University of Wisconsin Milwaukee UWM Digital Commons Theses and Dissertations December 2016 Characterization and Use of Folate Receptor Isoforms for Targeting of Epithelial and Myeloid Cells Sreya Biswas University of Wisconsin-Milwaukee Follow this and additional works at: https://dc.uwm.edu/etd Part of the Biology Commons, and the Molecular Biology Commons Recommended Citation Biswas, Sreya, "Characterization and Use of Folate Receptor Isoforms for Targeting of Epithelial and Myeloid Cells" (2016). Theses and Dissertations. 1352. https://dc.uwm.edu/etd/1352 This Dissertation is brought to you for free and open access by UWM Digital Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UWM Digital Commons. For more information, please contact [email protected]. CHARACTERIZATION AND USE OF FOLATE RECEPTOR ISOFORMS FOR TARGETING OF EPITHELIAL AND MYELOID CELLS by Sreya Biswas A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biological Sciences at The University of Wisconsin-Milwaukee December 2016 ABSTRACT CHARACTERIZATION AND USE OF FOLATE RECEPTOR ISOFORMS FOR TARGETING OF EPITHELIAL AND MYELOID CELLS by Sreya Biswas The University of Wisconsin-Milwaukee, 2016 Under the Supervision of Professor Douglas A. Steeber Folate receptor (FR) is a GPI-anchored glycoprotein with high binding affinity for folic acid. FR has two membrane-associated isoforms, α and β, that are overexpressed on epithelial and myeloid tumors, respectively. Normal cells may also exhibit FR expression at very low levels but interestingly, FR-α on normal cells is restricted to the apical surface i.e., away from the blood stream. This differential expression and orientation of the FR-α isoform on tumor cells has been exploited to selectively target and deliver conjugates (e.g., drugs, nanoparticles, liposomes) to tumor cells without harming neighboring healthy cells. However, the functions and use of FR-β as a potential target have not been explored, and its functions on myeloid cells remain largely unknown. Therefore, we investigated the functions of FR-β to determine its potential as a target in myeloid malignancies using a human myelomonocytic leukemia cell line, U937. FR-α studies were conducted using a murine epithelial breast carcinoma cell line, 4T1, and tumors harvested from 4T1 tumor-bearing mice. The isoforms were found overexpressed on tumor cells and tissues, both in vitro and in vivo, with no expression observed on corresponding normal cells. Studies conducted using folic acid-fluorochrome conjugates to determine intracellular receptor fate indicated that FR-β was not internalized into cells unlike FR-α. However, both isoforms ii exhibited strong binding to folic acid conjugates (e.g., fluorochromes, nanoparticles) thus indicating that they could be selectively targeted using folic acid-dependent methods. We also determined the potential of a novel histone deacetylase (HDAC) inhibitor (HDACi) as an anti-cancer agent that could be used along with folic acid for achieving better selectivity in targeting tumor cells. Preliminary studies showed that Compound 5 (Cpd5) is stable with strong anti-proliferative activities against human tumor cells. Cpd5 was also able to reduce the rate of 4T1 tumor growth in mice without inducing systemic toxicity in the animals. In addition, Cpd5 exhibited desirable pharmacokinetic properties and showed direct effects on acetylation levels of histone proteins. These studies not only provide insights into the functional differences associated with FR-α and FR-β isoforms, but also highlight the potential of future targeting strategies utilizing these to target both epithelial and myeloid malignancies with improved selectivity. iii © Copyright by Sreya Biswas, 2016 All Rights Reserved iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ……………………………………………………………...…viii LIST OF FIGURES ………………………………………………………………….......…...ix LIST OF TABLES …………………………………………………………………................xi LIST OF ABBREVIATIONS ……………………………………………………………….xii CHAPTER 1--- GENERAL INTRODUCTION ……………………………………………...1 Figures and Figure Legends .………………………………………………………….36 CHAPTER 2--- FOLATE RECEPTOR ISOFORMS α AND β HAVE DISTINCT EXPRESSION PROFILES AND EXHIBIT DIFFERENTIAL ACTIVITY Abstract ……………………………………………………………………………….39 Introduction ………………………………………………………………………….. 41 Materials and Methods ………………………………………………………………..45 Results ………………………………………………………………………………...51 Discussions ……………………………………………………………………………56 Figures and Figure Legends …………………………………………………………..59 CHAPTER 3 --- FR-α and FR-β ISOFORMS EXHIBIT DIFFERENT INTRACELLULAR FATE Abstract ……………………………………………………………………………….71 Introduction …………………………………………………………………………...73 Materials and Methods ………………………………………………………………..76 Results ………………………………………………………………………………...85 Discussions ……………………………………………………………………………97 Figures and Figure Legends ……………………………………………………..