)ORULGD6WDWH8QLYHUVLW\/LEUDULHV 2021 Investigating the Role of Gankyrin in Breast Cancer and its Potential as a Novel Therapeutic Target Jessica Margaret Jarnagin Follow this and additional works at DigiNole: FSU's Digital Repository. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS & SCIENCES INVESTIGATING THE ROLE OF GANKYRIN IN BREAST CANCER AND ITS POTENTIAL AS A NOVEL THERAPEUTIC TARGET By JESSICA MARGARET JARNAGIN A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for graduation with Honors in the Major Degree Awarded: Spring, 2021 The members of the Defense Committee approve the thesis of Jessica M. Jarnagin defended on April 2nd, 2021. ______________________________ Dr. Antonia Nemec Thesis Director ______________________________ Dr. Robert J. Tomko Jr. Committee Member ______________________________ Dr. Qian Yin Committee Member ______________________________ Dr. Scott Stagg Committee Member 2 INVESTIGATING THE ROLE OF GANKYRIN IN BREAST CANCER AND ITS POTENTIAL AS A NOVEL THERAPEUTIC TARGET 1. ABSTRACT 4 - 5 2. INTRODUCTION: 5 - 11 2.1 BREAST CANCER 5 - 6 2.2 THE UBIQUITIN-PROTEASOME SYSTEM 6 - 8 2.3 PROTEASOME UPREGULATION IN CANCERS 8 - 11 3. METHODS: 11 - 16 3.1 CELL LINES AND PLASMID CREATION 11 - 13 3.1.1 CELL LINES 11 - 12 3.1.2 PLASMIDS - 12 3.1.3 CLONING 12 - 13 3.1.3.1 CLONING pRVYtet-off-FLAG-hsGANKYRIN 3.1.3.2 CLONING pLJM1-HA-PSMC4 AND pLJM1-HA-psmc4(337-419) 3.1.3.3 CLONING pBabe-HA-PSMC4 AND pBabe-HA-psmc4(337-419) 3.2 PROTEIN EXPRESSION 13 - 14 3.2.1 DENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS 13 - 14 3.3 MAKING RETROVIRUS WITH GP2-293 CELLS 14 - 15 3.4 RETROVIRAL INFECTION 15 3.5 PEPTIDASE ACTIVITY ASSAY 15 - 16 3.6 ANCHORAGE INDEPENDENT GROWTH 16 4. RESULTS: 16 - 25 4.1 GANKYRIN IS EXPRESSED IN FLP-IN HEK293 CELLS 16 - 17 4.2 INCREASED PEPTIDASE ACTIVITY OF FLP-IN HEK293 CELLS OVEREXPRESSING GANKYRIN 18 4.3 EXPRESSION OF FLAG-GANKYRIN IN MCF10A CELLS 19 4.4 NO CHANGE IN PEPTIDASE ACTIVITY OF MCF10A CELLS OVEREXPRESSING GANKYRIN 19 - 20 4.5 ANCHORAGE INDEPENDENCE PHENOTYPE FOR MCF10A GANKYRIN AND EMPTY VECTOR CELLS 20 4.6 EXPRESSION OF HA-PSMC4 AND HA-PSMC4(337-419) IN MCF10A CELLS 21 - 22 4.7 EXPRESSION OF HA-PSMC4 AND HA-PSMC4(337-419) IN FLP-IN HEK293 CELLS 22 - 23 4.8 INCREASED PEPTIDASE ACTIVITY OF MCF10A CELLS EXPRESSING HA-PSMC4 23 - 24 4.9 ANCHORAGE INDEPENDENCE PHENOTYPE FOR MCF10A EMPTY VECTOR, GANKYRIN, PSMC4, AND GANKYRIN + PSMC4 CELLS 24 - 25 5. DISCUSSION 25 - 29 6. ACKNOWLEDGMENTS 29 - 30 7. REFERENCES 31 - 32 8. SUPPLEMENTAL INFORMATION: 33 8.1 TABLE 1 – PLASMIDS 33 3 1. Abstract Breast cancer is a heterogeneous disease and is typically accompanied by the activation of HER2, hormone receptors, and/or mutations in the BRCA genes. Treatment of breast cancer is dependent on the molecular features of the cancer, and response to treatment varies [1]. The oncogene gankyrin was found to be overexpressed in many molecular subtypes of breast cancer and correlated with poor prognosis [4]. Gankyrin is a chaperone protein that aids in the assembly of the 26S proteasome. The proteasome is a large ~70 subunit protease complex that drives cancer by destroying anti-growth and pro-death signals and is upregulated in many cancer types [25]. Because gankyrin is critical for proteasome assembly, we aimed to determine the role that gankyrin overexpression plays in contributing to proteasome upregulation. We generated both MCF10A breast epithelial cell lines and Flp-In HEK293 cell lines overexpressing gankyrin or empty vector which allowed us to test the effects of gankyrin overexpression on the peptidase activity of the proteasome and the occurrence of the anchorage independence phenotype. Using these cell lines, we conducted peptidase activity assays and determined that gankyrin overexpression leads to increased proteasome activity in Flp-In HEK293 cells. We also conducted anchorage independence assays, which showed that overexpression of gankyrin in MCF10A gankyrin cells lines led to increased occurrence of anchorage independence phenotypes. Additionally, during proteasome assembly gankyrin binds to the subunit PSMC4 and functions to prevent premature docking of PSMC4 onto the core particle, so we created a minimal part of PSMC4 (psmc4(337-419)) that sequesters gankyrin to interfere with proteasome assembly in a method called gankyrin trapping [2]. We attempted to generate both MCF10A cell lines and Flp-In HEK293 cell lines that expressed either gankyrin or empty vector and psmc4(337-419), PSMC4, or an empty pBabe or pLJM1 plasmid. However, the retroviral infection of Flp-In HEK293 cells with virus containing these plasmids was unsuccessful. Also, 4 HA-psmc4(337-419) was undetectable in MCF10A cells. Using the cell lines that were successfully created with full-length PSMC4, we conducted peptidase activity assays and anchorage independence assays which showed that overexpression of PSMC4 in MCF10A gankyrin cell lines led to increased proteasome activity and an increased occurrence of the anchorage independent growth phenotype. This data indicates that gankyrin-trapping with full length PSMC4 is not a valid mechanism for decreasing oncogenic activity. Altogether, this study indicates the significance of the role of gankyrin overexpression in proteasome upregulation, and that gankyrin is potentially a valid target for proteasome inhibition. 