Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Dada, Sarah
Master of Science - MSc
University of British Columbia
Cancer is a devastating disease that is responsible for the death of a quarter of Canadians. Cancer is the result of cells that gain genetic mutations or epigenetic changes that incur abnormal, uncontrolled proliferation. Oftentimes, such mutations give rise to the metastatic form of cancer, which is capable of moving throughout the body to distant sites. Adaptive immunity, including Cytolytic T cells (CTLs), are intended to kill tumours based on tumour specific antigen presented on Major Histocompatability Complex (MHC) Class I. However, metastatic tumours are capable of escaping such death through the downregulation of Antigen presentation machinery (APM), including MHC Class I. Here, we investigate the role of the small molecule Cannabigerol in upregulating class I MHC and altering cytokine signalling in metastatic tumours, and the consequent CTL tumour killing ex-vivo. Additionally, we determined the impact of Cannabigerol on immune infiltrates and tumour burden in vivo. Small molecules which enhance the efficacy of the CTL killing through upregulation of tumour-antigen bound MHC class I killing would be beneficial for therapeutic use through increased tumour killing, as well as reduced tumour metastasis, and would result in a prolonged host survival.
USE OF SMALL MOLECULE ACTIVITY IN UPREGULATION OF CLASS I MHC AND CHANGE IN TUMOUR CYTOKINE PROFILE IN METASTATIC MURINE EPITHELIAL CARCINOMA. by Sarah Dada B.Sc, Hon., Dalhousie University, 2016 A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2019 © Sarah Dada, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Use of small molecule activity in upregulation of Class I MHC and change in tumour cytokine profile in metastatic murine epithelial carcinoma. submitted by Sarah Dada in partial fulfillment of the requirements for the degree of Master of Science in Microbiology and Immunology Examining Committee: Dr. Wilfred Jefferies Supervisor Dr. Raymond Andersen Supervisory Committee Member Dr. Nathan Lack Additional Examiner Additional Supervisory Committee Members: Dr. Marc Horwitz Supervisory Committee Member Dr. Michael Gold Supervisory Committee Member iii Abstract Cancer is a devastating disease that is responsible for the death of a quarter of Canadians. Cancer is the result of cells that gain genetic mutations or epigenetic changes that incur abnormal, uncontrolled proliferation. Oftentimes, such mutations give rise to the metastatic form of cancer, which is capable of moving throughout the body to distant sites. Adaptive immunity, including Cytolytic T cells (CTLs), are intended to kill tumours based on tumour specific antigen presented on Major Histocompatability Complex (MHC) Class I. However, metastatic tumours are capable of escaping such death through the downregulation of Antigen presentation machinery (APM), including MHC Class I. Here, we investigate the role of the small molecule Cannabigerol in upregulating class I MHC and altering cytokine signalling in metastatic tumours, and the consequent CTL tumour killing ex-vivo. Additionally, we determined the impact of Cannabigerol on immune infiltrates and tumour burden in vivo. Small molecules which enhance the efficacy of the CTL killing through upregulation of tumour-antigen bound MHC class I killing would be beneficial for therapeutic use through increased tumour killing, as well as reduced tumour metastasis, and would result in a prolonged host survival. iv Lay Summary Cancer spread, named metastasis, accounts for up to 90% of deaths associated with cancer. Oftentimes, cancer has the ability to evade the immune system and travel throughout the body. Cancer does this by reducing a molecule on itself that the immune system can use to target and kill it, called Class I MHC. Cytolytic T Cells will kill cancer that has Class I MHC upregulated on it. This study focuses on using cannabinoids as therapeutics for cancer through the upregulation of Class I MHC, the change in cancer immune signalling profile, and other gene expression modifications which ultimately lead to cancer killing. v Preface Dr.Wilfred Jefferies was responsible for the idea of small molecule screening for use of Antigen Presentation Machinery upregulation, in collaboration with Dr.Raymond Andersen. Cell lines were kindly provided by Dr. Michal Šmahel’s laboratory. Dr.Lilian Nohara conceived of and performed the marine extract screening flow through via Cellomics and flow cytometry. This was further performed by Carola Dreier, Jessica O’Keefe-Morrice, Krysta Coyle and Daniel Joo Sung Shim. Previous work with Curcuphenol was performed by Samantha Ellis. Curcuphenol was fractionated and identified from the marine invertebrate extracts by Dr.David Williams. Lonna Munro assisted with in vivo Cannabigerol mouse experiments. All animal breeding (A14-0267, A18-298) and experimental (A15-0284) protocols were approved by the UBC Animal Care Committee, which is overseen by the Canadian Council on Animal Care (CCAC). Experiments were performed under the UBC Biosafety protocol B11-0066 and B17-0002. Sarah Dada performed all other work completed in this thesis. vi Table of Contents Abstract………………………………………………………………………...…iii Lay Summary……………………………………………………………...……...iv Preface……………………………………………………………………...……...v Table of Contents………………………………………………………...……….vi List of Tables………………………………………………………………...…....ix List of Figures……………………………………………………………….……..x List of Abbreviations……………………………………………………..……...xii Acknowledgements……………………………………………………...………xiv Dedication……………………………………………………...………...……….xv 1. Introduction…………………………………………………………………......1 1.1 Overview………………………………………………………………………………......1 1.2 Cancer ………………………………………………………………………………….....2 1.3 Cancer metastasis ………………………………………………………...…………….…2 1.4 Cancer and the immune system……………………………………………………………3 1.5 The Cytolytic T cell response………………………………………………………...……6 1.6 Antigen Presentation Machinery………………………………………………………......7 1.7 Epigenetic Modifications and Cancer ……….…………………………………………….8 1.8 Histone Deacetylases and HDAC inhibitors………………..…………….………………..8 1.9 Novel Therapeutics……………………………………………………….……….………9 vii 1.10 Cannabigerol as a Therapeutic………………………………..………....……….………9 2. Hypothesis and Objectives……………………………………………….……11 2.1 Hypothesis ………………………………………………………………………………..11 2.2 Objectives ……………………………………………………………………………..…11 2.2.1 Investigation of small molecules in MHC class I upregulation………………..……11 2.2.2 Investigation of small molecules in cytokine profile……………………………..…11 2.2.3 Investigation of small molecules on tumour burden in vivo……………………....…11 2.2.4 Investigation of small molecules on histone deacetylase function…………….....…11 3. Materials and Methods ………………………………………………..……...12 3.1 Cell lines…………………………………………………………………………...…..…12 3.1.1 Investigation of small molecules in MHC class I upregulation………………..……12 3.2 Upregulation of Class I MHC on A9 cells using small molecules………………………...12 3.3 Reverse Transcription and RT-qPCR ………………………………………………13 3.4 Cytokine Secretion Profile of A9 cells upon treatment of Small Molecules……………....14 3.5 Cytolytic T Cell assay…………………………………………………………...…..14 3.6 Tumour Trial……………………………………………………………………16 3.7 siRNA knockdowns HDACs in TC1 cells……………………………………….. ...18 3.8 Chromatin Immunoprecipitation Assay………………………………………….. ...18 4. Results…………………………………………..…………………………...…..19 4.1 MHC Class I and TAP expression in TC1 and A9 cells ……………………...……...19 viii 4.2 Cytokine Profile in Cannabigerol and Curcuphenol treated A9s……….......................20 4.3 CTL activity in association with Cannabigerol and Curcuphenol treated A9 cells……......21 4.4 Immune Cell Infiltrates and Tumour in Cannabigerol treated tumour-bearing mice……...21 4.5 Acetylation status of APM genes in Cannabigerol and Curcuphenol treated A9 cells using Chromatin Immunoprecipitation..........................................................................22 5. Discussion...........................................................................................................38 Bibliography...........................................................................................................53 ix List of Tables Table 1 Primer sequences used for Reverse transcription quantitative PCR………………13 Table 2 Antibody panel used for fluorescence activated cell sorting………………………..17 x List of Figures Figure 1 Histone Deacetylase Classification……………………………………………….….23 Figure 2 MHC class I expression in TC1 Primary cell lines and A9 metastatic cell lines…23 Figure 3 Effect of Cannabigerol and Curcuphenol on A9 cells……………………………...24 Figure 4 Effect of Cannabigerol on A9 cells…………………………………………………..24 Figure 5: mRNA expression TAP and MHC class I in Cannabigerol and Curcuphenol treated A9 cells………………………………………………………………………….25 Figure 6: A9 cytokine production upon treatment of Cannabigerol and IFN gamma…….25 Figure 7: A9 cytokine production upon treatment of Cannabigerol and IFN gamma…….26 Figure 8: A9 cytokine production upon treatment of Curcuphenol and IFN gamma….….27 Figure 9: A9 cytokine production upon treatment of Curcuphenol and IFN gamma……..28 Figure 10: A9 fractalkine production upon treatment of Cannabigerol and CCL2 production upon treatment of Curcuphenol….………………………………………29 Figure 11: A9 fractalkine production upon treatment of Cannabigerol and CCL2 production upon treatment of Curcuphenol..…………………………...……………29 Figure 12: CTL activity in relation to Cannabigerol, Curcuphenol, and IFN gamma treated A9 cells.……………………………...……………………………………………...…...30 Figure 13: CTL activity in relation to Cannabigerol, Curcuphenol, and IFN gamma treated A9 cells. ……………………………..…………………………………...……………...30 Figure 14: MHC Class I expression and cell death of A9 cells associated with primed CD8+ T cells……………………………..…………………………………...…………….…...31 Figure 15: Tumour volumes of mice treated with PBS, Cannabigerol, and TSA…………..32 xi Figure 16: Mouse weight and tumour volumes of mice treated with PBS, Cannabigerol, and TSA. ……………………………..…………………………………...…………….........32 Figure 17: Gating strategy associated with the in vivo mouse trial immune cell infiltrates………...…………………..…………………………………...……………...33 Figure 18: Percentage of lymphocytes present within the spleen of tumour bearing mice……………………………..…………………………………...……………...........33 Figure 19: Percentage of MHC class 2 positive, CD49 positive or CD3 positive live cells present within the spleen of tumour bearing mice. ……………...……………...……34 Figure 20: Percentage of lymphocytes present within the tumours of tumour bearing mice.…………...……………...……………...……………...……………...……………34 Figure 21: Percentage of CD49, B220 and CD3 positive live lymphocytes present within the tumours of tumour bearing mice…..……………...……………...……………...……35 Figure 22: Percentage of CD3, MHC2 positive live lymphocytes present within the tumour of tumour bearing mice..……………...……………...……………...……………...…35 Figure 23: Absolute Numbers of live lymphocytes present within the tumour of tumour bearing mice….……………...……………...……………...……………...……………36 Figure 24: HDAC 8 and 10 mRNA expression in Cannabigerol and Curcuphenol treated A9 cells.………...……………...……………...……………...……………..…………..36 Figure 25: Class I MHC protein expression associated with siRNA treated TC1 cells against HDAC 5, 8, or 10 and 7uL of siRNA treatment.……………...