Analyzing the relationship between PDM2 and B-catenin proteins in human cancers

Tumorigenesis is promoted by the manipulation of several co-factors and pathways, in which PKM2 and P-catenin proteins play a significant role in a variety of cancers. New research states that these two hegemonic cancer proteins directly bind with each other and may provide a new angle for cancer treatment if this interaction is better understood. There are many studies detailing the influence and characteristics of these two individual proteins in the cytosol and in cancer; however, it is only recently that cancer research has found a direct connection between these two cancer proteins. In order to further understand the relationship between PKM2 and P-catenin proteins, three Enzyme-Linked Immunosorbent Assays (ELISA) were performed to analyze the behavior of the recombinant versions of these proteins. These ELISA reactions produced positive correlations in binding between PKM2 and P-catenin recombinant proteins, confirming current cancer research and illuminating a new therapeutics. A better understanding of the relationship between PKM2 and P-catenin proteins may potentially allow the development of novel cancer therapeutics that will ultimately target tumors from a new angle.

ANALYZING THE RELATIONSHIP BETWEEN PKM2 AND 0-CATENIN PROTEINS IN HUMAN CANCERS by Kapil Sharma A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biomedical Engineering Approved: Sunil Sharma, MD, FACP, MBA Thesis Faculty Supervisor Patrick Tresco, PhD Chair, Department of Bioengineering Kelly W. Broadhead, PhD Honors Faculty Advisor Sylvia D. Torti, PhD Dean, Honors College April 2016 Copyright © 2016 All Rights Reserved 1 ABSTRACT Tumorigenesis is promoted by the manipulation of several co-factors and pathways, in which PKM2 and P-catenin proteins play a significant role in a variety of cancers. New research states that these two hegemonic cancer proteins directly bind with each other and may provide a new angle for cancer treatment if this interaction is better understood. There are many studies detailing the influence and characteristics of these two individual proteins in the cytosol and in cancer; however, it is only recently that cancer research has found a direct connection between these two cancer proteins. In order to further understand the relationship between PKM2 and P-catenin proteins, three Enzyme-Linked Immunosorbent Assays (ELISA) were performed to analyze the behavior of the recombinant versions of these proteins. These ELISA reactions produced positive correlations in binding between PKM2 and P-catenin recombinant proteins, confirming current cancer research and illuminating a new approach for cancer therapeutics. A better understanding of the relationship between PKM2 and P-catenin proteins may potentially allow the development of novel cancer therapeutics that will ultimately target tumors from a new angle. TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 4 RESULTS 8 DISCUSSION 11 REFERENCES 13 iii INTRODUCTION Cancer is one of the leading causes of death in the United States second to heart disease. Understanding the inner workings of metabolic pathways responsible for tumor cell development has been the main focus for cancer research for many years. It is known that cancer cells consume more glucose and produce a large amount of lactate even in a well-oxygenized environment, a process known as aerobic glycolysis or the Warburg effect [1, 2], Moreover, the Warburg Effect/aerobic glycolysis meets the demands of cancer and proliferating cells for macromolecular synthesis and energy production [7, 8] (1). These demands are achieved via manipulation of key proteins such as the pyruvate kinase M2 enzyme (PKM2). PKM2 is responsible for the irreversible transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP in order to produce ATP (2). Furthermore, cancer utilizes proteins like PKM2 to manipulate the ATP production and amino acid/lipid synthesis of metabolic pathways in favor of tumor cell development. It is, therefore, important to investigate methods to inhibit PKM2-like proteins in the cancer condition. Another prominent metabolic pathway that works in conjunction with PKM2 and aerobic glycolysis is the Wnt Signaling pathway. The Wnt signaling pathway is a signaling transduction pathway for proteins traveling from outside to inside the cell. Furthermore, its downstream transcriptional activator P-catenin is involved in various cellular processes including embryogenesis, cell proliferation, cell fate, adult stem cells differentiation, and oncogenesis. P-catenin mutations prevent its degradation, which leads to excessive stem cell renewal/proliferation that predisposes cells