lung cancer and may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in lung cancer cells.
Project description:BACKGROUND:The human coagulation trigger tissue factor (TF) is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi) technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo. METHODS:The specific small interfering RNA (siRNA) designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated. RESULTS:TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma. CONCLUSIONS:Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.
Project description:OTUD7B, a multifunctional deubiquitinylase, plays an essential role in inflammation and proliferation signals. However, its function in lung cancer remains largely unknown. The aim of this study was to evaluate the prognostic significance of OTUD7B in patients with lung adenocarcinoma and squamous carcinoma and to characterize its molecular mechanisms in lung cancer progression and metastasis. Two tissue microarrays containing 150 pairs of lung squamous carcinoma and matched adjacent non-cancer tissues, and one tissue microarray containing 75 pairs of lung adenocarcinoma and adjacent non-cancer tissues were included, and immunohistochemical staining was performed to assess the clinical relevance of OTUD7B in non-small cell lung cancer. OTUD7B is highly expressed in both lung squamous carcinoma and adenocarcinoma and correlates with a worse prognosis. MTT proliferation, colony formation, migration and invasion assays and immunoblotting assay in NCI-H358 and A549 cell lines suggested that OTUD7B enhances EGF-induced Akt signal transduction and promotes lung cancer cell proliferation and migration. Immunohistochemical staining of large-scale lung cancer subjects (171 cases) revealed positive correlation of OTUD7B and VEGF expression. ELISA and tube formation assay revealed OTUD7B promotes VEGF production and angiogenesis. NCI-H358 tumor model demonstrated OTUD7B is required for lung tumor progression by facilitating activation of Akt signaling. These findings collectively identified OTUD7B as an independent predictive factor for the prognosis of non-small cell lung cancer and revealed OTUD7B promotes lung cancer cell proliferation and metastasis via Akt/VEGF signal pathway.
Project description:Metastasis is the main cause of lung cancer-related death. The tumor microenvironment greatly contributes to tumor metastasis. Resistin, mainly secreted by tumor-associated macrophages in tumor tissues, is a 12.5-kDa cysteine-rich secretory protein that is found at significantly higher levels in the serum or plasma of cancer patients compared with healthy controls. In this study, we explored the expression and role of resistin in lung adenocarcinoma. Our study showed that resistin was strongly expressed in lung adenocarcinoma tissues and promoted the migration and invasion of lung adenocarcinoma cells in a dose-dependent manner. Toll-like receptor 4 (TLR4) was the functional receptor of resistin for migration and invasion in A549 cells. Src/epidermal growth factor receptor (EGFR) was involved in resistin-induced migration and invasion. Resistin increased the phosphorylation of EGFR through the TLR4/Src pathway. We also found that PI3K/nuclear factor (NF)-?B were the intracellular downstream effectors mediating resistin-induced migration and invasion. Taken together, our results suggested that resistin promoted lung adenocarcinoma metastasis through the TLR4/Src/EGFR/PI3K/NF-?B pathway.
Project description:Chondrosarcoma is the second most common primary malignant bone cancer, with potential for local invasion and distant metastasis. Chemokine CCL5 (formerly RANTES) of the CC-chemokine family plays a crucial role in metastasis. Angiogenesis is essential for the cancer metastasis. However, correlation of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is still unknown. CCL5-mediated VEGF expression was assessed by qPCR, ELISA, and Western blotting. CCL5-induced angiogenesis was examined by migration and tube formation in endothelial progenitor cells in vitro. CCL5 increased VEGF expression and also promoted chondrosarcoma conditional medium-mediated angiogenesis in vitro and in vivo. Stimulation of chondrosarcoma with CCL5 augmented PI3K and Akt phosphorylation, while PI3K and Akt inhibitor or siRNA abolished CCL5-induced VEGF expression and angiogenesis. We also demonstrated CCL5 inhibiting miR-200b expression and miR-200b mimic reversing the CCL5-enhanced VEGF expression and angiogenesis. Moreover, in chondrosarcoma patients showed the positive correlation between CCL5 and VEGF; negative correlation between CCL5 and miR-200b. Taken together, results demonstrate CCL5 promoting VEGF-dependent angiogenesis in human chondrosarcoma cells by down-regulating miR-200b through PI3K/Akt signaling pathway.
