TGF-?-mediated exosomal lnc-MMP2-2 regulates migration and invasion of lung cancer cells to the vasculature by promoting MMP2 expression.
ABSTRACT: Previous studies indicated that transforming growth factor (TGF)-?-mediated exosomal microRNAs (miRNAs) regulate the migration and invasion of lung cancer cells; however, whether and how TGF-?-mediated exosomal long noncoding (lnc) RNAs regulate migration and invasion of lung cancer cells remains unclear. Here, coculture experiments showed that TGF-? pretreatment increased the migration and invasion potential of lung cancer cells and TGF-? pretreated A549 cells increases vascular permeability. Furthermore, we found that TGF-?-mediated exosomes, as carriers of intercellular communication, regulated lung cancer invasion, and vascular permeability. Transcriptional analysis also revealed that lnc-MMP2-2 was highly enriched in TGF-?-mediated exosomes and might function by increasing the expression of matrix metalloproteinase (MMP)2 through its enhancer activity, with ectopic expression and silencing of lnc-MMP2-2 affecting lung cancer invasion and vascular permeability. Additionally, lnc-MMP2-2 and MMP2 expression was assessed semiquantitatively, and tissue-specific correlations between lnc-MMP2-2 and MMP2 expression were evaluated. These results suggested that exosomal lnc-MMP2-2 might regulate the migration and invasion of lung cancer cells into the vasculature by promoting MMP2 expression, suggesting this lncRNA as a novel therapeutic target and predictive marker of tumor metastasis in lung cancer.
Project description:Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-? (IFN?) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFN? induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.
Project description:Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin ?1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin ?1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3'-UTRs of integrin ?1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin ?1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin ?1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.
Project description:KLF2, a member of the Kruppel-like factor (KLF) family, is thought to be a tumor suppressor in many kinds of malignant tumors. Its functions in prostate cancer (PCa) are unknown. This study aimed to explore the role of KLF2 in the migration and invasion of PCa cells. The expression of KLF2 was measured by immunohistochemistry in PCa tissues and in paired non-tumor tissues. KLF2 and MMP2 expression in cells was measured by Western blot and RT-qPCR. Adenoviruses and siRNAs were used in cell function tests to investigate the role of KLF2 in regulating MMP2. Interactions between KLF2 and MMP2 were analyzed by a luciferase activity assay. The present study, for the first time, identified that KLF2 was downregulated both in PCa clinical tissue samples and in cancer cell lines. The overexpression of KLF2 inhibited the migration and invasion of PCa cells via the suppression of MMP2.This study demonstrates that KLF2 might act as a tumor suppressor gene in PCa and that the pharmaceutical upregulation of KLF2 may be a potential approach for treatment.
Project description:Histone deacetylase 6 (HDAC6) is a unique member of the histone deacetylase family. Although HDAC6 is mainly localized in the cytoplasm, it can regulate the activities of the transcription factors in the nucleus. However, a correlation of intracellular distribution of HDAC6 with tumor progression is lacking. In this study, we found that a low frequency of nuclear HDAC6-positive cells in tumors was associated with distant metastasis and a worse overall survival in 134 patients with non-small cell lung cancer (NSCLC). Ectopic expression of wild-type HDAC6 promoted migration and invasion of A549 and H661 cells. However, the enforced expression of nuclear export signal-deleted HDAC6 inhibited the invasion but not the migration of both cell lines. The inhibitory effect of nuclear HDAC6 on invasion was mediated by the deacetylation of the p65 subunit of nuclear factor-?B, which decreased its DNA-binding activity to the MMP2 promoter, leading to the downregulation of MMP2 expression. Our findings indicated that the loss of nuclear HDAC6 may be a potential biomarker for predicting metastasis in patients with NSCLC.
Project description:Restenosis limits the efficacy of vascular percutaneous intervention, in which vascular smooth muscle cell (VSMC) proliferation and activation of inflammation are two primary causal factors. Calpains influence VSMC proliferation and collagen synthesis. However, the roles of calpastatin and calpains in vascular restenosis remain unclear. Here, restenosis was induced by ligating the left carotid artery, and VSMCs were pretreated with platelet-derived growth factor (PDGF)-BB. Adenovirus vector carrying MMP2 sequence and specific small interfering RNA against calpain-1/2 were introduced. Finally, restenosis enhanced the expression of calpain-1/2, but reduced calpastatin content. In calpastatin transgenic mice, lumen narrowing was attenuated gradually and peaked on days 14-21. Cell proliferation and migration as well as collagen synthesis were inhibited in transgenic mice, and expression of calpain-1/2 and MMP2/transforming growth factor-?1 (TGF-?1). Consistently, in VSMCs pretreated with PDGF-BB, calpastatin induction and calpains inhibition suppressed the proliferation and migration of VSMCs and collagen synthesis, and reduced expression of calpain-1/2 and MMP2/TGF-?1. Moreover, simvastatin improved restenosis indicators by suppressing the HIF-1?/calpains/MMP2/TGF-?1 pathway. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin. Collectively, calpains inhibition plays crucial roles in vascular restenosis by preventing neointimal hyperplasia at the early stage via suppression of the MMP2/TGF-?1 pathway.
