MdmX Regulates Mitotic Spindle Polarity Of Human Breast .

MQP-BIO-DSA-6662MdmX Regulates Mitotic Spindle Polarity ofHuman Breast Cancer CellsA Major Qualifying Project ReportSubmitted to the Faculty of theWORCESTER POLYTECHNIC INSTITUTEin partial fulfillment of the requirements for theDegree of Bachelor of ScienceinBiology and BiotechnologybyKevin WoodsApril 25, 2013APPROVED:Stephen N. Jones, PhDDept. of Cell and Developmental BiologyUMASS Medical SchoolMAJOR ADVISORDavid Adams, PhDBiology and BiotechnologyWPI Project Advisor

ABSTRACTMdm2 and MdmX are oncoproteins that promote cancer by binding and inhibitingthe p53 tumor suppressor protein. Amplification and overexpression of the Mdm2 andMdmX genes has been observed in a significant fraction of human cancers, includingleukemia, neural tumors, sarcomas, and melanoma. However, MdmX (but not Mdm2)has also been recently shown to inhibit cell proliferation and increase genomic stability ina p53-independent manner in cancer cells and non-transformed cells of murine origin.This project investigates a putative tumor-suppressing role of MdmX in human cells byexamining the effects of MdmX on the genomic stability of human breast cancer cells.The results show a correlation between the presence of MdmX, decreased formation ofmultipolar spindles, and a concomitant increase in chromosome stability during cellmitosis. These findings indicate that MdmX may also have a p53-independent role insuppressing human breast cancer, and could provide a fundamental understanding of therole of genomic stability in neoplasia.2

TABLE OF CONTENTSSignature Page . . . 1Abstract . . 2Table of Contents . . . 3Acknowledgements . . 41.0 Background . . . 52.0 Project Purpose . . 143.0 Methods . . 154.0 Results . 215.0 Discussion . 28Bibliography 313

ACKNOWLEDGEMENTSI would first like to thank Dr. Stephen Jones for giving me the chance to study inhis lab this year. It was a truly great experience that developed me as a person and as aprofessional. He always made time to accommodate my project and offer me advice. Byrelating my research to the rest of the scientific world, Steve has taught me much that willrelate to my career in the future.I would also like to thank all the members of the Jones Lab: Chris, Mike, Marilyn,and Kathy, who were always so helpful whenever I had questions or struggled. Theyprovided a welcoming environment that made it exciting to learn and a pleasure to attendevery week. In particular, I would like to sincerely thank Zdenka, who instructed andmentored me throughout the length of the project. Despite her busy schedule, she alwaysmade time to meet with me when I had questions, edit and advise my paper/presentations,or show me how to do procedures I struggled with. When I made mistakes, she nevercriticized, but instead took the time to explain what went wrong and how to fix it in thefuture. Without her guidance, this project would not have happened and I am trulygrateful for her help this past year.Lastly, I would like to thank Dave Adams for helping me find an MQP andalways being there to answer my questions. He has given me advice not only with thisproject, but also regarding job and internship searches, on applying to graduate schools,and on setting personal life goals. Dave has been an amazing advisor and professor thatalways puts the students first, and I am glad I had the pleasure of working with him.4

1.0 BACKGROUND1.1 The p53 Tumor SuppressorThe p53 transcription factor regulates the expression of multiple genes whoseprotein products govern cell proliferation, apoptosis, cell metabolism, and the response ofcells to DNA damage, hypoxia, and other forms of cell stress. These p53-mediatedactivities regulate organismal tissue growth and homeostasis, metabolism, ageing, andtumor formation. Given its many important biological functions, it is not surprising thatp53 is one of the most studied proteins. This is especially true in the field of CancerBiology, as mutation of the p53 gene has been found in approximately half of all humancancers (Hollstein et al., 1991).Under physiological conditions, the p53 protein is present at very low levelswithin the cell. However, the p53 protein is stabilized and subjected to significant posttranslational modification following exposure of cells to DNA damage or other types ofstress. This stabilization of p53 leads to a rapid elevation of p53 protein levels, and thesubsequent modifications to the p53 protein enhances its activity as a transcription factor(Fig. 1). These inputs lead to transcriptional activation of p53 target genes such as Pumaor p21. Once expressed, these p53-downstream target proteins can induce apoptosis, cellcycle arrest, senescence, and/or activation of DNA repair. These responses occur in acell-context-specific manner, and are also partly dependent on the amplitude and natureof the genetic or cellular damage. In addition, p53 can induce expression of its ownnegative regulators, including Mdm2 and MdmX, thereby limiting the duration of the p53response within the cell.5

Figure 1: Overview of p53 Activation and Response. The p53 protein can beinduced by many different cellular stresses and in turn activates a variety ofresponses that impact the life cycle of a cell.1.2 Regulation of p53 by MDM proteinsThough p53 tumor suppressor activity is closely governed within the cell by manydifferent mechanisms, it is clear from genetic experiments that Mdm2 and MdmX, twoRING domain proteins, are the major regulators of p53 activities in mammalian cells(Jones et al., 1995; Parant et al., 2001). Mdm2 and MdmX are structural homologues thatcan bind to each other as well as to the p53 protein. These proteins serve to inhibit p53functions under normal physiological (unstressed) conditions (Wade et al., 2010), and areencoded by separate genes whose expression is transactivated by p53. Thus, the level ofp53 is auto-regulated in cells by the ability of p53 to induce its own negative regulators,Mdm2 and MdmX (Gannon and Jones, 2012).6

