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DOPAMINE D2 RECEPTOR ANTAGONISTS AS POTENTIAL

THERAPEUTICS FOR GLIOBLASTOMA MULTIFORME

A Dissertation in

Biomedical Sciences

Jillian S. Weissenrieder

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

May 2019

The dissertation of Jillian S. Weissenrieder was reviewed and approved* by the following:

Raymond J. Hohl Professor of Medicine and Pharmacology Dissertation Advisor Chair of Committee

Jeffrey D. Neighbors Assistant Professor of Pharmacology and Medicine

Kent E. Vrana Professor of Pharmacology

Xuemei Huang Professor of Neurology, Pharmacology, and Neurosurgery

David J. DeGraff Assistant Professor of Pathology, Surgery and Biochemistry & Molecular Biology

Ralph Keil Director, Biomedical Sciences Graduate Program

*Signatures are on file in the Graduate School.

ABSTRACT

Recent reports have suggested that dopamine D2 receptor antagonists may have potential to be repurposed as anticancer therapeutics. These compounds, including the phenothiazine chemotype and others, have a long history as therapeutics and some are approved in the US as antipsychotics in patients with schizophrenia. Thus, they have well- known pharmacokinetic, pharmacodynamic, and safety profiles that position them well for repurposing for other indications, such as anticancer treatment. While much is known about their safety and efficacy in the context of schizophrenia, little is known about their mechanism of action as anticancer agents.

There is a great disparity in compound affinity for the D2 (and D2-like) receptors and anticancer efficacy. Thus, we hypothesized that the anticancer activity of these compounds was due to an “off-target” effect, not direct action at the D2 receptor. Indeed, though these compounds have broad cytotoxicity in glioblastoma multiforme (GBM) monolayer cultures, these effects both require high, non-D2 selective concentrations and are apparently independent of D2 receptor signaling. We observe a dependence on calcium signaling to elicit this cytotoxicity which may be blocked by calcium chelation with

BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis

(acetoxymethyl ester)). Thus, we conclude that the mechanism for acute cytotoxicity of D2 receptor antagonists may require calcium overload.

On the other hand, GBM cells do express functional D2 receptors. The presence of these receptors may affect spheroid formation, a marker of stemness, at more selective concentrations of D2R modulators. This may provide another potential mechanism by which these compounds may provide therapeutic benefit at a lower and perhaps more

iii selective concentration. However, we did not see any changes in stemness markers at these concentrations, only distortions in spheroid-forming behaviors. These findings suggest that other factors, such as metabolism or cell-cell adhesion, are altered in D2-antagonist treated

GBM cells to contribute to these spheroid formation phenotypes. Overall, we conclude that

D2 receptor antagonists may provide some limited therapeutic efficacy for patients with few treatment options, but that this effect is not due to direct interactions with the D2 receptor.

TABLE OF CONTENTS

List of Figures ...... viii

List of Tables ...... ix

List of Abbreviations ...... x

Acknowledgements ...... xv

Chapter 1. The Need for Novel Cancer Therapeutics ...... 1

Introduction...... 1

Glioblastoma Multiforme ...... 2

Current Therapeutics ...... 4

Limitations of Current Treatment Options ...... 5

Repurposing/Repositioning Existing Compounds ...... 7

Conclusion...... 8

Chapter 2. The Role of the Dopamine D2 Receptor in Cancer ...... 9

Preface ...... 9

Abstract ...... 10

Introduction...... 11

Clinical studies of dopaminergic drugs and cancer ...... 13

Cancer and the “non-neuropharmacology” of dopamine receptor ligands ...... 15

D2R is a potential target for anticancer therapies...... 16

D2R receptor signaling mechanisms and cancer cell growth ...... 21

Autophagy may be affected by D2 antagonists...... 26

Lipid synthesis and trafficking are altered by D2R antagonist treatment...... 27

D2R antagonists may interact positively with other anticancer compounds...... 29

Critical interpretation and future directions ...... 30

Tables ...... 34

Figures ...... 43

Chapter 3. Dopamine D2 Receptor Antagonists in Glioblastoma ...... 46

Preface ...... 46

Abstract ...... 48

Methods...... 49

Results ...... 55

Conclusions ...... 59

Respective Contributions ...... 59

Figures ...... 60

Chapter 4. Dopamine D2 Receptor Modulation in Spheroids ...... 72

Preface ...... 72

Abstract ...... 73

Introduction...... 75

Materials/Methods ...... 77

Results ...... 79

Conclusions/Discussion ...... 83

Respective Contributions ...... 84

Figures ...... 85

Chapter 5. Dopamine D2 receptor antagonists and the isoprenoid pathway ..... 91

Preface ...... 91

Abstract ...... 92

Introduction...... 92

Methods...... 95

Results ...... 96

Conclusions ...... 99

Respective Contributions ...... 101

Figures ...... 102

Chapter 6. Future Directions ...... 107

Introduction...... 107

D2R antagonists and calcium signaling ...... 107

Immunological effects of D2R antagonists ...... 109

D2R antagonists and spheroids ...... 110

Anticancer efficacy of D2 antagonists in humans ...... 112

Central nervous system modulation of anticancer effects ...... 112

Conclusions ...... 113

Chapter 7. Conclusions ...... 114

Introduction...... 114

D2R antagonists elicit D2R-independent cytotoxicity in GBM cells...... 114

Conclusions: Providing Context ...... 116

References ...... 118

vii

LIST OF FIGURES

Figure 1. Organization of the dopamine receptor family...... 43

Figure 2. Forest plot of risk ratios from Table 2, by ID number...... 44

Figure 3. D2 antagonists affect vital metabolic processes in cancer...... 45

Figure 4. The dopamine D2 receptor is present and signals in GBM cell lines. ... 60

Figure 5. Cyclic AMP levels in GBM cells are reduced by D2 agonists...... 61

Figure 6. D2 antagonists induce cytotoxicity in GBM cell lines...... 62

Figure 7. Thioridazine induces apoptosis in GBM cell lines...... 64

Figure 8. D2R is not involved in cytotoxic responses to D2 antagonists...... 65

Figure 9. D2R antagonist cytotoxicity is independent of D2R...... 66

Figure 10. D2 antagonists except remoxipride alter calcium signaling in GBM. . 68

Figure 11. Calcium signaling is altered in D2 antagonist-treated GBM cells...... 70

Figure 12. Spheroid formation is altered by D2 modulators...... 85

Figure 13.Genetic modulation of D2R alters spheroid formation capacity...... 86

Figure 14. The D2R is involved in observed spheroid formation phenotypes...... 87

Figure 15. D2R modulators alter spheroid behaviors but not stemness markers. . 89

Figure 16. The isoprenoid biosynthetic pathway...... 102

Figure 17. Isoprenoid pathway inhibitors sensitize GBM cells to thioridazine. . 104

Figure 18. IBP intermediates do not strongly affect thioridazine sensitivity. .... 105

Figure 19. Methyl-schweinfurthin G sensitizes GBM cells to thioridazine...... 106

viii

LIST OF TABLES

Table 1. Timeline of D2 receptor pharmacology and early cancer findings...... 34

Table 2. Cancer risk in schizophrenia and Parkinson’s disease patients...... 35

Table 3. Ligand affinities of select D2 antagonists (nM)...... 37

Table 4. D2 antagonist IC50 values in cell culture...... 38

Table 5. Tolerated human plasma levels of selected D2 antagonists...... 40

Table 6. D2 antagonist efficacy in animal studies...... 41

LIST OF ABBREVIATIONS

5-FU 5-fluorouracil

7-AAD 7-aminoactinomycin D

ABCA1 ATP binding cassette protein A1

ABCG1 ATP binding cassette protein G1

ADAM A disintegrin and metalloproteinase protein

Akt Ak strain transforming (aka PKB, protein kinase B)

AM azoxymethyl ester

AML acute myelogenous leukemia

ANOVA analysis of variance test

APOE apolipoprotein E

ATCC American Type Culture Collection

ATF4 activating transcription 4 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis BAPTA-AM (acetoxymethyl ester)

BBB blood brain barrier

BRCA1/2 breast cancer gene ½ cAMP cyclic adenosine monophosphate

CDK2 cyclin dependent kinase 2

CEPIA calcium-measuring organelle-entrapped protein indicators

CHO Chinese hamster ovary

CI confidence interval

CNS central nervous system

CREB calcium responsive element binding protein

CSC cancer stem-like cells

Cyt C cytochrome C

D2R dopamine D2 receptor

DGBP digeranyl bisphosphonate

DRD1/2/3/4/5 dopamine receptor D1/2/3/4/5 gene

Dvl-3 disheveled-3

EC50 effective concentration 50%

EGF epidermal growth factor

EGFR epidermal growth factor receptor

EGFRvIII epidermal growth factor receptor variant III (active)

ER endoplasmic reticulum

ERK extracellular signaling regulated kinase

FDA Food and Drug Administration

FDPS farnesyl diphosphate synthase

FOH Farnesol

FOXO forkhead box O transcription factor

FPP farnesyl pyrophosphate

G1 growth 1 phase of cell cycle

GBM glioblastoma multiforme

GFAP glial fibrillary acidic protein

GFP green fluorescent protein

GGDPS geranylgeranyl diphosphate synthase

GGOH Geranylgeraniol

GGPP geranylgeranyl pyrophosphate

GPCR G-protein-coupled receptor

GSK3β glycogen synthase kinase 3 beta

HBSS Hank's buffered salt solution

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase hnPSC human neoplastic pluripotent stem cells

IC50 inhibitory concentration 50%

ICAM intracellular adhesion molecules

IDH1 isocitrate dehydrogenase 1

IL-6 interleukin 6

IP3R inositol-3-phosphate receptor

JAK Janus kinase

Ki inhibitory constant

KLF5 Kruppel like factor 5

LC3 microtubule-associated protein 1A/1B-light chain 3

LXRα/β liver X receptor alpha/beta

xii

MAPK mitogen activated protein kinase

MCU mitochondrial uniporter

MeSG methyl schweinfurthin G

MGMT O6-methylguanine-methyltransferase

MOPS 3-(N-morpholino)propanesulfonic acid

MTT 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide

MYC Myc protooncogene, bHLH transcription factor

NPC Niemann Pick type C

PBS phosphate buffered saline

PD Parkinson's disease

PDAC pancreatic ductal adenocarcinoma

PERK phosphorylated protein kinase RNA-like endoplasmic reticulum kinase

PHNO naxagolide hydrochloride (4-propyl-9-hydroxynaphthoxazine)

