Lung cancer is the common cause of cancer death. STAT3 (signal transducer and activator of transcription 3) has been reported to be an oncogenic transcription factor and high expression of STAT3 is associated with lung cancer progression. RECK (reversion-inducing cysteine-rich protein with Kazal motifs) is a tumor suppressor gene and a membrane-anchored glycoprotein that reduces the matrix metalloproteinases (MMPs)-induced destruction of extra-cellular matrix (ECM) and tumor metastasis. RECK also inhibits tumor angiogenesis. We have previously elucidated the transcriptional regulation of RECK gene. Recently, microRNAs (miRs) are shown to be key players in gene regulation and cancer progression. In this study, we try to elucidate whether ovexpression of STAT3 can affect microRNA expression to regulate RECK via post-transcriptional modulation. miR-17-92a cluster is a well-known oncomir which is highly expressed in lung cancer tissue. We find that miR-92a, a member of miR-17-92a cluster can target RECK 3¡¦UTR. In addition, our data suggest that STAT3 regulates the expression of miR-92a and inhibition of STAT3 can decrease miR-92a expression. Furthermore, overexpression of miR-92a can decrease RECK protein level. While knockdown of miR-92a expression in STAT3-overexpressing cell lines can restore RECK protein level, and reduce invasion and migration. Results of this study suggest that STAT3 up-regulates miR-92a to inhibit RECK expression and promote lung cancer metastasis.

Part I BCR-ABL fusion oncogene results fromt(9;22)(q34;q11) translocation of chromosome is the most common genetic abnormality found in chronic myeloid leukemia (CML) cells . The encoded protein of this fusion gene exhibits constitutively active tyrosinekinase activity which is required for the pathogenesis of CML. We addressed how BCR-ABL oncoprotein increased Skp2 expression. Treatment of Imatinib or LY294002 reduced Skp2mRNA in BCR-ABL-positive K562 cells. Knockdown of AKT by small hairpin RNAalso reduced Skp2 expression. We found that BCR-ABL up-regulated Skp2 via Sp1 because (1) the Sp1 site located at the −386/−380 promoter region was important for BCR-ABL-induced Skp2 promoter activity, (2) chromatin immunoprecipitation assay demonstrated that Imatinib inhibited the recruitment of p300 to the Sp1 site of Skp2 promoter and (3) knockdown of Sp1 reduced Skp2 expression in K562 cells. These results suggest that BCR-ABL controls Skp2 gene transcription via the PI3K/AKT/Sp1 pathway. In addition to transcriptional regulation of Skp2, Bcr-Abl also modulates Skp2 protein stability in these cells. Treatment of Bcr-Abl kinase inhibitor imatinib led to G1 growth arrest accompanied with reduced Skp2 expression. Interestingly, reduction of Skp2 protein occurred prior to down-regulation of Skp2 mRNA suggesting a post-translational control. The half-life of Skp2 protein was significantly attenuated in imatinib-treated cells. Knockdown of Bcr-Abl similarly caused Skp2 protein instability. The decrease of Skp2 was induced by increased protein degradation through the ubiquitin/ proteasome pathway. Our results demonstrated that imatinib treatment or Bcr-Abl knockdown reduced Emi1, an endogenous inhibitor of the E3 ligase APC/Cdh1 which mediated the degradation of Skp2 protein. We found that Emi1 stability was regulated by phosphorylation and mutation of tyrosine 142 significantly reduced the stability. Lines of evidence suggested Bcr-Abl-induced Emi1 phosphorylation was mediated by Src kinase. (1) Src inhibitor SU6656 inhibited Emi1 tyrosine phosphorylation in Bcr-Abl-positive K562 cells. (2) Transfection of v-Src rescued the reduction of Emi1 by imatinib. (3) Mutation of tyrosine 142 to phenylalanine (Y142F) abolished the phosphorylation of Emi1 by recombinant Src kinase. In addition, ectopic expression of wild type but not Y142F mutant Emi1 could counteract imatinib-caused G1 growth arrest. Collectively, our results suggest that Bcr-Abl fusion oncogene increases Emi1 phosphorylation and stability to prevent Skp2 protein degradation via APC/Cdh1-induced ubiquitination and to enhance proliferation of CML cells. Part II Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anti-cancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells. WA at low concentrations induced autophagy in imatinib-sensitive K562 cells. Co-treatment of chloroquine suppressed autophagy and switched WA-treated K562 cells to apoptosis. This data indicated that autophagy protected K562 cells from apoptosis induced by WA. However, we found that WA triggered caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect was associated with down-regulation of Akt activity. Treatment of the AKT inhibitor LY294002 also caused apoptosis in imatinib-resistant T315I-positive cells. Ectopic expression of constitutively active Akt reversed WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA repressed the Akt signaling pathway by decreasing Akt expression. We found that WA abolished formation of the hsp90/cdc37/Akt complex to cause Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induced AKT down-regulation and apoptosis in primary CML cells. Taken together, our results suggested that imatinib-resistant T315I-positive cells were more addicted to Akt-dependent survival pathway and were more sensitive to WA. Therefore, WA could be useful for the treatment of imatinib-resistant CML. Part III Suberoylanilide hydroxamic acid (SAHA) is undergoing clinical trial for the treatment of various cancers including chronic myeloid leukemia (CML). We study the potential miRNAs which involved in the anti-cancer effect of SAHA. Microarray analysis revealed that the expression of 57 and 63 miRNAs was significantly changed in K562 cells treated with SAHA for 8h and 24h respectively. Five miRNAs(miR-92a, miR-199b-5p, miR-223, miR-627 and miR-675) were highly expressed in K562 cells and continuously repressed by SAHA. miR-92a and miR-223 known to play important roles in normal and hematopoisis were further characterized. Up-regulation of miR-92a was found in K562 cells and in primary CML cells. Inhibition of miR-92a with SAHA led to increase of the tumor suppressor Fbxw7. Conversely, ectopic expression of pri-miR-92a reversed SAHA-induced apoptosis of K562 cells, increase of Fbxw7 3¡¦-UTR reporter activity and up-regulation of Fbxw7. Collecively, miR-92a is up-regulated in CML cells, and SAHA downregulated the expression of miR-92a to result in apoptosis of CML cells.