to tumorigenesis via P- 1 catenin nuclear accumulation (5). The healthy (left) and cancerous (right) conditions of the Wnt signaling pathway are highlighted in Figure 1 below: Figure 1. The healthy expression of Wnt signaling (left) displays P-catenin performing its cellular functions within the cytosol, following its appropriate degradation process. In the cancerous expression o f Wnt signaling (right), P-catenin escapes its degradation process, traverses into the nucleus, binds to DNA and various protein factors, and activates cancer-inducing genes (6). New research states that PKM 2 and P-catenin bind directly with each other. This binding interaction induces the translocation of PKM 2 with P-catenin into the nucleus, interacting with the DNA and activating a variety of cancer-inducing genes (3). It is still unclear, however, if PKM 2 and P-catenin have additional proteins or transcriptional co-factors involved in their binding. Moreover, it is unknown if the interaction of these two proteins require post-translational modifications in order to bind to each other (4). Furthermore, the binding between both of these flagship oncogenic proteins raises questions as to whether cancer therapeutics should be developed to target PKM 2 or p-catenin in the long 2 In order to further understand the relationship between PKM2 and P-catenin, we hypothesized that PKM2 recombinant proteins do bind with P-catenin and also bind to and outcompete P-catenin’s common co-factor Transducin (beta)-Like lX-linked protein (TBL1X). In order to test this hypothesis, we utilized enzyme-linked immunosorbant assays (ELISA) reactions to investigate the corresponding protein-protein interactions. These competitive ELISA reactions involve the stacking of PKM2, P-catenin, and TBL1 proteins, while raising the activation energy of the well plate environment via addition of a catalyst in order to measure the amount of proteins bound and unbound. The results displayed a positive correlation in PKM2/p-catenin binding and no correlation in PKM2/TBL1 binding. These reactions also showed that PKM2 binds to P-catenin ARM domain (AD), the middle region of the P-catenin protein. This particular finding highlights the therapeutic opportunity of the PKM2-P-catenin interaction if a cancer drug is to be made in order to inhibit the binding to diminish cancer. Furthermore, these results deliver more insight into the relationship between the oncogenic proteins PKM2 and Pcatenin and solidify the interconnection between their corresponding metabolic pathways: aerobic glycolysis and Wnt signaling. 3 METHODS To better define the relationship between PKM2 and P-catenin, recombinant PKM2 protein was produced and purified for testing for three sets of sandwich ELISA reactions. In these sets of reactions, the P-catenin recombinant protein was the independent variable; whereas, the PKM2 and TBL1-WT recombinant proteins were the dependent variables. Moreover, the concentration of P-catenin recombinant proteins remained the same in the all three sets of reactions, while the PKM2 and TBL1-WT recombinant proteins were distributed into six dilutions ranging from 0-10 micromolar and 0-1 micromolar respectively. Moreover in these ELISA reactions, the P-catenin or TBL1-WT protein (depending on the ELISA reaction) was added and bound to the 96well plate. The plate was then washed three times fifteen minutes in between with PBS 1% BSA in order to remove any unbound material. Diluted concentrations of PKM2 were then added to the well plate, then washed with PBS 1% BSA in the same manner. After this second wash, the secondary antibody, alpha-Rabbit HRP was added to well plate, then mixed in the cold storage overnight. After addition of the secondary antibody, the well was again washed in the same manner with PBS 0.05% Tween 20; 50 microliters of TMB was added to the plate in order to initiate a catalytic response; a color change was produced indicating potential binding of protein; 50 microliters of 2 M Sulfuric Acid was added five minutes after addition of TMB in order to officially stop the sandwich ELISA reaction. The first ELISA reaction was intended to see if PKM2 binds with P-catenin or TBL1-WT or both. In this first ELISA setup seen in Figure 2, PKM2 was added with diluted concentrations (total 50 micromolars); whereas, P-catenin and TBL1-WT 4 remained at a fixed concentration in each well (50 micromolar). The second sandwich ELISA reaction setup determined if PKM 2 directly binds with P-catenin ARM domain (P-catenin AD); P-catenin AD is the middle region of the P-catenin protein, which is involved in a variety of drug testing at the Center of Investigational Therapeutics (CIT) lab of the Huntsman Cancer Institute (HCI). This second set of ELISA reactions followed the same concentration distribution and protocol as the first set. The third and last sandwich ELISA reaction involved varying concentrations of PKM 2 and TBL1-WT proteins against P-catenin. This last ELISA reaction (setup seen in Figure 3) determined whether PKM 2 outcompetes TBL1-WT in protein binding with P-catenin or not. Fig. 2. 