Mei

Downregulation of miR-21 inhibits EGFR pathway and suppresses the growth of human glioblastoma cells independent of PTEN status

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that regulate gene expression after transcription. Aberrant expression of miRNAs has been shown to be involved in tumorigenesis. We showed that miR-21 was one of the most frequently overexpressed miRNA in human glioblastoma (GBM) cell lines. To explore whether miR-21 can serve as a therapeutic target for glioblastoma, we downregulated miR-21 with a specific antisense oligonucleotide and found that apoptosis was induced and cell-cycle progression was inhibited in vitro in U251 (PTEN mutant) and LN229 (PTEN wild-type) GBM cells; xenograft tumors from antisense-treated U251 cells were suppressed in vivo. Antisense-miR-21-treated cells showed a decreased expression of EGFR, activated Akt, cyclin D, and Bcl-2. Although miR-21 is known to regulate PTEN and downregulation of miR-21 led to increased PTEN expression both endogenously and in a reporter gene assay, the GBM suppressor effect of antisense-miR-21 is most likely independent of PTEN regulation because U251 has mutant PTEN. Microarray analysis showed that the knockdown of miR-21 significantly altered expression of 169 genes involved in nine cell-cycle and signaling pathways. Taken together, our studies provide evidence that miR-21 may serve as a novel therapeutic target for malignant gliomas independent of PTEN status.

MicroRNA-21 inhibitor sensitizes human glioblastoma cells U251 (PTEN-mutant) and LN229 (PTEN-wild type) to taxol

BackgroundSubstantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM.MethodsHuman glioblastoma U251 (PTEN-mutant) and LN229 (PTEN wild-type) cells were treated with taxol and the miR-21 inhibitor (in a poly (amidoamine) (PAMAM) dendrimer), alone or in combination. The 50% inhibitory concentration and cell viability were determined by the MTT assay. The mechanism between the miR-21 inhibitor and the anticancer drug taxol was analyzed using the Zheng-Jun Jin method. Annexin V/PI staining was performed, and apoptosis and the cell cycle were evaluated by flow cytometry analysis. Expression of miR-21 was investigated by RT-PCR, and western blotting was performed to evaluate malignancy related protein alteration.ResultsIC(50) values were dramatically decreased in cells treated with miR-21 inhibitor combine with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-21 inhibitor significantly enhanced apoptosis in both U251 cells and LN229 cells, and cell invasiveness was obviously weakened. Interestingly, the above data suggested that in both the PTEN mutant and the wild-type GBM cells, miR-21 blockage increased the chemosensitivity to taxol. It is worth noting that the miR-21 inhibitor additively interacted with taxol on U251cells and synergistically on LN229 cells. Thus, the miR-21 inhibitor might interrupt the activity of EGFR pathways, independently of PTEN status. Meanwhile, the expression of STAT3 and p-STAT3 decreased to relatively low levels after miR-21 inhibitor and taxol treatment. The data strongly suggested that a regulatory loop between miR-21 and STAT3 might provide an insight into the mechanism of modulating EGFR/STAT3 signaling.ConclusionsTaken together, the miR-21 inhibitor could enhance the chemo-sensitivity of human glioblastoma cells to taxol. A combination of miR-21 inhibitor and taxol could be an effective therapeutic strategy for controlling the growth of GBM by inhibiting STAT3 expression and phosphorylation.

Downregulation of miR-21 Enhances Chemotherapeutic Effect of Taxol in Breast Carcinoma Cells