…..103 CHAPTER 4--- DETERMINING THE POTENTIAL OF A NOVEL HISTONE DEACETYLASE INHIBITOR (HDACi) AS AN ANTI-CANCER AGENT Abstract ………………………………………………………………………….…..131 Introduction ……………………………………………………………………..…...133 Materials and Methods …………………………………………………………..…..136 Results …………………………………………………………………………..…...143 Discussions …………………………………………………………………………..147 Figures and Figure Legends …………………………………………………………150 CHAPTER 5--- CONCLUSIONS………………………………………………………......158 Figures and Figure Legends ………………………………………………………....165 v REFERENCES ……………………………………………………………………..………167 CURRICULUM VITAE ………………………………………………………………..….195 vi To my parents and my husband vii ACKNOWLEDGEMENTS I would sincerely like to express my heartfelt gratitude to Dr. Douglas Steeber for providing me with such a wonderful opportunity to work on this project. His guidance, persistence, and words of encouragement have been a constant source of inspiration for me, throughout my research. I have always tried to imbibe maximal knowledge on this subject through our scientific research discussions. I would also like to thank other members of my supervisory committee, Dr. Julie Oliver, Dr. Heather Owen, Dr. Mahmun Hossain and Dr. Reinhold Hutz for their valuable input. I thank Dr. Julie Oliver and her lab for sharing the tissue culture equipment, as well as Dr. Heather Owen for her help with the electron microscope. I would like to thank my lab members and the entire faculty, and staff in the department for their help and support in some way or the other during my doctoral research. Last but not the least, I would like to express my love and appreciation towards my parents, in-laws, and my husband for believing in my abilities, and for the endless support I received from them throughout my research work. viii LIST OF FIGURES Figure 1. Differential expression of FR-α in normal and cancer cells facilitates selective targeting…………………………………………………………………………………... 36 Figure 2. Receptor-mediated endocytosis of folic acid-conjugates ……………………... 37 Figure 3. FR-α expression on murine epithelial breast carcinoma cell line 4T1 ……….. 59 Figure 4. In vivo expression of FR-α on 4T1 tumor cells ……………………………….. 60 Figure. 5. Comparing FR-α expression in normal and tumor epithelial cells …………… 62 Figure 6. FR-β expression on U937 myeloid leukemia cell line ………………………… 63 Figure 7. FR-β expression on the Jurkat T-cell leukemia cell line………………………. 64 Figure 8. Comparing FR-β expression on normal peripheral blood monocytes to that of the U937 myelomonocytic leukemia cell line ……………………………………………. 65 Figure 9. FR-α and FR-β expression patterns on 4T1 and U937 cells …………………... 67 Figure 10. High-resolution imaging by confocal microscopy comparing FR-α and FR-β staining patterns ………………………………………………………………………….. 67 Figure 11. Effect of exogenous folate on FR expression ………………………………... 68 Figure 12. Comparing FR-α and FR-β activity in 4T1 and U937 cells ………………… 69 Figure 13. Synthesis of folic acid-conjugated fluorescent probes ……………………. 103 Figure 14. UV-Vis analysis of the BSA-FITC, BSA-TRITC, FA-BSA-FITC and FA- BSA-TRITC probes ……………………………………………………………………… 104 Figure 15. FA-BSA-FITC probe showed specific binding to FR-α in 4T1 cells ………... 105 Figure 16. FA-BSA-FITC probe showed specific binding to FR-β in U937 cells ……… 107 Figure 17. Comparing FR-α and FR-β internalization by high-resolution confocal microscopy ……………………………………………………………………………….. 110 Figure 18. The FA-BSA-TRITC probe showed strong staining on tumor sections harvested from 4-week 4T1 tumor-bearing mice ……………………………………........ 111 Figure 19. Free folic acid did not compete with the FA-BSA-FITC probe for binding to the FR-α isoform………………………………………………………………………….. 112 ix Figure 20. The FA-BSA-TRITC probe showed specific binding to the FR-β receptors on myeloid-derived suppressor cells (MDSCs) ………………………………………….. 113 Figure 21. The FA-BSA-Au18 conjugates showed specific binding to FR-α receptors on 4T1 cells ………………………………………………………………………………….. 117 Figure 22. The FA-BSA-Au18 conjugates were localized to spread areas of U937 cells ... 118 Figure 23. Comparing FR-α and transferrin internalization pathways ………………….. 120 Figure 24. Comparing FR-α internalization with dextran pinocytosis …………………... 121 Figure 25. FR-α binding activity was inhibited by cholesterol depletion ……………….. 122 Figure 26. Transferrin-mediated endocytosis was largely unaffected by cholesterol depletion in both 4T1 and U937 cells ……………………………………………………. 124 Figure 27. Pinocytosis in 4T1 and U937 cells was unaffected by cholesterol depletion ... 126 Figure 28. Cholesterol depletion inhibited FA-BSA-TRITC probe