96 well plate concentration breakdown of the first set o f sandwich ELISA reactions intended to measure PKM2 vs. P-catenin (orange arrow) and PKM2 vs. TBL1-WT (purple arrow). The arrows are representative of diluted concentration gradients. 5 Fig. 3. 96 well plate concentration breakdown of the third sandwich ELISA reaction displaying positive controls TBL1-WT vs. P-catenin (blue arrow), PKM2 vs. P-catenin (red arrow), and the competition experiment PKM2 & TBL1-WT vs. P-catenin (green arrow). The arrows are representative of diluted concentration gradients. Sandwich ELISA Reactions in a 96 Well Plate Preparation for a Sandwich ELISA procedure (stacking of proteins) were made in order to determine whether the PKM2 protein binds/co-precipitates with P-catenin and/or Transducin Beta-like 1-wild type (TBL1-WT), another oncogenic protein involved in transcriptional repression via nuclear receptors and transcription factors and known to co­ precipitate with p-catenin. A 96 well plate was utilized to execute the procedure; six decreasing concentrations of PKM2 protein ranging from 10 micro-molar to 0 micro­ molar were placed in the first three columns of the well plate; moreover, these 18 wells were intended for potential binding of PKM2 with TBL1-WT. A second set of 18 wells was prepared in the exact same manner within the 96 well plate and intended for potential binding of PKM2 with P-catenin. The correct PBS 1% BSA washing sequences and 6 primary and secondary antibodies were correctly implemented to the 96 well plate, following the standard protocol of a Sandwich ELISA procedure. Once the correct concentrations of proteins were injected and washed in the 96 well plate, tetramethylbenzidine (TMB) was added in order to instigate a binding reaction between the proteins. Immediately after addition of TMB, sulfuric acid was added in order to stop the reaction and ensure that TMB does not completely alter the chemical environment of the reaction. After addition of sulfuric acid, the well plate is placed in a color-sensing plate reader in order to determine the concentration of proteins bound. Overall, the process mentioned above was repeated two more times; the first ELISA reaction was intended to confirm a binding interaction with PKM2 and/or 0catenin and TBL1-WT; the second ELISA’s purpose was to identify if PKM2 binds to 0catenin ARM domain (middle region of the P-catenin protein), which may be significant for potential drug binding; the third ELISA reaction was intended to determine if PKM2 & TBL1 compete for binding with P-catenin. 7 RESULTS Figure 4 displays the results of the first set of ELISA reactions, which were required to confirm if PKM 2 binds to P-catenin. Moreover, Figure 4 depicts the correlations of measured concentrations of bound PKM 2 to P-catenin-WT (orange) and TBL1 WT (purple) respectively. Diluted concentrations of PKM 2 recombinant protein were placed into wells containing fixed concentrations of P-catenin WT (orange) and TBL1 WT (purple) in the well plate. Figure 4. First set of ELISA reactions; PKM2 vs. P-catenin-WT (orange) and PKM2 vs. TBL1WT (purple). Figure 5 portrays the results of the second set of ELISA reactions displaying the correlations of measured concentrations of bound PKM 2 to P-catenin WT (green) and Pcatenin AD (gold) respectively. Varying concentrations of PKM 2 recombinant protein were placed into wells containing fixed concentrations of P-catenin WT (green) and P- 8 catenin AD (gold) in the well plate. Moreover, Figure 5 highlights the relationship between PKM2 binding with P-catenin-AD. Saturation binding assay beta catenin VvT beta catenin AD PKM2 uM Figure 5. Second set o f ELISA reactions; PKM2 vs. P-catenin-WT (green); PKM2 vs. P-cateninAD (gold). Lastly, Figure 6 presents the results of the final set of ELISA reactions; this particular set was performed with the intention to determine whether PKM 2 competes with TBL1 for protein binding with P-catenin. Varying concentrations of PKM 2 (red) and TBL1 (blue) recombinant proteins were placed into wells containing fixed concentrations of P-catenin AD (all three sections) in the well plate. Furthermore, two positive controls were established: TBL1 vs. P-catenin-AD (blue) and PKM 2 vs. P-catenin-AD (red). Equal dilutions of both PKM 2 and TBL1 were added in this last set of wells in order to determine whether the two proteins compete for binding with P-catenin-AD. 9 Competition assay PKM2/ TBL1 WT x p-catenin AD 6 - 4 - 2 Log uM 0 2 Figure 6. Final set of ELISA reactions to determine competition between PKM2 ad TBL1 recombinant proteins; the positive controls include: TBL1 vs. P-catenin-AD (blue) and PKM2 vs. P-catenin-AD (red). The competition assay includes PKM2/TBL1 vs. P-catenin-AD (green). 