Histone Deacetylase-3/CAGE Axis Targets EGFR Signaling and Regulates the Response to Anti-Cancer Drugs

We have previously reported the role of miR-326-HDAC3 loop in anti-cancer drug-resistance. CAGE, a cancer/testis antigen, regulates the response to anti-cancer drug-resistance by forming a negative feedback loop with miR-200b. Studies investigating the relationship between CAGE and HDAC3 revealed that HDAC3 negatively regulated the expression of CAGE. ChIP assays demonstrated the binding of HDAC3 to the promoter sequences of CAGE. However, CAGE did not affect the expression of HDAC3. We also found that EGFR signaling regulated the expressions of HDAC3 and CAGE. Anti-cancer drug-resistant cancer cell lines show an increased expression of pEGFR^Y845. HDAC3 was found to negatively regulate the expression of pEGFR^Y845. CAGE showed an interaction and co-localization with EGFR. It was seen that miR-326, a negative regulator of HDAC3, regulated the expression of CAGE, pEGFR^Y845, and the interaction between CAGE and EGFR. miR-326 inhibitor induced the binding of HDAC3 to the promoter sequences in anti-cancer drug-resistant Malme3M^R cells, decreasing the tumorigenic potential of Malme3M^R cells in a manner associated with its effect on the expression of HDAC3, CAGE and pEGFR^Y845. The down-regulation of HDAC3 enhanced the tumorigenic, angiogenic and invasion potential of the anti-cancer drug-sensitive Malme3M cells in CAGE-dependent manner. Studies revealed that PKCδ was responsible for the increased expression of pEGFR^Y845 and CAGE in Malme3M^R cells. CAGE showed an interaction with PKCδ in Malme3M^R cells. Our results show that HDAC3-CAGE axis can be employed as a target for overcoming resistance to EGFR inhibitors.     

miR-326-Histone Deacetylase-3 Feedback Loop Regulates the Invasion and Tumorigenic and Angiogenic Response to Anti-cancer Drugs

Histone modification is known to be associated with multidrug resistance phenotypes. Cancer cell lines that are resistant or have been made resistant to anti-cancer drugs showed lower expression levels of histone deacetylase-3 (HDAC3), among the histone deacetylase(s), than cancer cell lines that were sensitive to anti-cancer drugs. Celastrol and Taxol decreased the expression of HDAC3 in cancer cell lines sensitive to anti-cancer drugs. HDAC3 negatively regulated the invasion, migration, and anchorage-independent growth of cancer cells. HDAC3 conferred sensitivity to anti-cancer drugs in vitro and in vivo. TargetScan analysis predicted miR-326 as a negative regulator of HDAC3. ChIP assays and luciferase assays showed a negative feedback loop between HDAC3 and miR-326. miR-326 decreased the apoptotic effect of anti-cancer drugs, and the miR-326 inhibitor increased the apoptotic effect of anti-cancer drugs. miR-326 enhanced the invasion and migration potential of cancer cells. The miR-326 inhibitor negatively regulated the tumorigenic, metastatic, and angiogenic potential of anti-cancer drug-resistant cancer cells. HDAC3 showed a positive feedback loop with miRNAs such as miR-200b, miR-217, and miR-335. miR-200b, miR-217, and miR-335 negatively regulated the expression of miR-326 and the invasion and migration potential of cancer cells while enhancing the apoptotic effect of anti-cancer drugs. TargetScan analysis predicted miR-200b and miR-217 as negative regulators of cancer-associated gene, a cancer/testis antigen, which is known to regulate the response to anti-cancer drugs. HDAC3 and miR-326 acted upstream of the cancer-associated gene. Thus, we show that the miR-326-HDAC3 feedback loop can be employed as a target for the development of anti-cancer therapeutics.     

miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3

We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3′-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.     

Roles and Mechanism of miR-199a and miR-125b in Tumor Angiogenesis

MicroRNAs (miRNAs) have been shown to be involved in different aspects of cancer biology including tumor angiogenesis. In this study, we identified that two miRNAs, miR-199a and miR-125b were downregulated in ovarian cancer tissues and cell lines. Overexpression of miR-199a and miR-125b inhibited tumor-induced angiogenesis associated with the decrease of HIF-1α and VEGF expression in ovarian cancer cells. Moreover, the levels of miR-199a and miR-125b were negatively correlated with VEGF mRNA levels in ovarian tissues. We further showed that direct targets of miR-199a and miR-125b HER2 and HER3 were functionally relevant. Forced expression of HER2 and HER3 rescued miR-199a- and miR-125b-inhibiting angiogenesis responses and Akt/p70S6K1/HIF-1α pathway. This study provides a rationale for new therapeutic approach to suppress tumor angiogenesis using miR-199a, miR-125b, or their mimics for ovarian cancer treatment in the future.     