The successful of anti-cancer treatment are often limited by the development of drug resistance. Recent work has highlighted the involvement of non-coding RNAs, microRNAs(miRNAs) in cancer development, and their possible involvement in the evolution of drug resistance has been proposed. In this study, we combine taxol chemotherapy and miR-21 inhibitor treatment via polyamidoamine (PAMAM) dendrimers vector to evaluate the effects of combination therapy on suppression of breast cancer cells. The 50% inhibitory concentration (IC50) values for taxol were significantly decreased to a greater extent in the cells transfected with miR-21 inhibitor compared with cells treated with taxol alone. Taxol treatment also increased the percentage of apoptotic breast cancer cells in miR-21 inhibitor transfected cells compared with control cells. Furthermore, treatment of the miR-21 inhibitor-transfected cells with the anti-cancer drugs taxol resulted in significantly reduced cell viability and invasiveness compared with control cells. These results indicated that the miR-21 plays an important role in the resistance of breast carcinoma cells to chemotherapeutic drugs. Therefore, miR-21 inhibitor gene therapy combined with taxol chemotherapy might represent a promising novel therapeutic approach for the treatment of breast malignancies.

Long non coding RNA MALAT1 promotes tumor growth and metastasis by inducing epithelial-mesenchymal transition in oral squamous cell carcinoma

The prognosis of advanced oral squamous cell carcinoma (OSCC) patients remains dismal, and a better understanding of the underlying mechanisms is critical for identifying effective targets with therapeutic potential to improve the survival of patients with OSCC. This study aims to clarify the clinical and biological significance of metastasis-associated long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in OSCC. We found that MALAT1 is overexpressed in OSCC tissues compared to normal oral mucosa by real-time PCR. MALAT1 served as a new prognostic factor in OSCC patients. When knockdown by small interfering RNA (siRNA) in OSCC cell lines TSCCA and Tca8113, MALAT1 was shown to be required for maintaining epithelial-mesenchymal transition (EMT) mediated cell migration and invasion. Western blot and immunofluorescence staining showed that MALAT1 knockdown significantly suppressed N-cadherin and Vimentin expression but induced E-cadherin expression in vitro. Meanwhile, both nucleus and cytoplasm levels of β-catenin and NF-κB were attenuated, while elevated MALAT1 level triggered the expression of β-catenin and NF-κB. More importantly, targeting MALAT1 inhibited TSCCA cell-induced xenograft tumor growth in vivo. Therefore, these findings provide mechanistic insight into the role of MALAT1 in regulating OSCC metastasis, suggesting that MALAT1 is an important prognostic factor and therapeutic target for OSCC.

Targeting STAT3/miR-21 axis inhibits epithelial-mesenchymal transition via regulating CDK5 in head and neck squamous cell carcinoma.

BackgroundAbnormal activation of STAT3 and miR-21 plays a vital role in progression and invasion of solid tumors. The cyclin-dependent kinase 5 (CDK5) is reported to contribute to cancer metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of STAT3/miR-21 axis and CDK5 in head and neck squamous cell carcinoma remains unclear.MethodsWe measured the expression of miR-21, CDK5 and EMT markers in 60 HNSCC tumor samples. We used Immunohistochemistry and in situ hybridization assay to examine the role of STAT3/miR-21 axis and CDK5 activation in the invasiveness of HNSCC. The clinical survival relevance was analyzed by Kaplan-Meier analysis and univariate/multivariate COX regression model. Multiple approaches including scratch, transwell chamber assay and other molecular biology techniques were used to validate the anti-invasion effect of targeting miR-21 in Tca8113 and Hep-2 cell lines in vitro. Furthermore, whether miR-21 depletion inhibits HNSCC invasion in vivo was confirmed in Tca8113 xenograft tumor model.ResultsThe expression of miR-21 and CDK5 were significantly correlated with lymph node metastasis in HNSCC. Hep-2 and Tca8113 cell lines showed co-overexpression of miR-21 and CDK5. WP1066 or asON-miR-21 treatment depleted miR-21 and CDK5 expression and significantly inhibited migration or invasion in Hep-2 and Tca8113 cells. The expression levels of CDK5/p35, N-cadherin, vimentin, β-catenin were inhibited while E-cadherin level was increased by miR-21 depletion in vitro and in vivo. Conversely, ectopic CDK5 overexpression significantly induced tumor cell motility and EMT. Moreover, ectopic CDK5 overexpression in Hep-2 and Tca8113 cells rescued the observed phenotype after miR-21 silencing or WP1066 treatment.ConclusionsmiR-21 cooperates with CDK5 to promote EMT and invasion in HNSCC. This finding suggests that CDK5 may be an important cofactor for targeting when designing metastasis-blocking therapy by targeting STAT3/miR-21 axis with STAT3 inhibitor or miR-21 antisense oligonucleotide. This is the first demonstration of the novel role of STAT3/miR-21 axis and CDK5/CDK5R1 (p35) in metastasis of HNSCC.

