Study of Cancer Related Proteins: Lrg-1 and Pd-L1

STUDY OF CANCER RELATED PROTEINS:

LRG-1 AND PD-L1

QIAOYUN ZHENG

Bachelor of Biological Pharmacy Beijing University of Chinese Medicine July 2009

Master of Science in Microbial and Biochemical Pharmacy Beijing University of Chinese Medicine July 2011

Submitted in partial fulfillment of requirements for the degree DOCTOR OF PHIOLOSOPHY IN CLINICAL-BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY May 2017

We hereby approve this dissertation for Qiaoyun Zheng Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for The Department of Chemistry And the CLEVELAND STATE UNIVERSITY College of Graduate Studies

. Dissertation Chairperson, Aimin Zhou, Ph.D.

. Department & Date

. Michael Kalafatis, Ph.D.

. Department & Date

. Xue-Long Sun, Ph.D.

. Department & Date

. Nolan Holland, Ph.D.

. Department & Date

. Baochuan Guo, Ph.D.

. Department & Date

Date of Defense: May 1st, 2017

ACKNOWLEDGEMENTS

I owe my gratitude to all those people who have helped me to accomplish this dissertation. I am grateful for their time and efforts that have contributed to my professional growth and education throughout my years as a graduate student.

First and foremost, I would like to give my utmost gratitude to my advisor, Dr. Aimin

Zhou for his guidance, support, encouragement, understanding and kindness that helped me complete this research. I have always admired Dr. Zhou’s extraordinary diligence, accountability, enthusiasm, friendliness and patience and he has inspired me in so many ways. His enduring enthusiasm ans rigorousness in scientific research will always inspire me throughout my life careers. Without his guidance and persistent support, this dissertation would not have been possible.

I would like to thank my committee members, Dr. Michael Kalafatis, Dr. Xue-long

Sun, Dr, Baochuan Guo, Dr. Ge Jin, and Dr. Nolan Holland, for their insightful comments and suggestions and valuable support for my projects and future career. I also would like to thank Dr. Bin Su for his help, support, advice, encouragement, understanding, kindness and patience. I admire Dr. Su’s helpfulness, outgoingness and care. He has been a devoted teacher, and a very pleasant person.

I would like to thank all past and current lab members in Dr. Zhou’s lab for their support and suggestions in my experiments and daily life. I really appreciate the harmonious environment we build in the lab.

Last but not the least, I would like to give my sincere acknowledgment to my family members, especially my twin sister, Qiaoxia Zheng. Their continuous encouragement, support and love are always the driving force for me to stay calm and work hard.

STUDY OF CANCER RELATED PROTEINS: LRG-1 AND PD-L1

QIAOYUN ZHENG

ABSTRACT

PROJECT I:

In this study, we used the proteomic method to identify a potent biomarker candidate as leucine-rich-α-2-glycoprotein-1 (LRG-1) for cancer from the urine of patients with hepatocellular carcinoma. Further screening revealed that LRG-1 was also present in the urine of patients with a wide range of cancer types and inflammation. We found that LRG-

1 could be secreted outside cells as a glycosylated form. To characterize LRG-1, we systematically studied the structure and function of this protein by using chemical N-link and/or O-link glycosylation inhibitors and site-directed mutagenesis, and showed that glycosylation of LRG-1 was able to prevent its degradation by a protease although its secretion was independent on glycosylation. Interestingly, cells deficient LRG-1 migrated faster than wild types cells and the expression of Cyclooxygenase-2 (COX-2) was down regulated in T cells after incubated with the glycosylated form of LRG-1, suggesting a role of LRG-1 in anti-inflammation. Our findings provide very useful information for developing LRG-1 as a noninvasive urinary biomarker for diagnosis of cancer and inflammatory diseases.

PROJECT II:

Programmed death-ligand 1 (PD-L1) on cancer cells interacts with programmed cell death protein 1(PD1) of immune cells, resulting in suppression of anti-tumor immunity. Various factors contribute to the high expression of PD-L1 in cancer cells and lead to the escaping from the immune system. Targeting the PD-L1 and PD1 pathway is a novel strategy to restore the immunity and re-activate the immune system to abolish the tumor. Currently, the main research focusing on the pathway uses antibody drugs to block the interaction between PD-L1 and PD1. There is yet no any research effort focusing on blockade of the

PD-L1 expression in cancer cells. Small molecule drugs that can downregulate the PD-L1 expression in cancer tissue have multiple advantages such as penetration of the blood brain barrier compared to antibody drugs. To search for such type of potential lead compounds, a luciferase-based reporter assay was designed, developed, and validated to identify small molecules that can manipulate the expression of PD-L1 in cancer cells. By screening 1680 small molecule compounds, several of them were found to affect the PD-L1 expression in cancer cells.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... iii

ABSTRACT ...... iv

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

ABBREVIATIONS ...... xiii

CHAPTER I ...... 1

PROJECT I. STUDY OF LEUCINE-RICH-A-2-GLYCOPROTEIN-1(LRG-1)

GLYCOSYLATION AND ITS BIOLOGICAL FUNCTION ...... 1

A1. Introduction ...... 1

A1.1 Cancer ...... 1

A1.2 Liver cancer...... 5

A1.3 Metastasis ...... 6

A1.4 Tumor biomarkers ...... 9

A1.5 LRG-1 ...... 11

A1.6 Glycosylation ...... 14

A2. Reagents and methods ...... 21

A2.1 Animal ...... 21

A2.2 Cell lines and cell culture ...... 21

A2.3 Western blot and antibodies ...... 23

A2.4 Urinary Excretion of LRG-1 in the Patients with Cancer ...... 25

A2.5 Immunofluorescence and Confocal Microscopy ...... 25

A2.6 LRG-1 gene knockout ...... 26

A2.7 Cell migration and wound healing study ...... 26

A2.8 PARP cleavage study ...... 27

A2.9 LRG-1 mutant construction ...... 28

A2.10 Effects of BZGalNac and Tunicamycin on LRG-1 secretion ...... 29

A2.11 Glycosidase digestion ...... 30

A2.12 Glyco-Protein Stain ...... 30

A2.13 Effects of LPS on LRG-1 secretion ...... 31

A2.14. Effect of LRG-1 on the Expression of COX-2 ...... 31

A2.15 Effect of LRG-1 on the expression of small GPTases ...... 31

A2.16 Quantification and Statistical Analysis ...... 32

A3. Results ...... 32

A3.1 LRG-1 is excreted in the urine of patients with cancers ...... 32

A3.2 LRG-1 is localized on ER ...... 35

A3.3 Distribution of LRG-1 in Mouse Organs ...... 37

A3.4 Expression of LRG-1 in different cancer cells ...... 39

vii

A3.5 Secreted LRG-1 is glycosylated, but not the intracellular form ...... 44

A3.6 Effect of glycosylation on LRG-1 secretion ...... 46

A3.7 Effect of glycosylation on LRG-1 stability ...... 49

A3.8 LRG-1 is associated with inflammation...... 52

A3.9 LRG-1 secretion is not the result of LPS stimulation ...... 54

A3.10 Effect of LRG-1 on the expression of COX-2 ...... 56

A3.11 lack of LRG-1 promotes cell migration ...... 59

A3.12 Cells defect LRG-1 Are more sensitive to an anti-cancer drug ...... 62

A3.13 Effect of LRG-1 on the expression of small GTPases ...... 64

A4. Discussion ...... 66

A5. Reference ...... 71

CHAPTER II ...... 83

PROJECT II. DEVELOPMENT AND VALIDATION OF A LUCIFERASE REPORTER

ASSAY TO IDENTIFY SMALL MOLECULES REGULATING PD-L1 EXPRESSION

IN CANCER CELLS ...... 83

B1. Introduction ...... 83

B1.1 PD-L1 ...... 83

B1.2 PD-L1 and cancer treatment strategy...... 84

B1.3 Small molecule drugs ...... 86

B2. Reagents and methods ...... 86

viii

B2.1 Compound libraries and other reagents ...... 86

B2.2 Cell lines ...... 87

B2.3 Western Blotting ...... 90

B2.4 Construction of the plasmid ...... 90

B2.5 Cell transfection ...... 91

B2.6 RT-PCR ...... 91

B2.7 96-Well Luciferase Assays ...... 92

B2.8 Cell viability analysis ...... 93

B3. Results ...... 94

B3.1 Identification of the suitable cancer cell lines for the transfection ...... 94

B3.2 Cell transfection and validation of the identified colonies ...... 96

B3.3 Pilot screening of the two compound libraries ...... 101

B3.4 The identified compounds affect PD-L1 level in cancer cell lines...... 108

B4. Discussion ...... 111

B5. Reference ...... 112

LIST OF TABLES

Table 1. Cell lines used in project 1…………………………………………………..…...22

Table 2. Antibodies used in project 1……………………………………………………24

Table 3. Cell lines used in project 2……………………………………………………...88

Table 4. Z factor of all the plates in the screening assay………………………….……...102

Table 5. The structures of the five identified compounds………………………….……107

LIST OF FIGURES

Figure 1-1. Percent distribution of the 10 leading causes of death, by sex: United States,

2014……………………………………………………………………………………….4

Figure 1-2. Five classes of glycosylation…………………………………….……..……15

Figure 1-3. Principles and steps of N-glycosylation……………………………………..17

Figure 1-4. Biosynthesis of mucin-type O-glycans……………………………………...20

Figure 2-1. LRG-1 pCMV / hygro – His plasmid map……………………………...... …28

Figure 3-1. The excretion level of LRG-1 in the urine of patients with certain cancer….34

Figure 3-2. Subcellular localization of LRG-1 in the cell……………………………….36

Figure 3-3. Distribution of LRG-1 in Mouse Organs……………………………………38

Figure 3-4. The levels of LRG-1 in cell lysate and conditional medium………………..41

Figure 3-5. Comparison of the LRG-1 level normal and cancerous cells……...... 43

Figure 3-6. Glycosylation of LRG-1 in cells…………………………………………….45

Figure 3-7. Effect of Glycosylation on the secretion of LRG-1………………………....47

Figure 3-8. LRG-1 Stability Study………………………………..……………………..51

Figure 3-9. LRG-1 is associated with inflammation…………..………………….……...53

Figure 3-10. LRG-1 Secretion is not the Result of LPS Stimulation……………..……...55

Figure 3-11. Effect of LRG-1 on the Expression of COX-2……………………………..57

Figure 3-12. LRG-1 impacts cell migration……………………………………………...61

Figure 3-13. LRG-1 impacts PARP cleavage ………………………………………..….63

Figure 3-14. Effect of LRG-1 on the expression of small GPTases…………………..…65

Figure 4-1. Expression of PD-L1 in various cancer cell lines…………………………...95

Figure 4-2. Schematic representation of the hygromycin resistant luciferase reporter plasmid,pGL4.26.luc2.minP.hygro…………………………..…………………….……99

Figure 4-3. Validation of the transfected Hey1B cells………………………………....100

Figure 4-4. High throughput screening assay results in the identification of several compounds that affect the luciferase activity………………………………….……...... 106

Figure 4-5. The identified five compounds including Brefeldin A (BFA, 1µM),

xii

ABBREVIATIONS

LRG-1 leucine-rich-a-2-glycoprotein 1

HCC hepatocellular carcinoma

HBV Hepatitis B virus

AFP alpha-fetoprotein

TGF-β Transforming growth factor beta

ASN asparagine

ECM cell-extracellular matrix

EMT epithelial-mesenchymal-transition

CRP C-reactive protein

ER endoplasmic reticulum

OST oligosaccharyltransferase

TGN Trans Golgi Network cDNA complementary deoxyribonucleic acid mRNA messenger ribonucleic acid rRNA ribosomal ribonucleic acid

FBS fetal bovine serum

FDA U.S. Food and Drug Administration

IFN interferon

LPS lipopolysaccharide

RNA ribonucleic acid

PBS phosphate buffer saline

xiii

RT-PCR reverse transcription PCR

SDS sodium dodecyl sulphate

PAGE polyacrylamide gel electrophoresis

BZGalNac Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside

PARP poly (ADP-ribose) polymerase

PD1 programmed cell death protein

PD-L1 programmed death-ligand 1

NFATc1 nuclear factor of activated T cells c1

IFN-γ interferon-γ

Th1 T helper cell type 1

PI3K phosphoinositide 3-kinase

STAT3 signal transducer and activator of transcription 3

BFA Brefeldin A

MTX methotrexate

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H- tetrazolium bromide

DMSO Dimethyl sulfoxide

xiv

CHAPTER I

PROJECT I. STUDY OF LEUCINE-RICH-A-2-GLYCOPROTEIN-

1(LRG-1) GLYCOSYLATION AND ITS BIOLOGICAL FUNCTION

A1. Introduction

A1.1 Cancer

Cancer is a class of diseases that result from uncontrolled growth and spread of abnormal cells. Uncontrolled spreading of cancerous cells leads to morbidity and mortality. There are over 100 types of cancers affect humans [1]. Based on the data cited from the Centers for

Disease Control and Prevention (CDC), cancer is the second leading cause of death worldwide [2]. Each year in the United States, more than 1.5 million people are diagnosed with cancer and more than 500,000 die from this disease.