……………..37 Figure 26: mRNA expression of HDAC siRNA knockdowns using siRNA in TC1 cell lines. xii List of Abbreviations ACK Ammonium Chloride Potassium APC Allophycocyanin APM Antigen Presentation Machinery ARU Animal research unit BSA Bovine Serum Albumin CBD Cannabidiol CBG Cannabigerol CFSE Carboxyfluorescein succinimidy chIP chromatin immunoprecipitation CTL Cytolytic T cells DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl Sulfoxide FACS Fluorescence Activated Cell Sorting FBS Fetal Bovine Serum FITC Fluorescein Isothiocyanate HDAC Histone Deacetylase HDACi Histone Deacetylase inhibitors IFN gamma Interferon gamma MHC Major Histocompatability Complex OT1 C57BL/6-Tg(TcraTcrb)1100Mjb/J Mice P+S Penicillin + Streptomycin PBS Phosphate Buffered Saline xiii PE Phycoerythrin PerCP peridinin-chlorophyll-protein RPM Revolutions per minute RPMI Roswell Park Memorial Institute Medium RT-qPCR Reverse Transcription quantitative polymerase chain reaction Siinfekl Siinfekl Ovalbumin Peptide siRNA small interfering RNA TAP Transporter associated with antigen processing TCR T cell receptor THC D-9Tetrahydrocannabadiol TSA Trichostatin A xiv Acknowledgements I would like to thank my committee members, Dr.Raymond Andersen, Dr.Marc Horwitz, and Dr.Michael Gold, as well as my lab members, especially Franz, Chahaat, Pablo, Shawna, Maria, Hitesh, Brett, Lonna, Cheryl, KB, Jeff, and Samantha. The many faculty members that have guided me along the way, and of course my supervisor Dr.Wilfred Jefferies for the opportunity to live and learn here. Thanks to Erin, Mariah, Caroline, Danny, Dan, Big Sean, Little Sean, and Nicole. Finally, I’d like to thank my kind, generous, and humorous peers in the Microbiology and Immunology, and Biochemistry and Molecular Biology departments. Your warmth makes my world go around. xv For my Mother, Humaira, my father, Nadeem, and my brother, Sameer. Thanks for being supportive, persistent, and nice, for giving me perspective, and for pointing out the joys in life. 1 1 Introduction 1.1 Overview Cancer causes the death of one in four Canadians1 and is the leading cause of death in persons aged 24-852. This represents a massive emotional burden for Canadians, as well as a substantial financial burden upon Canadian Health Services. Tumour metastasis involves the spreading of a primary tumour to the distant sites and is usually the ultimate cause of death associated with cancer3. During cancer metastasis, a change in gene expression oftentimes results in the downregulation of antigen presentation machinery (APM)4. Subsequently, the adaptive immunity that we depend on to kill tumours is left non- functional. Pertinently, the Cytolytic CD8+ T cells (CTLs) are unable to bind the Major Histocompatibility Complex (MHC) I tumour-antigen complex due to this downregulation of APM, resulting in failed tumour killing. This project focuses on the expression of MHC class I in A9 metastatic lung carcinoma and its subsequent impact on CTLs in tumour killing. Our lab found a small molecule, Cannabigerol, which upregulates MHC Class I on A9 cells. This upregulation of MHC class I was determined to cause the proliferation of associated CTLs ex-vivo. Finally, we looked at the role of Cannabigerol in vivo in reducing tumour burden through our suggested model of upregulation of class I MHC leading to CTL killing, as well as the profile of immune infiltrates induced by Cannabigerol. 2 1.2 Cancer Cancer diagnosis occurs in one in two Canadians, and half of these diagnosed cases result in death. Although prostate cancer is the most prevalent cancer in men, and breast cancer is the most prevalent cancer in women, ultimately over 25% of the deaths will be caused by lung cancer1. Cancer is the abnormal, unregulated proliferation of cancer cells 5. There are several hallmarks of cancer6, including the induction of angiogenesis, resistance to cell death, and replicative immortality. Cancer can be either a primary malignant tumour, and confined to a single region of the body, or metastatic. Metastatic tumours have the ability to both invade organs and spread to distant sites, usually through the circulatory or lymphatic systems. This movement is called metastasis. Invasion and metastasis is a key hallmark of cancer, and indeed the final cause of death from cancer about 90% of the time3. 1.3 Cancer Metastasis Our organs and tissues contain cells that are very specific to their environment; with the exception of bone-marrow derived and immune cells3, they do not generally move to different areas of the body. However, genomic and epigenomic instability can cause tumours to acquire mutations that give them the ability to permeate new areas of the body and withstand otherwise debilitating niches to continue growing3. Metastatic colonization is the movement of cancer from a primary to a secondary site and involves the survival and proliferation of disseminated cells1. Genes that mediate tumorigenic functions, such as immune evasion, are considered precursors to metastasis. To metastasize, tumours first gain genetic instability, as well as epigenetic changes which may result in genetic instability. This instability gives rise to tumour initiating gene 3 mutations and disseminations3. Genetic changes might include mutations that allow for increased angiogenesis, proliferation, or evasion of death and intravasation3, and epigenetic changes may include silencing of DNA repair genes or cell cycle regulators7, which can in turn cause changes in genetics. Gene expression signatures, and concurrent epigenetic patterns and chromatin structures7, serve as useful tools to predict outcome3. In cancer, epigenetic events can be reversed to restore the function of important control pathways in malignant, or nearly malignant cells. Metastatic dissemination occurs when a primary tumour is capable of permeating the bloodstream through the angiogenesis or nearby vasculature and circulating through the body. Oftentimes, the niche in which the tumours metastasize will express chemokines or cytokines which will benefit the survival and proliferation of tumour cells3. For this reason, changing the chemokine and cytokine profile of the area surrounding the tumour may be a beneficial method by which metastasis may be deterred. Due to the dissemination of a tumour ultimately leading to death, it would be beneficial for the host to elicit an adaptive immune response associated with the tumour antigen, which could track and kill metastasizing cells. 1.4 Cancer and The Immune System Following hints perhaps the immune system had a role to play in tumour development and growth, in 1957 Sir Macfarlane Burnet published a paper stating that he believed that there was a change in tumour cells that gave them the property to escape normal control the host previously had against tumour cells. This, alongside the work of Lewis Thomas8, culminated into the concept of “immune surveillance.” The idea was that perhaps tumours have specific antigen that the immune system would could eliminate, oftentimes before clinical signs existed. The most challenging component of this hypothesis was that it was hard to prove because it was 4 difficult to demonstrate the destruction of something that no longer existed. The work of Stutman in 1974 had momentarily extinguished the concept of T cells mediating immune surveillance9 by showing that there was no difference between the growth of tumours in athymic nude mice lacking T cells versus wild-type animals10. However, Stutman was unaware that he was studying tumours that lacked APM function and were therefore invisible to host T cell recognition. Thus, his conclusions were invalid. In 2001, a study done by R.D Schreiber’s group11 showed that immune-incompetent Rag knockout mice, which do not have T cells, B cells and NK T cells, or Stat-/- mice, which lack the IFN gamma receptor gene, developed more chemically induced sarcomas much more quickly than wild type mice, and that Rag 2-/- or double Rag 2 and Stat knockout mice developed tumours that were not due to viruses, and appeared similar to the development of human cancers9,11. This was seen as substantial evidence supporting immune surveillance9. However, since the animals that Schreiber’s group studied lacked T and B cells and NK T cells, in the context of Stutman’s study, the deduced conclusion would be that B cells and NK T cells mediate immune surveillance. Fortunately, Schreiber’s group referenced a paper published a year previously from the laboratory of W. A. Jefferies12. This paper repudiated Stutman’s study by examining the growth APM competent, chemically induced tumours in athymic nude mice lacking T cells versus wild-type animals. The Jefferies group demonstrated conclusively for the first time that T cells are required for immune surveillance, as is the expression of functional APM and MHC-I in a tumour. The three primary methods by which the immune system intervenes in tumour development are as follows: 1) Protection from viral infection which may cause tumour induction, 2) Reduction of inflammatory environment which benefits the tumour by rapid 5 elimination of pathogenesis, and subsequent reduction of inflammation 3) elimination of tumours by the immune system using markers of cellular stress or tumour specific antigen. This third point is deemed immune surveillance13. Jefferies and Schreiber’s models on immune surveillance are well accepted in cancer immunology. It is as follows14: There is an initial infiltration of innate lymphocytes, including NKT, NK, and gamma delta T cells. By an unknown mechanism, the innate lymphocytes recognize the tumour cells and begin producing IFN gamma. The IFN gamma will then induce chemokines from innate immune cells, which will elicit recruitment of macrophages, dendritic cells, and NK cells14. The IFN gamma also results in the activation of cytolytic activity by macrophages and NK cells, and directly effects the tumour through antiproliferative action. Through indirect and direct methods, the IFN gamma, therefore, causes tumour death14. Following tumour cell death, tumour debris is thought to be picked up by dendritic cells and brought the draining lymph node, where there is presented to the CD4+ and CD8+ T cells. The tumour specific T cells then home to the tumour using a chemokine homing gradient, where they proceed to kill tumour cells. When the concept of immune editing was founded, in which it was suggested the there is a state of equilibrium associated with the immune system reducing tumour burden to the extent that the immune system eliminates a tumour persistently. However, this same elimination exerts selective pressure. Eventually, the selective pressure progresses to create a tumour that can escape the immune system13. 6 1.5 The Cytolytic T cell Response The adaptive cellular immune response is antigen specific and retains memory against the specific antigen for which it was developed. The adaptive cellular immune response includes T lymphocytes, including the CTLs required for effector function15. T lymphocytes begin in the bone marrow, but migrate to the thymus for maturation. Even upon exiting the thymus, T cells are considered naïve. Within the thymus, the T cell will rearrange gene segments that allow for the creation of the T cell receptor (TCR), which consists of the TCR-alpha and TCR-beta chains. TCRs are able to recognize major histocompatibility molecules (MHC), which are membrane-bound glycoproteins that are important for intercellular recognition, as well as using antigen to distinguish between self and non-self15. MHC Class I, which is present on all nucleated cells, usually associates with CD8+ CTLs, and is used to determine the intracellular antigen of the cell presenting the antigen. MHC class II molecules interact with the CD4+ cells (T helper cells), and are usually present on antigen presenting cells (APCs)15. CD4+ cells need to be activated using professional antigen presenting cells (such as a dendritic cell), and naïve CD8+ cells need antigen presented by dendritic cells as well as co-stimulation by dendritic cells which have been by activated CD4+ cells15. Once activated, CTLs are quickly available to monitor and kill pathogens or tumours within the host should there be a reoccurrence of the threatening antigen. CTLs are known to give protection against viruses and other intracellular pathogens, and are thought to be associated with protection against spontaneous malignant tumours. It is also possible for MHC class I to be used by professional antigen presenting cells to present antigen to CTLs15. Activation of CTLs requires TCR ligation, co-stimulatory signals, such as CD28 binding of CD80 and CD86, 16 and IL-2 mediated signalling 15. This results in proliferation and 7 differentiation of CTLs. Antigen experienced T cells need little to no costimulation for function. CTLs may kill through binding of Fas ligand to Fas on the target cell, triggering the classical caspase cascade, the release of perforin and granzyme, and the secretion of IFN gamma, which will trigger indirect killing via MHC class I upregulation and respective death15. 