AC1MMYR2 impairs high dose paclitaxel-induced tumor metastasis by targeting miR-21/CDK5 axis

Paclitaxel (taxol) is a widely used chemo-drug for many solid tumors, while continual taxol treatment is revealed to stimulate tumor dissemination. We previously found that a small molecule inhibitor of miR-21, termed AC1MMYR2, had the potential to impair tumorigenesis and metastasis. The aim of this study was to investigate whether combining AC1MMYR2 with taxol could be explored as a means to limit tumor metastasis. Here we showed that abnormal activation of miR-21/CDK5 axis was associated with breast cancer lymph node metastasis, which was also contribute to high dose taxol-induced invasion and epithelial mesenchymal transition (EMT) in both breast cancer cell line MDA-MB-231 and glioblastoma cell line U87VIII. AC1MMYR2 attenuated CDK5 activity by functional targeting CDK5RAP1, CDK5 activator p39 and target p-FAK(ser732). A series of in vitro assays indicated that treatment of AC1MMYR2 combined with taxol suppressed tumor migration and invasion ability in both MDA-MB-231 and U87VIII cell. More importantly, combination therapy impaired high-dose taxol induced invadopodia, and EMT markers including β-catenin, E-cadherin and vimentin. Strikingly, a significant reduction of lung metastasis in mice was observed in the AC1MMYR2 plus taxol treatment. Taken together, our work demonstrated that AC1MMYR2 appeared to be a promising strategy in combating taxol induced cancer metastasis by targeting miR-21/CDK5 axis, which highlighted the potential for development of therapeutic modalities for better clinic taxol application.

Targeting EZH2 regulates tumor growth and apoptosis through modulating mitochondria dependent cell-death pathway in HNSCC

EZH2 is a negative prognostic factor and is overexpressed or activated in most human cancers including head and neck squamous cell carcinoma (HNSCC). Analysis of The Cancer Genome Atlas (TCGA) HNSCC data indicated that EZH2 over-expression was associated with high tumor grade and conferred poor prognosis. EZH2 inhibition triggered cell apoptosis, cell cycle arrest and decreased cell growth in vitro. MICU1 (mitochondrial calcium uptake1) was shown to be down regulated when EZH2 expression was inhibited in HNSCC. When the EZH2 and MICU1 were inhibited, HNSCC cells became susceptible to cell cycle arrest and apoptosis. Mitochondrial membrane potential and cytosolic Ca2+ concentration analysis suggested that EZH2 and MICU1 were required to maintain mitochondrial membrane potential stability. A xenograft tumor model was used to confirm that EZH2 depletion inhibited HNSCC cell growth and induced tumor cell apoptosis. In summary, EZH2 is a potential anti-tumor target in HNSCC.

Targeting HOTAIR Induces Mitochondria Related Apoptosis and Inhibits Tumor Growth in Head and Neck Squamous Cell Carcinoma in vitro and in vivo.