PI3K phosphoinositide-3 kinase

PKA protein kinase A

PKC protein kinase C

PMCA plasma membrane calcium ATPase

PP2A protein phosphatase 2A

PPARγ peroxisome proliferator-activated receptor gamma

PTEN phosphatase and tensin homolog

RB1 retinoblastoma 1

xiii

ROS reactive oxygen species

RTK receptor tyrosine kinase

SCZ Schizophrenia

SEM standard error of the mean

SERCA sarco-endoplasmic reticulum calcium ATPase

SOAT-1 sterol-O-acyltransferase 1

SOX2 SRY-box 2

SREBP sterol responsive element binding protein

SS squalene synthase

STAT signal transducer and activator of transcription

T-ALL T-cell acute lymphoblastic leukemia

TCF transcription factor

TG Thapsigargin

TMZ Temozolomide

TP53 tumor protein 53

TRAIL tumor necrosis factor-related, apoptosis-inducing ligand

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor

Wnt Wingless/Integrated

βArr2 β-arrestin-2

xiv

ACKNOWLEDGEMENTS

I would like to express my gratitude to the many individuals who supported and contributed to the body of work contained herein. Firstly, my advisor, Dr. Raymond J. Hohl provided critical insights and supported my studies, encouraging me to branch out and collaborate extensively. Secondly, I would like to thank Dr. Jeffrey D. Neighbors for his aid with experimental design and communication. My critical thinking, experimental design, and communication skills were also substantially improved as a result of the tireless assistance of Dr. Richard B. Mailman.

Similarly, I would like to extend my thanks to the remaining members of my committee: Dr. Kent E. Vrana, Dr. Xuemei Huang, and Dr. Dave Degraff. This work also required extensive collaboration. In that vein, I would like to thank Dr. Mohamed Trebak and Martin Johnson for their insight and training in calcium signaling, as well as Dr.

George-Lucian Moldovan for his aid in stable cell line development. The Flow Cytometry,

Microscopy, and Genomics Cores at the Penn State College of Medicine also provided resources critical for my research, for which I am grateful.

Finally, I would like to recognize my family and friends for their support during my graduate school career. Most notably, I would like to thank my husband, J. Dylan

Weissenkampen, for his unwavering support and tireless, cheerful assistance with manuscript edits. I would also like to thank my former teachers, including Wendy

Maganas, for believing in me and my former research advisor, Dr. Edward Rajaseelan, for pushing me to further my career in the sciences. This work was funded by, but may not represent the opinions of, the Penn State Cancer Institute, the Pritchard Distinguished

Graduate Fellowship, and the National Cancer Institute (T32CA060395-21A1).

CHAPTER 1. THE NEED FOR NOVEL CANCER THERAPEUTICS

Introduction

Cancer, a common disease characterized by abnormal cells dividing in an unregulated manner which can invade other tissues, will affect about one third of all men and women alive today (Sasieni et al., 2011; Lewis et al., 2017). Cancer cells provide their own growth signals, resist anti-growth signals, evade apoptosis, stimulate angiogenesis, metastasize to distant locations within the body, and replicate indefinitely (Hanahan and

Weinberg, 2000). Such uncontrolled growth can lead to symptoms such as tumor masses, difficulty with normal biological processes (such as digestion, urination, or breathing), fatigue, and eventually, death. It is thought that genetic predisposition, environmental exposures, and random mutational events in cells contribute to the pathogenesis of this disease.

Cancer has been known since at least the times of early Egypt, though the name

“cancer” comes from Hippocrates, who used the word “karkinos,” or crab, to describe the appearance of tumor masses with veins protruding out like crab legs (Hajdu, 2011). During these early times, attempts to treat cancer included rudimentary surgical resection, bloodletting, and humour-balancing treatments, which rarely helped and often could kill patients on their own (Hajdu, 2011). Today, cancer is better understood and treatment options are available to help extend survival and improve quality of life for many cancer patients, though not all will benefit from current therapeutics. However, there is still much that we do not understand about this disease. The limits of our understanding constrain our development of new therapeutics which could further improve patient outcomes. In this

dissertation, the need for new chemotherapeutic agents will be discussed, followed by the study of a potential novel therapeutic agent for glioblastoma multiforme (GBM).

Glioblastoma Multiforme

Cancer can affect many different systems within the body, and a cancer which starts in one organ will often metastasize to another. In the United States of America, the most common cancers affect the breasts, lungs, prostate, colon, skin, thyroid, liver, blood, and female reproductive tract (Jemal et al., 2017). However, the most lethal cancers are those of the pancreas, central nervous system, and lung (Jemal et al., 2017). These cancers are often aggressive, metastasizing and highly invasive (Hu et al., 2016; Popper, 2016;

Giovannetti et al., 2017). Resection is also complicated by the need to leave healthy tissue intact to maintain survival; while a patient can survive without a kidney, the brain and lungs are essential for survival.

Of particular interest for this work, glioblastoma multiforme (GBM) is a central nervous system cancer caused by the uncontrolled growth of glial cells (Thakkar et al.,

2014). Glial cancers, termed gliomas, are some of the most common central nervous system cancers, accounting for over three quarters of malignant CNS cancers (Ostrom et al., 2013).

GBM, also known as grade IV astrocytic glioma, accounts for roughly half of brain tumors and the majority of glioma deaths (Jemal et al., 2010; Louis et al., 2016; Rasmussen et al.,

2017). While less than 5 out of 100,000 people worldwide will develop this cancer, their median survival is less than two years (Thakkar et al., 2014; Louis et al., 2016; Batash et al., 2017). GBM is most common in patients over sixty years of age, though it can occur in children (van Tellingen et al., 2015; Alexander and Cloughesy, 2017). Although occurrence is most common in Caucasian populations, GBM can also occur in those of

Hispanic, African, and Asian ethnicities (Ostrom et al., 2018). Occurrence is 60% higher in males than in females (Ostrom et al., 2018).

Genetically, a number of mutations are common in GBM and may be tied to treatment response. Patients may be stratified based on their isocitrate dehydrogenase 1

(IDH1) and O6-methylguanine-methyltransferase (MGMT) status, which have been correlated with treatment response and overall prognosis (Parsons et al., 2008; Aldape et al., 2015). Another common mutation in GBM is the constitutively active epidermal growth factor variant III mutation (EGFRvIII) (Cancer Genome Atlas Research Network, 2008).

As in many cancers, tumor protein 53 (TP53), phosphatase and tensin homolog (PTEN), and retinoblastoma 1 (RB1) are also commonly mutated (Cancer Genome Atlas Research

Network, 2008). However, GBM is highly heterogeneous from patient to patient, with different driver mutations and different chemotherapeutic responses complicating therapy

(Cancer Genome Atlas Research Network, 2008). Even within a single tumor, clonal evolution leads to high levels of heterogeneity within the same tumor (Sottoriva et al.,

2013; Patel et al., 2014).

GBM tumors are particularly lethal due to their privileged location in the brain where they are subject to limited immune surveillance (Iwasaki, 2017). As these tumors grow, pressure increases within the brain and may cause severe headaches, seizures, memory loss, mood swings, and disruption of survival functions, leading to dysregulation of breathing, blood pressure, and body temperature (Alexander and Cloughesy, 2017).

These sequela often lead to death as tumor tissue infiltrates the brain more fully, though non-CNS metastasis is rare (Hsu et al., 1998). Critically, this is one of the few cancers that

3 does not typically lead to death through metastasis, instead causing death by putting pressure on key structural elements of the brain as the tumor expands.

Current Therapeutics

Currently, the standard of care for glioblastoma involves maximal surgical resection, followed by radiotherapy and concomitant and adjuvant chemotherapy with temozolomide (TMZ) (Alexander and Cloughesy, 2017). Alternating electric field therapy was also FDA-approved within the past decade for this indication, though it showed no greater efficacy than the standard of care and has not yet become a common treatment modality (Stupp et al., 2012).

As opposed to some other cancers, resection is still considered to be one of the most important parts of GBM treatment. Increased extent of resection is tied to increased survival, but at the cost of permanent neurological deficits (Chaichana et al., 2014). In general, GBM tumors are difficult to surgically resect due to the sensitivity of surrounding tissues and the typical diffuse nature of GBM tumor margins, which tend to invade along white matter tracts (Pessina et al., 2017). Recurrence is also still common, even in the context of full hemispherectomies (Dandy, 1928). Still, resection reduces tumor bulk, often lessening the severity of neurological symptoms and providing a significant, if small, increase in survival.

Radiotherapy plays a prominent role in GBM treatment as well. Focal irradiation of the tumor is often carried out as 30 fractions of 2 Gy exposures, for a total of 60 Gy

(Stupp et al., 2005; Alexander and Cloughesy, 2017). Studies suggest that increasing exposure to 70 Gy had no significant survival benefit (Nelson et al., 1988). Increasingly, this is both concomitant with, and followed by, treatment with TMZ chemotherapy.

Complicating treatment even more, the blood-brain barrier (BBB) excludes many chemotherapeutics from the brain (van Tellingen et al., 2015). The standard of care chemotherapeutic agent, TMZ, is able to access the brain at high doses, where it can methylate DNA, causing DNA damage and inducing cell death (Stupp et al., 2005). The protein MGMT removes this methylation, thus reducing TMZ efficacy to marginal, yet significant, levels (Hegi et al., 2005). In patients with MGMT promoter methylation, this protein is inactivated and TMZ response is more pronounced (Hegi et al., 2005). Due to compound toxicity (discussed below) and the low magnitude of response in patients who lack MGMT promoter methylation, there is some question as to the value of treatment of such patients with this medication (Alexander and Cloughesy, 2017). Nevertheless, regardless of MGMT status, patients are usually treated with TMZ.