1.6 Antigen Presentation Machinery Oftentimes, metastasizing cancers acquire the ability to evade the immune system, particularly through the downregulation of antigen presentation machinery (APM)2. The APM consists of several components, including the proteasome, the transporter associated with Antigen Processing 1&2 (TAP1&2), and MHC class I17. The disruption of any one of these components will result in the faulty presentation of endogenous tumour antigens on nascent MHC class I, and as such, improper recognition of the tumour by CD8+ T-cells. The APM of relevance to our cancer work presents internal endogenous peptides to a CTL3. Multi-catalytic proteasome complexes and cytosolic proteases degrade cytosolic internal peptides, and TAP-1 & 2 bring the product peptides to the endoplasmic reticulum (ER). Finally, at the ER, peptides are loaded on the class I MHC through association with the heavy chain of class I MHC, as well as the beta-2 microglobulin15. The peptides are required to be loaded onto MHC class I before MHC class I can be presented on the cell surface to cytolytic T-lymphocytes3. CTLs are critical for immune surveillance against tumours; abnormalities in expression or function of the APM may cause downregulation of cell surface expression of the MHC class I antigens that CTLs require3. Improperly functioning machinery appears in up to 90% of metastatic cancers4. Many tumours, including lung carcinoma, lack TAP-13. It has been shown that there is reduced TAP-1 transcription in TAP deficient cells correlated with low levels 8 of recruitment of histone acetyltransferase to the TAP-1 promoter17. TAP-1 downregulation is thought to allow for tumour evasion of the immune system. 1.7 Epigenetic Modifications and Cancer Epigenetic modifications can lead to changes in cell gene expression, as well as phenotype and chromatin remodeling. Epigenetic regulatory proteins may undergo loss or gain of function due to epigenetic aberrations, and this is known to lead to the onset of human disease, including cancer18. TAP 1 promoter acetylation on H3 is shown to have decreased during tumour progression to its metastatic form19, leading to the idea that other antigen processing machinery may have reduced acetylation during metastasis. 1.8 Histone Deacetylases and HDAC inhibitors Histone deacetylases (HDACs) are epigenetic erasers that remove acetylation marks from lysine residues on several proteins, including histones, which oftentimes results in transcriptional silencing. There are three classes of HDACs: Class I, Class II (a & b) and Class IV20. Class I HDACs are improperly regulated in cancer, showing overexpression associated with lung, prostate, breast and other cancers. Class I HDAC includes HDAC 1,2,3, and 8. Class II HDACS include HDAC 4, 5, 6, 7, 9 and 1021 (Figure 1). Trichostatin A and Vorinostat (SAHA) are well-known class I and II HDAC inhibitors19,22,23 . Vorinostat has been licensed for use in Canada against primary cutaneous T-cell lymphoma, indicating that molecules which upregulate the APM may be useful for reducing tumour load through increased immune surveillance. In addition to reversing the repressive state of the chromatin, using histone deacetylase inhibitors (HDACi) may promote immune recognition of tumours due to the epigenetic enhancement of 9 APM19, and subsequent increase in transcription and translation of APM. Due to the high importance of HDAC in cancer development, the practical application of HDAC inhibitors moved to the center of clinical and research attention. 1.9 Novel Therapeutics Screening for natural compounds for anti-cancer properties is a common method for finding potential cancer therapies, and is especially prominent for plant and animal extracts prevalent in the sea. Many anti-cancer clinical agents are coming from the concept that marine life cover’s 70% of the earth’s surface, and that this biodiversity gives rise to plants and animals which contain compounds that have therapeutic benefits24. S-(+)-curcuphenol was found was taken from a marine invertebrate screen25. S+ Curcuphenol was shown to restore APM26, including demonstrating an increase in TAP-1 and MHC-I expression in A9 cells. Analogs of the compound were shown to have reduced tumour burden and increase immune cell infiltration and immunogenicity of A9 metastatic tumours in mice26. Curcuphenol analogs were also found to change HDAC I/II activity, causing activation of HDAC 5 and 10, and inhibition of HDAC 826. 1.10 Cannabigerol as a Therapeutic D-9Tetrahydrocannabadiol (THC) has been studied for its anti-tumour effects, however, the psychotropic effects of THC prevent the use of it as a therapeutic. Alternative plant Cannabinoids, such as cannabidiol, are being studied for their anti-tumour effects27. While much of the research on minor phytocannabanoids focused on the CBD28. Cannabigerol is also a promising candidate for various therapeutics. Cannabigerol is a non-psychotropic cannabinoid which has been studied for its antitumour effect27. In mouse colon carcinogenesis, cannabigerol 10 has caused reduced tumour cell growth, promoted apoptosis of cacao-2 colorectal cancer cells (CRC), stimulated ROS production, and reduced cell growth in CRC cells29. However, this study was done in athymic nude mice, which lack T cells29. Similarly, a different cannabinoid, Cannabidiol has been seen to reduce tumour burden of xenographed and chemically induced tumours, however, this was thought to occur through direct killing or induce apoptosis of the human breast cancer cell line30. In fact, many studies appear to use immune deficient mice to study the effect of phyto-cannabanoids30. Hence, little is known about the place of the immune response to tumour metastasis in the presence of several cannabanoids, including Cannabigerol. 11 2 Hypothesis and Objectives 2.1 Hypothesis Use of the cannabinoid Cannabigerol will alter the presentation of class I MHC, and will allow for reduced tumour burden through increased immune cell infiltration, and more specifically CTL mediated killing. 2.2 Objectives 2.2.1 To investigate the effect of small molecules that upregulate MHC class 1 and the APM expression in metastatic murine lung carcinoma cell lines (A9), and subsequent CTL killing. 2.2.2 To investigate the effect of small molecules on cytokine production of A9 cells. 2.2.3 To investigate the effect of Cannabigerol small molecule on tumour burden and immune infiltration of metastatic tumours in immune-capable mice. 2.2.4 To investigate the effect of small molecules on histone deacetylase function and acetylation patterns of APM in metastatic tumour A9 cells and primary tumour TC1 cell lines. 12 3 Materials and Methods 3.1 Cell lines The primary TC1 tumour cell line, originating from a female C57Bl/6 mouse, was developed by the transformation of murine primary lung cells with the human papilloma virus type16 E6 and E7 oncogenes and activated H-ras (cell-division regulating GTP-ase)31. These cells have high TAP-1 and MHC class I levels4,19,31. The metastatic A9 tumour cells are the metastatic clones derived from the primary TC1 tumour; these cells are capable of metastasis when injected subcutaneously into mice, not just when injected into blood4. The metastatic A9 tumour has downregulated MHC class-I expression and APM components4,19. A9 and TC-1 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Catalog #11995-065) with 10% Fetal Bovine Serum (FBS) (Catalog #A3160401, Gibco). 3.1.1 Cell Harvesting TC-1 and A9 cells were washed with Phosphate Buffered Saline (PBS) pH7.4 and trypsinized (0.05%, Gibco) before adding an additional 66% of media and spinning at 1500 RPM for 4 minutes. Cells were then resuspended or lysed, depending on the experiment. 3.2 Upregulation of Class I MHC on A9 cells using Small Molecules All compounds, (Cannabigerol, Curcuphenol, and IFN gamma) were dissolved in 1% Dimethyl Sulfoxide (DMSO) (Catalog #276855, Sigma) and media (DMEM and 10% FBS) as the vehicle. 1x106 A9 cells were plated onto a 6 well plate (Catalog #cls3516, Corning) in two mLs of media. A9 cells were treated after 24 hours at various concentrations of Cannabigerol and Curcuphenol, 5.832x10-6 nmol IFN gamma or DMSO vehicle for 48 hours. Cells were then harvested from the 13 plate as per protocol and half of the cells were checked for MHC class I expression levels using FACS. The Anti-mouse H2KB APC antibody (Catalog# 116518, Biolegend) was used at 1:200 in FACS buffer (2%FBS in PBS). Viability was determined using the 7AAD viability dye (Table 2) in FACS buffer at 1:100 In 2mL of media, 0.275, 0.031. 0.47, 0.055, 0.058, and 0.11 umol of Cannabigerol were used, with a corresponding molarity of 13.75mM, 15.5mM, 23.5mM, 27.5mM, 29mM, and 55mM. In 2mL of media, 0.00143, 0.00167, and 0.00191 umol of Curcuphenol were used, with a corresponding molarity of 0.715mM, 0.835mM, and 0.955mM. 3.3 Reverse Transcription and RT-qPCR RNA was isolated using the RNeasy plus mini kit. RNA was reverse transcribed into cDNA using the superscript II reverse transcription kit (Catalog # 18064014, Invitrogen). Quantitative RT PCR was done using 10nm of primer and 1uL of BioRad SYBR Green master mix (Catalog#1725271, Biorad). RT-qPCR was done on7500 Fast Real-Time PCR System from Applied Biosystems 40 cycles (95C denaturing for 15 seconds, 60C annealing for one minute.) Primers are as follows: Table 1: Primer sequences used for RT-qPCR PRIMER TARGET Forward (5’->3’) Reverse (5’->3’) HDAC 5 5’ GCC TCG GAA CCC AAC TTA AA 3’ 5’ GGC AGA GAA GGA GAX GTA TAG A 3’ HDAC 8 5’ CCT GAT TGA CGG GAA GTG TAA 3’ 5’ GTG CAG GGA CAC AGT CAT AA 3’ HDAC 10 5’ CCA AAC ATC CCA AGC AGA AATA A 3’ 5’CAG CAC CAA CTC AGG ATC AA 3’ TAP 1 5’ TTA TCA CCC AGC AGC TCA GCC T 3’ 5’ TCA GTC TGC AGG AGC CGC AA 3’ B-Actin 5’ ATG GAT GAC GAT ATC GCT GC 3’ 5’ TTC TCC AGG GAG GAA GAT 3’ 14 3.4 Cytokine Secretion Profile of A9 cells upon treatment of Small Molecules All compounds, (Cannabigerol, Curcuphenol, IFN gamma were dissolved in 1% Dimethyl Sulfoxide (DMSO) (Catalog #276855, Sigma) in media (1%DMSO). 1x106 A9 cells were plated onto a 6 well plate in two mL of DMEM media. Twenty-four hours after seeding, cells were cultured at the optimum concentrations of Cannabigerol 0.055umol, Curcuphenol 0.00167 umol, or 5.832x10-6 nmol IFN gamma or 1% DMSO vehicle for 48 hours in 2mL media. Relative expression levels of 111 soluble mouse proteins including cytokines, chemokines and growth factors were evaluated using the Proteome Profiler Mouse XL cytokine array kit (R&D System, ARY028) following the manufacturer’s instructions. Spot densities on the array film were detected and quantified using Image J analysis software Image J protein analyzer add-on on a scanned version of the film. Quantification of the spot intensity in the arrays was conducted with background subtraction in ImageJ. To determine fold change, the treatment values of Curcuphenol or IFN gamma microarrays were divided by the DMSO negative control values of the concurring spots. A value of 1 was subtracted from the absolute value of this fold change, to correct the value of DMSO to “0”. The experiment was done in technical replicates. Key cytokines that showed changes in expression levels were further characterized by pathway analysis for over-represented pathway identification through Reactome Database release 65, Pathway Brower Version 3.5. 