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Shapiro, David J. Shapiro, David J. Kemper, Jongsook Kim Bolton, Eric Anakk, Sayeepriyadarshini Mahapatra, Lily 2016-07-07T19:52:38Z 2016-07-07T19:52:38Z 2016-01-25 2016-05 RNA-binding proteins control a variety of biological processes ranging from messenger RNA splicing to transport and translation. These post-transcriptional events are critical for proper cell function. One emerging class of proteins functions in several of these capacities. The VICKZ family of RNA-binding proteins is involved in translation control, mRNA localization and mRNA stability. I have studied the Insulin-like Growth Factor-2 mRNA-Binding Protein 1 (IGF2BP1/IMP-1/CRD-BP). IMP-1 exhibits an oncofetal pattern of expression, where it is expressed in embryonic development and its expression is repressed shortly after birth. However, the IMP-1 gene is reactivated in many different human cancers. Overexpression of IMP-1 leads to increased levels of proteins that promote tumor growth, metastasis, and resistance to anticancer drugs and is associated with a poor prognosis. IMP-1 enhances proliferation and migration of cancer cells by binding to and stabilizing mRNAs important in cancer, such as c-Myc. Although the role of c-Myc in cancer has been well established, it has remained an elusive therapeutic target because of its role as a transcription factor in non-neoplastic proliferating cells. Given its oncofetal pattern of expression, targeting IMP-1 presents a novel approach to targeting c-Myc. To identify new chemical entities with therapeutic potential in IMP-1 positive cancer, we carried out a pilot screen using an in vitro fluorescence anisotropy microplate assay (FAMA) and found that this approach was robust and appropriate for high throughput screening. We then carried out a high throughput screen of approximately 150,000 small molecules. Reported here is BTYNB, the first small molecule inhibitor of IMP-1, BTYNB decreases levels of IMP-1 target mRNAs, inhibits proliferation of IMP-1 positive cancer cells, and functions through the unique mechanism of decreasing oncogene mRNA stability. We believe that BTYNB not only can be developed as a potential therapeutic agent, but also serves as a useful molecular tool, with which we can probe the actions of IMP-1 in cancer cells. In addition to identifying and characterizing the first small molecule inhibitor of IMP-1, we were also interested in identifying novel molecular targets of IMP-1. Using in silico analysis of publicly available microarrays where IMP-1 was knocked down, we identified a panel of candidate target genes. Using qRTPCR and Western blot analysis, we then confirmed whether or not the mRNAs of candidate genes were decreased with IMP-1 knockdown and identified Protein Kinase C a (PKCa) as a new molecular target of IMP-1. Overall, this work has led to the identification and characterization of the first small molecule inhibitor of IMP-1 and has demonstrated that despite the fact that studies of the role of IMP-1 in cancer are rapidly expanding, there still remain novel molecular targets, such as PKCa, which may play critical roles in IMP-1 action in cancer cells. Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2016-07-07 without embargo terms The student, Lily Mahapatra, accepted the attached license on 2016-01-13 at 17:41. The student, Lily Mahapatra, submitted this Dissertation for approval on 2016-01-13 at 17:52. This Dissertation was approved for publication on 2016-01-25 at 11:57. DSpace SAF Submission Ingestion Package generated from Vireo submission #9035 on 2016-07-07 at 13:26:42 Made available in DSpace on 2016-07-07T19:52:38Z (GMT). No. of bitstreams: 2 MAHAPATRA-DISSERTATION-2016.pdf: 3924666 bytes, checksum: 4a284b57866a6d4b774a5580e0921638 (MD5) LICENSE.txt: 4211 bytes, checksum: b61fd50968120dc34a925f50fdc86d79 (MD5) Previous issue date: 2016-01-25 application/pdf http://hdl.handle.net/2142/90460 en Copyright 2016 Lily Mahapatra IM-1 Small Molecule Inhibitor Identification and characterization of a small molecule inhibitor of IMP-1 that decreases expression of IMP-1 target mRNAs and inhibits proliferation of IMP-1 positive cancer cells text text Molecular & Integrative Physl Molecular & Integrative Physi University of Illinois at Urbana-Champaign Dissertation Ph.D.