Limitations of Current Treatment Options

Like many other cancers, GBM treatment is fraught with complications due to off- target effects. When patients undergo standard-of-care therapy, they are exposing themselves to the possibility of negative effects from all three prongs of this treatment combination. Surgical resection engenders all of the typical potential adverse effects of surgery, including increased infection risk, pain, and prolonged healing time, but patients may also have permanent neurological damage due to this process (Chaichana et al., 2014;

Pessina et al., 2017). Similarly, patients can develop hematological toxicities, increased risk of infection, and profound fatigue when they undergo radiation therapy (Stupp et al.,

2005; dos Santos et al., 2015). Finally, like most compounds that rely on DNA damage to induce cytotoxicity, TMZ causes many side effects in normal tissue that may include hematological toxicities, nausea/vomiting, and neurological symptoms (Wick et al., 2012).

In combination, the risk of neurological issues, hematological toxicities, profound fatigue, and infection are greatly elevated, but survival is only increased by 6-12 months over untreated patients (Brodbelt et al., 2015). Thus, in many patients, life may be prolonged but at the detriment of quality of life.

A major limitation of GBM treatment is the relative lack of options available for patients. Few compounds are readily delivered to the tumor across the BBB, and tumor heterogeneity reduces the likelihood of a sustained response due to clonal evolution. That said, there are a few other options available to patients who are not eligible for, or interested in, standard of care treatments. As mentioned above, alternating electric current therapy has shown some efficacy, with fewer side effects than the standard of care (Stupp et al.,

2012). Similarly, other chemotherapeutic agents have been suggested as potential treatments for GBM, but are less common in the clinic; these include irinotecan, carboplatin, and paclitaxel, as well as bevacizumab. Most rely on DNA damage and thus have similar toxicities as TMZ though they may have varying delivery systems. As an example, direct implantation of carmustine wafers in the space left by surgical resection of tumor bypasses the BBB entirely and demonstrated some efficacy in a clinical trial

(Westphal et al., 2003).

Although targeted therapies have shown promise in many other forms of cancer, they have shown limited efficacy in GBM, largely due to compound delivery issues and tumor heterogeneity (Touat et al., 2017). For instance, immune checkpoint inhibitors are able to cross the BBB and show efficacy in preclinical models, but appear to elicit little response in clinical testing (Maxwell et al., 2017; Touat et al., 2017). Thus, the rise of

6 targeted therapies and personalized medicine has, as yet, had little impact on the treatment of GBM, leaving patients with few options and a poor prognosis.

Repurposing/Repositioning Existing Compounds

It is evident that new, more efficacious treatment options are needed for

GBM. Yet, the development of therapies is very costly and time-consuming, and the vast majority of candidate compounds fail in the clinic (Wong et al., 2018). This attrition is usually due to unforeseen toxicities or lack of sufficient efficacy (Harrison, 2016).

Repurposing compounds which are in advanced stages of development or are already FDA- approved for other indications reduces the time cost and financial burden of drug development significantly (Sleire et al., 2017). In cancer, gemcitabine was originally designed as an antiviral agent which was instead successfully repurposed as a chemotherapeutic. Another example is celecoxib, an anti-inflammatory cyclooxygenase inhibitor which was initially approved for the treatment of arthritis but is now approved for use in familial adenomatous polyposis and under study for anticancer efficacy (Sleire et al., 2017).

As noted previously, one of the greatest hurdles in treatment of GBM is in development of compounds which are both efficacious and capable of crossing the BBB.

This barrier is a significant impedance to drug development for this indication. However, a plethora of psychoactive drugs are available on the market which are known to cross the

BBB in order to elicit their effects. These include antipsychotic and mood-modulating compounds that are used to treat myriad disorders, from schizophrenia and bipolar disorder to depression and anxiety. These represent a potential pool of compounds to screen for anticancer efficacy. Indeed, compound screens have identified some of these compounds,

7 most notably the antipsychotics, as efficacious anticancer agents in cell culture (Rho et al.,

2011; Dolma et al., 2016). Most of the psychoactive compounds identified by these screens are known to antagonize the dopamine D2 receptor (D2R) and act as antipsychotic medications. If these compounds prove to have anticancer activity at safe dosages in human patients, their ability to cross the BBB would position them well for use in anti-GBM therapy.

Conclusion

D2R antagonists provide a potential source of FDA-approved compounds to reposition as anticancer agents for central nervous system cancers such as GBM. If efficacious, this would save time and money, delivering potential therapeutics to patients in a speedy and economical way. In the chapters that follow, the anticancer activity of these agents will be described. Chapter 2 will discuss the state of the literature as it pertains to the D2R and its pharmacological modulators. Then, Chapter 3 will describe the mechanism of action for D2R antagonists, such as thioridazine, in the context of GBM. The role of the

D2R in spheroid formation and the cancer stem cell phenotype is the topic of Chapter 4, while Chapter 5 covers the effects of mevalonate pathway modulation under treatment with

D2R inhibitors. Finally, Chapter 6 and Chapter 7 will discuss future directions and conclusions that can be drawn from this work.

CHAPTER 2. THE ROLE OF THE DOPAMINE D2 RECEPTOR IN

CANCER

Preface

A relatively small group of FDA-approved compounds are known to cross the blood-brain barrier in appreciable amounts. These compounds are of potential interest for repurposing as anticancer agents in cases where tumors exist on the protected side of this barrier, such as in gliomas (Alexander and Cloughesy, 2017; Touat et al., 2017). As previously mentioned, current chemotherapies for these cancers have severely limited efficacy due to reduced drug delivery. Compounds which are known to cross the BBB are thus well poised for repurposing for anti-CNS cancer therapeutics.

Recently, one class of these molecules, the dopamine D2 receptor antagonists, has received some interest as potential anticancer agents (Schalop and Allen, 2016). In the past thirty years, numerous research groups have independently identified D2 antagonists of varying chemotypes as effective anticancer agents in vitro and in vivo. The literature hints at a number of different mechanisms, but no solid conclusions have been made at this time due to the complex pharmacology of these compounds. This chapter reviews the current literature on this subject from a pharmacological perspective to provide a basis for future studies. Human correlational data and mechanistic data are emphasized, as they provide a basis for future work in the area.

Abstract

The D2R family is upregulated in many cancers and tied to stemness, while cancer risk may correlate with dopamine-related disorders such as schizophrenia and Parkinson’s disease. D2R antagonists also have anticancer efficacy in cell culture and animal models where they reduce tumor growth, induce autophagy, affect lipid metabolism, and cause apoptosis, among other effects. This has led to the hypothesis that D2R ligands are a novel approach to cancer chemotherapy, a particularly appealing concept because of the large number of approved and experimental drugs of this class. We review the current state of the literature, and the evidence for and against this hypothesis. When the existing literature is evaluated from a pharmacological context, one of the striking findings is that the concentrations needed for cytotoxic effects of D2R antagonists are orders of magnitude higher than their affinity for this receptor. Although additional definitive studies will provide further clarity, our hypothesis is that targeting D2–like dopamine receptors will not yield useful ligands for cancer chemotherapy.

Introduction

The concatenation of cancer and neuropharmacology

The serendipitous discovery of chlorpromazine (Delay et al., 1952; Delay and

Deniker, 1955) over sixty years ago may be considered a landmark in several ways. Besides offering the first effective treatment of some of the symptoms of schizophrenia, it opened new doors to an understanding of the chemoarchitecture of the brain, especially the role of dopamine (Carlsson et al., 1958; Carlsson and Lindqvist, 1963). This led to millions of people being treated with drugs that targeted dopamine receptors. In psychiatry, this complicated a decades-long debate about whether schizophrenia itself affected cancer risk

(for review, see Gulbinat et al., 1992). Gulbinat et al. (1992) noted that pharmacological mechanisms were of particular interest, especially because some phenothiazine-based drugs had antitumor activity in murine leukemia and melanoma, and high concentrations of the antipsychotics or their metabolites were found in the lung (Driscoll et al., 1978).

These latter findings might explain a lower occurrence of malignancies sometimes reported in schizophrenia. Conversely, because classical antipsychotics markedly increased serum prolactin due to antagonism of inhibitory dopamine receptors on anterior pituitary lactotrophs, this also might explain an increased risk of breast cancer in females (Gulbinat et al., 1992). These early observations led to the hypotheses, first suggested in 1972, that dopamine receptor ligands might be a potential therapeutic approach in cancer (Csatary,

1972).

Dopamine receptors

Dopamine receptors are members of the heptahelical G protein-coupled receptor

(GPCR) superfamily that is divided pharmacologically into two subfamilies (Figure 1):

”D1-like”; and “D2-like” (Garau et al., 1978; Kebabian and Calne, 1979). The molecular biology and pharmacology of these receptors have been the subject of numerous reviews and books (Neve and Neve, 1997; Mailman and Huang, 2007). Dopamine receptors are encoded by five genes, with DRD1 and DRD5 encoding the two D1-like receptors (D1 and

D5), and DRD2, DRD3, and DRD4 encoding four expressed mammalian proteins (D2long,

D2short, D3, and D4). D2long and D2short are splice variants from DRD2 and together are the most highly expressed of the D2-like receptors (Dal Toso et al., 1989; Giros et al., 1989;

Monsma et al., 1989b; Chio et al., 1990). As noted earlier, the first drugs that were shown to bind to dopamine receptors (e.g., chlorpromazine) were discovered serendipitously because of effects in controlling positive symptoms of schizophrenia. The target of early antipsychotic drugs was soon identified, then validated, via radioreceptor studies and receptor cloning (Burt et al., 1976; Seeman et al., 1976; Dal Toso et al., 1989; Giros et al.,

1989; Monsma et al., 1989a; 1990). When using drugs as research tools, it is imperative to understand the relative effects of a molecule on both primary and secondary targets; antipsychotics in particular have many off-target actions. In addition, although they may have selectivity for one subfamily of dopamine receptor, there is often much less selectivity for an individual member (e.g., D2 vs. D3 vs. D4). Thus, when we discuss clinical findings, reference to “D2” will be a reference to D2-like affinity unless otherwise specified.

There is a rich literature on both agonist and antagonist effects on dopamine receptors, but it has largely been focused on central nervous system modulation of dopamine function in the context of schizophrenia and other brain disorders (Neve and

Neve, 1997). In the periphery, dopamine is known to play an important role in cardiovascular control and kidney function. The notion that dopamine receptor ligands

12 might affect the biology of neoplastic cells independent of their actions on neurotransmission is provocative, and offers both a novel mechanism and the ability to both purpose and repurpose the huge libraries of dopaminergic ligands and drugs that have resulted from neuropharmacological drug discovery and development (Schalop and Allen,

2016). Thus, an examination of this arena is timely.