In the war on cancer，there are several schools of thought on the role of extrinsic and intrinsic factors in cancer etiology. The first stems from Nobel Prize winning German biochemist Otto Warburg, who first proposed the metabolic theory of cancer in 1924. The metabolic theory of cancer [3] contends that cancer begins with damage to the mitochondria.

The cell is then forced to shift energy production to non-oxidative metabolism

(fermentation) in order to survive. An essential discovery of this theory is one feature of cancer, damaged mitochondria and increased fermentation, which is present in all cancer types. Another is the fact that the greater the degree of fermentation displayed by a given cancer, the more aggressive the cancer is. Because mitochondria in a cancer cell are damaged and therefore are forced to generate energy by such an inefficient pathway, they have to consume much more glucose to remain viable. Seyfried further remarkably improved this theory by using nuclear transfer studies [4]. Another school of thought was pioneered by Bert Vogelstein and Cristian Tomasetti of Johns Hopkins University, who have put forth a mathematical analysis of the genesis of cancer that suggests many cases are not preventable [5 ] . Drawing on the published literature, Tomasetti and Vogelstein reasoned that the tissues that host the greatest number of stem cell divisions are those most vulnerable to cancer. Cancer is just “bad luck”. This theory assumes that about 70% of cancer variability is attributed to random errors arising during DNA replication in normal, noncancerous stem cells, i.e. to internal factors, which is impossible either to expect or to prevent [6]. A contrasting theory was put forth by Wu Song, who explained that intrinsic risk factors contribute only modestly (less than 10-30% of lifetime risk) to cancer development [7]. Song and his colleagues first demonstrated that the correlation between

2 stem-cell division and cancer risk does not distinguish between the effects of intrinsic and extrinsic factors. They showed that intrinsic risk is better estimated by the lower bound risk controlling for total stem-cell divisions. Finally, they showed that the rates of endogenous mutation accumulation by intrinsic processes are not sufficient to account for the observed cancer risks. Collectively, Song and his colleagues conclude that cancer risk is heavily influenced by extrinsic factors [7]. Significant research showed that more than half of cancer deaths could be prevented through healthy choices, vaccinations, and screening [8]. Despite which opinion researchers hold on cancer etiology, human beings still have a long way to go in the battle with cancer.

Figure 1-1. Percent distribution of the 10 leading causes of death, by sex: United States, 2014[9]

A1.2 Liver cancer

Liver cancer is the fifth most common cancer in men, the eighth most common cancer in women, and the 10th most common cancer worldwide. Further, liver cancer is a leading cause of cancer deaths worldwide. The general 5-year survival rate of liver cancer is only

18% [10]. More than 700,000 people are diagnosed with liver cancer and more than 600,000 deaths occur each year throughout the world [11]. From 2008 to 2012, liver cancer incidence increased an average of 2.3% per year overall, and the liver cancer-related death rate increased by an average of 2.8% per year among men and 3.4% per year among women.

In 2013, 21,143 men and 8,330 women were diagnosed with liver cancer, while 16,300 men and 7,732 women died from liver cancer in the United States alone. When compared with the United States, liver cancer is much more common in sub-Saharan Africa and

Southeast Asia. Among men, Asian/Pacific Islander men have the highest rates of getting liver cancer (19.1 per 100,000 men), followed by Hispanic men (19.0), black men (17.0),

American Indian/Alaska Native men (12.8), and white men (10.8). Among women,

Hispanic women have the highest rates of getting liver cancer (7.5 per 100,000 women), followed by Asian/Pacific Islander women (6.8), American Indian/Alaska Native women

(6.1), black women (5.3), and white women (3.9) [12].

Hepatocellular carcinoma (HCC) or malignant hepatoma is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection, such as hepatitis B or C, or alcoholic cirrhosis [13]. Although HBV infection represents the main cause of HCC worldwide, chronic alcohol consumption-related HCC is frequent and generally increasing [14]. Another big resource of liver cancer is metastasis of cancer from

5 elsewhere in the body, typically, cancerous colon, breast, or lung. Up to 70% of people with colorectal cancer eventually develop liver metastases [15] due to the direct blood supply from the portal vein. In the United States and Europe, secondary liver cancer (Liver metastases) is much more common than primary liver cancer.

Treatment options of HCC and prognosis are dependent on many factors, most importantly on the tumor size and staging. HCC prognosis is usually poor because only 10-20% of hepatocellular carcinomas can be removed completely by surgery. Chemotherapy and radiation treatments are not usually effective; however, they may be able to shrink large tumors in an effort to increase surgical success. Aggressive surgery or a liver transplant may successfully treat small or slow-growing tumors if they are diagnosed early. However, few patients are diagnosed early. If hepatocellular carcinomas cannot be completely removed by surgical methods, fatality usually results within 3-6 months.

Accordingly, the need still exits for additional diagnostic tests for estimating an individual’s risk of developing cancer. Diagnostic tests which evaluate validated clinical indicators independently of traditional cancer screening methods and risk factors, such as alcoholism in the case of HCC, are especially desirable.

A1.3 Metastasis

One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, which can then invade adjoining parts of the body and spread to other organs. This process is referred to as metastasis. The liver, lungs, lymph nodes, and bones are common areas of spread or metastasis.

Metastasis is responsible for as much as 90% of cancer-associated mortality [16]. In most cases, cancer patients with localized tumors have significantly better prognoses than those with disseminated tumors. Therefore, metastasis is of great importance to the clinical management of cancer rather than the primary tumor[17]. Metastatic relapse invariably portends a poor prognosis, as metastatic outgrowths become rapidly recalcitrant to pharmacological treatment, seed additional metastatic colonies, and eventually compromise the function of vital organs. Recent evidence suggests that the first stage of metastasis can be an early event[18] and that 60% to 70% of patients have initiated the metastatic process by the time of diagnosis [19]. Therefore, an improved understanding of the factors leading to tumor dissemination is of vital importance.

Metastasis is traditionally viewed as a linear series of discrete events or steps, collectively referred to as the invasion-metastasis cascade[20]. The ﬁrst step starts when cancer cells at the primary site of the tumor are dissociated from one another or from adjacent normal cells, induce partial degradation of the underlying basement membrane, and penetrate into the underlying interstitial matrix which is known as invasion. In the second step, tumor cells foster the development of a tumor vasculature in order to sculpt a permissive microenvironment, enter the microvasculature of the lymph and blood systems and disseminate through the bloodstream. The final step, while in the microcirculatory system of the target organ and inﬁltrating its stroma (extravasation), cancer cells adopt various strategies to survive and eventually outgrow into macroscopic lesions colonizing a secondary tumor (colonization) [21 ]. Recent studies have shed significant light on the

7 molecular mechanisms governing the invasion and dissemination phases of metastasis [22,

23]. A number of models have been proposed to explain the biological complexities of metastasis and the molecular mechanisms regulating tumor cell migration [24]. For example, in the fusion model, it is believed that a tumor cell must acquire certain characteristics of lymphoid cells to gain a fully metastatic phenotype which is achieved by nuclear transduction with cells of myeloid origin. A characteristic of malignancy is the presence of tumor DNA in the bloodstream, thus in the gene transfer model, it is elaborated that tumor

DNA which carries the somatic mutations associated with neoplasia, is taken to the secondary site. Subsequently, stem cells absorb the tumor DNA at the distant organ, which endow the stem cell with malignant properties. The Genetic predisposition model also sets forth its position on the mechanism of metastasis. The metastatic potential of any primary tumor is altered by the genetic background upon which it arises. It implicates that an individual will be more or less susceptible to tumor dissemination as a consequence of constitutional polymorphism. Such germ line variations influence all aspects of the metastatic cascade, including the expression of pro-metastatic gene expression signatures within the primary tumor. An important implication of the effect of genetic background is not only impacting on the primary tumor, but all of the tissues of the body. This would also potentially play a role in establishment of the microenvironment of both primary and metastatic tumor cells [24].

Although investigation of cancer cell metastasis has achieved a great progression in recent years, the molecular mechanisms underlying the invasive capacity of tumor cells and their ability to breach the endothelial cell barrier remains to be fully understood [25].

A1.4 Tumor biomarkers

A tumor marker is a substance produced by cancer, or by the host in response to cancer, or certain benign conditions, which is used to differentiate a tumor from a normal tissue [26].

These substances can be found in the blood, urine, stool, other bodily fluids, or tissues.

Tumor markers may be diagnostic of a tumor or reflective of tumor activity or volume.

Although an elevated level of a tumor marker may suggest the presence of cancer, this alone is not enough to diagnose cancer. Measurements of tumor markers are usually combined with other tests to diagnose cancer (e.g. biopsies). In some types of cancer, the level of a tumor marker reflects the stage of the disease and/or the patient’s prognosis. A decrease in the level of a tumor marker or a return to the marker’s normal level may indicate that the cancer is responding to treatment, whereas no change or an increase may indicate that the cancer is not responding. Tumor markers may also be measured after treatment has ended to check for recurrence.

An ideal tumor marker has some characteristics, such as suiting for a specific type of cancer, sensitive enough to detect small tumors, sufficient lead-time for alteration of natural course of disease, levels correlate with tumor burden, short half-life, simple and cheap test, and easily attainable specimens. In reality, this kind of tumor marker does not exist. While tumor marker tests can provide very useful information, they do have common limitations: many tumor markers may also be elevated in persons with diseases other than cancer; some tumor markers are specific for a particular type of cancer, while others are seen in several different types of cancer; not every person with a particular type of cancer will have an elevated level of the corresponding tumor marker; and not every cancer has a tumor marker

9 that has been identified as associated with it. Tumor markers alone are not diagnostic for cancer but can provide additional information (for some types of cancer) that can be considered in conjunction with a patient's medical history and physical exam as well as other laboratory and/or imaging tests.

Alpha-fetoprotein (AFP) is used to help diagnose liver cancer and follow response to treatment. AFP is a glycoprotein that is produced in early fetal life by the liver. Neonates have markedly elevated AFP levels (>100,000 ng/mL) that rapidly fall to below 100 ng/mL by 150 days and gradually return to normal over their first year [ 27 ] . AFP is usually undetectable in the blood of healthy adult men or women. AFP also can be produced by several tumors including hepatocellular carcinoma, hepatoblastoma, and nonseminomatous germ cell tumors of the ovary and testis. A number of studies report that approximately 70% of patients with hepatocellular carcinoma have elevated AFP concentrations.

However, elevated AFP concentrations are not only found in patient with HCC but also in patients with nonseminomatous testicular tumors, gastrointestin tumors, active liver disease and pregnancy. Thus, AFP has to be used together with iconography and pathology detection for early diagnosis of liver cancer in the clinical setting.

The success rate of therapeutic treatment for cancer can be greatly improved if the disease is diagnosed at an early stage. In general, cancer is diagnosed based on the symptoms, the results of a physical examination, and screening tests for the validated clinical indicators

(biomarkers) in the blood. Currently modern techniques such as mammograms, colonoscopy, and computed tomography are widely used for early cancer diagnosis [28].

However, these diagnostic methods are almost all site specific, invasive and quite expensive.

In recent years, noninvasive tests that detect the metabolites and excreted molecules in body wastes have shown the potential to be a simple, cheap and a reliable method for cancer diagnosis [29 ] . For example, cytology, growth factors and other molecules presented in sputum samples are used for diagnosis of lung cancer [30, 31]; a number of genetic, epigenetic or protein markers in stool have been identified for detecting colorectal cancer [32, 33]; and metabolites and macromolecules excreted in urine are interesting targets for prostate, renal and bladder cancers [34- 36].

A1.5 LRG-1

Leucine rich α-2-glycoprotein-1 (LRG-1, GeneBank accession number: NP_443204) is a serum glycoprotein in a size of about 36 kDa with 312 amino acids in which 66 of them are leucine and its functions are largely unknown. The secreted form of LRG-1 contains one attached galactosamine-based and four attached glucosamine-based oligosaccharides, and shows a molecular weight about 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [ 37 ]. The LRG-1-modified glycoprotein contains two intrachain disulfide bonds, and has a normal plasma concentration of 21-50 μg/mL[38 ] .

