Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Transcriptional Regulation of RKIP in Prostate Cancer Progression Submitted by: Sandra Marie Beach In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Kam Yeung, Ph.D. Academic William Maltese, Ph.D. Advisory Committee: Sonia Najjar, Ph.D. Han-Fei Ding, M.D., Ph.D. Manohar Ratnam, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: May 16, 2007 Transcriptional Regulation of RKIP in Prostate Cancer Progression Sandra Beach University of Toledo ACKNOWLDEGMENTS I thank my major advisor, Dr. Kam Yeung, for the opportunity to pursue my degree in his laboratory. I am also indebted to my advisory committee members past and present, Drs. Sonia Najjar, Han-Fei Ding, Manohar Ratnam, James Trempe, and Douglas Pittman for generously and judiciously guiding my studies and sharing reagents and equipment. I owe extended thanks to Dr. William Maltese as a committee member and chairman of my department for supporting my degree progress. The entire Department of Biochemistry and Cancer Biology has been most kind and helpful to me. Drs. Roy Collaco and Hong-Juan Cui have shared their excellent technical and practical advice with me throughout my studies. I thank members of the Yeung laboratory, Dr. Sungdae Park, Hui Hui Tang, Miranda Yeung for their support and collegiality. The data mining studies herein would not have been possible without the helpful advice of Dr. Robert Trumbly. I am also grateful for the exceptional assistance and shared microarray data of Dr. Mohan Dhanasekaran and Jianjun Yu who both work in the laboratory of Dr. Arul Chinnaiyan at the University of Michigan. Thank you to Dr. Sadik Khuder and Peter Basely in Bioinformatics for data processing discussions and statistical assistance. There have been many special people who have helped me with their scholastic advice, academic assistance, and moral support throughout my years of study. I will not spoil their humility by naming them, but I thank you all from the bottom of my heart. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS................................................................................................ ii TABLE OF CONTENTS................................................................................................... iii INTRODUCTION ...............................................................................................................1 LITERATURE.....................................................................................................................3 MATERIALS AND METHODS.......................................................................................29 RESULTS ..........................................................................................................................39 DISCUSSION....................................................................................................................97 SUMARY........................................................................................................................104 BIBLIOGRAPHY............................................................................................................105 ABSTRACT.....................................................................................................................122 iii INTRODUCTION Raf kinase inhibitor protein (RKIP) is a conserved, multifunctional protein that seems to have a role in the metastatic process in cancer. RKIP, or PEBP1, has been described as having functions in neuronal pathways, gonadal tissues, and cell signaling pathways [reviewed in (Keller et al., 2005; Odabaei et al., 2004)]. It was originally identified as a negative regulator of the mitogen-activated protein kinase cascade initiated by Raf-1 (Yeung et al., 1999). RKIP is also able to inhibit NF-kB activation and signaling (Yeung et al., 2001). G-protein signaling can also be facilitated by RKIP. RKIP inhibits GRK-2, which is involved in inactivating G-protein signaling [reviewed in (Goel and Baldassare, 2004)]. Recently, RKIP has been found to be downregulated in various cancers, including melanoma, breast, hepatocellular, and prostate (Chatterjee et al., 2004; Hagan et al., 2005; Lee et al., 2006; Park et al., 2005). Breast and prostate cancer metastases have been correlated with RKIP expression in human tissue samples as well (Fu et al., 2006; Hagan et al., 2005). Little is currently known about RKIP regulation. Putative transcription factors such as AP1 and YY1 were hypothesized to bind the RKIP promoter using computer database analysis, but these have yet to be tested (Odabaei et al., 2004). The downregulation of RKIP in cancer metastases suggests it is acted upon by a