Homeobox (HOX) transcript antisense RNA (HOTAIR), a long nuclear-retained noncoding RNA (lncRNA), is overexpressed in a variety of human cancers. Increasing evidence shows that HOTAIR plays a vital role in cancer initiation and progression by affecting cell cycle progress, apoptosis and invasion. However, whether HOTAIR serves as a target of therapeutic potential and the underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unclear. Thus, we employed a HOTAIR specific siRNA to deplete its expression in two human HNSCC cell lines, Tca8113 and Tscca. The flow cytometry (FCM) analysis showed that HOTAIR depletion induced tumor cell apoptosis in vitro. JC-1 probe examination showed that the mitochondrial membrane potential was changed significantly by HOTAIR blockage. Mitochondrial calcium uptake 1(MICU1) dependent cell death was induced by HOTAIR depletion. Protein expression analysis indicated that mitochondrial related cell death pathway (Bcl-2, BAX, Caspase-3, Cleaved Caspase-3, Cytochrome c) involved in HOTAIR dependent apoptosis process. Moreover, a Tscca derived xenograft tumor model was employed to further validate that injection of HOTAIR siRNA inhibited tumor growth. In summary, we suggested that HOTAIR inhibition could be developed as a new therapeutic in HNSCC treatments.

Reprogramming carcinoma associated fibroblasts by AC1MMYR2 impedes tumor metastasis and improves chemotherapy efficacy

Carcinoma associated fibroblasts (CAFs) produce a nutrient-rich microenvironment to fuel tumor progression and metastasis. Reactive oxygen species (ROS) levels and the inflammation pathway co-operate to transform CAFs. Therefore, elucidating the mechanism mediating the activity of CAFs might identify novel therapies. Abnormal miR-21 expression was reported to be involved in the conversion of resident fibroblasts to CAFs, yet the factor that drives transformation was poorly understood. Here, we reported that high miR-21 expression was strongly associated with lymph node metastasis in breast cancer, and the activation of the miR-21/NF-кB was required for the metastatic promoting effect of CAFs. AC1MMYR2, a small molecule inhibitor of miR-21, attenuated NF-кB activity by directly targeting VHL, thereby blocking the co-precipitation of NF-кB and ß-catenin and nuclear translocation. Taxol failed to constrain the aggressive behavior of cancer cells stimulated by CAFs, whereas AC1MMYR2 plus taxol significantly suppressed tumor migration and invasion ability. Remodeling and depolarization of F-actin, decreased levels of β-catenin and vimentin, and increased E-cadherin were also detected in the combination therapy. Furthermore, reduced levels of FAP-α and α-SMA were observed, suggesting that AC1MMYR2 was competent to reprogram CAFs via the NF-кB/miR-21/VHL axis. Strikingly, a significant reduction of tumor growth and lung metastasis was observed in the combination treated mice. Taken together, our findings identified miR-21 as a critical mediator of metastasis in breast cancer through the tumor environment. AC1MMYR2 may be translated into the clinic and developed as a more personalized and effective neoadjuvant treatment for patients to reduce metastasis and improve the chemotherapy response.

PTEN activation sensitizes breast cancer to PI3-kinase inhibitor through the β-catenin signaling pathway.

Combination therapy is considered a promising therapeutic modality in enhancing treatment efficacy. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is almost universally dysregulated in breast cancer, with specific occurrence of PTEN mutations; thus, it has become an attractive target for cancer treatment. However, the use of single targeted therapeutics against the PI3K/AKT pathway has demonstrated only modest clinical benefits. In this study, recombinant adenovirus-mediated gene transfer of PTEN (AD-PTEN) combined with treatment with LY294002 was utilized to evaluate the effects of suppression of breast cancer cell proliferation. Herein, we show that AD-PTEN significantly enhanced the sensitization of breast cancer cells to LY294002. The 50% inhibitory concentration (IC50) values of LY294002 were significantly decreased to a greater extent in cells transfected with combination therapy. In addition, treatment of AD-PTEN-transfected cells with LY294002 resulted in significantly reduced cell viability and invasion ability compared to single LY294002 treatment. Using western blotting, we found that combination treatment resulted in lower levels of phosphorylated AKTSer473 and GSK-3βSer9 than single treatment with LY294002. Furthermore, we showed a significant decrease in nuclear β-catenin, Fra-1, Tcf-4 and c-Myc by combination treatment. Our results indicate that AD-PTEN sensitization of breast cancer to LY294002 is achieved by increased GSK-3β activity, thus resulting in inhibition of the β-catenin signaling pathway.