Clinical studies of dopaminergic drugs and cancer

Correlative studies and case reports support a role for the D2 receptor in cancer development and treatment response.

To date, all antipsychotic drugs engage D2 receptors, usually as antagonists (Creese et al., 1976; Mailman, 2007; Boyd and Mailman, 2012b), whereas therapy for PD relies primarily on activation of dopamine receptors indirectly via levodopa, or directly by direct agonists (Mailman and Huang, 2007). The accepted targets of current dopamine agonists in PD have been the D2 and D3 receptors. Although some findings suggest a greater role for D1 receptors (Taylor et al., 1991; Mailman et al., 2001), the clinical data of relevance to this topic deals with D2R-targeted therapeutics.

Investigations into the relationship between D2R antagonists and cancer began almost as soon as these drugs were approved for psychiatric indications (Table 1), starting with isolated case reports of increased treatment response from cancer patients treated concurrently with antipsychotics (Osterman, 1961; Csatary, 1972; Eicke, 1973; Hercbergs,

1988). Correlative studies of cancer risk in the context of other diseases strengthened this anecdotal association (Table 2, Figure 2). By the 1980s, population-based correlative studies to determine cancer risk within groups of patients with schizophrenia and PD were underway. Many studies showed clear, significant differences in cancer development, yet

13 methodologies were quite variable, and cohorts often small. Some studies were prospective and followed matched cohorts, whereas others mined national healthcare databases. These differences complicate arriving at a unitary hypothesis.

Of particular note was a study of more than 100,000 age- and gender-matched, primarily Han Chinese schizophrenia patients in which both male and female subjects showed a strong inverse correlation for age and development of cancers (Wu et al., 2013).

One possible explanation for this trend is that older patient populations had undergone long-term treatment with neuroleptic agents that might have attenuated the increased risk inherent in schizophrenics. This study was limited, however, by the lack of ethnic diversity, as well as the lack of stratification for other risk factors, such as smoking status.

The D2 receptor is expressed in a number of cancer cell lines and in patient samples.

D2 receptor expression has been reported at both the mRNA and protein level in a variety of cancers. Increased immunohistochemical staining has been reported in cervical, esophageal, and lung cancers, often correlating with tumor grade or survival (Li et al.,

2006; Hoeppner et al., 2015; Kanakis et al., 2015; Mao et al., 2015; Cherubini et al.,

2016b). In acute myeloid leukemia (AML), D2R protein is also highly expressed. DRD2 mRNA levels are elevated in breast cancer (Pornour et al., 2014), ovarian cancer (Moreno-

Smith et al., 2011), glioma (Li et al., 2014), and neuroblastoma (Deslauriers et al., 2011).

Peripheral blood mononuclear cells of breast cancer patients express DRD1, DRD2, DRD3, and DRD4 mRNA (Pornour et al., 2014). Because the D3R and D4R have significant homology to the D2R and often recognize the same drugs, these D2-like receptors may also be relevant.

These studies, however, are often limited by small numbers of patient samples, a lack of blinding, and poor antibody specificity (Stojanovic et al., 2017). Moreover, few studies have ascertained both protein and mRNA levels of the D2R, and no histochemical studies have published replicate data with other probes to verify selectivity. Importantly, reported mRNA levels have typically been quite low, so large fold-changes in mRNA presence may have little functional impact. Most notably, many studies that have shown the anticancer efficacy of dopamine receptor ligands (e.g., the antipsychotics) have failed to show definitive presence of D2R protein or message. Critically, the literature lacks reports of D2R ligand binding or functional assays in the context of cancer.

Cancer and the “non-neuropharmacology” of dopamine receptor ligands

Some of the earliest indications of anticancer activity for D2R ligands were from

Driscoll et al. (1978) and Akiyama et al. (1986). Micromolar concentrations of phenothiazine antipsychotics reversed KB cell line resistance to doxorubicin, vinblastine, dactinomycin and daunorubicin in a non-calmodulin dependent manner (Akiyama et al.,

1986). In contrast, another study concluded that a reduced proliferative effect of the D2- like antagonists thioridazine and pimozide in MCF-7 cells was due to calmodulin antagonism (Strobl et al., 1990). Yet, Iishi et al. (1992) soon reported that the D2-like agonist bromocriptine promoted gastric carcinogenesis in a rat model, shortly followed by the suggestion of genetic linkage between the DRD2 gene and BRCA1-competent breast cancer (Cortessis et al., 1993). Whereas these early studies hinted at a potential role for

D2R antagonism in cancer development and treatment, there are some issues that should be considered in interpreting these data. In particular, the effects of the four antipsychotic

15 drugs noted above require concentrations two or more orders-of-magnitude higher than their KD (Table 3).

D2R is a potential target for anticancer therapies.

Since 2003, several screening studies identified D2R antagonists as potential therapeutics for cancer treatment based on their biological activity and/or presence in cancer cells. Like calmodulin inhibitors, phenothiazines selectively increased forkhead box

O (FOXO) transcription factor nuclear localization in 786-O renal cell adenocarcinoma cells (Kau et al., 2003), yet FOXO localization remained unchanged when treated with D2R antagonists of different chemotypes (i.e., clozapine and haloperidol) to control for off- target effects. Although this suggests that the D2R is not involved in the mechanism of action, it contrasts with previous reports noting that D2R agonist treatment increases phospho-Akt levels in neurons, an effect that would be expected to exclude FOXO proteins from the nucleus (Brami-Cherrier et al., 2002; Kihara et al., 2002). Nuclear localization and transcriptional activity of FOXO3 in the human breast cancer BT549 cell line, however, was increased by 5 μM concentrations of the calcium channel blocker bepridil or the antipsychotic trifluoperazine (Park et al., 2016).

An in silico screening approach suggested thioridazine may inhibit the Akt/PI3K pathway as well (Rho et al., 2011). Experimentally, thioridazine (20 μM) decreased PI3K pathway activation, inhibited cell cycle progression at G1, reduced cell viability, and induced apoptosis via caspase-3 cleavage over 24 h of treatment in a manner that was additive with paclitaxel and cisplatin. This suggested that phenothiazines could impact

Akt/PI3K signaling in a cell type specific manner, but target engagement was not verified and may not involve the D2R (Rho et al., 2011). More recently, Gutierrez et al. (2014)

16 performed dual screening in search of compounds that were toxic toward zebrafish thymocytes that overexpress MYC and synergized with Notch inhibitors in human T cell acute lymphoblastic leukemia (T-ALL) cells. They identified several phenothiazines

(including perphenazine and chlorpromazine) as potential anti-T-ALL treatments that bound protein phosphatase 2A (PP2A) (Gutierrez et al., 2014).

Two large-scale screens identified the D2R protein itself as a potential target that is up-regulated in pancreatic cancer and glioblastoma multiforme. The D2R and its associated

G protein Gαi2 were highly up-regulated in pancreatic ductal adenocarcinoma tissue samples (Jandaghi et al., 2016). In an shRNA screen to identify genes necessary for GBM cell line survival, the D2R was also identified (Li et al., 2014). Inhibition of D2R signaling with shRNA, siRNA, and several antagonists (i.e., spiperone, haloperidol, risperidone, and

L-741,626) reduced cell viability, proliferation, and clonogenicity in U87MG glioblastoma cells. To our knowledge, this was the only study to show that DRD2 knockdown reduces cell viability and tumor growth.

D2R antagonists reduce cell proliferation and induce apoptosis in vitro.

During the past twenty years, other studies also have identified D2R antagonists as potential anticancer therapeutics through in vitro studies utilizing cell lines and patient samples (Table 4). Phenothiazines, most notably thioridazine, have been suggested as anticancer therapeutics more often than other chemotypes, but haloperidol, pimozide, and olanzapine also have been studied. These compounds have been shown to reduce cell viability, induce apoptosis, cause necrotic cell death, induce cell cycle arrest, and alter protease activity (Figure 3). This anticancer activity is apparent in a broad range of cancer types, including sex-specific (Kang et al., 2012; Mao et al., 2015; Park et al., 2016; Ranjan

17 et al., 2016; Ranjan and Srivastava, 2016; Zhou et al., 2016), pancreatic (Ranjan and

Srivastava, 2016), nervous system (Gil-Ad et al., 2004; Daley et al., 2005; Levkovitz et al.,

2005; Shin et al., 2012; Shin et al., 2013; Li et al., 2014; Karpel-Massler et al., 2015), blood

(Zhelev et al., 2004), oral (Choi et al., 2014), lung (Yue et al., 2016), gastric (Mu et al.,

2014) and renal (Min et al., 2014) cancers, among others (Levkovitz et al., 2005; Nagel et al., 2012). Typical in vitro cell viability assay IC50 values for D2R antagonists range from

5-20 μM, yet D2R antagonists appear to be only modestly selective for cancer cells.

Fibroblasts were less sensitive to pimozide treatment than five different pancreatic cancer cell lines, but there was a trivial difference in IC50 (twofold selectivity, 10 vs. 20 μM)

(Jandaghi et al., 2016). Astrocytic cell lines were also less sensitive to haloperidol as compared to GBM cells (Li et al., 2014). These concentrations exceed the known maximum tolerated plasma concentrations in humans (Table 5), and suggest a narrow therapeutic window or even dose-limiting toxicity if applied to clinical use. In most cases, cytotoxic concentrations of these compounds are much higher (>100 fold) than would be expected for a D2R based mechanism, as determined from D2R receptor affinity (Table 3).

It is possible that this is due to differences in receptor environment or functional partners, but it is also important to consider other mechanisms, especially because of the multiple targets that high concentrations of these drugs might engage (Besnard et al., 2012).

In vivo models of cancer suggest efficacy of D2R antagonism.

Animal models of cancer have suggested that D2R antagonists might have chemotherapeutic utility (Table 6). Authors have reported significant reductions in tumor growth with D2R antagonist treatment in gastric, glial, ovarian, medulloblastoma, oral, lung, pancreatic, prostate, and breast cancer xenograft models. Many of these studies

18 observed evidence of Akt signaling inhibition and/or alterations in autophagic flux in vivo.