LRG-1 was first identified as a trace protein in human serum [39]. The function of LRG-1 remains largely unknown, but it is believed that LRG-1, a membrane-associated LRR

11 family member, may play a role in cell adhesion[40], cell migration [41] and regulating of glucan synthesis [42]. Other reports have shown that LRG-1 may also be involved in the

TGF-βR II signaling pathway[43]. The binding of serum LRG-1 to cytochrome c suggests that the protein plays a role in cell survival and apoptosis[44]. Most recently it has been found that LRG-1 promotes angiogenesis by directly binding to the TGF-β accessory receptor endoglin to activate the Smad1/5/8 signaling pathway [ 45 ]. Furthermore, the leucine-rich repeats and overexpression of LRG-1 in high-endothelial venules implicate the potential cell migration function of LRG-1 [46, 47]. Interestingly, LRG-1 is found to be at a significantly high level in sera and urine of certain patients, which has shown a promise as a biomarker for certain diseases based on qualitative assessments.

Fujimoto and Serada [48] illustrate that LRG was more useful than other biomarkers in discriminating between active and inactive diseases in human Rheumatoid Arthritis (RA) under conditions of IL-6 inhibition and in detecting joint inflammation in experimental arthritis. LRG-1 may serve as a convenient biomarker for RA disease activity during IL-6 blockade treatment [48]. Analyzing coronary sinus serum from patients with asymptomatic hypertension by using 2-dimensional difference gel electrophoresis and mass spectrometry,

Watson CJ, et al., demonstrated that LRG-1 could be a stronger marker of heart failure than

B-type natriuretic peptide (BNP) and this is independent of age, sex, creatinine, ischemia,

β-blocker therapy, and BNP [49]. Kentsis and Anupam [50, 51] reported that serum/urine LRG-

1 was elevated in pediatric appendicitis. Increased LRG-1 expression has been demonstrated by many researchers in several cancer types, such as ovarian cancer[52], non- small cell lung cancer[53-55], bladder cancer[56-58], biliary tract cancer[59], colorectal cancer[60],

12 pancreatic cancer[61], hepatocellular carcinoma (HCC) [62], etc. LRG-1 has been implicated as a serum and/or plasma biomarker for diagnosis [63, 64]. Its expression level and prognostic value have been revealed in endometrial carcinoma [65].

A1.6 Glycosylation

Post-translational modifications of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Glycosylation is a form of co- translational and post-translational modification, which serves a variety of structural and functional roles in membrane and secreted proteins [66]. Glycosylation plays important roles in protein’s folding, stabilities [67], cell-cell adhesion [58], optimizing glycoprotein-based drugs, underpins the ABO blood group system [68], immune recognition [69], etc. There are five classes of glycans, which are identified in Figure. 1-2 below:

Figure 1-2. Five classes of glycosylation. 1) N-linked glycans which attached to nitrogen of asparagine or arginine side-chains. N-glycosylation takes place during translation of target proteins by addition of glycan structures to the amino group of asparagine (ASN) residues at the consensus motif asparagine-X-serine/threonine (NXS/T) in which X is any amino acid except proline. 2) O-linked glycans which attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side- chains, or to oxygens on lipids such as ceramide. 3) Glypiation, which is the addition of a

GPI anchor that links proteins to lipids through glycan linkages. 4) C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain.

5) Phospho-glycans which linked through the phosphate of a phospho-serine.

In this study, we only focus on N-linked glycans and O-linked glycans. Others will not be addressed here. N-linked glycosylation is a very common form of glycosylation and plays a key role in the folding of many eukaryotic glycoproteins and for cell-cell, cell- extracellular matrix (ECM) attachment and contributing to epithelial-mesenchymal- transition (EMT). The N-linked glycosylation process occurs in the lumen of the endoplasmic reticulum (ER) of eukaryotes and archaea, but rarely in bacteria. In addition to its function in protein folding and cellular attachment, the N-linked glycosylation can modulate a protein's function, in some cases acting as an on-off switch [70]. N-glycosylation also has strong impact on cell signaling, cell growth and survival, facilitating tumor- induced immunomodulation and metastasis [71, 72].

The biosynthesis of N-linked glycans includes 3 major steps [64]: synthesis of dolichol- linked precursor oligosaccharide; en bloc transfer of precursor oligosaccharide to protein, and processing of the oligosaccharide. The first two steps occur in the ER. Subsequent processing and modification of the oligosaccharide chain are carried out in the Golgi apparatus. The synthesis of glycoproteins is spatially separated in different cellular compartments. Therefore, the type of N-glycan depends on its accessibility to the different enzymes present in these cellular compartments [73]. Figure 1-3 below demonstrates the N- glycan steps.

Figure 1-3. Principles and steps of N-glycosylation. 1). Synthesis of an oligosaccharide precursor consisting of 14 monomers bound to a dolichol anchor in the ER. Enzymes

17 involved are DPAG1, ALG1-14 etc. 2). Transfer of the precursor to a suitable ASN residue within the nascent polypeptide by the oligosaccharyltransferase (OST) protein complex which consists of various subunits, among them STT3A and STT3B, which form the catalytic center, ribophorin I and II (RPN1, RPN2), OST48, N33, IAP and DAD. 3).

Trimming of the glycan precursor in the ER and exit to the Golgi: cleavage of 3 terminal glucose residues by DCS1 and GANAB, and one mannose by ER mannosidase resulting in high-mannose glycans. 4). Cleavage of part of the mannose residues by Golgi mannosidases, followed by branching and chain extension by different monosaccharides, i.e. GlcNAc (MGAT family), Gal (B3GALT1 etc.), Fucose (FUT’s), sialic acid (ST6GAL or ST3GAL) [74].

O-linked glycosylation is the attachment of a sugar molecule to an oxygen atom in an amino acid residue in a protein, which occurs in the Golgi apparatus in eukaryotes, archaea and bacteria. O-Linked glycans are generally classified by their core structure due to its heterogeneity. Non-elongated O-GlcNAc groups have been recently shown to be related to phosphorylation states and dynamic processing, which contributes to cell signaling transduction in cells [75]. O-Linked glycans are prevalent in most secretory cells and tissues.

They are present in high concentrations in the zona pellucida surrounding mammalian eggs and may function as sperm receptors (ZP3 glycoprotein) [76]. O-Linked glycans are also involved in hematopoiesis, inflammation response mechanisms, and the formation of ABO blood antigens [77].

The O-glycosylation is a covalent post-translational modification in which monosaccharides are attached to serine (Ser) and/or threonine (Thr) residues of specific proteins by an O-glycosidic bond. O-Linked glycosylation does not require a consensus sequence and no any oligosaccharide precursor is required for protein transfer. The synthesis of O-glycans takes place in the Golgi apparatus, and is depicted in Figure 1-4 below. The most common type of O-linked glycans is initiated by the activity of polypeptide-GalNAc-transferases (pp-GalNAcTs, GALNTs) that link a single N- acetylgalactosamine (GalNAc) residue to Ser or Thr, thus forming the Tn antigen, which are commonly referred to as mucin-type glycans. Other O-linked glycans include glucosamine, xylose, galactose, fucose, or manose as the initial sugar bound to the Ser/Thr residues.

Figure 1-4. Biosynthesis of mucin-type O-glycans. A) A single α-linked GalNAc is transferred to Ser/Thr via the activity of ppGalNAcTs (GALNTs) forming the Tn antigen.

The elongation of the Tn antigen results in sialyl-Tn antigen through ST6GalNAc-I or T antigen (core 1) due to T-synthase/COSMC. The T antigen can further branch in sLea/sLex,core 2 and sialyl-T antigen through the action of certain glycosyltransferases or sialyltransferases. Core 3 is initiated via the activity of C2GnT, which in turns form core 4 and further extends structures. The synthesis of core 1 and core 2 starts in the cis-Golgi and the extension of core structures are completed in medial-Golgi and trans-Golgi. The elongation of further complex core structures (core 3, core 4 and sialyl-T antigen etc.) occurs mostly in the trans-Golgi.

A2. Reagents and methods

A2.1 Animal

Male C75BL/6J mice (6 to 8 weeks old) were from Dr. Aimin Zhou’s lab and housed in a temperature-controlled room with a 12-h light/dark cycle and allowed food and water at libitum at Cleveland State University. All procedures were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the

National Institutes of Health and the protocol was approved by the Committee on the Ethics of Animal Experiments of Cleveland State University.

A2.2 Cell lines and cell culture

All cell lines used in the study are shown in Table 1. All cell lines were grown in complete

RPMI media (Cleveland Clinic, OH), supplemented with 10% fetal bovine serum (FBS,

PAA Laboratories, MA), 100 unit/ml of penicillin and 100 µg/ml of streptomycin(Cleveland Clinic, OH). Cultures were maintained in a humidified incubator at 37°C in 5% CO2.

Table 1. Cell lines used in project 1

Cell line Origin of Tumor or Cell Type Source

MCF-7 Mammary Gland/Breast ATCC

MDA-mb-231 Mammary Gland/Breast ATCC

SK-Br-3 Mammary Gland/Breast ATCC

HT-29 Colon ATCC

T84 Colon ATCC

NCIH292 Lung ATCC

Hey-1B Ovary Dr. Aimin Zhou’s lab

SK-hep-1 Liver / Ascites ATCC

HepG2 Liver ATCC

HEK293 Embryonic Kidney ATCC

RCC45 kidney ATCC

J774 Murine macrophage ATCC

Jurkat leukaemic T-cell ATCC

HL60 Human promyelocytic leukemia cells ATCC

U937 Lymphocyte (Histiocytic Lymphoma) ATCC

THP-1 Monocyte (Acute Monocytic Leukemia) ATCC

K562 Bone Marrow (Chronic Myelogenous ATCC Leukemia)

A2.3 Western blot and antibodies

Proteins were separated by SDS–PAGE. Gels were transferred onto a polyvinylidene difluoride membrane (PVDF) membrane (Fisher Scientific, IL). The membranes were blocked with 5% non-fat milk in PBST (PBS containing 0.2% (v/v) Tween 20) and incubated with first antibodies 1hr at room temperature. Antibodies used for Western blotting are described in Table 2. The membranes were then washed with PBST three times and incubated with an anti-rabbit/mouse IgG secondary antibody conjugated with HRP for

1hr at room temperature. After the washing step, these immunoreactive bands were detected by a chemiluminescence method, according to the manufacturer’s specification

(Thermo Fisher, IL). Densitometry was performed using ImageJ software (National

Institutes of Health).

Table 2. Antibodies used in project 1

Antigen Species Source LRG-1 rabbit polyclonal Cat#: HPA 001888, Sigma Aldrich LRG-1 rabbit polyclonal Cat#: HPA 001889, Sigma Aldrich LRG-1 Mouse monoclonal Cat#: ab57992, Abcam GAPDH mouse monoclonal Cat#: sc-32233, Santa Cruz Biotechnology His-tag mouse monoclonal Cat#: A00186-100, Genscript PARP Rabbit monoclonal Cat#: 9532S, Cell Signaling Calreticulin Rabbit polyclonal Cat#: ab2907, Abcam Anti-mouse IgG, HRP- Horse Cat# 7076S, Cell Signaling linked Antibody Anti-rabbit IgG, HRP- Goat Cat# 7074S, Cell Signaling linked Antibody Anti-Rabbit IgG H&L Goat Cat#: ab150077, Abcam (Alexa Fluor® 488) Anti-Mouse IgG H&L Goat Cat#: ab150114, Abcam (Alexa Fluor® 555)

A2.4 Urinary Excretion of LRG-1 in the Patients with Cancer

We collected urine of patients with leukemia, lymphoma, prostate cancer, lung cancer, kidney cancer, colon cancer, liver cancer, and healthy individuals. Each urine sample (30

μL) was directly loaded and fractionated on SDS-PAGE gels. Then the excretion level of

LRG-1 in different patient’s urine was examined by using Western blot assay.

A2.5 Immunofluorescence and Confocal Microscopy

To determine the localization of LRG-1 in the cells, HepG2 cells grown on coverslips were rinsed twice with PBS, and fixed with freshly prepared 4% paraformaldehyde at 37°C for

15 min. Rinsed the cells twice with PBS, and then permeabilized cells with 0.2% Triton X-

100 for 10 min on ice. Cells were then rinsed with PBS and blocked with 10% BSA for 30 min. The sections were incubated with antibodies in PBS and 1% BSA for 2h at RT (or overnight at 4°C), rinsed three times with PBS (3 min/time) and then incubated with various Alexa Fluor-conjugated secondary antibodies (Table 2) for 1h in dark at room temperature. After rinsed three times with PBS (3 min/time), coverslips were mounted onto slides with anti-fade mounting medium and sealed with nail polish. Confocal imaging was carried out with a Zeiss LSM710 microscope with a 63X, 1.4 N.A. oil immersion objective lens using ZEN2010 software or on a Leica TCS SP5 Confocal microscope with a 63x

1.4NA Plan Apochromat oil immersion objective lens using LAS AF version 1.3.1 build

525 software.