3.5 Cytolytic T Cell assay 1x106 A9 cells were plated onto a 6 well plate in two mL of RPMI media (Advanced RPMI-1640 Medium (Catalog# 12633020, Gibco), 100U/mL of Penicillin-Streptomycin (P+S) (Catalog# 15 15070063, Thermofisher), 1% L-Glutamine (Catalog #25030081, Gibco), and 10%FBS). A9 cells were treated with Cannabigerol 0.055umol, Curcuphenol 0.00167 umol, 5.832x10-6 nmol IFN gamma, or 1% DMSO vehicle in 2mL of media. Following 24 hours, siinfekl OVA Peptide (Siinfekl) (Catalog #257-264, Genscript), was added to the A9 cells. Following an additional 24 hours, CD8+ T cells were collected from OT1 mouse spleens. Spleens were collected and passed through a 100 micron cell strainer (Catalog #352260, Falcon). Red blood cells were removed from the spleen isolate using Red Blood Cell ACK lysis buffer (Catalog#A10492-01, Gibco). Enrichment for CD8+ T cells was done using CD8+ Untouched Mouse CD8+ cells Dynabeads (Catalog #11417D, Thermofisher), as per manufacturer protocol. RPMI media was removed from the A9 cells, and A9 cells were washed three times with PBS before being replaced with RPMI media. An extra well of untreated A9 cells was counted and used as a standard count. CD8+ T cells were counted and then treated with CFSE (Catalog #79898, Biolegend) per manufacturer protocol, before being co-cultured with A9 cells to a 1:1 ratio or a 1:5 ratio. T cells for the positive control were stimulated 24 hours later using CD28 Monoclonal Antibody clone 37.51 (Catalog # 14-0281-86, eBioscience) at a concentration of 5ug/mL and CD3e Monoclonal Antibody clone 145-2C11 at 10ug/mL, (eBioscience Catalog # 14-0031-86). CD8+ T cells were collected from the cell media, and spun down at 1500 RPMI for use with FACS, and A9s were harvested for FACS. CD8+ T cells were stained with CD8 PE-efluor 610 antibody (Table 2). A9 Cells were stained using PE H-2KB antibody (Table 2), and 7AAD viability dye (Table 2) in FACS buffer. 16 3.6 Tumour Trial C57BL/6 mice from the Jackson Laboratories were maintained in the Animal Research Unit (ARU) animal facility at the University of British Columbia at 8-10 weeks of age. The tumour trials were approved by the Animal Care Committee of the University of British Columbia. Animals were maintained and euthanized in accordance with the guidelines of the Canadian Council for Animal care. 1x105 A9 cells were injected in a volume of 50uL of PBS into the right flank of each mouse subcutaneously. Intraperitoneal injections were done daily starting day 7 of Cannabigerol (Catalog# 20164, Cayman Chemical Company) at 10mg/kg, Trichostatin A (TSA)(Catalog #28552-5MG, Sigma-Aldrich) at 0.5mg/kg dissolved in 1%DMSO and PBS, or vehicle (0.2% DMSO, 5% Bovine Serum Albumin (BSA), in PBS). Cannabigerol was dissolved In 0.2% DMSO, 5% Bovine Serum Albumin, in PBS as per Valdeolivas et al32. Weighing was done 3 times a week and during tumour measurements, and tumour measurements were done upon the appearance of the tumour, on days 12 and 13 of the trial. Tumour volume was found using calipers to measure tumour dimensions. Tumour volume=(length2 x width)/2, with the longest axis of the tumour considered “length”. Mice were sacrificed at day 10, 11, and 12, as was dictated by subscribed humane endpoints, in which mice experienced tumour ulceration. 17 Table 2 : Antibody panel used for FACS during mouse tumour trial Target Fluorophore (Anti-mouse) Clone Catalog # Company Ratio used CD3E APC-eFluor 780 145-2C11 47-0031-82 Invitrogen 1:200 CD11C eFluor 506 N418 69-0114-82 Invitrogen 1:200 CD4 Alexafluor 700 GK1.5 56-0041-82 Invitrogen 1:200 CD69 PE-Cyanine7 H1.2 F3 25-0691-82 Invitrogen 1:200 CD45R FITC RA3-6B2 11-0452-82 Invitrogen 1:200 MHCII eFluor 450 AF6-120.1 48-5320-82 Invitrogen 1:200 CD49B APC DX5 12-5971-82 Invitrogen 1:200 CD11B SuperBright 645 M1/70 64-0112-82 Invitrogen 1:200 CD62L SuperBright 702 MEL-14 67-0621-82 Invitrogen 1:200 MHC Class I (H-2KB) PE AF6-88.5.5.3 12-5958-82 Invitrogen 1:200 Viability Dye 7AAD PerCP Cyanine5.5 - 420404 Biolegend 2:200 CD8A PE-EF610 53-6.7 61-0081-82 Invitrogen 1:200 Super Bright Staining Buffer - SB-440-75 eBioscience 50:200 Half of the tumours were removed from the mouse and immediately placed in digestion media (RPMI Medium 1640 (Catalog #22400-089, Gibco) with 10% FBS and Penicillin + Streptomycin (P+S), with the addition of 10% collagenase hyaluronidase (Catalog #07912), 15% DNAse I (Catalog # 07900, Stemcell)). Tumours were chopped in 1mL of digestion media, and placed at a temperature of 35C for 30 minutes, before being passed through a 100 micron cell strainer (Catalog #352260, Falcon). Cells from these tumours were stained in FACS buffer using the panel from Table 2, before being taken to FACS on the Attune 2 FACS machine. The second half of the tumours were placed in Neg-50 (Catalog#6502, Richard Allan Scientific) for freezing and further histology. The lungs, adrenal glands, and liver were collected and placed in formalin for further histology. All spleens were collected and passed through a 100 micron cell strainer. Red blood cells were removed from the spleen isolate using Red Blood Cell (ACK) lysis buffer 18 and the remaining cells were stained using the Table 2 panel in FACS buffer before being examined on the Attune 2 FACS machine. 3.7 siRNA knockdowns HDACs in TC1 cells TC1 cells were plated 24 hours before transfection in media (DMEM and 10%FBS) at a concerntration1x106 TC1 cells per well, in a 6 well plate. TC1 cells were transfected with 7ul of siRNAs HDAC5 (Catalog sc-35543, Santa Cruz), HDAC8 (Catalog sc-35549, Santa Cruz), HDAC10 (sc-72308, Santa Cruz) or Control siRNA-A (sc-37007, Santa Cruz). The siRNA was put in 150 uL of Opti-MEM Reduced serum medium (Catalog# 31985062, Gibco) for 5 minutes. Concurrently, 12.5 uL of Lipofectamine RNAiMAX (Catalog#13778150, Lipofectamine Thermofisher) or Lipofectamine 2000 reagent (Catalog #11668027, Lipofectamine Thermofisher) was placed in 150uL of Opti-MEM for 5 minutes. The siRNA and lipofectamine in optiMEM were then mixed together and incubated for 10 minutes before placing into each TC1 well. 48 hours following transfection, TC1s were harvested from the plate and half of the cells were checked for MHC expression levels using the anti-mouse H2KB APC antibody (Catalog #116518, Biolegend) and viability using the 7AAD viability dye using FACS; the other half of the TC1 cells were lysed for quantitative RT-PCR using the RNeasy plus mini kit (Catalog #74134, Qiagen). 3.8 Chromatin Immunoprecipitation Assay 1x106 A9 cells were plated onto a 6 well plate in two mL of DMEM media. Twenty-four hours after seeding, cells were cultured at the optimum concentrations of Cannabigerol 0.055umol, Curcuphenol 0.00167 umol, or 5.832x10-6 nmol IFN gamma or 1% DMSO vehicle in 2mL of 19 media for 48 hours. Cells were harvested using trypsin, and were pelleted at 200g for 5 mins before being resuspended in PBS. Cell viability was determined using Trypan Blue dye (Catalog# 15250061, Gibco) and cell counting slides (Catalog#1450015, Biorad) with Biorad TC20 automated cell counter. Once the viability was above 90%, the cells were quantified and 130K live cells were transferred into a new tube. The cells were pelleted at 500g for 6 minutes at 4C. Cells were put into Chip-seq lysis buffer (provided by Dr.Martin Hirst lab) (Confidential) and mixed up and down three times before being placed immediately in liquid nitrogen for 15-20 seconds. Cells were then immediately transferred to dry ice and stored in the -80C until they were ready for transport. Cells were transported to Dr.Martin Hirst’s lab on dry ice. 4 Results 4.1 MHC Class I and TAP expression in TC1 and A9 cells MHC Class I expression is higher in TC1 primary cell lines than A9 metastatic cell lines, as was anticipated by previous research (Figure 2). Cannabigerol and Curcuphenol (Figure 3) were seen to increase the abundance of MHC class I protein in A9 cells, as seen by FACS, and an ideal concentration to promote increased expression of MHC class I, but not kill the A9 cells was found to be 0.055umol (Figure 3, 4) in 2mL media. Optimum concentration to balance MHC class I upregulation and viability was at 0.055 umol for Cannabigerol and 0.00167 umol for Curcuphenol (Figure 3) in 2mL of media. MHC class I protein expression was seen to be higher on A9 cells treated with Cannabigerol, as determined by the higher APC fluorescence associated with MHC class I using FACS. There was an increase in A9 MHC class I protein expression upon treatment of cannabigerol and curcuphenol (figure 3a,b). Treatment with Cannabigerol up to 0.058umol did 20 not appear to induce cell death in 2mL of media, as there were similar numbers of live cells among treated and untreated cells, however upon doubling the optimum concentration there appeared to be a drastic amount of cell death. At half of the optimal concentration, there appeared to be little upregulation of class I MHC, however Cannabigerol appeared to induce a significant increase in class I MHC relative to A9 cells treated with DMSO (vehicle) (Figure 4) at the optimum concentration, reflecting a p-value of <0.0001 upon use of a one way ANOVA. IFN gamma was a successful positive control, inducing a high amount of MHC class I protein expression. TAP and MHC class I mRNA appeared to increase upon these treatments with optimum concentrations of Cannabigerol and Curcuphenol, as well (Figure 5). 4.2 Cytokine Profile in Cannabigerol and Curcuphenol treated A9 cells Cannabigerol and Curcuphenol were also found to cause a change in cytokines involved in inflammation, migration, growth and differentiation, angiogenesis, immune regulation, leukocyte development and metabolism (Figures 6,7,8,9). There was a notable increase of Fractalkine upon treatment with Cannabigerol (Figure 10A,11A) and a decrease of CCL2 upon treatment with Curcuphenol (Figure 10B,11B) in the supernatant of the A9 cells relative to DMSO (vehicle treated) A9 cells. Other cytokines of importance to our lab that were upregulated upon the use of Cannabigerol, including IL-28A, CCL22, FGF-21 (figure 6C,7C), and IL-33 (figure 6A,7A). Additionally, there was downregulation of IL-6 (figure 6D,7D), and IL-11 (figure 6A,7A). Finally, there was an increase in VEGFA (Figure 6D). Upon treatment of Curcuphenol, A9 cells appear appeared to express higher levels of IL1b (Figure 8A,9A), and IL28A/B (Figure 8D,9D), as well as a much stronger increase in CD40 21 (Figure 8D,9D). There was a decrease in the production of IGDBP3 (Figure 8A,9A), LDLR (Figure 7B), as well as MMP2 and MMP3 (Figure 8C,9D). 4.3 CTL activity in association with Cannabigerol and Curcuphenol treated A9 cells Ex vivo, OT1 (ova-peptide siinfekl specific) CD8+ T cells were found to show increased proliferation when their co-cultured metastatic tumour cells were treated with Cannabigerol for 48 hours prior to co-culture, suggesting that the T cells are activated and would have increased cytolytic T cell activity upon use of Cannabigerol and Curcuphenol (Figures 12,13). There was an increase in the MHC class I on the A9 cells associated with the OT1 T cells (Figure 14a), with the exception of a duplicate of Curcuphenol. This duplicate likely had low T cell proliferation due to the high amount of A9 death in this sample (Figure 14b). 4.4 Tumour Burden and Immune Cell Infiltrates in Cannabigerol treated tumour-bearing mice In vivo, the tumour burden (Figure 15, 16b) did not appear to decrease upon treatment with Cannabigerol, relative to the PBS control, although several immune cell infiltrates were found within the tumours of all treatment groups (Figures18-22), most notably there was high numbers of NK cells into the tumours (Figure 23b). There was little change in mouse weight for the duration of the trial (Figure 16A). Trichostatin A is a known HDAC inhibitor which the Jefferies lab has previously shown to reduce tumour burden in mice.19 22 4.5 Acetylation status of APM genes in Cannabigerol and Curcuphenol treated A9 cells using Chromatin Immunoprecipitation ChIP sequencing samples of the Cannabigerol and Curcuphenol treated A9 were submitted several times to interrogate the H3K27 residue for acetylation status, however inadequate quality of sample prevented reliable results from being obtained. Cannabigerol and Curcuphenol were also seen to upregulate HDACs 8 and 10 in the treated A9 metastatic cells (Figure 23). 