2. Introduction 2.1 Breast Cancer Breast cancer is the second most common cancer found in women and is characterized by malignancies of the breast tissue. The main components of this disease are mutations of BRCA genes, activation of estrogen and/or progesterone receptors, and the activation of the human epidermal growth factor receptor 2 (HER2) encoded by the gene ERBB2 [1]. Breast cancer can either develop sporadically or be genetically linked, however only 10% of breast cancers are inherited [5]. If treated in the early stages of carcinogenesis prior to metastasis, there is a 70-80% chance of full recovery. However advanced/metastatic breast cancer is currently considered incurable [1]. The most commonly amplified genes are often targeted for treatment development, and include TP53, PTEN, ERBB2, BRCA 1/2, and FGFR1 [6]. Over the last 20 years, treatment of breast cancer has had improved outcomes due to the current focus on the heterogeneity of the disease. Treatments have evolved to target the specific combination of BRCA mutations, estrogen/progesterone receptor activation, and HER2 activation that exists in each person diagnosed with the disease. However, studies have shown that most breast cancers are caused by 5 the accumulation of many mutations, which can make targeting treatments difficult to develop [7]. Thus, it is crucial to develop new therapies independent of mutation status. Recently, the oncogene gankyrin, a chaperone protein that aids in the assembly of the proteasome, was found to be upregulated in breast tumors and its overexpression in breast epithelial cell lines induced a tumorigenic phenotype. Gankyrin overexpression occurs in the early stages of breast cancer and is correlated to poor prognosis and metastasis [33]. Although it was found to be associated with HER2 expression, gankyrin overexpression was found in breast cancer regardless of HER2 and ER/PR status, including triple-negative breast cancer, an aggressive cancer with poor treatment options and prognosis [4] [34]. 2.2 The Ubiquitin-Proteasome System The ubiquitin proteasome system (UPS) is the primary mechanism for protein degradation in eukaryotic cells. Proteins that are destined for degradation are modified by the addition of a chain of the protein ubiquitin (Ub). The polyubiquitin (polyUb) chain signals the protein for delivery to the 26S proteasome. Figure 1. Structure of the Human 26S Proteasome The proteasome consists of two main parts: the 19S regulatory particle (RP) and the 20S core particle (CP). The RP is responsible for binding incoming 6 substrates, removing the polyubiquitin chain, unfolding the substrate, and translocating it into the interior of the CP. The CP houses the peptidase active sites in its interior and cleaves the substrate into short peptides. The proteasome is a large 2.5 mDa multi-subunit protease that can be divided into the 19S regulatory particle (RP) and 20S core particle (CP) (Figure 1). The RP can be further divided into the RP lid and RP base. The RP lid consists of nine regulatory particle non-ATPase subunits, and deubiquinates the substrate. The RP base is made up of a heterohexameric ATPase ring and four non-ATPase subunits. The base unfolds the substrate using energy derived from ATP. The CP consists of four stacked heteroheptameric rings resulting in the formation of a barrel-like shape. The peptidase active sites are located within the CP and cleave the substrate into short peptides [2] These peptidase active sites include the PSMB5 subunit which has chymotrypsin- like activity, as it cleaves after amino acids with hydrophobic side chains; the PSMB6 subunit which has caspase-like activity, as it cleaves after amino acids with acidic side chains; and the PSMB7 subunit which has trypsin-like activity, as it cleaves after amino acids with basic side chains [32]. The proteasome contains ~70 subunits that must assemble correctly in order to function properly. Proteasome assembly is driven by intrinsic features of several proteasome subunits themselves, as well as extrinsic, dedicated assembly chaperone proteins. This process is highly regulated, but not well understood. The assembly of the RP base subcomplex is aided by four chaperone proteins under normal growth: PAAF1, gankyrin, S5b, and p27 (Rpn14, Nas6, Hsm3, and Nas2 in yeast, respectively). These chaperones function in part to stabilize base assembly intermediates, and to regulate attachment of the lid and CP to the base. However, the mechanism by which they are evicted to form mature proteasomes is not well understood. Gankyrin has a long crescent-like shape that allows it to bind to the C-terminus of the ATPase base subunit PSMC4.