We performed a double staining with LRG-1 and calreticulin (as listed in Table 2), a resident protein (ERp60) in endoplasmic reticulum.

A2.6 LRG-1 gene knockout

H292 lung cancer cells were seeded in a 24-well plate at 0.5 × 105 cells/well and cultured for 24h to reach 70% confluence. Then cells were transfected with the LRG-1-HDR

CRISPR Knockout plasmid (Santa Cruz Biotechnology, TX) by using Lipofectamine®

2000 Reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA).

Briefly, 1μg LRG-1-HDR CRISPR Knockout plasmid DNA or 1 μl of Lipofectamine®

2000 Reagent diluted into 50 μl Opti-MEM reduced serum medium (Invitrogen, Carlsbad,

CA, USA) was mixed after incubating for 5 min. The mixture was incubated at room temperature for additional 30 min and carefully added to the cells. Then, the cells were incubated for 4h at 37 °C and the medium was changed to complete RPMI 1640 medium containing 10% FBS. Cells were maintained with completed medium at 37 °C for 2-3 days. Stably transfected clones were established by selection with puromycin (Thermo

Fisher Scientific, IL). The LRG-1 expression in each picked clone was examined by

Western blot assay.

A2.7 Cell migration and wound healing study

For Transwell chamber assay, H292 control cells or LRG-1-knock out H292 cells (5 x 105) in the serum free medium were added into the upper chambers of 8μm Transwells

(ThinCertTM-24 Well) (VWR, Bridge Port, NJ) pre-coated with 20μg/ml fibronectin

(Thermo Fisher Scientific, CA) for overnight at 4oC. In the lower chambers, 500μL serum- free RPMI, serum-free RPMI supplemented with 10% FBS or 100ng/ml of glycosylated

LRG-1 were added respectively, and the cells were allowed to migrate at 37°C for 6h. At the end of the assay, the filter side of the up chamber was cleaned with a cotton swab and

26 the migrated cells were fixed by 10% formalin for 5 min and then stained with Coomassie blue G250. The membranes of the insert were cut off and photographed. Five fields per transwell were photographed at x 200 magnification under a microscope and the cells in each image were counted.

For the scratch wound-healing assay, 1×106 H292 control cells or LRG-1-knock out H292 cells were seeded into a 12-well plate with complete RPMI medium. After cell attached to the bottom of the plate to form a monolayer cell surface, rinsed the cells twice with PBS and then scratched a straight line across each well of the 12-well plate using a pipette tip perpendicular to the bottom of the well. Make sure the cell free area had sharp clear edges.

Gently washed out detached cells and replenished the well with fresh RPMI with 10% FBS and then took photos of the gaps. After incubation for additional 24h, cells were fixed with

4% paraformaldehye for 20 min and photos of the gap distance were taken once again.

A2.8 PARP cleavage study

Wild type H292 cells or LRG-1-knock out H292 cells were grown in RPMI (Cleveland

Clinic, OH) supplemented with 10% fetal bovine serum (PAA Laboratories, MA) and

o antibiotics in a humidified atmosphere of 5% CO2 at 37 C. For treatment, cells were grown to 90% confluence and incubated with 10μM doxorubicin (DOXO) for 24h. PARP cleavage was measured by Western-blot analysis for PARP1.

A2.9 LRG-1 mutant construction

A2.9.1 Plasmid map and construction

Human LRG-1 cDNA was purchased from Sino Biological Inc (Human LRG-1 Gene cDNA Clone / ORF Clone, pCMV / hygro – His, Beijing, China). GeneArt® Site-Directed

Mutagenesis System was purchased from Thermo Fisher (USA).

Plasmids expressing LRG-1 single-site mutants T37A, N79Q, N186Q, N269Q, N325Q, two-site mutant N269Q- N325Q, three-site mutant N186Q- N269Q-N325Q, and four-site mutant N79Q- N186Q- N269Q-N325Q were generated by PCR-based mutagenesis using

WT LRG-1 plasmid as a template (Fig. 2-1). All LRG-1 proteins expressed by these plasmids had a C-terminal His tag for protein detection.

Figure 2-1. LRG-1 pCMV / hygro – His plasmid map

A2.9.2 Expression and Analysis of LRG-1 Proteins

Plasmids were transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen |

Thermo Fisher Scientific, CA), the same method as described in 2.5. The conditional medium was collected after 48-60h. Secreted LRG-1 proteins were denatured in a buffer with (reducing) 2.5% β-mercaptoethanol and separated by SDS-PAGE. Expressed LRG-1 proteins were analyzed by Western blotting using an anti-His antibody (Table 2) and quantitation was performed by densitometry.

A2.10 Effects of BZGalNac and Tunicamycin on LRG-1 secretion

To inhibit O-glycosylation in cells, we used O-linked oligosaccharide synthesis inhibitor

BZGalNac (Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside), which inhibits UDP-

GlcNAc:GalNAc-β1,3-N-acetylgucosaminyl-transferase activity. HepG2 cells were grown in 10cm dishes. We added BZGalNac (Sigma Aldrich) at different concentrations (0, 0.74,

1.84 and 2.76mM) to the cells in separate dishes and cultured them for 24h. To inhibit N- glycosylation in cells, we used Tumicamycin, a commonly used ER stress inducer, which inhibits GlcNAc phosphotransferase (GPT) that catalyzes the transfer of N- acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol phosphate in the first step of glycoprotein synthesis. We added Tunicamycin (Sigma Aldrich) at different concentrations (0, 1, 2, 3 and 4ug/mL) to the cells in separate dishes and cultured them for

24h. We collected the conditional media and analyzed LRG-1 level by Western blotting using an anti-LRG-1 antibody.

A2.11 Glycosidase digestion

To investigate the N-link galactosamine oligosaccharides in human LRG-1, we carried out glycosidase digestion experiments using PNGase F (Sigma Aldrich, MO). In general,

HepG2 conditional medium containing LRG-1 was collected and was digested with

PNGase F according to the manufacturer’s instructions. Firstly, 100 μg HepG2 cell lysate was adjusted to 35μL with water. Then, 10 μL of PBS 0.25 M, pH 7.5, and 2.5 μL of 2%

SDS with 1M 2-mercaptpethanol were added. The reaction mixture was incubated at 100°C for 5min. After the reaction mixture was cooled down to room temperature, 2.5 μL of 15%

(v/v) Triton-X-100 and 2.0 μL of PNGase F (500units/mL) (Sigma-Aldrich, MO) were added to the sample. The reaction mixture was incubated at 37 °C for 3h. We analyzed the digested samples by Western blotting using an anti-LRG-1 antibody.

A2.12 Glyco-Protein Stain

HepG2 cells were grown in RPMI supplemented with 10% FBS, cells and conditional medium from each dish were collected at different time points. The cells were lysed with cell lysis buffer. Proteins were separated by SDS–PAGE. Glyco-Protein staining was conducted by following the protocol provided with the glyco-staining kit (G-Biosciences,

St. Louis, MO). Briefly, SDS-PAGE gels were firstly fixed by 3% acetic acid for 30 minutes and then rinsed by 50% methanol for 3 times. The Glyco-Oxidizing reagent (25mL) was used to oxidize the gel. The gel was incubated with the Glyco-Oxidizing reagent for 15 min, followed by 3 times of washing with 50% methanol. After washing, the gel was stained with 25mL of the Glyco-stain solution for 15 min, followed by using 25mL of

Glyco-reduction reagent for 5min. Glycoproteins were observed as magenta bands after 3 times of washing with 50% methanol.

A2.13 Effects of LPS on LRG-1 secretion

Raw cells and Jurkat cells were grown in RPMI supplemented with 10% FBS. Raw Cells were treated with Lipopolysaccharide (LPS) with final concentration as 1.0 ug/ml for 24 hours. For Jurkat cells, we added LPS to at different concentrations (0, 10, 50, 100,

500ng/mL) and incubated the cells for 24 hours. Cell were harvested and lysed with cell lysis buffer. The expression level of LRG-1 in the cell lysate was examined by using western blotting assay. Conditional media and were also collected to analyze the expression level of LRG-1.

A2.14. Effect of LRG-1 on the Expression of COX-2

Raw cells and Jurkat cells were grown in RPMI supplemented with 10% FBS. To investigate the effects of LRG-1 on the expression of Cyclooxygenase (COX-2), an inflammatory enzyme, cells were incubated with 0.5ug/ml LPS and 20ug/ml Non- glycosylated or Glycosylated LRG-1 protein simultaneously, for 24h. PBS was used as control in this experiment. Cell pellets were collected and lysed with cell lysis buffer and the expression level of COX-2 was examined by Western blotting analysis.

A2.15 Effect of LRG-1 on the expression of small GPTases

To investigate if LRG-1 plays any role on the expression of small GPTases, wide type and

LRG-1 gene knockout H292 cells were treated with 5µM of Statin for 24h. After statin treatment, cells were collected and lysed with cell lysis buffer and the expression level of

CDC42 was tested by Western blotting analysis.

A2.16 Quantification and Statistical Analysis

Details of statistical tests used to determine significance are provided in the figure legends.

A3. Results

A3.1 LRG-1 is excreted in the urine of patients with cancers

It is obvious that the successful rate of therapeutic treatment for cancer can be greatly improved if the disease is diagnosed at an early stage. In general, cancer is diagnosed based on the symptoms, the results from a physical examination and screening tests for the validated clinical indicators (biomarkers) of certain cancer in blood. Currently modern techniques such as mammograms, colonoscopy, and computed tomography are widely used for early cancer diagnosis [40]. However, these diagnostic methods are, for most of them if not all, are a site specific, invasive and often expensive. Recent years, noninvasive tests by detecting the metabolites and excreted molecules in body wastes have shown the potential to be developed as a simple, economic and reliable method for cancer diagnosis.

To determine if there are any proteins differentially excreted in the urine from patients with hepatocellular carcinoma (HCC) and health people, we performed a proteomic analysis for the profile of the excreted proteins and successfully identified LRG-1 as one of the unique proteins present in the urinary samples from cancer patients (data not shown). The results

32 were further confirmed by Western Blot analysis. To examine if LRG-1 is present in the urine of patients with other cancer types, we analyzed the urinary samples from patients with leukemia, prostate cancer, lung cancer and colon cancer. As shown in Fig. 3-1, LRG-

1 was present in the urine of most cancer patients at a significantly high level in compared to that from health people, suggesting LRG-1 may be a general biomarker for all kinds of cancer.

Figure 3-1. The excretion level of LRG-1 in the urine of patients with certain cancer.

The excretion level of LRG-1 in the urine of patients with certain cancer was determined by using western blot analysis with a polyclonal antibody to human LRG-1. The samples were from: A) and B). healthy individuals. A108: leukemia patient sample; C). patients with hepatocellular carcinoma (HCC); D). patients with colon cancer; E. patients with leukemia, lymphoma, prostate cancer, lung cancer or kidney cancer.

A3.2 LRG-1 is localized on ER

The physiological role of LRG-1 is largely unknown. The subcellular location of a protein in the cell can be used to predict, at least in part, the function of a protein. To determine the localization of LRG-1 in the cells, we performed immunostaining for LRG-1 in cells by using a polyclonal antibody against human LRG-1 and its subcellular location was analyzed by Confocal Microscopy. Interestingly, our result showed that LRG-1 is co- localized with an ER marker (Calreticulin) (Figure 3-2), suggesting that LRG-1 may be localized in ER although its role in ER needs to be further investigated.

Figure 3-2. Subcellular localization of LRG-1 in the cell

A3.3 Distribution of LRG-1 in Mouse Organs

We also determined the distribution of LRG-1 in different organs. In this experiment, adult mouse tissues were collected and lysed in the buffer containing 50 mM Tris-HCl (pH 8.0),

150 mM NaCl, 1% Nonidet P-40 (vol/vol), and protease inhibitors (Sigma, 1:100). The expressing level of LRG-1 in each organ was analyzed by Western blot analysis. As shown in Fig. 3-3, a high level of LRG-1 expression was found in the brain, liver, and kidney, but it is much less expressed in the heart, pancreas, and colon. However, this protein is barely detectable in the thymus, lung and spleen.