Project description:Purpose:Metastasis is a crucial cause of the high mortality in patients with lung cancer. Long non-coding RNAs (lncRNAs) are emerging as important players in the development and progression of human cancers. Here, we aimed to identify metastasis-associated lncRNA and to study its roles in the migration and invasion of lung cancer cells. Materials and Methods:We screened differentially expressed lncRNAs between high- and low-metastatic lung cancer cell lines by using microarray and identified the target lncRNA TM4SF1-AS1. The effect of the TM4SF1-AS1 on the invasion and migration was evaluated through the wound healing experiment and transwell assay. The expression of related genes was assessed by RNA sequence and Western blotting. Results:TM4SF1-AS1 was highly expressed in high metastatic lung cancer cell line, and it was also significantly up-regulated in lymph node metastatic lung cancer and was associated with lymph node metastasis. Overexpression of TM4SF1-AS1 promoted the migration and invasion of lung cancer cells. Overexpression of TM4SF1-AS1 decreased the expression of E-Cadherin and increased the expression of Vimentin, Snail and Twist, while knockdown of TM4SF1-AS1 exhibited the opposite trend. Furthermore, RNA sequence analysis revealed that some signaling pathways, including PI3K/AKT signaling pathway, were enriched upon TM4SF1-AS1 overexpression. Western blotting further confirmed that the PI3K/AKT signaling pathway was activated by TM4SF1-AS1. Conclusion:This study illustrates that TM4SF1-AS1 promotes the migration and invasion of lung cancer cells by activating the PI3K/AKT signaling pathway. TM4SF1-AS1 might be a novel target of molecular treatment for lung cancer.
Project description:Lung adenocarcinoma (LUAD) is the main subtype of lung cancer. In this study, we found that RBP Mex3a was significantly upregulated in LUAD tissues and elevated Mex3a expression was associated with poor LUAD prognosis and metastasis. Furthermore, we demonstrated that Mex3a knockdown significantly inhibited LUAD cell migration and invasion in vitro and metastasis in nude mice. Transcriptome sequencing indicated that Mex3a affected gene expression linked to ECM-receptor interactions, including laminin subunit alpha 2(LAMA2). RNA immunoprecipitation (RIP) assay revealed Mex3a directly bound to LAMA2 mRNA and Mex3a increased the instability of LAMA2 mRNA in LUAD cells. Furthermore, we discovered that LAMA2 was surprisingly downregulated in LUAD and inhibited LUAD metastasis. LAMA2 knockdown partially reverse the decrease of cell migration and invasion caused by Mex3a knockdown. In addition, we found that both Mex3a and LAMA2 could influence PI3K-AKT pathway, which are downstream effectors of the ECM-receptor pathway. Moreover, the reduced activation of PI3K-AKT pathway in caused by Mex3a depletion was rescued by LAMA2 knockdown. In conclusion, we demonstrated that Mex3a downregulates LAMA2 expression to exert a prometastatic role in LUAD. Our study revealed the prognostic and prometastatic effects of Mex3a in LUAD, suggesting that Mex3a can serve as a prognostic biomarker and a target for metastatic therapy.
Project description:All-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance.We performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RAR? to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RAR?-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RAR?2 and p53. In contrast, over-expression of the inactive form of Akt restores RAR?2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes.Our results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.
Project description:BACKGROUND:MiR-125 has been shown to be involved in a variety of cancers, including cervical cancer (CC). Here, our goal was to explore miR-125 functional role and molecular mechanism in cervical cancer development and progression. METHODS:qRT-PCR was employ to detect miR-125 and VEGF mRNA expression. Western blot was applied for testing protein levels (VEGF, E-cadherin, N-cadherin, vimentin, AKT, p-AKT, PI3K, and p-PI3K). MTT and transwell assays were used for detecting cervical cancer cell progression, including cell viability, migration, and invasion. RESULTS:We observed that miR-125 was downregulated, whereas VEGF was upregulated in cervical cancer tissues and cell lines (CaSki and SiHa). MiR-125 inhibited the proliferation, invasion, and migration by targeting VEGF in cervical cancer. Moreover, miR-125 negatively regulated VEGF expression in cervical cancer tissues. Finally, we demonstrated that miR-520d-5p inhibited the activation of PI3K/AKT signaling pathway. CONCLUSION:In conclusion, the findings demonstrated that miR-125 inhibited cervical cancer progression and development by suppression VEGF and PI3K/AKT signaling pathway.
Project description:Tumor metastasis is the most lethal and debilitating process that threatens cancer patients. Among the regulators involved in tumor metastasis, lysyl oxidase (LOX) is an important contributor for tumor invasion, migration and the formation of the pre-metastatic niche. Although the relationship between LOX and poor prognosis of lung patients has been preliminary reported, the mechanism remains poorly understood. Here, we found that LOX overexpression is closely related to the survival of lung adenocarcinoma patients but not squamous cell carcinoma patients. Moreover, we confirmed that LOX expression is regulated by the activation of epidermal growth factor receptor (EGFR) via the PI3K/AKT, MEK/ERK, and SAPK/JNK signaling pathways in non-small cell lung cancer (NSCLC). Meanwhile, the study also suggested that the traditional anti-fibrosis drug silibinin inhibited NSCLC cell migration in an EGFR/LOX dependent manner. In addition, an orthotopic implantation metastasis model also confirmed that the EGFR inhibitor WZ4002 and silibinin decreased tumor metastasis through the EGFR/LOX pathway. Altogether, this study revealed that LOX expression is regulated by the EGFR pathway and this may account for the anti-cancer metastasis effects of silibinin, indicating LOX as a potentially therapeutic target for NSCLC treatment.
Project description:Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.