Project description:The transforming growth factor-? (TGF-?)/Smads signal plays an important role in cancer metastasis by mediating the epithelial-mesenchymal transition (EMT) in cancer cells. lnc-TSI is a recently identified long noncoding RNA that negatively regulates the TGF-?/Smads signal. The present study was conducted to test the hypothesis that lnc-TSI inhibits metastasis in clear cell renal cell carcinoma (ccRCC) by regulating the TGF-?/Smad3 pathway. Herein, we show that lnc-TSI was upregulated in ccRCC cells and tissue and was associated with activation of the TGF-?/Smads signal. Depleting lnc-TSI enhanced tumor cell invasion and metastasis in vitro and ccRCC lung metastasis in vivo, whereas overexpressing lnc-TSI inhibited ccRCC cell invasion and tumor metastasis. Mechanistic studies indicated that lnc-TSI specifically inhibited the phosphorylation of Smad3 and subsequent EMT by binding with the MH2 domain of Smad3 to block the interaction between Smad3 and TGF-? receptor I in ccRCC cells. In a cohort of 150 patients with ccRCC, expression of lnc-TSI in tumors was negatively correlated with phosphorylated (p)Smad3 and activated EMT markers. Patients with expression of tumor lnc-TSI greater than or equal to the median at radical nephrectomy had a higher survival rate compared to those with lnc-TSI below the median during follow-up. These findings reveal a new regulatory mechanism of ccRCC metastasis and suggest a potential molecular target for the development of anti-cancer drugs.
Project description:Background:Thymosin ?1 (T?1) is one of the most commonly used immunomodulators for metastatic non-small-cell lung cancer (NSCLC) patients in many countries. Despite the identification of the direct suppression on cancer cell proliferation, little is known about its effect on metastasis and metastasis-related signaling such as matrix metalloproteinases (MMPs) and programmed cell death ligand 1 (PD-L1). Materials and methods:NSCLC cells with distinguishing PD-L1 expression levels were treated with T?1. siRNAs were used to knockdown PD-L1. Cell migration and invasion abilities were evaluated by wound-healing and transwell assays. The xenograft model by BALB/c nude mice was constructed to test the inhibitory effect of T?1 on metastasis in vivo. The expression levels of metastasis-related signaling pathways and key molecules were assessed by Western blot (WB) and quantitative reverse transcriptase PCR (qRT-PCR). Results:T?1 significantly suppressed cell migration and invasion in PD-L1 high-expressing H1299, NL9980, and L9981 cells but not in PD-L1 low-expressing A549 or SPC-A-1 cells. This difference was demonstrated by mouse model in vivo as well. Knocking down of PD-L1 significantly impaired the inhibition of cell migration and invasion caused by T?1 treating in PD-L1 high-expressing cells. Besides, T?1 inhibited the activation and translocation of STAT3 and the expression of MMP2 in PD-L1 high-expressing NSCLC cells. Moreover, the treatment of STAT3 activator colivelin could partly reverse the T?1-induced MMP2 suppression and the migration phenotype. Conclusion:T?1 significantly suppresses migration and invasion in PD-L1 high-expressing NSCLC cells compared with PD-L1 low-expressing NSCLC cells in vitro and in vivo, through the downregulation of STAT3-MMP2 signaling. These different responses to T?1, together with the depiction of T?1-induced signaling changes, suggest a potential benefit of T?1 for PD-L1-positive NSCLC patients, enlightening the combination of T?1 with target therapy or immune checkpoint inhibitors.
Project description:The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient's prostate cells.
Project description:Long non-coding (lnc)RNA mortal obligate RNA transcript (MORT) is inhibited in numerous types of cancer in humans, indicating its role as a tumor suppressor. The present study demonstrated downregulation of lncRNA MORT in the tumor tissues of patients with colon cancer. The expression of MORT in tumor tissues was linearly associated with its expression levels in plasma. Low MORT expression was associated with low overall survival rate. Moreover, the overexpression of MORT resulted in decreased, whereas treatment with transforming growth factor ?1 (TGF-?1) resulted in increased, invasion and migration rates of colon cancer cells. In addition, TGF-?1 treatment attenuated the inhibitory effect of MORT overexpression on the invasion and migration rates of colon cancer cells. The overexpression of MORT inhibited TGF-?1 expression in colon cancer cells, whereas treatment with TGF-?1 failed to affect the expression of the lncRNA. Therefore, it is postulated that MORT inhibits invasion and migration colon cancer cells by inactivating TGF-?1.
Project description:Background:It is known that secreted protein acidic and cysteine rich (osteonectin), cwcv and kazal-like domains proteoglycan 2 (SPOCK2) plays a significant role in the development and progression of several human cancers; however, the role of SPOCK2 in prostate cancer (PCa) remains unclear. This study aimed to find the role and mechanism of SPOCK2 in the development and progression of PCa. Methods:The messenger ribonucleic acid (mRNA) expression of SPOCK2 in PCa tissue was detected by real-time polymerase chain reaction (PCR). Upregulation of the SPOCK2 gene was achieved using the DU145 and LNCaP cells by transfecting the cells with SPOCK2 recombinant fragment. Cell invasion and migration ability were detected by transwell chamber and wound healing assay. The expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase 2 (MMP2) in the cells was detected by Western Blot and zymography gel assay. Results:The mRNA level of SPOCK2 was significantly lower in the PCa tissue compared to benign prostate hyperplasia. Upregulation of SPOCK2 inhibited cell invasion and migration in DU145 and LNCaP cells, inhibited the expression of MT1-MMP and MMP2 and, inhibited activation of MMP2 in DU145 and LNCaP cells. Conclusion:SPOCK2 is associated with the progression of PCa. Upregulation of SPOCK2 can inhibit PCa cell invasion and metastasis by decreasing MT1-MMP and MMP2 gene expression and decreasing MMP2 protein activation.