Figure 3-3. Distribution of LRG-1 in Mouse Organs

A3.4 Expression of LRG-1 in different cancer cells

To determine the expression of LRG-1 in different cancer cells, we analyzed the level of

LRG-1 in the cell extracts from a variety of cancer cell types including MCF-7 (human breast cancer cell line), MDA-mb-231(human breast cancer cell line), SK-Br-3(human breast cancer cell line), Hey-1B (human ovarian cancer cell line), HT-29 (human colon cancer cell line), H292 (human lung cancer cell line), T84 (human colon cancer cell line),

SK-hep-1(human liver cancer cell line), and HepG2 (human liver cancer cell line). As shown in Fig. 3-4A, LRG-1 is almost equally expressed in all types of cancer cells we examined. Since LRG-1 was found to be excreted in the urine of patients with cancer, we attempted to examine if LRG-1 is able to be secreted in the medium culturing the cells. In this study, cells (2.5×106) of each cell type were seeded in 10cm cell culture and grown in

RPMI supplemented with 10% fetal bovine serum. The culture medium was taken from each culture dish after the cells were grown for 48hrs. The level of LRG-1 in the medium was determined by Western Blot analysis. Surprisingly, the secretion level of LRG-1 in different cell types was significantly distinguished although it could be secreted from most of the cancer cells (Fig. 3-4B). Apparently HepG2 cells are the best cells able to secrete

LRG-1 in medium. To determine the secretion of LRG-1 in the medium is time dependent, both cells and media of the HepG2 cultures were collected at different time points, followed to be analyzed for the level of LRG-1. As expected, the level of LRG-1 in the medium was dramatically increased along with the time extension although its intracellular level remained unchanged (Fig. 3-4C). Interestingly, the size of LRG-1 secreted in the medium is about 45kDa whereas intracellular LRG-1 is about 36 KDa, suggesting that LRG-1 is subjected to posttranslational modification before secretion. The time dependent secretion

39 of LRG-1 was further confirmed by both Coomassie Blue staining and Western blot analysis as shown in Fig. 3-4D.

Figure 3-4. The levels of LRG-1 in cell lysate and conditional medium. The levels of

LRG-1 in cell lysate and conditional medium. The level of LRG-1 in cell extracts and media was determined by Western blot analysis using a polyclonal antibody to human

LRG-1 and Coomassie blue staining. A). The level of LRG-1 from cell extracts. B). The secretion level of LRG-1 in cell culture media. C). The levels of LRG-1 in HepG2 cell extracts and cell culture medium. D). HepG2 cell culture media.

However, the level of LRG-1 expression in normal cells was apparently lower that in cancer cells (Fig. 3-5) although this observation needs to be further demonstrated.

Figure 3-5. Comparison of the LRG-1 level normal and cancerous cells

A3.5 Secreted LRG-1 is glycosylated, but not the intracellular form

A mentioned above, intracellular LRG-1 was found to be significantly smaller in size than that secreted in the medium, suggesting LRG-1 underwent posttranslational modification.

It has been reported that LRG-1 is a glycoprotein [41]. To determine if the secreted LRG-1 is a glycosylated form, the proteins in the cultured media and cell lysates were separated by SDS-polyacrylamide gel electrophoresis and subsequently stained with a glycoprotein staining kit. As shown in Fig. 3-6A, LRG-1 secreted in the media was apparently glycosylated. To further confirm the observation, we compared the size of LRG-1 from different sources: isolated by using cytochrome C beads from human serum (Abcam, MA), the recombinant protein from E.coli (Novus Biologicals, CO), HepG2 cell lysate and conditional medium. Since the posttranslational modification process only occurs in eukaryotes, not in bacteria, recombinant LRG-1 should not be glycosylated and has a molecular weight of about 34 KDa according to the gene sequence information. As expected, the molecular weight of LRG-1 from human serum and HepG2 cell conditional medium was about 45KDa whereas the recombinant LRG-1 was about 34 KDa.

Interestingly, the intracellular form of LRG-1 displayed a molecular weight about 36 KDa, implicating that the protein inside cells is partially glycosylated.

Figure 3-6. Glycosylation of LRG-1 in cells. The glycosylation status of LRG-1 inside and secreted outside the cells was determined by staining with a glycoprotein staining kit

A) and Western blot analysis B).

A3.6 Effect of glycosylation on LRG-1 secretion

The secreted form of LRG-1 is fully glycosylated. To determine if glycosylation impacts the secretion of LRG-1 out of the cell, we treated the cells with tunicamycin (N- glycosylation inhibitor) and BZGalNac (O-glycosylation inhibitor) (Sigma-Aldrich, MO) and examined the expression and secretion of LRG-1. HepG2 cells were incubated with different concentrations of tunicamycin, and/or BZGalNac for 48h. Conditional media and cell lysates from each treatment were analyzed by Western blotting. As shown in Fig.3-7A, the expression of LRG-1 was not altered in the presence of either tunicamycin (up to

4μg/ml) or BZGalNac (up to 2.76mM). However, although the concentration of tunicamycin or BZGalNac was sufficient for inhibiting N- and O-glycosylation in living cells, LRG-1 was still able to be secreted from the cells, suggesting that LRG-1 glycosylation is not responsible for the secretion of this protein. Furthermore, the gradually reduced molecular weight of LRG-1 was observed upon the treatment with different types and concentrations of the inhibitors. Based on the sequence information and structural analysis, there are one O-link glycosylation site and four N-link glycosylation sites on

LRG-1. We performed site-directed mutagenesis to determine how important each or a combination of the O- and N-glycans to the secretion of LRG-1. These constructs were transfected into Hek293 cells and the expressed mutants inside the cell and secreted in the medium were analyzed by Western blot analysis. As shown in Fig. 3-7C & 7D, neither single or multi-glycan deletion stopped LRG-1 secretion. Above all, glycosylation is not responsible for the secretion of LRG-1 outside of the cell.

Figure 3-7. Effect of Glycosylation on the secretion of LRG-1. A) and B). Effect of N- link or O-link glycosylation inhibitors on the secretion of LRG-1. C). Effect of single glycan mutation on the secretion of LRG-1. D). Effect of multiply glycan mutations on the secretion of LRG-1.

A3.7 Effect of glycosylation on LRG-1 stability

Posttranslational modification of a protein in blood and urine is one of the natural ways to prevent the protein from degradation or at least it can extend its half-life. To examine the effect of glycosylation on LRG-1 stability, HEK293 cells were transfected with a plasmid construct fused human LRG-1 cDNA with the His6 tag. Stable cell lines were established by selecting the cells with hygromycin (Fig. 3-8A). We used tunicamycin (N-glycan inhibitor) and BZGAlNac (O-glycan inhibitor) to treat the transfected HEK293 cells for 24 hours. The conditional medium was collected and incubated at 37°C over time, or treated by α-chymotrypsin, a proteolytic enzyme which is acting in the digestive systems of many organisms, over concentration. Glycosylated and Non/partial-glycosylated LRG-1 were analyzes by Western blotting. As shown in Fig.3-8B and 8C, LRG-1 was partially inhibited in the presence of tunicamycin and BZGAlNac, as showed by a smaller molecular mass of

LRG-1. Partial inhibition of N-link and O-link glycosylation allowed us to obtain enough

LRG-1 from conditional medium for LRG-1 stability experiments. We used densitometry to calculate the ratio of fully glycosylated and non/partial glycosylated LRG-1 in α- chymotrypsin digestion experiment by using ImageJ software. The result showed that the non/partial glycosylated form of LRG-1 was less stable than fully glycosylated LRG-1 (Fig.

3-8C and 8D).

Figure 3-8. LRG-1 Stability Study. A). the transfection of His-tagged human LRG-1 plasmid to HEK293 cells. B). Transfected HEK293 cells were treated with glycosylation inhibitors: Tunicamycin and BZGalNAC. The conditional media were incubated at 37°C in a water bath. C). Conditional media were digested with α-chymotrypsin at different concentration. D). The ratio of glycosylated and partially-glycosylated LRG-1 in a- chymotrypsin digestion was calculated by using densitometry.

A3.8 LRG-1 is associated with inflammation

It is reported that LRG-1 is expressed during granulocyte differentiation [80]. To investigate if LRG-1 is involved in inflammatory response, ultra-pure Escherichia coli LPS (Invitrogen,

San Diego, CA) in the phosphate-buffered saline solution (PBS) was intraperitoneally (i.p) injected into mice (N=4) at a final concentration of 1.0 g/kg and PBS alone was used as control (N=3). The urine of treated mice was collected before and after 24hr injection.

Blood samples were collected in 24 hr post injection. The levels of LRG-1 in the urine and blood samples from the mice were separated by SDS-PAGE gel, followed by Western blot assay. Interestingly we found that the secreted level of LRG-1 in the urine of mice under

LPS stimulation was significantly increased although LRG-1 was undetectable before injection and in the control group (Fig. 3-9A). The increased levels of LRG-1 were also observed in plasma samples from the mice treated with LPS (Fig. 3-9B).

C-reactive protein (CRP) is a biomarker in the blood for inflammation and a high level of

CRP indicates an inflammatory response. To determine if LRG-1 coexists with CRP in the inflammatory condition, we analyzed the level of LRG-1 in the urine from patients with a high level of CRP in their plasma. As shown in Fig. 3-9C, the level of LRG-1 in the urine was consistent with the level of CRP in the plasma as determined by Western blot analysis.

The higher the CRP level in the plasma was, the higher the LRG-1 level in the urine was.

These findings suggest that LRG-1 may be a biomarker in inflammatory responses.

Figure 3-9. LRG-1 is associated with inflammation. LRG-1 is associated with inflammation. A). LPS induced LRG-1’s excretion into the urine of mice. B). LRG-1 level in mice plasma was elevated after LPS injection. C). LRG-1 is excreted in the urine of patients with inflammation in a significant level.

A3.9 LRG-1 secretion is not the result of LPS stimulation

From the previous results described at the section3.8, we demonstrated LRG-1 could be secreted into the urine in response to inflammatory stimulation in vivo. It is well known that macrophages and T cells play a vital role in promoting inflammation. To examine if

LPS stimulate LRG-1 secretion in vitro, we treated mouse Raw264.7 macrophage cells with 1.0µg/mL LPS for 24 hours and examined the level of LRG-1 in the conditional media.

As shown in Fig.3-10A, LRG-1 in the medium of the cells treated with LPS was not detectable. This observation suggested that LPS could not stimulate LRG-1 secretion from macrophage cells. To investigate if LPS stimulate the LRG-1 secretion in other immune cells, we firstly screened the LRG-1 secretion and expression level in J774 (mouse macrophage), Jurkat (human Lymphocyte), HL60 (human promyeloblast), U937 (human monocyte), THP-1 (human monocyte), and K562 (human lymphoblast) cells. Apparently the secretion of LRG-1 among these cells was significantly different (Fig. 3-10B). However,

J774 and Jurkat did not secrete any LRG-1 in the media no matter with or without LPS stimulation although the level of intracellular LRG-1 was similar to that in other cell types.

To determine if the expression and secretion levels of LRG-1 in Jurkat cells line is inducible by LPS, we treated the cells with LPS at difference concentrations of 1, 10, 50, 100 and

500ng/ml. cell pellets and conditional media were collected after 24 hours incubation.

LRG-1 levels in both cell lysate and conditional media were analyzed by using western blotting. As shown in Fig. 3-10.C, LPS treatment did not impact the level of LRG-1 in both the conditional media and inside the cells.

Figure 3-10. LRG-1 Secretion is not the Result of LPS Stimulation. A). Raw cells were treated with LPS and Non-glycosylated/ glycosylated LRG-1 protein. B). The expression and secretion level of LRG-1 in different hemocytes. C). Jurkat cells were treated with LPS at different concentrations.

A3.10 Effect of LRG-1 on the expression of COX-2

Chronic inflammation is recognized as a vital factor for cancer progression. Release of inflammatory molecules generates microenvironment which is highly favorable for development of tumor, cancer progression and metastasis [81]. Cyclooxygenase-2 (COX-2) is a key mediator of inflammatory pathways and its elevated expression has been found in several other human cancer as well [82 , 83 ] . The relationship between inflammation and cancer in general is well documented. Since studies have shown that LRG-1 is involved in the TGF-β signaling pathway in angiogenesis and TGF-β displays a role in anti- inflammation through down-regulating the level of COX-2, we investigated the direct role of LRG-1 and glycosylation in COX-2 induction. In these experiments, we treated

Raw264.7 and Jurkat cells with 0.5µg/mL LPS in combining with 20µg/mL Non- glycosylated or glycosylated LRG-1 protein for 24 hours. Subsequently the COX-2 level in the cells was measured by using Western blotting assay. As shown in Figure 3-11A, although COX-2 was induced to a significant high level in the presence of LPS, LRG-1, neither non-glycosylated nor the glycosylated form, showed any impact on COX-2 expression. Jurkat cells have a certain level of endogenous COX-2, but did not response to

LPS treatment. However, the expression of COX-2 was down regulated in approximately

25% when Jurkat cells were treated only with glycosylated LRG-1, as shown in Figure 3-

11.C and D. The result suggests that LGR-1 may have a certain role in anti-inflammation.

Figure 3-11. Effect of LRG-1 on the Expression of COX-2. The expression level of

COX-2 in the cells treated with LPS in the presence of either Non-glycosylated or glycosylated LRG-1. A) Raw cells; B). Jurkat cells; and C). The statistical analysis of the expression level of COX-2 in Jurkat cells.