23 Figure 1: HDAC classification (adapted from Lucio-Eterovic, 2008) Figure 2. MHC class I expression in TC1 Primary cell lines and A9 metastatic cell lines 0500010000150002000025000Cell lineMean Fluoresence of APCMHC class I expressionTC1A9 24 Figure 3. Effect of the a) Cannabigerol on A9 cells treated for 48 hours in 2 mL of media at concentrations of 0.047 umol, 0.055 umol, and 0.58 umol and b) Curcuphenol on A9 cells at 0.00143, 0.00167, and 0.00191 umol on MHC class I protein expression using FACS. Frequency of live cells was found using 7AAD viability dye. Figure 4: Effect of the a) Cannabigerol on A9 cells treated for 48 hours at concentrations of 0.027 umol, 0.055 umol, and 0.11 umol in 2mL of media. MHC class I protein upregulation determined using FACS was found to be statistically significant with a p-value of less than 0.0001 between DMSO treated cells and Cannabigerol treated cells at a concentration of 0.055 umol in 2mL of media while using an ordinary one-way ANOVA, as denoted by ****. IFN gamma treatment was at 5.832x10-6 in two mL of media. A9 UntreatedDMSOIFN GammaCannabigerol 0.0275 umolCannabigerol 0.055 umolCannabigerol 0.11 umol050010001500020406080100umol of tested CompoundMHC I ExpressionEffect of Compounds on MHC I expression and Cell DeathMean MHCLive CellsFrequency of live cells**** 25 Figure 5: Treatment with a) Cannabigerol at 0.055 umol in 2mL of media and b) Curcuphenol at 0.00167 umol in 2mL of media causes increased mRNA expression of TAP, MHC class I, as found by quantitative PCR. Figure 6: Fold change of A9 cell cytokine production in supernatant upon treatment of 0.00875 mg/mL Cannabigerol (0.05529 umol) and 0.1 ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells in 2mL of media. A, B, C) Fold change of cytokines present on microarray D) Cytokines implicated in the IL4, 10, and 13 pathways. Data was determined using Proteome Profiler Mouse XL cytokine array kit and Image J protein analyzer add-on. Pathway analysis was determined using Reactome Database release 65, Pathway Brower Version 3.5. 26 Figure 7: Alternative representation of Figure 6. Fold change of A9 cell cytokine production in supernatant upon treatment of 0.00875 mg/mL Cannabigerol (0.05529 umol in 2mL of media) and 0.1ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. A, B, C) Fold change of cytokines present on microarray D) Cytokines implicated in the IL4, 10, and 13 pathways. Data was determined using Proteome Profiler Mouse XL cytokine array kit and Image J protein analyzer add-on. Pathway analysis was determined using Reactome Database release 65, Pathway Brower Version 3.5. 27 Figure 8: Fold change of A9 cell cytokine production in supernatant upon treatment of Curcuphenol (0.00167 umol in 2mL of media) and 0.1 ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. A, B, C) Fold change of cytokines present on microarray D) Cytokines implicated in the IL4, 10, and 13 pathways. Data was determined using Proteome Profiler Mouse XL cytokine array kit and Image J protein analyzer add-on. Pathway analysis was determined using Reactome Database release 65, Pathway Brower Version 3.5. 28 Figure 9: Alternative representation of Figure 8. Fold change of A9 cell cytokine production in supernatant upon treatment of 0.00875 mg/mL Cannabigerol (0.05529 umol in 2mL of media) and 0.1 ug/mL (100 ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. A, B, C) Fold change of cytokines present on microarray D) Cytokines implicated in the IL4, 10, and 13 pathways. Data was determined using Proteome Profiler Mouse XL cytokine array kit and Image J protein analyzer add-on. Pathway analysis was determined using Reactome Database release 65, Pathway Brower Version 3.5. 29 Figure 10 a) Fold change of A9 cell Fractalkine production in supernatant upon treatment of 0.00875 mg/mL Cannabigerol (0.05529 umol in 2mL of media) and 0.1ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. B) Fold change of A9 cell CCL2 production in supernatant upon treatment of 0.0.1827 mg/mL Curcuphenol (0.00167 umol) and 0.1 ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. Figure 11: Alternative representation of Figure 10. a) Fold change of A9 cell Fractalkine production in supernatant upon treatment of 0.00875 mg/mL Cannabigerol (0.05529 umol in 2mL of media) and 0.1 ug/mL (100 ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. B) Fold change of A9 cell CCL2 production in supernatant upon treatment of 0.0.1827 mg/mL Curcuphenol (0.00167 umol) and 0.1ug/mL (100ng/mL) (5.832x10-6 nmol) IFN gamma relative to DMSO (vehicle)-treated A9 cells. 30 Figure 12: CD8+ T cells from siinfekl-primed OT1 mice in response to siinfekl peptide presented on MHC class I of A9 cells. A9 cells were treated with 0.055 umol of Cannabigerol, 5.832x10-6 nmol mL of IFN gamma (Positive control), or 0.00167 umol Curcuphenol in 2mL of media. T cells were labeled with CFSE proliferation dye, which is reduced within the progeny cells as the generations progress. The positive control consisted of use of CD3 and CD28 antibody in a well of only T cells, to induce proliferation. Figure 13: Second experiment using CD8+ T cells from siinfekl-primed OT1 mice in response to siinfekl peptide presented on MHC class I of A9 cells. A9 cells were treated with 0.055 umol of Cannabigerol, 5.832x10-6 nmol mL of IFN gamma positive control, or 0.00167 umol Curcuphenol in 2mL of media. The negative control is named “CFSE and siinfekl CD8+ T cells”. T cells were labeled with CFSE proliferation dye, which is reduced within the progeny cells as the generations progress. 31 Figure 14: a) MHC Class I expression of A9 cells associated with CD8+ T cells from siinfekl-primed OT1 (Figure 10 above). A9 cells were treated with 0.055 umol of Cannabigerol, 5.832x10-6 nmol mL of IFN gamma, or 0.00167 umol Curcuphenol in 2mL of media.b) Viability of A9 cells associated with CD8+ T cells from siinfekl-primed OT1 (panel A). 7AAD viability dye is associated with increased cell death. This demonstrates that the reduced MHC class I present on the Curcuphenol sample in purple (panel A) is likely due to A9 cell death as a result of Curcuphenol treatment. 32 Figure 15: Tumour volumes (cm3) of mice in PBS negative control, Cannabigerol treatment, and TSA positive control (n=8). A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. Mice were treated with 10mg/kg Cannabigerol, 0.5mg/kg of TSA, or vehicle through IP injection daily, and were sacrificed based on humane endpoint. Slope was determined by average ((x2-x1)/y2-y1)) where x is time and y is average tumour volume. Figure 16 : a)Mouse weight did not drastically decrease for the duration of the mouse trial among treatment groups. N=8 per group up to and including day 12; n=8 for Cannabigerol and PBS vehicle on day 13 and n=6 for TSA group on day 13. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. Mice were treated with 10mg/kg Cannabigerol, 0.5mg/kg of TSA, or vehicle through IP injection daily, and were sacrificed based on humane endpoint. B) Tumour volumes (cm3) of mice in PBS negative control, Cannabigerol treatment, and TSA positive control (n=8) on days 5 and 6 of treatment (day 12 and 13 of the trial). 33 Figure 17: Gating strategy associated with the in vivo mouse trial immune cell infiltrates. Figure 18: Percentage of lymphocytes present within the spleen of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS when gating, as well as live lymphocyte and B220 positive, MHC1 positive and MHC2 positive percentages of live lymphocytes. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. 34 Figure 19: Percentage of a) MHC class 2 positive or CD49 positive or b) CD3 positive live cells present within the spleen of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS when gating. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. Figure 20: Percentage of lymphocytes present within the tumour of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS when gating, as well as live lymphocyte MHC 1 positive percentages of live lymphocytes. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. 35 Figure 21: Percentage of CD49, B220 and CD3 positive live lymphocytes present within the tumour of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS when gating. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. Figure 22: Percentage of CD3, MHC2 positive live lymphocytes present within the tumour of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS of the previous gate. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. 36 Figure 23: Absolute Numbers of live lymphocytes present within the tumour of tumour bearing mice (n=8 for PBS negative control and Cannabigerol treatments, n=6 for TSA positive control) relative to number of events found during FACS of the previous gate. A9 tumours were present in mice for 7 days before IP treatment, and 4-5 days before sacrifice. Figure 24: HDAC 8 and 10 mRNA expression in a) cannabigerol treated A9 cells (0.055 umol in 2mL of media) and b) curcuphenol treated (0.00167 umol in 2mL of media) A9 cells. S18 gene expression was used to quantify the relative mRNA amount. 37 Figure 25: Class I MHC protein expression associated with siRNA treated TC1 cells against HDAC 5, 8, or 10 and 7uL of siRNA treatment in 2mL of media. There appeared to be higher MHC expression upon knockdown of the HDACs. Figure 26: mRNA expression of HDAC siRNA knockdowns using siRNA in TC1 cell lines. 38 5 Discussion Overall, we saw that MHC Class I was more prevalent on TC1 primary cells than A9 metastatic cells (Figure 2). MHC class I was upregulated on A9 metastatic tumour cells upon the use of Cannabigerol or Curcuphenol (Figures 3,4), and this upregulation of MHC Class I caused the proliferation of co-cultured CD8+ Cytolytic T cells (Figures 12,13). We also found that A9s treated with Cannabigerol had a varied cytokine secretion profile, producing cytokines that are associated with leukocyte migration (Figure 4A, Figure5). Although Cannabigerol did not immediately reduce tumour burden in vivo (Figures 15,16B), there appeared to be increased infiltration of NK cells in the tumour and evidence for adaptive immunity (Figure 23). Cannabigerol appeared to increase HDAC 8 activity, as well as TAP and Class 1 MHC in A9 metastatic cell lines (Figure 24). Finally, there may be an increase of MHC Class I and TAP upon knockdown of HDAC 5, 8, 10 in primary TC1 cell lines (Figures 27,28). Knowledge gained from this thesis will allow us to determine if Cannabigerol is a novel therapeutic to reduce cancer metastasis, and presents a starting point for which a mechanism for tumour antigen presentation and subsequent cancer killing can be found. MHC class I surface protein expression on the TC1 primary cell line was increased relative to that of the metastatic A9 derivative cell line when using FACS (Figure 2). This was confirmation of what was expected, following several previous studies17, which showed corresponding decreased levels of TAP in A9 cells17. The downregulation of MHC class I suggests that the metastatic A9 cells are less susceptible to CTL killing and that A9s would be better at escaping immune surveillance, most pertinently the immune surveillance of CD8+ cell-mediated immunity. There was an increase in MHC class I protein expression upon A9 cell surface associated with an increase in treatment concentration of Cannabigerol and Curcuphenol (Figure 3A,3B). 