In an OVCAR-3 murine xenograft model, 10 mg/kg of thioridazine, trifluoperazine, or chlorpromazine reduced tumor growth, but an equivalent dose of fluperazine was found to be toxic to the animals (Choi et al., 2008), again suggesting a narrow therapeutic window.

A dose of 300 μg/day thioridazine or 400 μg/day mepazine reduced tumor size by half in

OCI-Ly10, but not in Su-DHL-6, xenograft models (Nagel et al., 2012). These doses led to compound plasma levels of 200 ng/mL, well below the achievable plasma level of 2,000 ng/mL in humans.

In summary, many animal studies have suggested that D2R antagonists are efficacious in reducing tumor size and prolonging survival in xenograft models. In general, plasma and tumor drug concentrations were not quantified, but they may be expected to be well above selective concentrations. When measured in one study, plasma levels were, however, less than those achievable in human patients (Table 5) (Nagel et al., 2012).

Unfortunately, toxicity of some compounds was observed. Therefore, D2 receptor involvement is difficult to ascertain based solely on pharmacological data. Ideally, such findings would be corroborated by studies which employ genetic methods in order to identify a target. To our knowledge, only one study has probed the role of DRD2 in a xenograft model in this way. In this study, a doxycycline-inducible DRD2 knockout in

U87MG intracranial xenografts prevented tumor growth in Nu/Nu mice, providing support for a role of D2R in cancer growth (Li et al., 2014). While most of these studies were carried out in the context of immunodeficient mice, it is also tempting to speculate on the effects that D2R modulators may have on the immune system through both indirect and direct means (i.e., through psychoactive effects or through direct interaction with immune cells).

D2R antagonists are associated with anti-CSC activity.

D2R expression is also implicated in stem-like cells (CSCs), hypothesized slow- cycling cells that promote tumor growth, chemoresistance, and metastasis. One in silico study using the Connectivity Map identified phenothiazines, notably trifluoperazine, as potential therapeutic agents capable of reversing stem-like gene expression profiles (Yeh et al., 2012). Trifluoperazine concentration-dependently induced apoptosis in a patient derived, gefitinib-resistant lung cancer cell line, and reduced clonogenicity in a number of other patient-derived lines, regardless of epidermal growth factor (EGFR) status. In a green fluorescent protein (GFP) reporter-based screen for Oct4 and Sox2 in human neoplastic pluripotent stem cells (hnPSCs), thioridazine appeared to target CSCs with an EC50 of 7

μM; prochlorperazine and fluphenazine were also identified, but not further characterized in this work (Sachlos et al., 2012). D2R antagonists, including thioridazine (10 µM), reduced cell number and colony forming units in AML samples and hPSCs (Sachlos et al.,

2012). This work was the first to conclude that D2R activity contributes to the survival and function of CSCs, employing both agonists and antagonists to examine this possibility. In glioblastoma CSCs, similar results were seen for the D2R functionally selective/partial agonist aripiprazole (10 μM) (Suzuki et al., 2016) as well as the D2R antagonists thioridazine and trifluoperazine and the selective D4 antagonists PNU 96415E and L-

741,742 (Dolma et al., 2016). Taken together, all of these results suggest that D2R is expressed in CSCs and may impact stemness.

D2R receptor signaling mechanisms and cancer cell growth

STAT and RTK signaling

Signal transducer and activator of transcription (STAT) proteins are attractive therapeutic targets because of their role in cellular proliferation and angiogenesis. In a screen for potential STAT5 inhibitors using chronic myelogenous leukemia cell lines, the

D2R antagonist pimozide (5-10 µM) decreased STAT5 phosphorylation and function, even downstream of potent oncogenic activation (Nelson et al., 2011). Moreover, pimozide inhibited interleukin-6 (IL-6)-induced growth and migration via inhibition of STAT3 in prostate cancer cells (Zhou et al., 2016). It is unknown if other D2R antagonists inhibit

STAT directly, but they reduce pro-neoplastic receptor tyrosine kinase (RTK) signaling upstream of Janus kinase (JAK)/STAT. The D2R agonists quinpirole (10 μM) and pramipexole (10 μM) both increased phosphorylation of extracellular signaling regulated kinase (ERK) and RTK EGFR in a D2R-dependent manner (Yoon and Baik, 2013).

Antagonism with thioridazine reduced vascular epithelial growth factor receptor (VEGFR) phosphorylation and VEGF availability (Park et al., 2014). These studies suggest an

RTK/JAK/STAT mechanism or downstream effect of D2R antagonists and a possible role for D2R in pro-neoplastic EGFR signaling.

Wnt

The Wingless/Integrated (Wnt) pathway affects development, carcinogenesis, and stem-like behavior, and is reportedly inhibited by D2R antagonists. In a patient-derived lung cancer cell line, trifluoperazine concentration-dependently inhibited transcription factor (TCF)-mediated transcription (Yeh et al., 2012), with the decreases in Wnt signaling being concomitant with the induction of cytotoxicity. Such findings are supported by an in

21 silico docking and network analysis study identifying the Wnt pathway protein glycogen synthase kinase 3 beta (GSK3β) as potentially affected by phenothiazine treatment (Qi and

Ding, 2013). Spiperone (10 μM) had similar effects, but researchers observed that these were not mediated by D2R, serotonin, or σ1/2 receptor activity via comparison to selective receptor modulators, but may involve intracellular calcium signaling and protein kinase C

(PKC) (Lu and Carson, 2009). Furthermore, D2R and Wnt5a co-immunoprecipitated from

3 HEK293T cells with a KI (inhibition constant) of 165 nM for competition with [ H]- spiperone, suggesting a possible direct interaction (Yoon et al., 2011). The quinpirole- induced upregulation of Wnt pathway protein disheveled-3 (Dvl-3) induces ERK activation

-/- in mesencephalic neuronal culture, but did not occur using cells from D2R mice (Yoon et al., 2011). These data suggest that the D2R may interact with the Wnt pathway in neuronal cells and that D2R antagonists can decrease Wnt signaling, but further studies are needed to see if this is more broadly applicable to the malignant phenotype.

PI3K

The phosphoinositide 3 kinase/Ak strain transforming (PI3K/Akt) pathway, a critical regulator of the cell cycle, has been suggested as a target pathway for D2R antagonists in cancer-related cell lines. In Chinese hamster ovary (CHO) cells expressing the human D2R, dopamine and quinelorane activated the PI3K pathway by increasing phospho-Akt (at both Ser-473 and Thr-308) and GSK-3β (at Ser-9) levels, with maximal effects at 10 μM (Mannoury la Cour et al., 2011). Pertussis toxin, as well as D2R antagonists, blocked this, suggesting a dependence on D2R-linked G protein signaling.

When receptor internalization was blocked with phenylarsine oxide, phosphorylation levels were reduced by half. Similarly, disruption of cholesterol-rich lipid rafts with

22 methyl-β-cyclodextrin inhibited phosphorylation. These latter data suggest that both G protein and β-arrestin signaling are important. Increased Akt phosphorylation was PKC- and calmodulin-dependent, and GSK-3β phosphorylation was due, at least in part, to Akt activity. Thus, there is the potential for these mechanisms to affect cancer cell growth, proliferation, and metabolism via Akt downstream effectors, including transcription factors

(like FOXO). In vivo, 25 mg/kg thioridazine given every third day to 2774-xenografted

(ovarian cancer) nude mice reduced phosphorylation levels of PI3K, Akt, phosphoinositide-dependent protein kinase (PDK1) and mammalian target of rapamycin

(mTOR) (Park et al., 2014). In normal rat brain, however, D2R antagonist raclopride (3 mg/kg/day) enhanced phosphorylation at both Thr308 and Ser473 of Akt which indicates activation, but did not alter total Akt protein levels. In the same model, the agonist quinpirole reduced phosphorylation (Sutton and Rushlow, 2012). In normal brain, Akt phosphorylation is reduced by D2 receptor activation in a β-arrestin-2 (βArr2) mediated manner involving a complex with protein phosphatase 2A (PP2A) (Sotnikova et al., 2005).

Antagonism may increase the overall level of Akt phosphorylation or block cell sensitivity

-/- to βArr2-mediated Akt regulation (Beaulieu et al., 2004). D2R mouse striatal lysates have increased Akt phosphorylation at Thr-308 both basally and in response to amphetamine (3 mg/kg) challenge (Beaulieu et al., 2007). Overall, it appears PI3K signaling is increased by D2R agonists but reduced by D2R antagonists in malignant tissues, whereas the opposite may be true in normal tissues.

Thioridazine (15 μM) induced apoptosis and inhibited the PI3K/Akt pathway in endometrial and cervical cancer cell lines (Kang et al., 2012), and at similar concentrations had effects resembling PI3K/Akt inhibition (Rho et al., 2011), decreasing PI3K activity by

60%, inducing G1 arrest after 24 h treatment, reducing cell viability by half at 48 hours, and inducing apoptosis. Phosphorylation of Akt, mTOR, and GSK-3β were also reduced by several antidopaminergic phenothiazine drugs at low micromolar concentrations in epidermal growth factor (EGF)-stimulated OVCAR-3 ovarian cancer cells, although the concentration-response relationship did not parallel D2R affinity (Choi et al., 2008). PI3K activation was unaffected by these phenothiazines.

MAPK/ERK

The mitogen activated protein kinase (MAPK)/ERK pathway, known to be involved in cancer cell survival and proliferation, was inhibited in U87MG and A172 glioma cell lines by four different D2R antagonists, albeit at relatively high concentrations

(spiperone and haloperidol at 5 μM, risperidone and L-741,626 at 10 μM) (Li et al., 2014).

MAPK8 and MAPK10 were also identified as potential targets by a correlational in silico docking and network analysis study of phenothiazines, including chlorpromazine, fluphenazine, and trifluoperazine (Qi and Ding, 2013). This may involve a cascade wherein peroxisome proliferator-activated receptor gamma (PPARγ) interaction affects MAPK8 status, leading to a protein kinase modulated alteration of activity in downstream effectors cyclin dependent kinase 2 (CDK2) and GSK3β (see section on Wnt signaling). In normal rat and mouse brain slices, the D2 agonist quinpirole (60 μM) increased MAPK and calcium responsive element binding (CREB) phosphorylation, with effects blocked by the D2 antagonist eticlopride (40 μM), the calcium chelator BAPTA/AM, or the PKC antagonist

Go6976 (Yan et al., 1999). Although these investigators did not directly assay G protein activity, they hypothesized a role for Gαq activation (Yan et al., 1999), although the D2- like receptors normally are not considered to couple readily to this α-subunit. Due to the

24 heterogeneous nature of the system and use of healthy tissue, these findings may or may not have any relationship to the behavior of cancer cells exposed to ligands that modulate

D2R function.