Mdm2 inhibits p53 by binding with p53 and masking the p53 transactivationdomain and by altering p53 stability (Kubbutat et al., 1997). Specifically, Mdm2 acts asan E3 ubiquitin ligase, placing ubiquitin moieties onto the p53 protein thereby targetingp53 for proteosomal degradation. Both transactivation domain masking and ubiquitinstimulating Mdm2 activities prevent p53 from functioning as a transcription factor tolimit cell growth and promote apoptosis. In contrast, MdmX inhibits p53 by binding top53 and masking its transactivation domain without destabilizing p53 (Francoz et al.,2006). Together, the two proteins negatively regulate p53 (Fig. 2).Reduced amounts of Mdm2 or MdmX within a cell lead to higher basal levels ofp53 activity. Likewise, preventing the ability of these MDM proteins to complex withp53 also promotes p53 stabilization and/or p53 activation, and many of the cell stresssignals that promote p53 up-regulation in a cell do so by phosphorylating Mdm2, MdmX,or p53, thereby altering Mdm-p53 signaling (Wade et al., 2010; Gannon and Jones,2012). Once stress signaling subsides, the MDM proteins are free to complex and inhibitp53 activity, thus returning p53 levels back to unstressed levels within the cell.Conversely, up-regulation or overexpression of Mdm2 or MdmX can result in decreasedlevels of p53 within the cell, and Mdm2 and MdmX gene amplification andoverexpression promotes cancer in mice (Jones et al., 1996; Marine and Jochemsen,2005) and is frequently associated with human cancers. Thus, extensive efforts have beenfocused on developing inhibitors of Mdm2-p53 and MdmX-p53 signaling for humancancer therapy.7

Figure 2: Regulation of p53 by Mdm2 and MdmX. The Mdm proteins worktogether to negatively regulate the tumor suppressor p53. Amplification or overexpression of Mdm2 and MdmX are seen commonly in human tumors.1.3 MdmX Suppresses Tumorigenesis in p53-null MiceThe oncogenic potential of Mdm2 and MdmX is well established by numerousstudies in the literature (Oliner et al., 1992; Francoz et al., 2006). However, in 2008,MdmX was surprisingly found to also play a role in suppressing tumorigenesis in p53deficient mice (Matijasevic et al., 2008a). Mice that are deleted for both p53 and MdmXdevelop tumors at a significantly faster rate than mice lacking only p53. Even miceheterozygous for MdmX display tumorigenesis at a delayed rate compared to p53-nullmice homozygous for MdmX. Fifty percent of p53-null mice develop tumors by 25weeks of age, while fifty percent of double-null mice present with tumors at 20 weeks(Matijasevic et al., 2008a).At the cellular level, the loss of MdmX increases the growth potential in p53deficient mouse tumors cells. Tumor cells lacking MdmX divide faster and accumulate tohigher densities, suggesting that MdmX is at least partly responsible in controllingproliferation when p53 is absent. A reintroduction of MdmX into double-null mouse cells8

reverses the increase in growth potential back to levels equivalent to p53-null cells(Matijasevic et al, 2008a).Cells harvested from thymic stromal tumors of p53-null mice show that MdmXhas a profound impact on chromosome stability and mitotic polarity. FACS analysis andmitotic spreads reveal higher chromosome numbers and DNA content when MdmX ispresent (Matijasevic et al, 2008a). It is hypothesized that MdmX prevents chromosomeloss due to its ability to promote bipolar mitosis when p53 is missing. Bipolar mitosis, orthe even segregation of chromosomes in two directions during mitotic division, is seenmore frequently in mouse tumor cells containing MdmX than in those p53-deficient cellslacking MdmX. Conversely, there is a greater than 50% decrease in the number of cellswith multipolar spindles when MdmX was re-introduced to MdmX/p53 double-nullmouse tumor cells (Matijasevic et al, 2008a).The reintroduction of MdmX into MdmX/p53-null mouse tumor cells decreasesproliferation rate, the occurrence of multipolar spindles and stabilizes the number ofchromosomes in these cells. These effects may be the reason for the observed suppressionof tumorigenesis in p53-deficient mice. Analysis of mouse embryonic fibroblasts showedthat MdmX promotes bipolar mitosis and genetic stability in a p53-independent manner(Matijasevic et al., 2008b).In contrast to what was seen with MdmX, cells deleted for Mdm2 displayed nosignificant difference on proliferation, chromosome stability or bipolar mitosis in p53deficient mouse cells (Matijasevic et al., 2008a). Furthermore, exogenous MdmX wascapable of suppressing cell proliferation, chromosomal loss, and multipolar mitosis intriple deleted cells (MdmX, Mdm2 and p53-deleted), indicating that Mdm2 is not9

required for these p53-independent functions of MdmX (Blodgett, 2012). However, it isunclear if these p53-independent and Mdm2-independent functions of MdmX existwithin human cells.1.4 Cancer and Genomic StabilityPrior to the onset of mitosis in eukaryotes, a cell will have duplicated itscentrosome and genomic DNA during S phase. During the early phases of mitosis, thechromosomes condense and align along the metaphase plate. Microtubule spindle fibersattach to the centrosomes and the kinetochore protein structures that form at thecentromere of each sister chromatid, linking the chromatids to the two centrosomes(hence “bipolar”) located at opposite poles of mitotic spindle and normally creating ahighly symmetrical structure (Fig. 3). The chromosomes are then diametrically separatedduring anaphase to yield equal proportions of genetic material to each daughter cell.10