MicroRNAs are emerging as key players in the regulation of the post-myocardial infarction (MI) environment. We previously identified that matrix-treated hearts had down-regulated expression of miR-92a, a miRNA with inflammatory and migratory effects that is normally up-regulated after MI. We have shown that type I collagen matrix treatment at 3h post-MI leads to less inflammation and improved cardiac function, but the underlying mechanisms remain to be better characterized. The goal of this study was to elucidate a possible role of miR-92a in the anti-inflammatory/pro-wound healing effect of matrix treatment post-MI. C57BL/6J mice underwent LAD ligation to induce MI. Hearts were removed at 4h, 1d, 3d, and 7d post-MI and RNA was extracted from infarct and peri-infarct tissue. PCR analysis revealed that hearts injected with matrix at 3h post-MI resulted in significantly decreased miR-92a at 4h, 1d, and 3d compared to non-injected animals at each time point (p<0.0001) and PBS injected animals at 4h and 7d (p<0.004). Several targets of miR-92a and regulators of macrophage polarization were found to be up-regulated (p<0.05) early in MI indicating early amelioration of inflammatory processes. In vitro, macrophages cultured on matrix also had decreased expression of miR-92a compared to cultures on tissue culture poly styrene (TCPS) (p<0.001). Integrins α5 (ITGAα5) and αV (ITGAαV), involved in cell-matrix interactions, as well as inflammatory regulators S1PR1 and SIRT1 were identified as putative miR-92a targets. When miR-92a is over-expressed in macrophages, ITGα5 (p=0.0002), ITGαV (p=0.02), and S1Pr1 (p<0.0001), and SIRT1 (p=0.03) all had decreased expression. STAT3 and IL-10 were found to be moderately down-regulated. In evaluating macrophage phenotypes, M2 macrophages had reduced miR-92a expression on matrix compared to M1 macrophages. The migration of M2 macrophages into the matrix is increased compared to M1 macrophages. We report that the beneficial effects of matrix treatment post-MI may be mediated, at least in part, through its ability to regulate miR-92a and pro-wound healing mechanisms in macrophages. These results present the matrix as a novel non-pharmacological approach to locally regulate miRNAs in vivo for reducing inflammation and protecting the myocardium post-MI.

Myocardial infarction (MI), a late-stage event of many cardiovascular diseases (CVD), results in cardiomyocyte death, myeloid cell recruitment to promote cellular debris removal and excessive cardiac remodeling affecting architecture and function, which can ultimately lead to heart failure. Currently, the use of biomaterials to intervene on the hostile post-MI environment and promote myocardial healing is being investigated to restore cardiac function. It has been shown that an injectable collagen matrix improves cardiac repair by altering macrophage polarization, reducing cell death and enhancing angiogenesis, leading to a reduction in infarct size and improved cardiac function when delivered at 3 hours post-MI. MicroRNAs (miRNA) “fine tune” gene expression by negatively regulating the translational output of target messenger RNA (mRNA). As such, miRNAs present interesting therapeutic opportunities for the treatment of MI. However, the delivery of miRNA mimics and/or inhibitors can be complicated by degradation and off target effects. The objectives of this thesis were to determine how the matrix may regulate endogenous miRNAs and to explore the biomaterial’s ability to deliver therapeutic miRNAs. It was shown that matrix treatment of MI mouse hearts resulted in altered expression of 119 miRNAs, some of which had functions linked to the beneficial effects of matrix treatment. Of particular interest, miR-92a was down-regulated within the infarct and peri-infarct cardiac tissue 2 days after matrix treatment (delivered at 3-hours post-MI) compared to PBS treatment. In in vitro cultures, the matrix down-regulated miR-92a levels in macrophages but did not significantly alter miR-92a expression in endothelial cells, circulating angiogenic cells or fibroblasts. In addition, using an in vitro model system, it was shown that the matrix may have the potential to deliver functional therapeutic miRNAs to cells; however further experimental optimisation is required to confirm these results. Therefore, collagen matrix treatment may be a promising approach to regulate and/or deliver miRNAs for protecting the myocardial environment and improving function of the infarcted heart.