Calcium signaling

D2R signaling and antagonist treatments both alter calcium signaling. Wolfe et al.

(1999) found that both the long and short D2R isoforms interacted with Gαo to reduce high- voltage-activated calcium channel activity. In wild-type astroglia, dopamine signaling is capable of both increasing and reducing intracellular calcium levels in a manner dependent on local neural type in brain slices (Jennings et al., 2016). Dopamine D2/D3 receptors were involved in the negative regulation of Ca2+ in this study.

The calcium channel blocker bepridil and the D2R antagonist triflupromazine had similar effects on PI3K signaling through FOXO3 in MDA-MB-231 breast cancer cells

(Park et al., 2016). FOXO3 activity was required to reduce colony formation with both trifluoperazine and bepridil, and FOXO3-regulated proteins D2R, Kruppel-like factor 5

(KLF-5) and c-MYC were downregulated by treatment with either drug. In vivo, 10 mg/kg trifluoperazine or bepridil three times a week significantly reduced tumor volume of MDA-

MB-231 xenografts in female athymic (nu/nu) mice (Park et al., 2016). A calmodulin mechanism was posited for both compounds, but not explored experimentally.

In pancreatic cancer lines MiaPaCa-2 and Panc-1, 10 μM pimozide or L-741,626 increased intracellular calcium levels sharply within seconds of treatment and concentration-dependently increased phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (PERK), suggesting an increase in endoplasmic reticulum

(ER) stress (Jandaghi et al., 2016). Protein kinase A (PKA) phosphorylation activity was

25 also modestly increased. Caspase activity upon treatment with pimozide was reduced by around 25% when activating transcription 4 (ATF4) was silenced with shRNA, further supporting the involvement of the unfolded protein response. Similar results were found for haloperidol, except IC50 values were increased and fibroblasts seemed even more resistant. Overall, it appears that multiple chemotypes of D2R antagonists can alter intracellular calcium levels and initiate cellular stress in cancer cells.

Autophagy may be affected by D2 antagonists.

Numerous studies have suggested that D2R antagonists are able to induce autophagic cell death in the context of in vitro and in vivo studies of cancer. One trifluoperazine derivative, A4, increased reactive oxygen species (ROS), DNA damage, and autophagic cell death, while also causing apoptosis and activating AMPK (Wu et al.,

2016). AMPK phosphorylation increases were also seen in D2R antagonist treated GBM stem cell cultures (Cheng et al., 2015). In SH-SY-5Y neuroblastoma cells, sertindole, pimozide, and trifluoperazine were identified as autophagy-inducing agents by a large- scale fluorescence-based screen (Shin et al., 2012). Increases in GFP-LC3 puncta were sertindole concentration- and time- dependent; autophagosome formation was also verified by electron microscopy. LC3 cleavage was responsive to 3-methyladenine, suggesting autophagic induction was partially regulated by the PI3K pathway. Conditional siRNA knockdown of the essential autophagic protein, ATG5, reduced autophagosome formation, enhanced cell viability, and reduced LC3 cleavage under treatment with 10 μM sertindole.

A fluorescence assay that included ROS scavengers indicated a partial role for reactive oxygen species in the cytotoxicity of sertindole. Similar results have been reported in glioma cell lines (Shin et al., 2013; Cheng et al., 2015). Although autophagy can contribute

26 to D2 antagonist-mediated cell death, D2 activity does not appear to be involved in this mechanism since thioridazine reduced D2R protein levels and increased autophagy, while trifluoperazine reduced D2R protein levels and did not increase autophagy at the same concentrations.

Lipid synthesis and trafficking are altered by D2R antagonist treatment.

An early study reported that chlorpromazine (10 μM) inhibited both sphingomyelinase activity and esterification of cholesterol in human fibroblasts in a manner comparable to 10 µM W-7, a known calmodulin antagonist (Masson et al., 1992).

Chlorpromazine treatment resulted in accumulation of unesterified cholesterol in lysosomal vacuoles reminiscent of a Niemann-Pick type C (NPC) lipidosis phenotype.

(Masson et al., 1992). Similar results were seen with 10-50 μM haloperidol, and concomitant insulin receptor signaling inhibition was reversed by cholesterol addback, suggesting lipid raft disruption (Sanchez-Wandelmer et al., 2010).

Other antipsychotics like haloperidol (10 μM) and clozapine (30 μM) increased cholesterol and fatty acid synthesis enzyme mRNA by 2-4-fold in GaMg glioma cells at 5-

10 h (Ferno et al., 2006). Sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and

SREBP-2), sterol-responsive transcription factors that regulate these genes, were upregulated at the protein level, supporting the idea that antipsychotic treatment may upregulate lipogenesis via SREBP signaling. Cholesterol-related mRNAs, including 3- hydroxy-3-methylglutaryl-CoA reductase (HMGCR), apolipoprotein E (APOE), ATP binding cassette A1 (ABCA1), liver X receptor alpha/beta (LXRα/β), and Niemann-Pick disease type C (NPC1/2) were increased after 24-48 h treatment with clozapine (25 μM), haloperidol (10 μM), olanzapine (10 μM), or imipramine in GaMg cells (Vik-Mo et al.,

2009). Protein levels of ApoE also increased in GaMg and HepG2 human hepatocellular carcinoma cells. Message level increases were more striking in glial cell cultures, suggesting the activation of LXR and its downstream targets may occur as an effect of earlier SREBP-modulated lipogenesis within the cell (Ferno et al., 2006). Lipogenesis and adequate cholesterol stores are essential for cancer cell survival, particularly in the case of glioma which are highly sensitive to exogenous cholesterol levels and LXR activity (Villa et al., 2016).

Although haloperidol and pimozide treatment (10 μM) slightly increased the expression of some SREBP-responsive genes, they also disrupted cholesterol trafficking, causing intracellular accumulation of unesterified cholesterol in intracellular puncta in

CHO-7 cells (Kristiana et al., 2010). Despite increases in active SREBP-2, cholesterol synthesis was ablated under treatment with these compounds. Aripiprazole, clozapine, quetiapine (all 10 μM), olanzapine, risperidone, and ziprasidone (25 μM) showed similar behavior, suggesting that the effect may be mediated by D2R or another common target of these compounds. Kristiana et al. (2010) posited that the intracellular trafficking of cholesterol was disrupted by these drugs, inhibiting SREBP cleavage activating protein

(SCAP) activation of SREBP and sterol O-acyltransferase 1 (SOAT-1) esterification of cholesterol. Similarly, 10-50 μM haloperidol reduces biosynthesis of cholesterol in SH-

SY-5Y cells while generating a buildup of sterol precursors (Sanchez-Wandelmer et al.,

2010). Risperidone, ziprasidone, and clozapine (5-25 µM) also induced buildup of sterol intermediates in HepG2 cells (Canfran-Duque et al., 2013).

Clearly, numerous chemotypes of D2R antagonist can reduce cellular cholesterol levels, disrupt lipid rafts, and alter lipid trafficking. These effects have, however, not been

28 shown to be the cause of D2R antagonist induced cytotoxicity; it is possible that lipid alterations are due to cellular coping mechanisms to deal with other types of stress, such as ROS or autophagic stress. Indeed, these lipid phenotypes indicate that cancer cells treated with these compounds behave as though they are lipid-starved and frustrated in their attempts to synthesize more.

D2R antagonists may interact positively with other anticancer compounds.

Studies also indicate that D2R antagonists can be additive with common chemotherapeutics. Aripiprazole sensitized CSC-enriched cultures to gemcitabine, 5- fluorouracil (5-FU), and cisplatin treatment in an additive manner (Suzuki et al., 2016).

Similarly, the pro-apopotic effects of trifluoperazine were synergistic with cisplatin (10

μM) and gefitinib (2.5-10 μM) in a patient derived lung cancer cell line (Yeh et al., 2012).

Tumor volume and weight of G362 GBM xenografts were decreased in mice treated with

20 mg/kg of either PNU 96415E or L-741,742 over control, though the difference in size is not large (Dolma et al., 2016). L-741,742 treatment on its own failed to improve survival of xenografted mice, but survival increased following co-treatment with temozolomide as compared to treatment with temozolomide alone. Similarly, thioridazine increased the efficacy of cytarabine in leukemia (Dolma et al., 2016), and cisplatin or paclitaxel in ovarian cancer (Rho et al., 2011). In treatment-resistant endometrial cancer cell lines ISK and KLE, combination treatment with 20 μM medroxyprogesterone acetate and 10 μM thioridazine reduced cell viability by half after 4 d (Meng et al., 2016). Such observations could potentially be explained by inhibition of P-glycoprotein (P-gp) or other efflux pumps associated with drug resistance, as suggested by the fact that thioridazine sensitizes chemoresistant oral squamous cancer cells (KBV20C) to vinblastine due to inhibited P-gp

29 efflux (Choi et al., 2014). Similarly, ATP binding cassette G2 (ABCG2) mediated chemoresistance in MDR cells is reduced by 10 μM of D3 antagonists PG01037, NGB2904,

SB277011A, and U99194 (Hussein et al., 2017).

Critical interpretation and future directions

As the literature currently stands, evidence is suggestive, but by no means conclusive, of an anticancer role for D2R antagonists. Correlative studies of patients with schizophrenia and PD, case studies of cancer patients under concomitant antipsychotic therapy, and repeated hits by unbiased screens support the notion that D2R may have a significant role in cancer development and may be a reasonable therapeutic target. Also,

D2R antagonists of varying chemotypes have anticancer activity both in vitro and in vivo, where they induce apoptosis, autophagic cell death, and cell cycle arrest (Figure 3). In some studies, they also induce CSC differentiation and/or disrupt cholesterol trafficking and synthesis. Such effects are favorable for anticancer therapies, especially since these compounds are modestly selective for cancer cells over normal cell type controls of various lineages.