39 An optimum concentration for future experiments was chosen based on the concentration of Cannabigerol or Curcuphenol which induced MHC class I on A9 cells but did not cause a large amount of cell death. Optimum concentration was found to be 0.055 umol of Cannabigerol and 0.00167 in umol of Curcuphenol in 2mL of media. An increase in MHC class I expression in A9 metastatic tumour cells upon treatment with Cannabigerol and Curcuphenol (Figures 2,3) showed similar levels to that of the primary cancer TC1 Class I MHC protein expression (Figure 2A, 2B), and is a promising discovery. The upregulation of Class I MHC suggests that if CTLs were primed against A9-specific antigen and as such given license to kill A9 tumour cells, the tumour would be much more susceptible to CTL killing. Additionally, it is possible that low levels of tumour cell death caused by Cannabigerol or Curcuphenol may be beneficial to the adaptive immune system, as it may rise to A9 cell debris. The A9 cell debris, including A9 specific antigen, may be picked up by dendritic cells and presented to CD8+ and CD4+ naïve T cells for licensing to kill against that particular tumour antigen. Figure 4 demonstrates that there is a significant increase in class I MHC upon optimum treatment of Cannabigerol (0.055 umol 2mL of media) relative to that of the vehicle (DMSO) treated cells, with a p-value of <0.0001 while using an ordinary one-way ANOVA. Although the class I MHC expression is not as high with Cannabigerol as with IFN gamma, they are inducing at least this same pathway. The gives rise to the possibility that Cannabigerol could elicit a similar response in the tumour environment as IFN gamma, which is known for recruiting innate immune cells and activating the cytolytic activity of NK and macrophages cells14. Figure 4 also demonstrates that there is a high amount of cell death at double the optimum concentration (0.11 umol in 2mL of media), which suggests that Cannabigerol is directly killing the tumour cells. This is supported by Borrelli et al33, who found that their colorectal cancer cells were directly killed by Cannabigerol. At a concentration 40 of half of the optimum concentration (0.0275 umol in 2mL of media), there was very little cell death; the level of cell death was comparable to that of DMSO treated or untreated cells. However, at this half-optimal concentration there was little upregulation of class I MHC relative to DMSO-treated or untreated A9 cells. Figure 5 shows a quantitative polymerase chain reaction (RT-qPCR) reflective of the mRNA expression of A9 cells treated with Cannabigerol, Curcuphenol, IFN gamma, or DMSO vehicle at optimum concentrations. The RT-qPCR does not appear to demonstrate the upregulation of class I MHC to the extent that FACS does (Figures 2.5). There appears to be slight upregulation of TAP and MHC class I mRNA upon treatment with Cannabigerol and Curcuphenol (Figure 5), while there is obvious protein upregulation of MHC class I (Figure 2). Although it may be expected that there would be upregulation of both TAP and MHC class I at the same time, as they are both antigen presentation machinery, it is not necessarily guaranteed that these transcripts are coupled in their production, as seen during the screening of other compounds (unpublished, Jefferies lab). When looking at genes involved in tumorigenesis, it is relevant to look at the cytokine signature of metastatic A9 cells. During metastasis, tumour cells accumulate mutations; subsequently, there is pressure from the immune system for a tumour to gain a beneficial mutation, which is then passed on to its progeny3. As tumour cells migrate, they find a promising niche in which to grow, and, additionally, they create an environment for themselves which aids their growth and maintains their immune evasion3. Some of the beneficial mutations that tumour cells obtain will change the cytokines that they release to create a better growth environment and to continue avoiding the immune system. If a compound was able to cause a change in cytokines released by a tumour, it may be possible to change the tumour environment to an environment 41 which is beneficial for the host. This may include an environment which has increased immune infiltration, lower angiogenesis, or reduction of other tumour-aiding cytokines. Using a protein microarray allowed us to determine the effect of Cannabigerol in a diverse spectrum of molecular functions, including cellular growth, differentiation, migration, disease-associated pathways and immune signalling pathways. Overall, Cannabigerol and Curcuphenol seemed to enhance production of leukocyte migration cytokines. Most evidently, there was a notable increase of Fractalkine upon treatment with Cannabigerol (Figure 10A). Fractalkine CXC3CL1 is the only member in its chemokine subclass and has a membrane-bound and soluble form34. Fractalkine has a soluble and membrane-bound form. The membrane form functions as an adhesion molecule for leukocytes which can be induced by TNF alpha, IFN gamma, and IL-1; the soluble form is a chemoattractant for monocytes, NK cells, and T cells34. The drastic increase in Fractalkine (CXC3CL1) (Figure 10A) upon treatment with Cannabigerol gives evidence that Cannabigerol could change the tumour cytokine secretion profile, and as such the tumour niche, so that there would be an increase in NK cells, monocytes, and T cells. This is especially interesting given the high absolute number of NK cells that were present in the tumour for the Cannabigerol-treated cells (Figure 23). Other cytokines of importance to cancer that were upregulated upon the use of Cannabigerol included IL-28A, CCL22, FGF-21 (Figure 6C), and IL-33 (Figure 6A). IL-33 expression is associated with the upregulation of MHC class I and APM4 and is decreased during metastasis4. The ability of Cannabigerol to increase IL-33 alarmin would indicate another method by which tumours may be able to upregulate the APM and MHC Class I in the tumour environment, and another method by which Cannabigerol may mediate killing. Additionally, CCL22 is usually secreted in response to IFN gamma and TNF alpha or IL-435 and is associated 42 with the induction of chemotaxis of T cells by the binding of CCR4. The production of CCL22 by Cannabigerol-treated A9s could provide a method by which the T cells could be recruited to the site. Interestingly, there was downregulation of IL-6 (Figure 6D). IL-6 is associated with differentiation of naïve CD4+ cells against a specific antigen, and of differentiation of CD8+ naïve cells into CTLs36. Also of note, but to a lesser extent, the cannabigerol-treated A9 cells also appeared to produce IL1RN, an IL-1 antagonist37, which would be a negative regulator of inflammation. As well, there was increase an in VEGFA. VEGFA is a crucial gene for the formation of blood vessels and angiogenesis (VEGFA NIH)38, during which the new blood vessels supply nutrients to a tumour and increase tumorigenesis3. In the future, it might be interesting to look at whether there was increased angiogenesis within Cannabigerol treated mice. Finally, Fibroblast growth factor (FGF-21), which was increased by Cannabigerol is associated with favorable (less) cancer risk in several cancers, including colon, prostate, and breast, as well as higher T-Reg activity39, which indicates that the increase in FGF caused by Cannabigerol may reduce tumorigenesis or metastasis. IL-28 was increased in the supernatant of both Cannabigerol and Curcuphenol treated A9 cell cultures, but to a larger extent in Cannabigerol treated A9 culture. IL-28 is a type III IFN which is induced by innate immune cells. Wongthida et al.40 have found that the use of Vesicular stomatitis virus (VSV) to induce the production of IL-28 in the area of the tumour microenvironment was able to sensitize tumour cells to NK cells, and cause NK cells to recognize and activate40. It may be possible to use Cannabigerol or Curcuphenol to sensitize immune cells to a tumour, in hosts that are immune-competent. 43 There was a strong decrease of CCL2 upon treatment with Curcuphenol (Figure 10B) in the supernatant of the A9 cells relative to DMSO (vehicle) treated A9 cells, as well as a visible decrease in MMP (Matrix metallopeptidase) 2 and 3 and IGFBP (Insulin like growth factor binding protein). Matrix metalloproteinases are involved in the breakdown of the extracellular matrix in physiological processes such as angiogenesis and tissue remodeling41. MMP2, in particular, is known to promote breast cancer cell migration42 and is considered a breast cancer prognosis marker43. MMP2 is also associated with colorectal cancer invasion and growth41. It is possible that the Curcuphenol-induced reduction of the production of this cytokine is beneficial against metastasis by corrupting a tumour’s opportunity to migrate, based on MMP2’s known function. CCL2 is a controversial monocyte attracting chemokine. It was previously thought to denote host immunity against tumours, however, in the past few years, CCL2 has been considered to contribute to tumorigenesis. CCL2 has been found to be constitutively produced in many cancer types. Although there is infiltration of the monocytes to the site of a tumour, there are several complex interactions between monocytes, tumour cells, and many other cell types, including stromal cells, which overall contribute to a tumorigenic environment44. Curcuphenol greatly reduced the production of CCL2 from A9 tumour cells, suggesting a therapeutic benefit against tumours through the reduction of monocyte infiltration to the tumour site, and the subsequent consequences. Curcuphenol also induced an increase in inflammatory cytokine IL1B. OT1 mice have CD8+ T cells that are primed to bind siinfekl peptide, and allow us to determine the functionality of MHC Class I presentation. This OT1 CD8+ T cell assay demonstrates that there is functional MHC class I presentation to CTL cells (Figure 12,13). This will result in the proliferation of CTLs and the presumed killing of the MHC class I expressing cells, which are A9 cells in this case. The presence of A9 cells with upregulated Class I MHC in 44 the same co-culture as the CTLs shows the association of the increased MHC class I with the proliferation of the co-cultured CTLs, which is further confirmation that binding of MHC class I to CTLs is causing the increased proliferation. This is further evidence that in a tumour setting, the use of Cannabigerol may cause upregulation of MHC class I and cause the CTLs to successfully kill the tumour cells through their proliferation and activation. Figure 14a demonstrates that the A9 treated with the Curcuphenol, represented by the purple fluorescence, did not have an increase in class I MHC. Upon further investigation, these particular Curcuphenol-treated cells, seen in teal in figure 14b, have died. This suggests that the MHC class I expression in this particular sample is not representative. In one particular experiment, IFN gamma did not appear to upregulate MHC Class I in this particular experiment (Figure 12), however this is likely due to use of the final amount of IFN gamma within the aliquot being less potent. In the future, it would be pertinent to look at the granzyme B released by CD8+ T cells that have proliferated in the OT1 assay, as well as upregulation of FAS molecules, as perhaps these signal increased cell killing functionality. It would also be beneficial to look at perforin release during this assay. Perforin is associated with cytolytic CD8+ T cells, as well as NK cells. Loss of perforin has been associated with the increase of tumour growth45. IFN gamma works on CD8+ T cells to enhance killing46,47 and is also known to increase MHC class I. Previous studies have shown reduced tumour burden upon an increase in IFN gamma, and mice with an IFN gamma receptor knockout48,49showed a 10-20 times increase in tumour induction through Mucin-like Cancer associated Antigen (MCA). Similarly, mice with a knockout of the of the IFN gamma alpha subunit receptor 1 enhanced tumour burden, and experienced reduced immunogenicity of their tumours49. Finally, Rag 2-/- mice, which lack NKT, T, and B cells were reported to have a higher tumour burden14. This evidence appears to point to 45 the importance of adaptive immunity in tumour reduction, but more specifically of CD8+ killing via MHC class I tumour antigen presentation as an important method