A3.11 Lack of LRG-1 promotes cell migration

LRG-1 is found to be highly expressed in cancer cells and clinical cancer specimens (Fig.

3-4 and 3-1). To determine if LRG-1 impacts cancer cell migration, we knocked out LRG-

1 in NClH292 cells, a human lung mucoepidermoid carcinoma cell line. In this experiment,

NClH292 cells were transfected with the human LRG-1 CRISPR/Cas9 KO Plasmid (Santa

Cruz: sc-405371, CA), followed by selection with puromycin (Santa Cruz Biotechnology,

Santa Cruz, CA). The level of LRG-1 in the clones was determined by Western blot analysis.

As shown in Fig. 3-13A, the expression of LRG-1 has been completely deleted in some colonies.

Upon identification LRG-1 knockout clones, we performed a cell migration assay. In wound healing assay, LRG-1 knock out cells migrated significantly faster than the control cells as shown if Fig. 3-12B. This observation was confirmed by a trasnswell cell migration assay (Fig.3-12.C). The results suggest that deficiency of LRG-1 may promote cell migration although the molecular mechanism is largely unknown.

Figure 3-12. LRG-1 impacts cell migration. A) LRG-1 knockout colonies selection. B)

Wound healing assay. C) Transwell cell migration assay. The migrated cells were stained with Coomassie-blue at magnification x 200.

A3.12 Cells defect LRG-1 Are more sensitive to an anti-cancer drug

Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a nuclear enzyme involved in DNA repair, DNA stability, and transcriptional regulation. Accumulating evidence showed that cleavage in PARP is a hallmark of apoptosis. Apoptosis usually results from the induction of caspase activity which PARP into an 89 KDa fragment. As shown in Figure 3-13, while PARP cleavage was clearly detected in LRG-1-KO (LRG-1 knock out) when cells treated with Doxorubicin (DOX) which induces apoptosis at 1µM.

While only a slight PARP cleavage was seen in the control when the cells were treated with

DOX at the same concentration. Likewise, more PARP cleavage were seen in the LRG-1 knock out cells when treated with 5µM DOX, compare to the control cells. These observations suggest that the deficiency of LRG-1 is more sensitive to apoptotic drugs.

This novel discovery may be advantageous in cases of malignancies that resist apoptosis induced by classical cancer agents.

Figure 3-13. LRG-1 impacts PARP cleavage

A3.13 Effect of LRG-1 on the expression of small GTPases

Small GTP-binding proteins (also known as small GTPases) are essential for multiple cellular processes such as cell proliferation (Ras), dynamics of the cytoskeleton (Rho and

Rop), membrane trafficking (Arf and Rab) and nucleo-cytoplasmic transport (Ran). They are known to be involved in numerous physiological processes including embryogenesis, establishment and/or maintenance of polarity, adhesion, migration and differentiation of numerous cell types [84]. Lovastatin, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, can induce apoptosis through the Rac1/Cdc42/JNK pathway [85]. To investigate the effect of LRG-1 one the expression of small GTPases, wide type and LRG-1 knock out

H292 cells were treated with 10 μM lovastatin for 24 hours. The expression level of CDC42 was examined by western blotting. As shown in Figure 3-14, the expression level of

DCD42 in wide type H292 cells was twofold greater extent compared to the LRG-1 deficiency H292 cells, approximately. Deficiency of LRG-1 prevent the expression of

CDC42 that stimulated by Lovastatin. LRG-1 promotes angiogenesis through upregulating the TGF-β1 pathway [86 ] . TGF-β-induced epithelial-mesenchymal transition (EMT) and

EMT-associated responses such as cell migration, invasion, and metastasis in cancer. EMT is considered a prerequisite for cells to adopt a motile and invasive phenotype and eventually become metastatic. A major regulator of EMT and metastasis in cancer is TGF-

β and its specific functions on tumor cells are mediated besides Smad proteins and mitogen- activated protein kinases (MAPKs) by small GTPases of the Rho/Rac1 family [87]. Our finding indicated that targeting the relationship between LRG-1 and small GTPases may allow for selective interference with pro-oncogenic TGF-β responses to aid in anti-cancer treatments.

Figure 3-14. Effect of LRG-1 on the expression of small GPTases

A4. Discussion

Glycosylation is a critical function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus, including cell attachment to the extracellular matrix and protein-ligand interactions in the cell. This study evaluated the effects of glycosylation on LRG-1’s secretion and stability. Studies reveals that glycans are able to function as protein “on and off” switches or as “analog regulators” to fine-tune protein function [88]. A great example of a glycan functioning as an “on-off” switch is the cutaneous lymphocyte-associated antigen (CLA) which is expressed by skin-infiltrating T cells. CLA mediates lymphocyte migration to the skin through its interaction with E- selectin which is present on endothelial cells within the skin. Actually, CLA is an inducible carbohydrate modification of P-selectin glycoprotein ligand-1 (PSGL-1). Interestingly,

PSGL-1 only binds to E-selectin and “turns on” its skin-homing function after sialyl-Lewis

X-decorated [70]. We show LRG-1 mainly exists two forms: a 36KD intracellular form and a 45KD secreted form. In Fig.3-7B, the molecular weight of LRG-1 in the conditional medium digested with PNGase F, an amidase of the peptide-N4-(N-acetyl-beta- glucosaminyl) asparagine amidase, is approximately 34-35 KD which is the same as the size of secreted LRG-1 after treated with Tunicamycin, an inhibitor of glucosamine phosphotransferase (GPT), indicating N-link glycosylation is the main reason of LRG-1’s molecular weight change. This observation is further demonstrated by glycan-staining as shown in Fig. 3-6A. Conversely, when the intracellular form of LRG-1 from HepG2 cell lysate was treated with PNGase F (Fig.3-7B), it maintained the same molecular weight as non-treated (36KD). However, this molecular weight is 2 KD higher than secreted form of

LRG-1 with all N-glycan digested (34-35KD), implicating that the intracellular form of

LRG-1 is subjected to O-link glycosylation or LRG-1 may contain a signal peptide which is a key determinant for targeting itself to the secretory pathway. To further investigate the secretory pathway of LRG-1, we treated HepG2 cells with monensin [89], a drug blocks the transport of a newly synthesized protein from the medial to the trans cisternae of the Golgi apparatus and also suppresses the addition of galactose to O-linked carbohydrates. Under this circumstance, the size of secreted LRG-1 in the conditional media was 44KD, which was the same size as the cells were treated with O-link glycosylation inhibitor BZGalNac, confirming that O-link glycosylation of LRG-1 takes place in the trans Golgi. However, neither Tunicamycin nor BZGalNac could stop LRG-1 from secreting. Furthermore, we employed mutagenesis of glycosylation sties on LRG-1 to investigate the mechanism by which N- and/or O- glycans regulate LRG-1’s secretion and its biological function. Results show that absence of individual glycan does not prevent the secretion of LRG-1, qualitatively and quantitatively. Nonetheless, studies have shown that the number of N- glycans displays on a protein correlated directly with folding and secretion rates of a protein, and impacts the yield of its active form [90]. Assays by using glycan site mutants showed that LRG-1 still could be secreted even after all glycan (N79, N186, N269 and

N235) deleted. Interestingly, the secretion rate was increased if two (N269, N235) or three glycan sites (N186, N269 and N235) were mutated, suggesting the N-link glycosylation site N186 and N269 may regulate the secretion rate of LRG-1. The regulation mechanism warrants an additional study. Over all, in this study, we illustrate that glycosylation does not impact LRG-1’s secretion by using glycan inhibitors and glycan site mutants.

Glycosylation is not an “on and off” switch for LRG-1 secretion, but it regulates its secretion rate.

A few studies demonstrate that glycosylation impacts protein stability [91-93]. In Fig.3-8B, it showed that both non-glycosylated and glycosylated form of LRG-1 protein are quite stable in the conditional media at 37 °C, which are clinically relevant to cancer patients with LRG-1 present in their urine. However, when LRG-1 protein was digested with α- chymotrypsin, the intracellular form of LRG-1 was less stable than fully glycosylated

LRG-1, suggesting glycosylation may prevent LRG-1 from protease degradation.

Many tumor markers are produced as a result of inflammation. It also reported that LRG1 is expressed during the granulocyte differentiation [80]. Granulocytes, also known as polymorphonuclear (PMN) leukocytes, are characterized by stained granules within their cytoplasm under a microscope. The granules contain enzymes that primarily damage or digest pathogens and release inflammatory mediators into the bloodstream [ 94 ].

Granulocytes include neutrophils, basophils, eosinophils, and mast cells. Study showed that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment [95]. In this study, we show that LRG-1 could be secreted into urine under inflammatory stimulation. This result was consistent with the level of LRG-1 in the urine of patients with a high level of serum CRP, suggesting that LRG-1 may be considered as biomarker in monitoring inflammation.

However, LPS stimulation in macrophage or lymphocyte was not responsible for LRG-1 secretion.

Macrophages, a type of phagocytes against bacterial or fungal infection and other very

68 small pathogens, are usually the first responder in pathogen elimination. Besides its canonical role in innate immune system, other functions of macrophages have recently been ascribed in both innate and adaptive immunity [96 , 97 ] . A number of macrophage granule proteins have been reported to display anti-microbial properties as well as the capacity to regulate the function of other immune cells. Prostaglandin E2 (PGE2) modulates a variety of physiological processes including the production of inflammatory cytokines. There are two cyclooxygenase (Cox) enzymes, Cox-1 and Cox-2, that are responsible for initiating PGE2 synthesis. These isozymes catalyze identical biosynthetic reactions but are regulated by different mechanisms in the cell. It has reported that infiltration of COX-2–expressing macrophages is a prerequisite for IL-1β–induced neovascularization and tumor growth [98]. In this study, we investigated if LRG-1 affects the expression and secretion of COX-2 in macrophages. Our data show that neither glycosylated nor the non-glycosylated LRG-1 showed any impact on COX-2 expression under LPS stimulation in macrophage. In contrast, fully glycosylated LRG-1 could down regulate the expression of COX-2 under LPS stimulation up to approximately 25% in

Jurkat cells. This result suggests that fully glycosylated LRG-1 may play a role in anti- inflammatory response.

The biological function of LRG-1 is largely unknown. Poly (ADP-ribose) Polymerase

(PARP) Cleavage is the hallmark event of apoptosis. Interestingly, Doxorubicin more efficiently induced apoptosis in LRG-1 KO H292 cells than that in H292 wild type cells as reflected by increased fragments of PARP. The data suggest that deficiency of LRG-1 improves cell sensitivity to apoptotic drugs. Wound healing and transwell assay results

69 showed that deficiency of LRG-1 promotes cell migration. It seems the involvement of

LRG-1 in cell migration is through regulating the expression of small GPTases. It has been well known that small GPTases play a pivotal role in regulating cell migration. In addition,

Small GTPases, particularly the Rho/Rac1 family, [99] mediate the TGF-β signaling pathway.

TGF-β is a major regulator of TGF-β-induced epithelial-mesenchymal transition (EMT) and metastasis in cancer. It has been reported that LRG-1 promotes angiogenesis through upregulating the TGF-β1 pathway [100]. Due to the complexity of the small GTPase pathway, which contains a multitude of growth factors and cytokines, a causative effect cannot be a result of any single factor or regulatory protein. The mechanism of LRG-1 on cell migration and cell sensitivity remains largely unknown. However, our findings may allow for selective interference of LRG-1 to aid anti-cancer treatments.

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5543.

CHAPTER II

PROJECT II. DEVELOPMENT AND VALIDATION OF A

LUCIFERASE REPORTER ASSAY TO IDENTIFY SMALL

MOLECULES REGULATING PD-L1 EXPRESSION IN CANCER

CELLS

B1. Introduction

B1.1 PD-L1

Programmed cell death protein (PD1) is a member of the immunoglobulin superfamily, and interacts with programmed death-ligand 1 (PD-L1) or PD-L2, resulting in suppression of

83 immune response, which is a critical pathway for some tissues to diminish the unwanted immune attacking [1, 2]. Therefore, the expression level of PD1, PD-L1 or PDL-2 in different tissues or immune cells has a direct impact to the interaction between the immune cells and corresponding organs. There are many transcriptional factors that are involved in the regulation of PD-1 expression. For example, the mutation of nuclear factor of activated T cells c1 (NFATc1) has been reported to cause a complete loss of PD-1 expression in T cells

[3]. Besides PD-1, the expression of its ligand PD-L1 and PD-L2 can be rapidly upregulated on various tissues [4-8]. The increased expression results in diminished interaction between immune cells and tissues. The expression of PD-L1 and PD-L2 on murine macrophages could be up-regulated by both T helper cell type 1 (Th1) cytokines and Th2 cytokines, whereas on human macrophages, interferon-γ (IFN-γ) could only up-regulates

PD-L1 expression, and PD-L2 expression is enhanced by interleukin-4 (IL-4) [3, 9-12]. These findings indicate that the regulation of the expression of PD1, PD-L1 or PD-L2 is very complex. However, the elucidation of the regulation mechanism may provide many novel targets, which could result in new drug development that target PD1, PD-L1 or PD-L2 expression. The increase of the expression of both PD-1 and PD-L1/L2 could suppress the immune response of many organs in the body [1]. On the other hand, targeting these signals to decrease the expression of PD1, PD-L1 or PD-L2 could enhance the immune response theoretically.

