A Dissertation entitled Characterization of the CXCR4-LASP1-eIF4F Axis in Triple-Negative Breast Cancer by Cory M Howard Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences ___________________________________________ Dayanidhi Raman, B.V.Sc., Ph.D., Committee Chair ___________________________________________ Amit Tiwari, Ph.D., Committee Member ___________________________________________ Ritu Chakravarti, Ph.D., Committee Member ___________________________________________ Nagalakshmi Nadiminty, Ph.D., Committee Member ___________________________________________ Saori Furuta, Ph.D., Committee Member ___________________________________________ Shi-He Liu, M.D., Committee Member ___________________________________________ Amanda C. Bryant-Friedrich, Ph.D., Dean College of Graduate Studies The University of Toledo August 2020 © 2020 Cory M. Howard This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Characterization of the CXCR4-LASP1-eIF4F Axis in Triple-Negative Breast Cancer by Cory M. Howard Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences The University of Toledo August 2020 Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are still lacking. In addition, patient mortality mainly results from the metastasized lesions. Therefore, there is an unmet need to develop novel therapies against metastatic TNBC (mTNBC). CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to an altered pro-oncogenic proteome that facilitates breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 specifically associated with eIF4A, a mRNA helicase, in a CXCL12-dependent manner via a proximity ligation assay. We validated this finding through many approaches including co-immunoprecipitation and GST- iii pulldown assays. Furthermore, we showed an association with eIF4B, an ancillary protein that enhances the helicase activity of eIF4A. Activation of CXCR4 signaling by its ligand, CXCL12, increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins implicating a role for LASP1 in mediating the signaling from CXCR4. This impaired ability of eIF4A was confirmed by a previously established luciferase reporter assay which harbors a 5'-leader dependent on the helicase activity of eIF4A. Finally, depletion of LASP1 sensitizes 231S cells to the pharmacological inhibition of eIF4A by Rocaglamide A as evident through reduced expression of BIRC5, ROCK1, Cyclin D1, and Mdm2 which are downstream effectors of eIF4A and serves as an endogenous measure of the level of its activity. Importantly, the viability of 231 cells is compromised with the concomitant depletion of LASP1 and the pharmacological inhibition of eIF4A. Overall, our work identified that the CXCR4- LASP1 axis is a novel mediator in oncogenic protein translation through activation of the helicase activity of eIF4A. Next, we attempted to identify novel small molecule inhibitors against the helicase activity of eIF4A which is part of the CXCR4-LASP1-eIF4F axis. To accomplish this goal, a (CGG)4-tdTomato-luciferase reporter system was established to determine the helicase activity of eIF4A in three distinct TNBC cell lines. The Prestwick Chemical Library, consisting of mostly FDA-approved and off-patent drugs, was then screened. This led to identification of cardiac glycosides as potential inhibitors of eIF4A- mediated translation. We hypothesize that cardiac glycosides inhibit the expression of eIF4A through decreases in c-Myc. The combination of rocaglamide A and a standard iv cardiac glycoside such as digoxin exhibited synergistic anti-cancer activity against TNBC cells in vitro. Thus, digoxin and other related cardiac glycosides could be potentially harnessed to target the helicase activity of eIF4A in order to disrupt the CXCR4-LASP1- eIF4F axis in mTNBC. v Table of Contents Abstract .............................................................................................................................. iii Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................. xiii List of Figures .................................................................................................................. xiv 1 Breast Cancer Research and Treatment ...................................................................1 1.1 Epidemiology ...................................................................................................1 1.2 Subclassification of Breast Cancer and its Clinical Importance ......................3 1.3 Triple-Negative Breast Cancer and Treatment ...............................................4 1.4 Subtypes of Triple-Negative Breast Cancer ....................................................6 1.5 The Phosphatidylinositol 3-kinase (PI3K) Pathway and Breast Cancer ..........8 1.6 Future Perspectives on the Research and Treatment of Triple-Negative Breast Cancer .................................................................................................10 2 CXCR4 and Cancer................................................................................................12 2.1 Chemokine Receptors in Cancer Biology ......................................................12 2.1.1 Chemokine Receptors: An Overview .............................................12 2.1.2 Chemokine Receptors and Cancer Biology ....................................14 2.2 CXCL12–CXCR4 and Breast Cancer ............................................................15 2.2.1 Cellular Signaling ...........................................................................16 2.2.2 Tumor Microenvironment ...............................................................17 vi 2.2.3 Cancer Stemness .............................................................................20 2.2.4 Atypical Chemokine Receptor 3 (CXCR7) ....................................21 3 LIM and SH3 Protein 1 ..........................................................................................23 3.1 Introduction ....................................................................................................23 3.1.1 LASP1 Phosphorylation..................................................................25 3.1.2 Physiological Functions ..................................................................26 3.2 LASP1 and Cancer .........................................................................................27 3.2.1 LASP1 and the Metastatic Cascade ................................................28 3.2.2 Uncovering a Unique Role for LASP1 in Chemotaxis ...................29 3.2.3 LASP1 and the PI3K-Akt-mTOR Pathway ....................................29 3.2.4 LASP1 and Epigenetics ..................................................................30 3.2.5 Regulation of LASP1 Expression Levels .......................................31 4 A Key Role for the eIF4F Complex in Cancer ......................................................35 4.1 Introduction ....................................................................................................35 4.1.1 mRNA Structure and Function .......................................................37 4.2 The 5’Leader Sequence..................................................................................37 4.2.1 5′ Terminal Oligopyrimidine (5’TOP) Motifs ................................37 4.2.2 (NGG)4 Motifs: G-quadruplexes vs. Classical Secondary Structures ........................................................................................38 4.2.2.1 G-quadruplexes ................................................................39 4.2.2.2 Translation Initiator of Short 5’UTR (TISU)...................40 4.2.3 Translation Initiator of Short 5’UTR (TISU) ..................................42 4.3 eIF4F and Cancer ...........................................................................................42 vii 4.3.1 eIF4E ...............................................................................................42 4.3.2 eIF4A ..............................................................................................45 4.3.3 eIF4G ..............................................................................................49 4.3.4 eIF4B...............................................................................................51 4.3.5 eIF4H ..............................................................................................53