10 DISCUSSION The overall objective of this study investigates the unique interactions between key cancer proteins involved in two of the most interconnected pathways in the body: Aerobic glycolysis and Wnt signaling. The corresponding flagship proteins of these two pathways, Pyruvate Kinase M2 enzyme (PKM2) from the glycolysis pathway, and 0catenin & TBL1 proteins from Wnt signaling pathway play significant roles in a variety of human cancers. Moreover, since cancer breaks the homeostasis of normal protein function, PKM2 acts as an agent of tumor cell nourishment, while P-catenin behaves as an agent of tumor cell growth. Recent research confirms that PKM2 and P-catenin from both of these hegemonic pathways mentioned above, specifically interact with each other in the nucleus and manipulate the DNA, activating a myriad of cancers in the body. Given a new insight to treat cancer, these protein interactions are analyzed via the utilization of Enzyme-linked Immunosorbent Assay (ELISA) reactions. Therefore in this project, three sets of ELISA reactions (for each of the hypotheses) confirmed positive correlations in binding between PKM2 and P-catenin; however, there was no significant binding between PKM2 and TBL1 proteins as seen in Figure 4. Furthermore, the fact that PKM2 binds to P-catenin via an ELISA reaction confirms what literature has been stating recently: the PKM2 and P-catenin interaction is a new player in the current cancer research game (4). PKM2 plays a pivotal role in cancer due to its interaction with P-catenin. Our second set of results confirms that PKM2 displays a positive correlation in binding between PKM2 and P-catenin AD seen in Figure 5. The strong affinity and binding towards the ARM domain of P-catenin (middle region), provides a beginning start of where exactly the PKM2 protein is probable to 11 bind; moreover, if a therapeutic is made to inhibit the ARM domain of the P-catenin protein, it is possible that a PKM2 and P-catenin interaction may not occur. Furthermore, the last ELISA reaction seen in Figure 6 displays a slightly larger positive curve of PKM2-P-catenin binding than the TBL1- P-catenin interaction, indicating that PKM2 slightly outcompetes TBL1 for P-catenin protein binding. This last piece of data strengthens the importance of the PKM2-P-catenin interaction in cancer, solidifying a potentially new angle to treat cancer. There are certain limitations present in this particular project. It is foremost evident that only the recombinant proteins of PKM2 and P-catenin have been confirmed to react with each other; the in vivo conditions have not yet been designed or conducted. Furthermore, it is mandatory that other procedures alongside ELISA reactions are performed in order to detect any hidden complexity between the specific interaction. Moreover, it is imperative to note that for further legitimization, animal testing is ultimately required in order for the project to progress to the next level. ELISA reactions only offer a basic step to any investigation; the following results are effective in pointing researchers to a new direction of cancer therapeutic development. Overall, the oncogenic proteins, PKM2 and P-catenin, provide researchers a unique and exciting opportunity for treating cancer at a novel angle. This particular research strengthens the views of current literature, highlighting PKM2’s edification as a pedestal cancer protein ideal for therapeutic targeting. 12 REFERENCES 1. N. Wong, J. De Melo and D. Tang, 'PKM2, a Central Point of Regulation in Cancer Metabolism', International Journal of Cell Biology, vol. 2013, pp. 1-11, 2013. 2. N. Wong, D. Ojo, J. Yan and D. Tang, 'PKM2 contributes to cancer metabolism', Cancer Letters vol. 356, no. 2, pp. 184-191,2015. 3. P. Hartz, 'OMIM Entry - * 179050 - PYRUVATE KINASE, MUSCLE; PKM', Omim.org, 2015. [Online]. Available: http://www.omim.org/entry/179050. [Accessed: 15- Jul- 2015]. 4. W. Yang, Y. Xia, H. Ji, Y. Zheng, J. Liang, W. Huang, X. Gao, K. Aldape and Z. Lu, 'Nuclear PKM2 regulates P-catenin transactivation upon EGFR activation', Nature, vol. 478, no. 7375, pp. 118-122,2011. 5. B. MacDonald, K. Tamai and X. He, 'Wnt/p-Catenin Signaling: Components, Mechanisms, and Diseases', Developmental Cell, vol. 17, no. 1, pp. 9-26,2009. 6. C. Lovly, W. Pao and J. Sosman, 2015. 13