Yet, although these compounds have effects and can affect many signaling pathways, the role of the D2R itself is still unclear. One major factor is that invariably the concentrations required to induce cytotoxicity are many orders of magnitude higher than the KD for this receptor. At these concentrations, this class of drug has many off-target actions. As approved drugs, there is a great deal of data regarding pharmacokinetics, pharmacodynamics, and toxicity profiles, which when considered in the light of the modest selectivity in cell culture studies, suggests that it may be difficult to achieve circulating plasma levels sufficient for meaningful anticancer activity (Table 5). Maximal circulating

30 levels are reported as concentrations of parent compound, although some of these compounds would also be present as active or inactive metabolites which may or may not have anticancer activities. Many D2R antagonists also have profound side effects that included marked increase in serum prolactin, large increases in body weight and metabolic syndrome, neurological side effects, and potentially fatal cardiac complications like torsades de pointes that results from QT prolongation. Although some of these are quite serious, they may be tolerable in patients with cancers that are unresponsive to other therapies, especially if the side effects are reversible. The question is whether there is an adequate therapeutic window and an adequate degree of efficacy.

Another issue arising from the high concentrations necessary for anticancer effects is that of target determination; it is far from clear that the D2R is a valid anticancer target based on pharmacological studies alone. Aside from the studies of Li et al. (2014) with

GBM, there is little in vitro or in vivo data to suggest that alteration of D2R levels can affect cell growth, viability, or response to D2R antagonist treatment. Studies to determine the role of the D2R will require both understanding of basic principles of pharmacology and the use of orthogonal approaches to decrease the likelihood of erroneous conclusions. Thus, if the D2R is hypothesized to be the target by which an antipsychotic drug kills or growth- inhibits cancer cells, then rigorous evidence must be provided to demonstrate that the receptor is both expressed on the cell type of interest and the principal target that needs to be engaged. Ideally a combination of approaches such as receptor binding assays, western blot, immunosorting analysis, mRNA quantification, molecular ablation, and the like, are needed to provide a rigorous test of the underlying hypothesis. Without these types of data, assigning activity to a specific target is risky.

Interestingly, a small molecule inhibitor of tumor necrosis factor-related, apoptosis- inducing ligand (TRAIL), ONC201, reduced proliferation and viability in HCT116 gastric cancer cells (Allen et al., 2016; Kline et al., 2018). ONC201 itself is a selective inhibitor of D2R, but its cytotoxicity was not eliminated by D2R knockdown or knockout. These results decrease the probability of a primary role of the D2R. Indeed, novel phenothiazine derivatives have been shown to have many potential anticancer activities aside from the established activities on calmodulin, dopamine receptors, and other known psychiatrically relevant targets. These include antioxidant ability, inhibition of tubulin polymerization, and inhibition of farnesyl transferase (Prinz et al., 2011; Baciu-Atudosie et al., 2012; Engwa et al., 2016; Ghinet et al., 2016).

In summary, we were attracted to this topic because it seemed like an excellent example of potential for drug repurposing with a known target (i.e., D2R) for which dozens of drugs are approved, and for which there are probably thousands of experimental compounds that already exist. If the D2R is a viable target, such a wealth of compounds and data would be a very fertile field for study. Yet, our attempt at a critical view of the literature has altered our initial opinion, such that we believe it is likely that the actions of

D2R antagonists both in vivo and in vitro are in most cases unlikely to involve effects mediated primarily by the D2 receptor. We recognize that much of the literature could be interpreted as arguing for the opposite hypothesis, but it would be useful if this generates controversy that leads to hypothesis-driven studies using orthogonal approaches and varying structural series of D2R antagonists. Such rigorous pharmacological and biochemical studies would settle this matter. Whether our supposition is correct or not, the

32 field will benefit from a clear resolution of these questions, and the knowledge might impact on the development of new therapeutic paradigms.

Tables

Table 1. Timeline of D2 receptor pharmacology and early cancer findings.

Year Event Source 1950 Chlorpromazine synthesized (Delay et al., 1952); Sigwald and Bouttier 1952 Chlorpromazine identified as antipsychotic (1953) Reactive oxygen species (ROS) are associated with 1959 antipsychotics at millimolar concentrations. (Dawkins et al., 1959) First published case reports of increased sensitivity to 1961-1988 chemotherapy with concurrent antipsychotic treatment. (Osterman, 1961) 1976-1979 2 dopamine receptor families identified (D1 like, D2 like) 1986 Phenothiazines can reverse doxorubicin resistance in KB cells. (Akiyama et al., 1986) 1988-1989 D2 receptor cloned (human and rodent). Pimozide and thioridazine reduce breast cancer cell 1990 proliferation (Strobl et al., 1990) Radiation sensitization of bone marrow under concurrent 1991 chlorpromazine treatment. (Jagetia and Ganapathi, 1991) Bromocriptine (D2 agonist) increases cancer growth and 1992 proliferation (rat gastric carcinogenesis model). (Iishi et al., 1992) DRD2 gene linked to breast cancer via linkage study in a single 1993 family lacking BRCA1 deficiency. (Cortessis et al., 1993) D2R are present and inducible by retinoic acid in SH-SY-5Y 1994 neuroblastoma cells. (Farooqui et al., 1994)

Table 2. Cancer risk in schizophrenia and Parkinson’s disease patients.

# Study Ratio 95% Type Cancer type Cohort Age Sex Diag- Note CI (n) nosis 1 (Barak et al., 0.58 0.48- SIR all types 3226 M/F SCZ 2005) 0.69 2 0.6 0.39- SIR Breast 1247 F SCZ 0.90 3 (Becker et al., 0.77 0.64- IRR all types 2993 M/F PD 2010) 0.92 4 0.47 0.25- IRR Lung 2993 M/F PD 0.86 5 0.33 0.18- IRR lymphoma/leukemia 2993 M/F PD 0.61 6 1.7 0.62- IRR Melanoma 2993 M/F PD 4.67 7 (Driver et al., 0.85 0.59- adjusted RR all types 487 M/F PD Prospective 2007) 1.22 8 0.32 0.07- adjusted RR Lung 487 M/F PD Prospective 1.53 9 0.54 0.14- adjusted RR Colorectal 487 M/F PD Prospective 2.16 10 6.15 1.77- adjusted RR Melanoma 487 M/F PD Prospective 21.37 11 (Jansson and ~.33 combined IR all types 406 M/F PD Jankovic, 1985) 12 (Jespersen et al., 0.73 0.63- adjusted OR Prostate 45429 M PD case control 2016) 0.83 13 (Lichtermann et 1.17 1.09- SIR all types 26996 M/F SCZ al., 2001) 1.25 14 2.17 1.78- SIR Lung 26996 M/F SCZ not controlled for smoking 2.6 15 (Wu et al., 2013) 0.92 0.9- SIR all types 102202 All M/F SCZ Declines with age 0.96 16 1.97 1.85- SIR all types 102202 20- M/F SCZ 2.33 29 17 0.68 0.65- SIR all types 102202 60- M/F SCZ 0.78 69 18 0.36 0.34- SIR all types 102202 >70 M/F SCZ 0.45 19 (Hamaue et al., <1 SIR all types 246 M/F PD retrospective, not significant (small 2000) sample size) 20 (Moller et al., 0.88 0.8-1.0 relative risk all types 7046 M/F PD national cohort 1995) 21 0.29 0.2-0.4 relative risk Lung 7046 M/F PD 22 1.96 1.1-3.2 relative risk Melanoma 7046 M/F PD 23 0.42 0.2-0.7 relative risk Bladder 7046 M/F PD

24 (Munk- 0.9 IRR all types 6152 all M/F SCZ Jorgensen and Mortensen, 1989) 25 0.76 IRR all types 2956 all M SCZ 26 1.06 IRR all types 3196 all F SCZ 27 0.38 IRR respiratory 6152 all M/F SCZ 28 (Eaton et al., 0.33 0.12- IRR Prostate 38 all M SCZ Treated with high dose neuroleptics (e.g. 1992) 0.94 chlorpromazine) 29 (Mortensen, 0.79 SIR all types 9156 all M/F SCZ with SCZ diagnosis 1994) 30 1.06 SIR all types 5658 M SCZ 31 0.68 SIR all types 5658 M SCZ before SCZ diagnosis 32 0.77 SIR all types 3498 F SCZ before SCZ diagnosis 33 0.86 SIR all types 3498 F SCZ 34 (Wang et al., 2.16 1.55- Adjusted OR all types 6211 all M/F PD Patients prescribed ergot-derived 2015b) 2.99 dopamine agonists

Table 3. Ligand affinities of select D2 antagonists (nM).

