20.109(F07): Rosa and Vivian's Research Proposal

20.109 Module 3: The Research Proposal

Project Overview
Studies have shown a link between cancer formation and the over-expression of genes producing IAPs, inhibitor-of-apoptosis proteins. An example of these IAPs is XIAP which is an X-linked inhibitor-of-apoptosis protein. In this study we aim to silence the expression of the gene producing XIAP by designing an siRNAs that targets the mRNA of XIAP in human lung cancer cells. With this form of RNAi we hope to silence the over-expression of XIAP.

Background
Cancer is associated with excessive cell proliferation and increased migration of cells into surrounding tissue. Apoptosis, the mechanism a cell uses to induce its own death, is deregulated in cancerous cells. Inhibitors of Apoptosis proteins (IAPs) are over-expressed in many cancers and are thought to be involved in cancer progression. IAPs most commonly suppress apoptosis by directly inhibiting caspases. Caspases are a class of proteases that lie dormant in the cell until activated by cell death pathways, after which they form part of a death-inducing signaling complex. By suppressing IAP expression, we can increase the rates of apoptosis.

IAP inhibition has potential as an attractive alternative for cancer treatment. Cancer treatments such as chemotherapy and radiation therapy are problematic because they may damage cell DNA, creating mutant cells and increasing the chance of a cell developing resistance to the therapies. Also, because IAP over-expression and over-activation disrupts normal cell death processes, it can cause resistance to cancer therapies that regulate apoptosis by restoring mitochondrial function.

The IAP family of proteins includes X-linked inhibitor of apoptosis (XIAP), human IAP1, human IAP2, Testis-specific IAP, BIR-containing ubiquitin conjugating enzyme (BRUCE), Survivin (TIAP) and Livin (KIAP). All contain a BIR domain, the site by which IAP directly inhibits caspases. XIAP is thought to be up-regulated in cancer cells more than other IAPs because their long UTR regions cause them to undergo a 5' cap-independent translation. Thus XIAP can still be expressed in stress conditions that would block cap-dependent translation. XIAP is up-regulated in a variety of cancers, including lung, pancreatic, and breast cancers.

Research Problem & Goals
We will use carrier THCO to introduce siRNAs targeting XIAP mRNA in human lung cancer cells. Depending on the success of this experiment in lung cancer cells, we will use the same method to transfect siRNAs into mice in which cancer has been induced. Previous studies have used RNAi to down-regulate XIAP expression in cultured MCF-7 breast cancer cells using other methods of transfection. We will use multifunctional carrier (MFC), 1,4,7-triazanonylimino-bis[N-(oleicyl-cysteinyl-histinyl)-1- aminoethyl)propionamide] (THCO), to introduce siRNA into the cells because of its pH-sensitive membrane disruption and ability to form complex nanoparticles with siRNA to protect it from nuclease degradation. Current siRNA carriers are similar to those used for DNA delivery. These cationic carriers exploit the long, negatively-charged DNA’s binding to the positive charges on the carriers; siRNAs are shorter and form less stable complexes with these carriers, presenting a problem for siRNA stability inside the cell. Thus we hope that by using a THCO carrier for siRNA targeting XIAP, we can effectively inhibit XIAP expression in cancerous cells.

Resources
Cummins JM, Kohli M, Rago C, Kinzler KW, Vogelstein B, Bunz F. X-Linked Inhibitor of Apoptosis Protein (XIAP) Is a Nonredundant Modulator of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Mediated Apoptosis in Human Cancer Cells. CANCER RESEARCH 64: 3006–3008.

Hunter AM, LaCasse EC, Korneluk RG. The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis (2007) 12:1543–1568.

Papetti M, Skoultchi AI. Reprogramming Leukemia Cells to Terminal Differentiation and Growth Arrest by RNA Interference of PU.1. Mol Cancer Res (2007) 5(10.

Wanga XL, Nguyena T, Gillespieb D, Jensenb R, Lu XR. A multifunctional and reversibly polymerizable carrier for efficient siRNA delivery. Biomaterials (2007) 29: 15–22.