B1.2 PD-L1 and cancer treatment strategy.

During the past decade, the PD-1/PD-L1 pathway has been found to play a crucial role in regulating tumor escaping form the immune recognition and attacking [2, 13, 14]. PD-L1 and

PD-L2 expression are up-regulated in a variety of human cancer tissues [13-16]. More specifically, PD-L1 is highly expressed in many types of solid tumors, whereas PD-L2 is highly expressed in certain subsets of B cell lymphomas. Over-expression of PD-L1 in immunogenic tumor cells facilitates them to escape from host T cell immunity, which significantly enhances their invasiveness in vivo. There are many factors contributing to the high expression of PD-L1 in tumor tissues. For instance, it has been reported that PI3K,

STAT3, and IFN-γ are involved in the upregulation of PD-L1 in tumors [3, 17-20]. However, the whole signaling pathways that drive the expression of PD-L1 are still unclear yet.

Based on the discovery, the blockade of PD-1 interaction with PD-L1 could potentiate the immune cells to recognize and attack cancer cells. Many monoclonal antibodies targeting

PD-1/PD-L1 pathway have been developed so far for the treatment of various types of cancer [13, 21]. Among these anti-PD-1 antibodies, nivolumab and pembrolizumab have been approved by the US Food and Drug Administration (FDA) for the treatment of patients with metastatic melanoma [ 2, 15]. An anti-PD-L1 antibody atezolizumab has been approved by FDA for urothelial carcinoma treatment. It has been well-documented that PD-L1 in glioblastoma is highly expressed, which makes these antibody drugs potentially useful for the treatment of glioblastoma in the future [19, 22]. However, the antibody drugs cannot penetrate the blood brain barrier more effectively, which limits the applicable to the glioblastoma treatment. Other strategies that could interfere with PD-L1 in glioblastoma should be investigated to overcome the blood brain barrier penetration issue.

B1.3 Small molecule drugs

Small molecule drugs have many advantages compared to antibody drugs. For example, they have better capability to cross blood brain barrier, which will make them useful in the treatment of glioblastoma and other solid tumors as well. In addition, the regulation of the expression of PD-L1 in cancer cells is not well understood yet. If small molecules that manipulate the expression of PD-L1 could be identified, they could be used as tool drugs to further elucidate the signaling pathways that drive the expression of PD-L1. A clear understanding of the regulation may provide new targets for drug development that target the expression of PD-L1 in cancer treatment. We hypothesize that some small molecules might be able to affect the promoter upstream signals of PD-L1 and change the expression of PD-L1. In this study, a luciferase report assay was designed herein, and two compound libraries were used to validate the effectiveness of the model as a potential high throughput screening assay for the future application. The screening led to the identification of several compounds that could affect the expression of PD-L1 in cancer cells.

B2. Reagents and methods

B2.1 Compound libraries and other reagents

Library of Pharmacologically Active Compounds LOPAC1280 (currently n = 1280 compounds), a collection of pharmacological active molecules that have been approved by

FDA or in the clinical trial for certain disease treatment, was obtained from Sigma-Aldrich

(St. Louis, MO). Another natural product compound library (n = 400 compounds) was purchased from Cleveland clinic core facility. The positive control IFN-γ and the identified several compounds including Brefeldin A (BFA), Methotrexate (MTX), Digoxin, Digitoxin

86 and Quabain were acquired from Sigma-Aldrich we well. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H- tetrazolium bromide (MTT) and other chemical reagents were from VWR

(Radnor, PA).

B2.2 Cell lines

All cell lines used in the study are shown in Table 3. The cells were maintained in

RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-

Glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin-streptomycin. FBS was heat inactivated for 30 min in a 56 ºC water bath before use. Cell cultures were grown at 37 ºC, in a humidified atmosphere of 5% CO2 in a Hereaus CO2 incubator.

Table 3. Cell lines used in project 2

Cell line Origin of Tumor or Cell Type Source

MDA-MB-468 Mammary Gland/Breast, Adenocarcinoma ATCC

MDA-mb-231 Mammary Gland/Breast, Adenocarcinoma ATCC

SK-Br-3 Mammary Gland/Breast, Adenocarcinoma ATCC

H522 Lung, Non-Small Cell Lung Cancer ATCC

H292 Lung, Mucoepidermoid Pulmonary ATCC Carcinoma A549 Lung, Carcinoma ATCC

Ovcar3 Ovary, Adenocarcinoma ATCC

SKOV3 Ovary, Adenocarcinoma ATCC

HT-29 Colon, Colorectal Adenocarcinoma ATCC

SW837 Rectum, Adenocarcinoma ATCC

PC-3 Prostate, Adenocarcinoma ATCC

LNCaP Prostate, Carcinoma ATCC

DU145 Prostate, Carcinoma ATCC

Dr. Yan Xu Hey-1B Ovary Carcinoma Indiana University School of Medicine SK-hep-1 Liver / Ascites, Adenocarcinoma ATCC

HepG2 Hepatocellular Carcinoma ATCC

Hep3B Hepatocellular Carcinoma ATCC

U87 Brain, Likely Glioblastoma ATCC

T98G Glioblastoma Multiforme ATCC

A172 Glioblastoma ATCC

U373 Glioblastoma astrocytoma ATCC

RCC45 Renal cell carcinoma ATCC

RCC28 Renal cell carcinoma ATCC

T24 Urinary Bladder, Transitional Cell ATCC Carcinoma HeLa Cervix, Adenocarcinoma ATCC

Jurkat leukaemic T-cell ATCC

HL60 Human promyelocytic leukemia cells ATCC

U937 Lymphocyte (Histiocytic Lymphoma) ATCC

THP-1 Monocyte (Acute Monocytic Leukemia) ATCC

K562 Bone Marrow (Chronic Myelogenous ATCC Leukemia)

B2.3 Western Blotting

To determine the effect of the compounds on the PD-L1 expression, cancer cells were treated with different agents at indicated concentrations respectively for 24 h. Total cell lysates were extracted using M-PER reagent (Pierce). Equal amounts of proteins (50 μg) samples were separated on 12% SDS-polyacrylmide gel and transferred onto a polyvinylidene diflouride (PVDF) membrane (Pall Cooperation, FL.). After blocking for 1 hour, the membrane was incubated in PBST containing 5% BSA and primary antibody specific to PD-L1 or GAPDH (Cell signaling, MA) overnight at 4°C. HRP-conjugated anti- rabbit IgG or anti-mouse IgG (Cell signaling, MA) were used as secondary antibody and incubated at room temperature for 1h. The membrane was incubated in ECL plus reagent

(GE health) and then exposed to hyper film. The results are based on three independent experiments, and only one of the representative bands is presented.

B2.4 Construction of the plasmid

Human genomic DNA was isolated from human ovarian cancer Hey 1B cells by using a

Wizard® Genomic DNA Purification Kit. The 3-kb of 5’-flanking promoter region was amplified from genomic DNA by PCR using an upstream primer, 5’-TGA CTC GAG ACA

CAT ATA GGA TGT GAG-3’; a downstream primer, 50-TCT CTC GAG CCC AAA

GAA AGG GTG TAG-3’; The amplified fragment was digested with Sac I and Xho I, and subcloned into pGL4.26 luciferase vector (Promega, WI). The inserted fragment was sequenced to verify the sequence information. The constructed plasmid was transfected into Hey 1B cells using lipofectamine 2000 (Invitrogen, NY), followed by selection in

90 medium containing hygromycin. The activity of the PD-L1promoter was confirmed by luciferase activity.

B2.5 Cell transfection

Hey1B cells were seeded in a 24-well plate at 0.5 × 105 cells/well and cultured for 24 hours to reach 70% confluence. Transfection was performed by using Lipofectamine® 2000

Reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA).

Briefly, 500ng plasmid DNA or 1 μL of Lipofectamine® 2000 Reagent diluted into 50 μl

Opti-MEM reduced serum medium (Invitrogen, Carlsbad, CA, USA) was mixed after incubating for 5 min, respectively. The mixture was incubated at room temperature for additional 20 min and carefully added to the cells. Then, the cells were incubated for 4 hrs at 37 °C and the medium was changed into complete RPMI 1640 medium containing 10%

FBS, 1%P/S, and L-glutamine. Cells were maintained with completed medium at 37 °C for

2-3 days. In order to obtain a stable transfection, the transfected cells were then incubated with complete medium supplemented with 250 μg/ml hygromycin B (Invitrogen) and were examined every 2 days until hygromycin B resistant colonies remained in the plates, they were considered as stably transfected cells and were screened for luciferase and PD-L1 expression.

B2.6 RT-PCR

Total RNA was isolated using the TRIzol reagent according to the manufacturer’s protocol.

Total RNA pellets were dissolved in Nuclease-free water and quantitated using a spectrophotometer. The quality of RNA samples was determined by electrophoresis

91 through agarose gels and staining with ethidium bromide; the 18S and 28S rRNA bands were visualized under ultraviolet light. Isolated total RNA (2 μg) was treated with DNase

I Amplification grade, according to the recommended protocol to eliminate any DNA before reverse transcription. Then 1.5 µg RNA/sample was amplified using a one-step RT-

PCR kit (Qiagen). PDL1 forward primer, 5′-AAACAATTAGACCTGGCTG-3′, and PDL1 reverse primer, 5′-TCTTACCACTCAGGACTTG-3′. cDNA was amplified in a Techne

TC-412 Thermal Cycler under the following conditions: reverse transcription for 30 min at 50°C, denature for 15 min at 95°C, 30 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for

1 min, followed by a final extension at 72°C for 10 min. PCR products were analyzed on a 1.0% agarose gel stained with ethidium bromide (Thermo Fisher Scientific, IL, USA).

B2.7 96-Well Luciferase Assays

The transfected Hey1B cells were harvested, counted and 10,000 cells in 100 μl of medium were added to each well of a 96-well white-wall-clear bottom plate (BD Biosciences, NJ) in RPMI-1640 + 10% FBS. The medium was replaced the next day with medium containing the test compounds with or without hormone. After 24 hours, the medium with

20 µM of compounds were added, which diluted the drug concentration to 10 µM eventually. After another 24 hours, the medium was aspirated off, and 50 μL of lysing buffer (Promega, WI) was added. To help lysing the cells, the plate was placed on a shaker for 5 min. Then 10 μL of the luciferase assay substrate (Promega Corporation, WI) were added, and the plate was read with GloMax 2020 Luminometer.

B2.8 Cell viability analysis

The effects of compounds on Hey1B cell viability were assessed using the MTT assay in four replicates. Cells were grown in RPMI1640 medium in 96-well, flat-bottomed plates for 24 h, and were exposed to various concentrations of the compounds dissolved in DMSO

(final concentration 0.1%) in media for 48h. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. The medium was removed, replaced with

200 µl of 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide in fresh media, and cells were incubated in the CO2 incubator at 37°C for 2 h. Supernatants were removed from the wells, and the reduced MTT was solubilized in 200 µl/well DMSO.

Absorbance at 570 nm was determined on a plate reader. Statistical and graphical information was determined using GraphPad Prism software (GraphPad Software

Incorporated) and Microsoft Excel (Microsoft Corporation).

B3. Results

B3.1 Identification of the suitable cancer cell lines for the transfection

First, a cell line with high level of PD-L1 is critical to establish the high throughput screening assay. High level of PD-L1 protein needs more active promoter activation, and the same promoter is very likely to drive a high level of luciferase expression after transfection. To identify such a cell line, the PD-L1 protein levels of 27 different cancer cell lines were examined with western blot assay. The results exhibited in Figure 1 reveal that PD-L1 expression varies dramatically from cell line to cell line. There are 8 cell lines including, a breast cancer cell line MDA-MB-231, an ovarian cancer cell line Hey1B, a prostate cancer cell line DU145, three kidney cancer cell lines RCC28, RCC45 and T24, two glioblastoma cell lines U87 and U373 that showed relatively higher level of PD-L1 compared to the rest of the other cell lines. To further narrow down to the best cell line, lower amount of protein samples were loaded in the western blot assay to identify the cell line that has the most abundant PD-L1 expression. The results showed that Hey1B, an ovarian cancer cell line, has the most abundant PD-L1 protein. In addition, Hey1B is a fast- growing cancer cell line, which makes it a good model for high throughput screening.