repressor that is upregulated during the metastatic process. Such a repressor may be Snail. The zinc finger transcription factor Snail is a potent repressor of E-cadherin and is involved in the 1 epithelial-to-mesenchymal transition phenotype found in cancer progression (Barbera et al., 2004; Barrallo-Gimeno and Nieto, 2005; De Craene et al., 2005). The aim of this study was to analyze the relationship between Snail and RKIP. We found that Snail represses RKIP in prostate cancer cell lines in vitro. Furthermore, we correlated RKIP downregulation with Snail in a meta-analysis of prostate cancer microarray studies. Finally, we utilized the microarray datasets to determine genes clustered with RKIP, and are potentially coregulated with RKIP. 2 LITERATURE REVIEW RKIP is a phosphatidylethanolamine-binding protein. RKIP in mammals RKIP belongs to a highly conserved family of phospholipid-binding proteins, the phosphatidylethanolamine-binding proteins (PEBP). PEBPs are represented in eukaryotes, bacteria, and archae with no significant sequence homology to other proteins (NCBI, 2003; NIH, 2004). Mammalian PEBPs have been categorized into four subfamilies based on sequence identity: PEBP1,2,3, or 4 (Simister et al., 2002). Humans have two identified PEBPs, hPEBP1 and hPEBP4. hPEBP1 was identified in a yeast two-hybrid screen as a Raf-1 interacting protein and was designated RKIP (Yeung et al., 1999). Human RKIP is located on chromosome 12 (12q24.23) and is composed of four exons. The RKIP mRNA is 1507 base pairs which is translated into a 187 amino acid protein with a molecular mass of 21-23 kDa. A simple search on OMIM search found no human diseases mapping to this location (Hamosh and Hartz, 1966-2004). hPEBP4 has approximately 33 residues in its N-terminus that is not included in hPEBP1, as well as two insertions and one deletion in the amino acid sequence (Simister et al., 2002). hPEBP4 was recently cloned from human bone marrow stromal cells and found to interfere with Ras/Raf/MEK/ERK signaling. hPEBP4 appears to promote resistance to tumor necrosis factor (TNF) α –induced apoptosis (Wang et al., 2004). 3 PEBP has been discovered and studied in several mammalian species. In 1980 a protein named “h3” was purified from human brain (and later determined to be hPEBP1) (Bollengier and Mahler, 1980). The molecule was identified as phosphatidylethanolamine protein after its discovery in bovine brain as a soluble basic protein (Bernier and Jolles, 1984; Bernier et al., 1986). The rat homolog of PEBP1 was found by Grandy et al. by morphine affinity chromatography and determined to be related to the bovine and human PEBP/h3 (Grandy et al., 1990; Seddiqi et al., 1994). PEBP members have also been determined in mouse and monkey (Simister et al., 2002). RKIP is localized in the cytoplasm and at the plasma membrane in many different cell types (Simister et al., 2002). RKIP and its mammalian homologs are widely expressed in tissues; it has been detected in lung, oviduct and ovary, mammary glands, uterus, prostate epithelium, thyroid, mesenteric lymph node, megakaryocytes of the heart, spleen, liver, and epididymis, testis, spermatids, Leydig cells, steroidogenic cells of the adrenal gland zona fasiculata, small intestine, plasma cells, and neural cells such as brain oliodendricytes, Schwann cells, and Pukinje cells (Frayne et al., 1998; Fu et al., 2003; Katada et al., 1996; Moore et al., 1996; Schoentgen and Jolles, 1995). Theroux et al. measured the highest expression of mouse RKIP in brain and testes tissues (Theroux et al., 2007). The mouse PEBP family includes PEBP2, which is testes-specific and believed to play a role in spermatogenesis (Hickox et al., 2002). Simister et al. had identified mouse PEBP members besides RKIP and PEBP2 (Simister et al., 2002). However, recent analysis of PEBP expression in the RKIP knockout mouse has shown that only RKIP and PEBP2 exist; other potential gene candidates were determined to be 4 silent pseudogenes as their was no protein or RNA products detected for them (Theroux et al., 2007). RKIP in non-mammals In addition to mammals, PEBP family members have been identified and described in various other species, but the cellular and molecular function of species- specific PEBPs is not clear. The yeast Saccharomyces cerevisiae has the phosphatidylethanolamine-binding protein called Tfs1p, which was identified as a suppressor of the cdc25-1 mutant (Robinson and Tatchell, 1991). Later reports described Tfs1 as Ic, an inhibitor of the classical member of the serine carboxypeptidase family, carboxypeptidase Y (Bruun et al., 1998; Mima et al., 2003). Tfs1 was shown to inhibit the yeast GTPase-activating protein Ira2, and to