of immune surveillance. This is especially pertinent in cells prone to immune evasion through a genetic or epigenetic mutation and gives us reason to push forward in our studies of CTL-based cancer killing studies. A9 cells were injected subcutaneously in mice, and mice were treated 7 days later with either Cannabigerol, TSA, or Vehicle. Mouse weight and tumour volume appeared similar among Cannabigerol treated and Vehicle treated groups (Figure 16). There appeared to be no difference in the percentage of immune infiltrates in either the spleen or the tumour of Cannabigerol, TSA, or Vehicle treated (Figures 18-20 ), however there were much higher absolute numbers of lymphocytes in the Cannabigerol treated mouse tumours relative to the vehicle treated tumours (Figure 20). A high number of the spleen lymphocytes were alive during the time of FACS, and many almost all of these were MHC Class I positive (Figure 18). A9 cells will appear on FACS as a similar size and granularity as lymphocytes, and as such, they are difficult to separate based on size and shape alone. However, the high percentage of MHC class I present on all cells gated in the “lymphocyte” area means that there would likely be class I MHC on the A9 tumour cells present. Over half of the lymphocytes present in the spleen were B220 positive, which is a B cell marker. A high number of cells were MHC class II positive, with a small percentage of these cells presenting as CD11b and/or CD11c positive, insinuating that they may be dendritic cells (CD11c) or macrophages (CD11b), although further markers would be needed to ascertain their cell type. Finally, nearly 40% of live cells were CD3+ positive cells, with these being split between CD4+ slightly more heavily than CD8+ T cells. The CD8+ T cells that were present showed signs of activation (Figure 19B), with the CD62L and CD69 markers. NK cells represented about 5 percent of the spleen lymphocytes, with a very small percentage of 46 CD49+ cells being activated (Figure 19A). In the tumour, there was no difference between the treatment groups in infiltration, with low percentages of B220 positive cells (associated with B cells), CD3+ (T cells), and CD49+ (NK) cells. There was a high percentage of MHC II positive cells, especially MHCII CD11c+ positive cells, which are markers associated with dendritic cells. This is a positive sign, as it indicates that there might be MHCII positive cells, perhaps dendritic cells, that could be picking up debris from the area of the tumour and bringing it to the lymph node to professionally present to T cells. This could be the start of the adaptive immunity. There were also phagocytes (CD11b+) present in the tumours of all mouse treatments, which is indicative of future T cell recruitment, as phagocytes generate danger signals which will recruit the T cells to the region15. There were higher numbers of lymphocytes in the Cannabigerol-treated mouse tumours. Perhaps the tumour density was different due to Cannabigerol treatment, and as such there was more infiltration of lymphocytes overall in the tumour. Finally, there was found to be high absolute numbers of NK cells in the Cannabigerol-treated mouse group tumours. perhaps cytokines were produced in the tumour which caused higher recruitment of the NK cells or MHCII positive cells in that treatment, or perhaps these cells were the first to arrive at the scene. It is important to note that although a previous group showed a reduced tumour burden in nude mice upon the use of the same dose of Cannabigerol, it is possible that this is via direct killing29. In such a case, the Cannabigerol would be killing more cells than just the tumour cells. This reiterates the value of looking at the changing immune cell population within the drug-treated mice, as we are trying to find an immune-based therapeutic. We also looked at the effect of Cannabigerol on HDAC 8 and 10 activity. An possible increase in HDAC 8 and 10 mRNA upon A9 treatment Cannabigerol and Curcuphenol, as well 47 as MHC class I suggests that there is a relationship between the HDAC activity and the APM pathways (Figure 24), however the increase in mRNA is not very strong. It is interesting that both HDAC 8 and 10 are increased alongside class I MHC expression upon treatment with IFN gamma (Figure 24). This data is different than the data presented in Figure 22 and 23, which are the results of the HDAC knockdown of HDACs in TC1 primary cells. Knockdown of HDACs 5, 8, and 10 in TC1 primary cell line all showed an increase of MHC class I, when viewing external class I MHC via FACS (Figures 25 and 26). There are a few reasons that the A9 metastatic cell results may have deviated from the TC1 form. Remember, A9 cells have picked up many mutations that distinguish from the TC1 cells in the time that they have metastasized. The metastatic form A9 cell line may have mutated HDAC activity further downstream, and as such the increased HDAC activity might not necessarily reduce MHC class I in the metastatic cells. This would allow for the immune escape in the metastatic form. It is also possible that the A9 metastatic form has mutations in other pathways that lead to the upregulation of MHC class I. Another possibility is that Cannabigerol is acting on another molecule, such as IL-33 (which is known to increase class I MHC, and is reduced in metastatic form), which may lead to the increase MHC class I. Finally, although there is an increase in HDAC 8 and 10 mRNA, this may not necessarily correlate to activity increase: you may need other proteins present to enhance their activity, such as kinases. However, the PKA/cAMP is known to repress MHC class I transcription (in thyroid cells)50. cAMP/PKA is also known to lead to the phosphorylation of HDAC 5 at serine 520, preventing its export to the cytoplasm, and thereby its function51. Additionally, cAMP is known to cause the degradation of HDAC 852. Knowing this, I expected the HDAC knockdowns to be associated with lower class I MHC. It appears that although cAMP downregulates MHC I and 48 decreases HDAC 5 and 8 activities, HDAC 5, 8, and 10 are also repressors of class I MHC in TC1 primary cell lines. When these HDACs are knocked down, MHC is increased. There is an amino acid sequence homology of only about 68 amino acids between HDAC 5,8, and 1053. While there is high homology between the C terminus of the HDAC 5 and the N- terminus of HDAC 10, The homology does not contain the serine residue known to be phosphorylated in the serine 280 of HDAC 5 upon alignment. In my experiments, MHC class I expression is upregulated in HDAC 5, 8, and 10 knockdowns on primary tumour cells. This suggests that the HDACs are involved in repressing MHC class I through a hereby unknown pathway functioning in the tiny conserved region of these HDACs, or through different signalling pathways. The idea that HDACs may be functioning unique pathways may be supported by the abundance of different types of HDACs, their different placements within the cell, and the variable methods of catalytic function. However, in such a case, one would wonder how cancers would mutate to increase the function of several HDACs, in order to reduce the MHC class I during metastasis. Perhaps simply increasing the activity on a single HDAC is enough to reduce the MHC class I for immune escape. IFN gamma is an important cytokine involved in upregulation of class I MHC and used in several experiments as a positive control. It would be beneficial for therapeutic purposes if Cannabigerol showed a similar role to IFN gamma because IFN gamma is well established as a cytokine that is so pivotal to the recruitment of dendritic cells, macrophages, and NK cells14. Similar to IFN gamma, Cannabigerol shows promise in recruiting leukocytes to the area of the tumour. In conclusion, NK cell infiltration and activation were increased in tumours in response to Cannabigerol treatment. It is possible that this NK activity is increased in response to a 49 Cannabigerol-induced alteration in chemokine profile, in which A9s produce cytokines associated with a pro-inflammatory response and leucocyte migration. This altered chemokine profile may promote NK infiltration and activation. Additionally, the cytokine profiler data shows that Cannabigerol causes A9 tumour cells to produce cytokines that promote T cell migration. It is possible that the cytokines promote increased T cell migration to the tumour area in vivo, and that if the tumour trial had continued longer, there may have been an increase in CTL infiltration to the tumour, as well as increased CTL proliferation and killing of the tumour cells. This is suggested by the Cannabigerol-induced increase MHC class I on surface A9s, which was shown to occur in vitro, and the increased CD8+ T cell proliferation associated with the Cannabigerol-treated A9s ex-vivo. In the future, our lab will look at adaptive immunity during a similar Cannabigerol-treatment mouse tumour trial. To do so, we will use a reduced number of A9 cells during the initial tumour inoculation so that the mice live longer while there is a visible tumour present, before the tumour perfusion causes humane endpoint, as it oftentimes does. We may also decide to begin Cannabigerol treatment earlier in the trial, to give the Cannabigerol ample opportunity to elicit changes in immunity. These changes will help us determine if the adaptive immune response that we predict to be incurred by Cannabigerol, in reference to CTLs, would indeed occur. The information garnered from this trial, however, informs us of the possible mechanisms of Cannabigerol treated immunity and the earlier stages of tumour infiltration. The presence of NK and macrophages is associated with early stages of immunity, before the infiltration of CD8+ Cytolytic T cells15. This is beneficial in its own right so that we can get a better story of the steps in which there proceeds to be cancer reduction. In reference to the epigenetic changes incurred by the Cannabigerol treatment in relation to A9 and TC1 cells, there is still much to be done with HDAC 5,8, and 10. The immediate 50 results show that the knockdown of these HDACs might incur an increase of class I MHC, but not enough is known on HDACs to determine if perhaps these HDACs can compensate for one another. Indeed, HDAC 10 is simply HDAC5 with an additional pseudo repeat, which gives rise to the question of shared functionality. Future learning on HDAC 5, 8, and 10 is necessary, as the MHC class I pathway is vital to immune surveillance, and it is evident that the knockdown associated with these genes increase class I MHC. HDACs, after all, are epigenetic mechanisms, and like other epigenetic targets, these can be compensated for therapeutically to reduce their effect. The Chromatin Immunoprecipitation (ChIP) assay to determine acetylation status with A9 cells treated with Cannabigerol or Curcuphenol would give us more information about the acetylation status of the antigen presenting machinery genes under treatment. If indeed, there is too much deacetylation, and the reduction of this deacetylation benefits the induction of APM, it would be worthwhile to find drugs that reduce the HDAC activity, as Cannabigerol and may do, and which Curcuphenol is known to do for some HDACs26. Unfortunately, the quality of the samples was not achievable to surmise information from ChIP sequencing, and as such the sequencing results are inconclusive. Finally, it would be expected that since Cannabigerol and Curcuphenol seem to induce similar effects in the change in protein expression of class I MHC and TAP, that they may incur similar epigenetic changes on these genes as well. It is possible that these compounds may reduce the methylation that causes silencing on the MHC, and that this may reduce the production of MHC class I. This would play right into the concept named by Setaidi at al.17 that there is decreased acetylation on APM genes in A9 cells, as methylation is oftentimes doing the same thing of