A networks method for ranking microRNA dysregulation in cancer

Background Despite the lack of agreement on their exact roles, it is known that miRNAs contribute to cancer progression. Many studies utilize methods to detect differential regulation of miRNA expression. It is prohibitively expensive to examine all potentially dysregulated miRNAs and traditionally, researchers have focused their efforts on the most extremely dysregulated miRNAs. These methods may overlook the contribution of less differentially expressed but more functionally relevant miRNAs. The purpose of this study was to outline a method that not only utilizes differential expression but ranks miRNAs based on the functional relevance of their targets. This work uses a networks based approach to determine the sum node degree for all experimentally verified miRNA targets to identify potential regulators of prostate cancer initiation, progression and metastasis. Results Here, we present a method for identifying functionally relevant miRNAs that contribute to prostate cancer development. This paper shows that miRNAs preferentially regulate highly connected, central proteins within a protein-protein interaction network. Known targets of miRNAs differentially regulated during prostate cancer progression are enriched in pathways with known involvement in tumorigenesis. To demonstrate the applicability of our method, we utilized a unique model of prostate cancer progression to identify five miRNAs that may contribute to the oncogenic state of the cell. Three of these miRNAs have been shown by other studies to have a role in cancer but their exact role in prostate cancer remains undefined. Conclusion Developing methods to determine which miRNAs to carry forward into biological and biochemical analyses is important as traditional approaches often overlook miRNAs that contribute to oncogenesis. Our method applied to a model of prostate cancer progression was able to identify miRNAs with roles in prostate cancer development

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression

Delivery of small molecule and RNA for the treatment of type 1 diabetes and prostate cancer

The aim of this thesis is to develop combination therapy using a small molecule and RNA including siRNA, shRNA, or miRNA inhibitor for the treatment of type 1 diabetes and prostate cancer. New amphiphilic biodegradable polymers capable of co-delivering small hydrophobic molecules and RNAs or human bone marrow-derived mesenchymal stem cell (hBMSC) for co-delivery of an shRNA and a miRNA inhibitor were used as drug delivery platform. The drug delivery properties were evaluated in vitro and in vivo islet transplantation, subcutaneous and orthotopic prostate cancer models. In Chapter 1, an overview of prostate cancer, the role of miRNA and the way for regulating miRNA as well as the design of delivery systems is given. I also introduce a brief background of type 1 diabetes and the treatment methods. Chapter 2 discussed the treatment of early stage or advanced prostate cancer using a luteinizing hormone release hormone (LHRH) conjugated micelles for target delivery of CBDIV17 (a bicalutamide analog) or combination therapy using CBDIV17 and embelin (a XIAP inhibitor). In Chapter 2, subcutaneous model was used to prove our concept. Our results indicated that LHRH conjugated micelles carrying CBDIV17 or both CBDIV17 and embelin inhibited tumor cell growth in vitro and in vivo. Chapter 3 reports the newly screened small molecule named rubone as a miR-34a modulator for combination therapy with paclitaxel to treat chemoresistant prostate cancer. This compound was first characterized for miR-34a modulation efficacy in paclitaxel resistant prostate cancer cell lines including DU145-TXR and PC3-TXR. The miR-34a downstream protein level and combination therapy efficacy were also evaluated. The biodegradable copolymer poly (ethylene glycol)-block-poly (2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol) (PEG-PCD) were used to co-deliver both drugs in an orthotopic prostate tumor model after characterizing the drug delivery properties. This combination therapy using rubone as a miR-34a modulator reversed the chemoresistance of prostate cancer and significantly inhibited paclitaxel-resistant tumor growth in vivo. Finally, we summarized the results for prostate cancer treatment and gives suggestions for further research. Chapter 4 provided the background information about islet transplantation for treating type 1 diabetes. In Chapter 4, we constructed plasmid encoding shRNA against Fas receptor and miRNA inhibitor for downregulating miR-375. This plasmid was transfected to hBMSCs as an RNA delivery vehicle and hBMSC transferred these two small RNAs to human islet by direct touch and exosome. This stem cell-based gene therapy and cell therapy suppressed islet apoptosis and promoted islet function in vitro and in a humanized NOD scid gamma (NSG) mouse model. The immune reaction after islet transplantation was suppressed by intravenous injection of hBMSC and peripheral blood mononuclear cells (PBMC) co-cultured exosomes. We obtained a 100% insulin independence after humanization by intraperitoneal injection of PBMC. Chapter 5 summarizes the results of this thesis and gives suggestions to further research

Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids

Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs are small, single-stranded RNA molecules that affect protein expression at the translational level, and dysregulation can dramatically affect cell metabolism. Comparison of 736 microRNA expression levels between the poorly metastatic SV40T immortalized prostate epithelial cell line P69 to its highly tumorigenic and metastatic subline M12 identified 231 miRs that were overexpressed and 150 miRs that showed loss of expression in the M12 cell line. Further evaluation of fourteen identified miRs was accomplished using other prostate cell lines as well as laser-capture microdissected prostate samples. Inhibition of miR-147b was found to affect proliferative, migratory and invasive capabilities of M12 cells, and reduced tumour growth in nude athymic mice. AATF, an activator of the cell-cycle inhibitor p21, was identified as a target. Overexpression of miR-9 was found to affect the epithelial to mesenchymal transition through suppression of e-cadherin, a protein characterized as lost in EMT, as well as suppression of SOCS5, an attenuator of JAK-STAT signaling. Inhibition of miR-9 resulted in reduction of migratory and invasive potential, and significant reduction of tumorigenesis and metastases in male nude athymic mice. miR-17-3p was previously identified as down-regulated in prostate cancer and loss of miR-17-3p shown to cause vimentin transcriptional activation. Reverse phase microarray analysis (RPMA) identified c-KIT as a potential second mRNA target for miR-17-3p. miR-17-3p was shown to modulate not only protein levels, but also messenger RNA levels of c-KIT. Four miR-17-3p binding sites in the c-KIT mRNA were identified. Thus, a number of microRNAs involved in prostate cancer were identified, and their targets found to be highly relevant to tumour progression and could potentially be used as targets for therapy or diagnostics. Stability of microRNAs in forensically relevant biological fluids was evaluated through heat treatment, ultraviolet radiation, and chemical treatment. The dried body fluids showed some susceptibility to harsh treatment, but in most cases microRNAs were still detectable in the samples. microRNAs could represent a highly stable species for body fluid identification methods in forensic science

An integrative workflow to study large-scale biochemical networks

I propose an integrative workflow to study large-scale biochemical networks by combining omics data, network structure and dynamical analysis to unravel disease mechanisms. Using the workflow, I identified core regulatory networks from the E2F1 network underlying EMT in bladder and breast cancer and detected disease signatures and drug targets, which were experimentally validated. Further, I developed a hybrid modeling framework that combines ODE- with logical-models to analyze the dynamics of large-scale non-linear systems. This thesis is a contribution to interdisciplinary cancer research