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AT-Dissertation 11-4-20.pdf (2.01 MB)

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Discovery of the CXCR4-dependent LASP1-Ago2 Interaction and its role in Triple-Negative Breast Cancer

Tilley, Augustus Michael Clifford
http://rave.ohiolink.edu/etdc/view?acc_num=mco1604505776727473

Abstract Details

2020, Doctor of Philosophy (PhD), University of Toledo, Biomedical Sciences (Cancer Biology).
Breast cancer continues to be the most prominent and devastating malignancy in women in the United States, with over 250,000 estimated new cases and an estimated 40,000 deaths. The challenge brought by the high number of cases is exacerbated by the fact that breast cancer is a very complex disease with widely varying subtypes. Most standard of care therapies have been designed to treat cases positive for overexpression of the hormone receptors estrogen and progesterone. This leaves a gap in treatment for other types of breast cancer including the “triple-negative” subtype (TNBC), which is negative for both hormone receptors and the growth factor receptor HER2. Of the breast cancer types triple-negative is the most aggressive and has the worst prognosis. The severity of the disease coupled with the lack of effective therapies drives the need for research into new prospective therapeutic targets. One such target is the chemokine receptor CXCR4 and its downstream signaling pathway. CXCR4 has been implicated in several processes critical to breast cancer progression and metastasis. The primary function of CXCR4 is to facilitate directional cell migration down the CXCL12 chemokine gradient. Upon binding, CXCR4 is activated, and as it is Gi-coupled receptor, it activates and dissociates the heterotrimeric G-protein Giinto Gi and GisubunitsBoth Gi and Gi activate a variety of downstream pathways including c-Src, MAPK, and NF-κB. These pathways mediate many steps involved in metastasis such as cell migration and survival outside of the primary site. While CXCR4 is a desirable target for treatment of metastatic breast cancer, its function in normal immune cell maturation makes it challenging to target in a clinical setting. This poses the need to identify actionable targets downstream of CXCR4 that are specific to breast cancer. LIM and SH3 Protein 1 (LASP1) is an adaptor protein that has been shown to be critically involved in CXCR4-depedent Matrigel invasion of breast cancer cells. LASP1 has several known associate proteins including filamentous actin (f-actin), zona-occludens protein 2 (ZO-2) and zyxin. Importantly, LASP1 can directly bind to the C-terminus of the CXC chemokine receptors 1-4 (CXCR1-4). These binding partners each have an influence over cell motility through the cell cytoskeleton (F-actin) and focal adhesion dynamics (ZO-2 and zyxin). Recent work has found that CXCR4 activation and binding to LASP1 has an impact on the two known phosphorylation sites on LASP1, tyrosine 171 and serine 146. Although this body of knowledge illustrates how LASP1 affects different proteins involved in specific cell functions, it does not explain how the loss of LASP1 prevented CXCR4-dependent Matrigel invasion. Here we aim to identify a novel and critical LASP1-dependent node in downstream CXCR4 activity. Previously, our laboratory had identified components of the RNA-induced silencing complex (RISC) as prospective LASP1 binding partners. Central to the RISC is the Argonaute family of proteins, which utilize microRNAs (miRNAs) to identify RNA targets for repression or degradation by the other components of the complex. Here we study specifically Argonaute 2 (Ago2) as it is the only Argonaute family member to have endonuclease activity, thereby giving it the ability to directly cleave RNA targets. Using variants of the MDA-MB-231 cell line, we were able to show that LASP1 interacts with Ago2 endogenously through direct binding. We also revealed that the interaction dynamics are determined by the phosphorylation status of LASP1, where Ago2 prefers to interact with LASP1 that is not phosphorylated on S146 but is phosphorylated on Y171. This interaction was also found to have functional consequences in the context of RNAi. We demonstrate that by altering the phosphorylation status of LASP1 can alter the activity of the tumor suppressor miRNA Let-7a. This in turn caused a significant change in protein expression of Let-7a’s targets related to metastasis. In summary, we have found a novel LASP1 binding partner that has broad functional consequences and connects CXCR4 to the critical cell regulator Ago2. In the future we hope this work can be built upon to better understand the underlying mechanism, and hopefully establish it as a therapeutic target to offer a more specific treatment for metastatic TNBC patients.
Dayanidhi Raman, PhD (Advisor)
Kathryn Eisenmann, PhD (Committee Member)
Malathi Krishnamurthy, PhD (Committee Member)
Ivana de la Serna, PhD (Committee Member)
Kam Yeung, PhD (Committee Member)
154 p.
Biomedical Research; Cellular Biology; Molecular Biology

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Citations

  • Tilley, A. M. C. (2020). Discovery of the CXCR4-dependent LASP1-Ago2 Interaction and its role in Triple-Negative Breast Cancer [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1604505776727473

    APA Style (7th edition)

  • Tilley, Augustus. Discovery of the CXCR4-dependent LASP1-Ago2 Interaction and its role in Triple-Negative Breast Cancer. 2020. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1604505776727473.

    MLA Style (8th edition)

  • Tilley, Augustus. "Discovery of the CXCR4-dependent LASP1-Ago2 Interaction and its role in Triple-Negative Breast Cancer." Doctoral dissertation, University of Toledo, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=mco1604505776727473

    Chicago Manual of Style (17th edition)

mco1604505776727473
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This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.
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