Figure 4-1. Expression of PD-L1 in various cancer cell lines. A) Pilot detection of the

PD-L1 expression in 27 cancer cell lines; B) Further examination of the PD-L1 expression in several cell lines that showed relatively higher expression. The results are representative of three independent experiments and the representative one or the mean are presented.

B3.2 Cell transfection and validation of the identified colonies

The PD-L1 promoter driven luciferase plasmid is indicated in Figure 4-2. Hey1B cells were stably transferred with the plasmid. The cells were treated with hygromycin b to select the surviving colonies. Within the survived colonies, two colonies #9-3 and #9-4 showed high luciferase activity, suggesting that the PD-L1 promoter driven luciferase expression in the cells was successful (Figure 4-3A). In addition, the two colonies gave much higher luciferase activity after IFN-γ treatment, suggesting that the promoter driven luciferase activity responded to the upstream signals. Meanwhile, it is necessary to determine if the

PD-L1 expression driven by this promoter still responses to IFN-γ treatment after the transfection. It has been well documented that IFN-γ could induce PD-L1 expression in many cell lines. The assay is developed to identify compounds that could interfere with the promoter upstream signals, and subsequently affect the promoter driven gene expression.

From results in Figure 3A, the artificially transfected promoter worked well. However, it is necessary to examine whether the parental promoter still responses to the external stimulating factors to increase the PD-L expression after the transfection. Herein, IFN-γ was used to treat the cells and the PD-L1 expression was determined. The results revealed that the PD-L1 expression in the parental cells, negative control cells, and selected colonies all responded to IFN-γ stimulation very well (Figure 4-3B). The PD-L1 levels were all significantly increased after the treatment, suggesting that the transfection did not affect the original PD-L1 promoter. To verify that all the PD-L1 protein change happened transcriptionally, RT-PCR experiment was performed (Figure 4-3.C). The results demonstrate that the mRNA of PD-L1 was all increased after the IFN-γ treatment, suggesting that the increased PD-L1 is from transcriptional up-regulation. One of the

96 colonies #9-4 always showed better response to IFN-γ treatment in all the experiments.

Therefore, we finally identified colony #9-4 as the cells for the high throughput screening assay development and validation. The colony showed nearly double of the luciferase reading after the IFN-γ treatment compared to the no treatment control (Figure 4-3. A), which match the western blot results of the increased PD-L1 expression after IFN-γ treatment. The vehicle negative control cells did not respond to IFN-γ stimulation in terms of the luciferase activity. All the results demonstrated that the promoter driven luciferase expression was successful installed in the cells and responded to the external stimulation very well.

Figure 4-2. Schematic representation of the hygromycin resistant luciferase reporter plasmid, pGL4.26.luc2.minP.hygro. Location of the luc and amp genes are estimated based on restriction digest patterns. A) Diagram of pGL4.26 [PD-L1 promoter-luc2/minP/Hygro]. PD-L1 promoter fragment cDNA is amplified from plasmid pGL4.26 with primers containing Xho I at 5’ and 3’ ends. The primers will generate two Xho I sites. B) Multiple cloning region of the pGL4.26[luc2/minP/Hygro]

Vector.

Figure 4-3. Validation of the transfected Hey1B cells. A) Identification of colonies that show higher luciferase reading. After IFN-γ treatment, the cells showed increased luciferase activity, indicating that the transfected promoter works well; B) Verification of the identified colonies that show high luciferase activity still respond to IFN-γ stimulation for PD-L1 expression; C) RT-PCR determination if the stimulation of PD-L1 by IFN-γ is from transcriptional level. The experiment was independently repeated for three times and the representative one or the mean are presented.

100

B3.3 Pilot screening of the two compound libraries

The identified cells were further used to develop the high throughput screening assay. For the 96 well plate, 10000 cells per well and 24 hours treatment was the best condition for the assay. Two compound libraries were used for the screening, including LOPAC (1280

FDA approved drugs or drug candidates in clinic trails) and a natural product library with

400 compounds. In each 96 well plate, IFN-γ was used as the positive control and DMSO was the negative control. All the compounds were tested at one dose (10 µM). The Z-factor is a well-used parameter to measure the statistical effectiveness of the high throughput screening assay. For an assay development study, it is necessary to examine the Z-factor of the experiment. The Z-factor is defined with four parameters: the means (µ) and standard deviations (σ) of both the positive (p) and negative (n) controls (µp, σp, and µn, σn). The Z value is calculated with the formula: Z=1-[3(σp+ σn)/(µp- µn)]. If the Z value is between 0.5 and 1, then the assay is an effective one. If the Z value is below 0.5, then the assay is less effective. After the pilot screening of the 1680 compounds, the Z value was calculated for each 96 well plate for the positive control IFN-γ (3 wells) and the negative control vehicle

DMSO (3 wells). The result is listed in table 4. It is noticed that all the Z factors in the experiments are above 0.5, suggesting that the assay development is successful.

101

Table 4. Z factor of all the plates in the screening assay.

The negative control is DMSO and positive control is IFN-γ and all the controls run in

triplicate.

Plate number Z factor Compound library 1 0.913 LOPAC 1 2 0.683 LOPAC 2 3 0.703 LOPAC 3 4 0.894 LOPAC 4 5 0.631 LOPAC 5 6 0.724 LOPAC 6 7 0.766 LOPAC 7 8 0.585 LOPAC 8 9 0.682 LOPAC 9 10 0.913 LOPAC 10 11 0.791 LOPAC 11 12 0.825 LOPAC 12 13 0.747 LOPAC 13 14 0.892 LOPAC 14 15 0.707 LOPAC 15 16 0.622 LOPAC 16 17 0.798 Natural product 1 18 0.915 Natural product 2 19 0.679 Natural product 3 20 0.905 Natural product 4 21 0.727 Natural product 5 22 0.956 Natural product 6 Average 0.775

102

With the 1680 tested compounds, the majority of them did not affect the luciferase activity (Figure 4-4.A). However, there are some compounds that showed stimulating effect to increase the luciferase activity, which is not what we try to search for. In addition, when the compounds with the stimulating effect were further tested at 1 µM, the change of the luciferase activity was diminished. It is unclear how these compounds enhanced the luciferase activity at higher concentration, but it is not worthy further investigation at this stage. Some compounds did show suppressing activity to the luciferase activity (Figure 4-4.A). Unfortunately, they showed cytotoxicity to the cells as well, which was indicated by the change of the cell morphology after the treatment.

The decreased luciferase activity could just be a reflection of the lower cell viability at this concentration. The compounds with higher cytotoxic effect were excluded from further study. After dose justification, there are five compounds that still significantly decreased luciferase activity, but did not affect the cell viability at the same concentration (Figure 4-4.B, 4-4.C). The structures of the five compounds are listed in table 5. These five compounds were reported to show different biological activities.

Brefeldin A is a lactone antibiotic and inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus. Methotrexate is a chemotherapy agent and immune system suppressant. The other three compounds, Digoxin, Digitoxin, and Ouabain all have steroid structures, and are cardiac glycosides. Digoxin and Digitoxin are widely used in the treatment of late stage heart failure. It is unclear if these reported biological activities of these compounds have any correlation to the decreased luciferase activity in the experiment. The compounds may also affect other cellular machinery that might

103 be related to the regulation of the PD-L1 promoter activity and then the promoter driven luciferase activity, which is exactly what the assay is designed for.

104

105

Figure 4-4. High throughput screening assay results in the identification of several compounds that affect the luciferase activity. A) Screening of all the 1680 compounds;

B) Five compounds decrease luciferase activity; *P <0.01 vs. DMSO by unpaired t test, #P

<0.05 vs. DMSO by unpaired t test. C) The compounds did not affect the cell viability at the same concentration, indicating that the decreased luciferase activity is not due to the lower cell viability. The experiment of the five compounds was independently repeated for three times and the representative one or the mean are presented.

106

Table 5. The structures of the five identified compounds.

Compound name Structures Molecular Reported biological weight activity Brefeldin A(BFA) 280.36 A lactone antibiotic and inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus Methotrexate(MTX) 454.44 An antimetabolite of the antifolate, used as a chemotherapy agent and immune system suppressant Digoxin 780.94 A cardiac glycoside, widely used to treat heart failure.

Digitoxin 764.98 A cardiac glycoside, widely used to treat heart failure.

Ouabain 728.77 A cardiac glycoside, used as a tool to investigate the mode of action of cardiotonic steroids.

107

B3.4 The identified compounds affect PD-L1 level in cancer cell lines.

The five compounds significantly decreased the luciferase activity in the Hey1B-#9-4 ovarian cancer cells. Theoretically, they will suppress PD-L1 expression based on our hypothesis. These compounds were tested with western blot assay to examine their effect on PD-L1 expression in the same cells. Very opposite to our hypothesis, these compounds significantly stimulated PD-L1 expression in the cells (Figure 4-5.A). To confirm whether this is a unique phenomenon in Hey1B cells, the compounds were also tested with T24 kidney cancer cells as well (Figure 4-5.B). The results revealed that the activity was consistent in the two cell lines.

To determine if the regulation of PD-L1 is from transcriptional level, RT-PCR experiment was performed with both Hey1B and T24 cells. The results showed that the compounds could enhance the mRNA levels of PD-L1 (Figure 4-5.C & 5D), suggesting that these compounds did affect the PD-L1 expression from transcriptional level. Taking all the results together, we speculate that the inconsistent of the luciferase activity and the PD-L1 expression very likely is due to the simplified promoter of PD-L1 that drives luciferase gene activation. The sequence of the promoter in our study to drive luciferase expression may not be the full promoters used by PD-L1. The screening based on the luciferase activity results in identification of compounds that could decrease the promoter activity and subsequently decrease luciferase activity. However, this decreased of luciferase could only reflex the narrowed promoter. The whole promoter of PD-L1 might be more broad and complex than the reported one, and the PD-L1 expression could be multiple pathways controlled. The designed PD-L1 promoter driven luciferase expression in our system may

108 mimic only a small part of the complicated regulation network. Blockade of this promoter activity may quickly activate the feedback loop, which could stimulate other part of the unknown promoters that even further increase the PD-L1 transcription to compensate the blockade of the experimental used promoter in our model.

The results demonstrate that the regulation of PD-L1 is much more complex than what has been documented. It is possible that some feedback loops are involved when the inhibition of the transcription of PD-L1 takes place. However, for the luciferase, the feedback mechanism enhanced stimulation could not enhance the luciferase gene activation.

Therefore, the identified compounds decreased the luciferase activity, whereas increased

PD-L1 expression.

The results overall suggest that the whole promoters for the transcriptional regulation of

PD-L1 are much more complicated than the current understanding. More research is needed to further elucidate the whole promoters and other factors that regulate PD-L1 expression.

109

A B

C D

Figure 4-5. The identified five compounds including Brefeldin A (BFA, 1µM),

Methotrexate (MTX, 1µM), Digoxin (0.1µM), Digitoxin (0.1µM) and Quabain (0.1µM) increase PD-L1 expression from transcriptional level, although they decrease luciferase activity. A) The compounds increase PD-L1 expression in Hey1B cells; B) The compounds increase PD-L1 expression in T24 cells; C) The compounds increase mRNA of PD-L1 in Hey1B cells; D) The compounds increase mRNA of PD-L1 in T24 cells. IFN-

γ (2000u/mL) was used as the positive control. The experiments were independently repeated for three times and the representative ones are presented.

110

B4. Discussion

To search for small molecules that can regulate PD-L1 expression in cancer cells, a reporter luciferase assay was developed with Hey1B ovarian cancer cells transfected with a PD-L1 promoter driven luciferase gene. The cells responded well to IFN-γ stimulation for the luciferase activity. Two small molecule compound libraries were used for the high throughput screening, and several compounds were identified that decreased luciferase activity. However, these compounds were found to stimulate PD-L1 expression, which is opposite to the hypothesis. Further investigation revealed that the promoter used to drive the luciferase expression may not be the whole promoter region of PD-L1 that is actually not fully understood yet. The blockade of the current promoter decreased the luciferase activity. Whereas the PD-L1 expression was increased after the blockade of this know promoter with the identified compounds, which is likely due to the feedback mechanism to compensate the blockade of the promoter. The reflect arc even increased the PD-L1 expression through other part of the whole PD-L1 promoters. Overall, our current assay is effective to search for small molecules that could interfere with the current reported PD-

L1 promoter. The pilot study demonstrates that it is feasible to use the luciferase reporter assay to identify such novel agents to regulate PD-L1 expression. After the whole promoters of PD-L1 are fully elucidated in the future, better assay could be developed based on our current work.

111

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