reducing gene transcription that decreased acetylation that would do. 51 The major caveat of this tumour trial was that it ended earlier than we had anticipated; it appeared that the A9 clone being used was much more aggressive than earlier A9 clones, and as such, there was a reduction in the length of the tumour trial and our opportunity to look at change in adaptive immunity, and less dosing of Cannabigerol than may have been necessary to reduce the tumour. We also took the adrenal gland, liver, and lungs, which are common targets of metastasis, and froze them. In the future, it would be beneficial to look at the histology, to determine if there were tumour or immune cell infiltrates in these areas which are frequently metastasized, and if this changed upon treatment with Cannabigerol. Unfortunately, there was no determining tumour marker by which we could check for metastasis or tumour specific upregulation of class I MHC. Our lab is working towards creating a stable A9 clone containing a GFP vector, with which a tumour could be tracked. Using this, we could look more easily at metastasis, as it would be possible to take several organs, such as the lymph nodes, adrenal glands, livers and lungs, and use FACS to determine how much of a metastatic tumour had metastasized to that area. This could be used with other immune cell markers, to determine if immune infiltration was happening in the areas in which the tumour cells metastasize concurrently. It would be possible to determine if the Cannabigerol decreased metastasis or increased immunity, especially in innate NK cells, and adaptive CTLs. It would be beneficial to look at perforin and granzyme B in the future to determine the CTL activation. In the future, it would also be beneficial to look at fractalkine secretion from tumours in mice, to determine if there was an increase in secretion in vivo. It would be prudent to knockdown the CB1R and CB2R to test if the Cannabigerol upregulation of MHC class I occurs despite the knockdowns. Since it is known that Cannabigerol works through these receptors, one would suspect that the Cannabigerol’s effect of decreasing MHC class I54. Finally, further 52 characterization of the NK cells using a FACS panel more specific to this cell type would be interesting to look at. After looking at the results received during this tumour trial, I would create the pane based on the length of the trial. If it were a short trial, I would further characterize innate cells, such as dendritic cells, monocytes/macrophages, and NK cells, or adaptive cells, pertinently CD4+ helper and CD8+ CTLs and their activation and homing markers if it were a longer trial. Keep in mind, the tumour only provides enough cells for a single panel on FACS, and as such you would have to choose your markers wisely. My project initially centred on investigating the HDACs inhibitors, and the acetylation status associated with cannabinoids as well as its mode of action. However, it became primarily concerned with the effect of cannabinoids in the upregulation of class I MHC to reverse the downregulation of class I MHC, and immune evasion, commonly associated with metastatic cancer. Determining the mechanism that causes reduction of immune surveillance through downregulation of APM is an important step towards using these compounds to enhance immune recognition of tumours. It will also provide us with protein targets that will help restore the original APM function, and possibly lead to therapeutics to minimize cancer metastasis. This knowledge could be considered for not only cancer therapy, but a greater understanding of immune surveillance will aid in vaccine development. 53 Bibliography 1. Canadian Cancer Statistics - Canada.ca. Available at: https://www.canada.ca/en/public-health/services/chronic-diseases/cancer/canadian-cancer-statistics.html. (Accessed: 11th December 2018) 2. Leading Causes of Death in Canada, 2009. Symbol of Statistics Canada (2009). Available at: https://www150.statcan.gc.ca/n1/pub/84-215-x/2012001/hl-fs-eng.htm. (Accessed: 11th December 2018) 3. Nguyen, D. X. & Massagué, J. Genetic determinants of cancer metastasis. Nat. Rev. Genet. 8, 341–352 (2007). 4. Saranchova, I. et al. Discovery of a Metastatic Immune Escape Mechanism Initiated by the Loss of Expression of the Tumour Biomarker Interleukin-33. Sci. Rep. 6, 30555 (2016). 5. Cooper, G. The Cell: A Molecular Approach. (Sinauer Associates, 2000). 6. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: The next generation. Cell 144, 646–674 (2011). 7. Jones, P. A. & Baylin, S. B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002). 8. Thomas, L. Discussion. Cell. Humoral Asp. Hypersensitive States H. S. Lawe, 529–532. (1959). 9. Fridman, W. H. Immuno-Oncology Immunoediting: Birth of Modern From Cancer Immune Surveillance to Cancer. (2018). doi:10.4049/jimmunol.1800827 10. Stutman, O. Tumor Development after 3-Methylcholanthrene in Immunologically Deficient Athymic. Source: Science, New Series 183, (1974). 54 11. Shankaran, V. et al. IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity. Nature 410, 1107–1111 (2001). 12. Alimonti, J. et al. TAP expression provides a general method for improving the recognition of malignant cells in vivo. Nat. Biotechnol. 18, 515–520 (2000). 13. Swann, J. B. & Smyth, M. J. Immune surveillance of tumors. J. Clin. Invest. 117, 1137–46 (2007). 14. Dunn, G. P., Bruce, A. T., Ikeda, H., Old, L. J. & Schreiber, R. D. Cancer immunoediting: from immunosurveillance to tumor escape. Nat. Immunol. 3, 991–998 (2002). 15. Andersen, M. H., Schrama, D., thor Straten, P. & Becker, J. C. Cytotoxic T Cells. J. Invest. Dermatol. 126, 32–41 (2006). 16. Beyersdorf, N., Kerkau, T. & Hünig, T. CD28 co-stimulation in T-cell homeostasis: a recent perspective. ImmunoTargets Ther. 4, 111–22 (2015). 17. Setiadi, A. F. et al. Epigenetic Control of the Immune Escape Mechanisms in Malignant Carcinomas. Mol. Cell. Biol. 27, 7886–7894 (2007). 18. Falkenberg, K. J. & Johnstone, R. W. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 13, 673–691 (2014). 19. Setiadi, A. F. et al. Epigenetic Enhancement of Antigen Processing and Presentation Promotes Immune Recognition of Tumors. Cancer Res. 68, 9601–9607 (2008). 20. Lucio-Eterovic, A. K. et al. Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas. BMC Cancer 8, 243 (2008). 21. Benedetti, R., Conte, M. & Altucci, L. Targeting Histone Deacetylases in Diseases: Where Are We? Antioxid. Redox Signal. 23, 99–126 (2015). 55 22. Richon, V. M. Cancer biology: mechanism of antitumour action of vorinostat (suberoylanilide hydroxamic acid), a novel histone deacetylase inhibitor. Br. J. Cancer 95, S2–S6 (2006). 23. Kim, H.-J. & Bae, S.-C. Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs. Am. J. Transl. Res. 3, 166–79 (2011). 24. Simmons, T. L., Andrianasolo, E., McPhail, K., Flatt, P. & Gerwick, W. H. Marine natural products as anticancer drugs. Mol. Cancer Ther. 4, 333–42 (2005). 25. Crier, C. Investigation of extract influence on antigen presentation in Nef-expressing dendritic cells and cancer cells. (University of Applied Sciences Mannheim, 2011). 26. Ellis, S. Induction of the antigen presentation machinery using novel small molecules. (University of British Columbia, 2017). 27. Ligresti, A. Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma. J. Pharmacol. Exp. Ther. 318, 1375–1387 (2006). 28. Deiana, S. et al. Plasma and brain pharmacokinetic profile of cannabidiol (CBD), cannabidivarine (CBDV), Δ9-tetrahydrocannabivarin (THCV) and cannabigerol (CBG) in rats and mice following oral and intraperitoneal administration and CBD action on obsessive-compulsive behavi. Psychopharmacology (Berl). 219, 859–873 (2012). 29. Romano, B. et al. Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol. Phytomedicine 21, 631–639 (2014). 30. Ladin, D. A., Soliman, E., Griffin, L. & Van Dross, R. Preclinical and Clinical Assessment of Cannabinoids as Anti-Cancer Agents. Front. Pharmacol. 7, 361 (2016). 31. Šmahel, M. et al. Immunisation with modified HPV16 E7 genes against mouse oncogenic 56 TC-1 cell sublines with downregulated expression of MHC class I molecules. Vaccine 21, 1125–1136 (2003). 32. Valdeolivas, S. et al. Neuroprotective Properties of Cannabigerol in Huntington’s Disease: Studies in R6/2 Mice and 3-Nitropropionate-lesioned Mice. Neurotherapeutics 12, 185–199 (2015). 33. Borrelli, F. et al. Colon carcinogenesis is inhibited by the TRPM8 antagonist cannabigerol, a Cannabis-derived non-psychotropic cannabinoid. Carcinogenesis 35, 2787–2797 (2014). 34. Jones, B. A., Beamer, M. & Ahmed, S. Fractalkine/CX3CL1: a potential new target for inflammatory diseases. Mol. Interv. 10, 263–70 (2010). 35. Lacy, P. Eosinophil Cytokines in Allergy. Cytokine Eff. Funct. Tissues 173–218 (2017). doi:10.1016/B978-0-12-804214-4.00011-7 36. Tanaka, T., Narazaki, M. & Kishimoto, T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol. 6, a016295 (2014). 37. Al-Moundhri, M. S. et al. Combined polymorphism analysis of glutathione S-transferase M1/G1 and interleukin-1B (IL-1B)/interleukin 1-receptor antagonist (IL-1RN) and gastric cancer risk in an Omani Arab Population. J. Clin. Gastroenterol. 43, 152–6 (2009). 38. VEGFA gene - Genetics Home Reference - NIH. Available at: https://ghr.nlm.nih.gov/gene/VEGFA. (Accessed: 28th November 2018) 39. McCarty, M. F. GCN2 and FGF21 are likely mediators of the protection from cancer, autoimmunity, obesity, and diabetes afforded by vegan diets. Med. Hypotheses 83, 365–371 (2014). 40. Wongthida, P. et al. Type III IFN Interleukin-28 Mediates the Antitumor Efficacy of 57 Oncolytic Virus VSV in Immune-Competent Mouse Models of Cancer. Cancer Res. 70, 4539–4549 (2010). 41. Dong, W. et al. Matrix metalloproteinase 2 promotes cell growth and invasion in colorectal cancer. Acta Biochim. Biophys. Sin. (Shanghai). 43, 840–848 (2011). 42. Xu, X. et al. Matrix metalloproteinase-2 contributes to cancer cell migration on collagen. Cancer Res. 65, 130–6 (2005). 43. Li, H., Qiu, Z., Li, F. & Wang, C. The relationship between MMP-2 and MMP-9 expression levels with breast cancer incidence and prognosis. Oncol. Lett. 14, 5865–5870 (2017). 44. Yoshimura, T. The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments. Cytokine 98, 71–78 (2017). 45. Russell, J. H. & Ley, T. J. L YMPHOCYTE -M EDIATED C YTOTOXICITY. Annu. Rev. Immunol. 20, 323–370 (2002). 46. Zhou, F. Molecular Mechanisms of IFN-γ to Up-Regulate MHC Class I Antigen Processing and Presentation. Int. Rev. Immunol. 28, 239–260 (2009). 47. Whitmire, J. K., Tan, J. T. & Whitton, J. L. Interferon-gamma acts directly on CD8+ T cells to increase their abundance during virus infection. J. Exp. Med. 201, 1053–9 (2005). 48. Kaplan, D. H. et al. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. U. S. A. 95, 7556–61 (1998). 49. Dighe, A. S., Richards, E., Old, L. J. & Schreiber, R. D. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors. Immunity 1, 447–56 (1994). 50. McCormack, M. et al. HLA-A*3101 and Carbamazepine-Induced Hypersensitivity 58 Reactions in Europeans. N Engl J Med 364, 1134–43 (2011). 51. Ha, C. H. et al. PKA phosphorylates histone deacetylase 5 and prevents its nuclear export, leading to the inhibition of gene transcription and cardiomyocyte hypertrophy. Proc. Natl. Acad. Sci. 107, 15467–15472 (2010). 52. Park, J.-Y. & Juhnn, Y.-S. cAMP signaling increases histone deacetylase 8 expression via the Epac2–Rap1A–Akt pathway in H1299 lung cancer cells. Exp. Mol. Med. 49, e297–e297 (2017). 53. Protein BLAST: search protein databases using a protein query. Available at: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. (Accessed: 17th May 2018) 54. Navarro, G. et al. Cannabigerol action at cannabinoid CB1 and CB2 receptors and at CB1-CB2 heteroreceptor complexes. Front. Pharmacol. 9, 1–14 (2018).
Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Use of small molecule activity in upregulation of Class I MHC and change in tumour cytokine profile in metastatic murine epithelial carcinoma.
Text