MicroRNA-122 Inhibits Tumorigenic Properties of Hepatocellular Carcinoma Cells and Sensitizes These Cells to Sorafenib

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.     

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M^R) and the human hepatic cancer cell line SNU387 (SNU387^R) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387^R cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M^R cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.     

Tubulin Beta3 Serves as a Target of HDAC3 and Mediates Resistance to Microtubule-Targeting Drugs

We investigated the role of HDAC3 in anti-cancer drug-resistance. The expression of HDAC3 was decreased in cancer cell lines resistant to anti-cancer drugs such as celastrol and taxol. HDAC3 conferred sensitivity to these anti-cancer drugs. HDAC3 activity was necessary for conferring sensitivity to these anti-cancer drugs. The down-regulation of HDAC3 increased the expression of MDR1 and conferred resistance to anti-cancer drugs. The expression of tubulin β3 was increased in drug-resistant cancer cell lines. ChIP assays showed the binding of HDAC3 to the promoter sequences of tubulin β3 and HDAC6. HDAC6 showed an interaction with tubulin β3. HDAC3 had a negative regulatory role in the expression of tubulin β3 and HDAC6. The down-regulation of HDAC6 decreased the expression of MDR1 and tubulin β3, but did not affect HDAC3 expression. The down-regulation of HDAC6 conferred sensitivity to taxol. The down-regulation of tubulin β3 did not affect the expression of HDAC6 or MDR1. The down-regulation of tubulin β3 conferred sensitivity to anti-cancer drugs. Our results showed that tubulin β3 serves as a downstream target of HDAC3 and mediates resistance to microtubule-targeting drugs. Thus, the HDAC3-HDAC6-Tubulin β axis can be employed for the development of anti-cancer drugs.     

MiR-200c Increases the Radiosensitivity of Non-Small-Cell Lung Cancer Cell Line A549 by Targeting VEGF-VEGFR2 Pathway

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT), is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

Hypoxia-Induced Aggressiveness of Pancreatic Cancer Cells Is Due to Increased Expression of VEGF,IL-6 and miR-21, Which Can Be Attenuated by CDF Treatment

Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC) cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.     

Colorectal Cancer Migration and Invasion Initiated by microRNA-106a

MicroRNAs have been implicated in the regulation of several cellular signaling pathways of colorectal cancer (CRC) cells. Although emerging evidence proves that microRNA (miR)-106a is expressed highly in primary tumor and stool samples of CRC patients; whether or not miR-106a mediates cancer metastasis is unknown. We show here that miR-106a is highly expressed in metastatic CRC cells, and regulates cancer cell migration and invasion positively in vitro and in vivo. These phenotypes do not involve confounding influences on cancer cell proliferation. MiR-106a inhibits the expression of transforming growth factor-β receptor 2 (TGFBR2), leading to increased CRC cell migration and invasion. Importantly, miR-106a expression levels in primary CRCs are correlated with clinical cancer progression. These observations indicate that miR-106a inhibits the anti-metastatic target directly and results in CRC cell migration and invasion.     

Reverse Correlation between MicroRNA-145 and FSCN1 Affecting Gastric Cancer Migration and Invasion

MicroRNAs (miRs) play important roles in modulating gene expression during the processes of tumorigenesis and tumor development. Previous studies have found that miR-145 is down-regulated in the stomach neoplasm and is related to tumor migration and invasion. However, both the molecular mechanism and function of miR-145 in gastric cancer remain unclear. The present study is the first demonstration of the significant down-regulation of miR-145 expression in infiltrative gastric cancer compared to expanding gastric cancer. Additionally, correlation analyses revealed strong inverse correlations between miR-145 and FSCN1 expression levels in infiltrative gastric cancer. Furthermore, we demonstrated that miR-145 directly targets FSCN1 and suppresses cell migration and invasion in gastric cancer. Knocking down the expression of FSCN1 led to the suppression of migration and invasion in gastric cancer cells, and re-expressing FSCN1 in miR-145-overexpressing cells reversed their migration and invasion defects. Thus, we concluded that miR-145 regulates cell migration and invasion in gastric cancer primarily by directly targeting FSCN1.