Reference Reference Reference Referenc Reference Compound D2 Ligand Source D1 Ligand Source D3 Ligand Source D4 e Ligand Source D5 Ligand Source 0.9 (Besnard et 3H- (Besnard et (Shapiro et al., 3H- (Besnard et 3H- (Besnard et Aripiprazole 3H-NMSP 387 9.7 3H-NMSP 514 1676

5 al., 2012) SCH23390 al., 2012) 2003) NMSP al., 2012) SCH23390 al., 2012) (Besnard et 3H- (Besnard et 3H- 3H- (Besnard et 3H- (Besnard et Chlorpromazine 2 3H-NMSP 112 1.3 (Seeman, 2006) 24 133

al., 2012) SCH23390 al., 2012) raclopride NMSP al., 2012) SCH23390 al., 2012) 77. 3H- (Millan et 3H- (Toll et al., 3H- (Millan et al., Clomipramine 219 50.1 6 spiperone al., 2001) SCH23390 1998) spiperone 2001) 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et Clozapine 431 3H-NMSP 189 340 nemonaprid (Seeman, 2006) 39 235

al., 2012) SCH23390 al., 2012) NMSP al., 2012) SCH23390 al., 2012) e 3H- 0.5 (Besnard et 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et Fluphenazine 3H-NMSP 24 0.3 nemonaprid (Seeman, 2006) 36 12

4 al., 2012) SCH23390 al., 2012) NMSP al., 2012) SCH23390 al., 2012) e 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et Haloperidol 2 3H-NMSP 83 23 nemonaprid (Seeman, 2006) 15 147

al., 2012) SCH23390 al., 2012) NMSP al., 2012) SCH23390 al., 2012) e 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et 3H- (Besnard et Olanzapine 72 3H-NMSP 58 40 nemoniprid (Seeman, 2006) 19 90

al., 2012) SCH23390 al., 2012) NMSP al., 2012) SCH23390 al., 2012) e ROTH BL, 3H- (Burt et al., 3H- (Burt et al., 3H- Penfluridol 5.6 1600 31 ET AL., haloperidol 1976) dopamine 1976) spiperone

1995 (Burt et al., 3H- 1976; 3H- (Billard W, et 3H- (Seeman, Perphenazine 1.4 3H-NMSP 29.9 1.1 nemonaprid (Seeman, 2006) 32

Kroeze et SCH23390 al., 1984) spiperone 2006) e al., 2003) 125I- 0.6 (Kroeze et 3H- (Toll et al., (Sokoloff et al., (Burstein et Pimozide 3H-NMSP >104 11 iodosulprid 1.8 R-SAT 5 al., 2003) SCH23390 1998) 1992) al., 2005) e 125I- Prochlorperazin 3H- (Seeman, (Sokoloff et al., 3H- (Seeman, 4 1.8 iodosulprid 70

e spiperone 2006) 1992) spiperone 2006) e 3H- (Besnard et 3H- (Besnard et 3H- Besnard et 3H- (Besnard et Risperidone 4.9 3H-NMSP 60.6 5.2 nemonaprid (Seeman, 2006) 18.6 16

al., 2012) SCH23390 al., 2012) NMSP al., 2012) SCH23390 al., 2012) e 3H- 3H- 214 (Grandy et 3H- (Sunahara et >10 (Neumeyer et al., 3H- Van Tol, HH 3H- (Sunahara et SCH 23390 domperidon 0.35 nemonaprid 3560 0.3

5 al., 1989) SCH23390 al., 1991) 000 2003) spiperone et al., 1991 SCH23390 al., 1991) e e 3H- (Sunahara et (Cussac et al., 0.1 (Grandy et 3H- 0.27 3H- 3H- Tang L, et al. 3H- (Sunahara et Spiperone domperidon 577 al., 1991; Toll 2000; Neumeyer 4 4500

25 al., 1989) SCH23390 5 spiperone spiperone 1994 SCH23390 al., 1991) e et al., 1998) et al., 2003) (Grandy et al., 1989; 3H- (Besnard et 3H- 3H- (Besnard et 3H- (Besnard et

Thioridazine 10 3H-NMSP 89 5.2 (Seeman, 2006) 17 216

Besnard et SCH23390 al., 2012) spiperone NMSP al., 2012) SCH23390 al., 2012) al., 2012) (Seeman et Black: human receptor/tissue (Kroeze et 3H- (Burt et al., 3H- al., 1997; Red: rat receptor/tissue Trifluoperazine 1.3 3H-NMSP 740 44 al., 2003) dopamine 1976) spiperone Besnard et Blue: calf receptor/tissue al., 2012)

Table 4. D2 antagonist IC50 values in cell culture.

IC50 Experiment Compound (uM) type Cell Line Source Note aripiprazole 10-100 trypan blue PANC-1, PSN-1, A549 (Ikai et al., 2016) chlorpromazine 5-10 MTT U87MG, GBM8401 (Cheng et al., 2015) found autophagy induction SK-MEL-28, HT29, Colo205, chlorpromazine 4.8-14.5 MTT SW480,HCT116,MCF7 (Choi et al., 2008) chlorpromazine 8 CellTiter-Blue KOPT-K1 (Gutierrez et al., 2014) chlorpromazine 10 CCK-8 U87 MG (Shin et al., 2013) K-562, Daudi, Raji, BALL-1, MOLT-4, HPB-ALL, CCRF- chlorpromazine ~10 CellTiter-Glo HSB-2 (Zhelev et al., 2004) fluphenazine 5-10 MTT U87MG, GBM8401 (Cheng et al., 2015) SK-MEL-28, HT29, Colo205, fluphenazine 3.9-7.9 MTT SW480,HCT116,MCF7 (Choi et al., 2008) SK-MEL-28, HT29, Colo205, haloperidol >25 MTT SW480,HCT116,MCF7 (Choi et al., 2008) haloperidol 5-15 Clonogenicity LN18,U87MG, T98G (Li et al., 2014) cancer stem cell selective (8 fold L-741,742 >=50 MTT BJ,U2OS,Daoy (Dolma et al., 2016) selectivity) L-741,742 1.56 MTT G380 (Dolma et al., 2016) (Karpel-Massler et al., olanzapine 25-79.9 MTT U87MG, A172, SC38, SC40 2015) MDA-MB-231, HCC-1806, (Ranjan and Srivastava, penfluridol ~5 MTT 4T1 2016) (Ranjan and Srivastava, penfluridol 3,4,5 MTT Panc-1, AsPC-1, BxPC-3 2016) perphenazine 5-10 MTT U87MG, GBM8401 (Cheng et al., 2015) perphenazine 7 CellTiter-Blue KOPT-K1 (Gutierrez et al., 2014) sulforhodamine Panc-1, CFPAC-1, Capan- pimozide 7-15 B 1,BxPC-3, MiaPaCa-2 (Jandaghi et al., 2016) pimozide ~10 MTT LNCaP, PC3M, 22RV1 (Zhou et al., 2016) cancer stem cell selective (8 fold PNU 96415E >50 MTT BJ,U2OS,Daoy (Dolma et al., 2016) selectivity) PNU 96415E 1.56 MTT G380 (Dolma et al., 2016) prochlorperazine >10 MTT U87MG, GBM8401 (Cheng et al., 2015) thioridazine 5-10 crystal violet NCI-N87, AGS (Mu et al., 2014)

K-562, Daudi, Raji, BALL-1, MOLT-4, HPB-ALL, CCRF- thioridazine ~10 CellTiter-Glo HSB-2 (Zhelev et al., 2004) thioridazine 5-10 MTT U87MG, GBM8401 (Cheng et al., 2015) thioridazine 12.5-17.5 EZ-Cy Tox KB, KBV20C (Choi et al., 2014) SK-MEL-28, HT29, Colo205, thioridazine 3.8-6.7 MTT SW480,HCT116,MCF7 (Choi et al., 2008) neutral red/ thioridazine 11.2-15.1 alamar blue C6, SHSY-5Y (Gil-Ad et al., 2004) neutral red/ thioridazine 41.3 alamar blue primary mouse brain (Gil-Ad et al., 2004) HeLa,C33A, Caski, HEC-1-A, thioridazine ~15 MTT KLE (Byun et al., 2012) thioridazine SiHa (Mao et al., 2015) thioridazine MTT ISK, KLE (Meng et al., 2016) slowed growth over 96 h at 10 µM thioridazine (Min et al., 2014) sensitized to TRAIL at 10 µM thioridazine ~15 MTT NCI-N87, AGS (Mu et al., 2014) ABC-DLBCL lines (HBL-1, thioridazine ~10 MTT Ocl-Ly3, U2932, TMD8) (Nagel et al., 2012) GCB-DLBCL lines (BJAB, Su- thioridazine >10 MTT DHL-6, Su-DHL-4) (Nagel et al., 2012) thioridazine 20 MTT SKOV-3 (Rho et al., 2011) thioridazine 1-10 CFU AML blasts (Sachlos et al., 2012) also reduces stemness flow cytometry (Annexin V/ thioridazine 10-20 PI) 4T1 (Yin et al., 2015) thioridazine ~10 MTT NCI-H1299, 95-D (Yue et al., 2016) trifluoperazine >10 MTT U87MG, GBM8401 (Cheng et al., 2015) SK-MEL-28, HT29, Colo205, trifluoperazine 4.3-7.7 MTT SW480,HCT116,MCF7 (Choi et al., 2008) trifluoperazine ~7 MTT MDA-MB-231, BT549 (Park et al., 2016) trifluoperazine 10-15 MTT Ca922 (Wu et al., 2016) trifluoperazine >10 MTT A549, H1975 (Yeh et al., 2012) K-562, Daudi, Raji, BALL-1, MOLT-4, HPB-ALL, CCRF- trifluoperazine ~10 CellTiter-Glo HSB-2 (Zhelev et al., 2004) trifluoperazine 5-10 MTT H69, U1285, U-1906, U-2020 (Zong et al., 2014)

Table 5. Tolerated human plasma levels of selected D2 antagonists.

[C] plasma in humans (nM) Compound Max Min Source thioridazine 2,699 270 (Smith et al., 1984) chlorpromazine 1,548 101 (Chetty et al., 1999) pimozide 32 2 (Kerbusch et al., 1997) olanzapine 40 31 (Kassahun et al., 1997) haloperidol 67 11 (Froemming et al., 1989) clozapine 4,525 1,007 (Guitton et al., 1998)

Table 6. D2 antagonist efficacy in animal studies.

Dose Compound (mg/kg) Timing Model Efficacy Source Note MNNG induced Wistar ~2.5 fold increase in bromocriptine 1-2 qd rats (gastric cancer) tumor number (Iishi et al., 1992) agonist OVCAR-3 xenograft, 64% tumor growth (Choi et al., chlorpromazine 10 qdX5 nude mice suppression 2008) 43.5% inhibition of (Shin et al., chlorpromazine 20 qd U87MG xenograft tumor growth 2013) OVCAR-3 xenograft, (Choi et al., fluphenazine 10 qdX5 nude mice toxicity – ND 2008) MiaPaCa-2 xenograft in ~50% decrease in tumor (Jandaghi et al., haloperidol 10 qd NSG mice mass 2016)

no significant reductions haloperidol 10 qd U87MG xenograft in tumor size or survival (Li et al., 2014) synergy with AG1478 cooperated with twofold increase in ticlopidine to enhance survival time over (Shchors et al., autophagy and increase imipramine 40 qd GRLp53het mice control 2015) survival 40.9% reduction in G362 xenograft, flank tumor mass, prolonged (Dolma et al., L-741,742 20 qd and intracranial survival 2016) synergy with TMZ 4T1 orthotopic mammary xenografts, 49% reduction in tumor (Ranjan and penfluridol 10 qd female Balb/c mice size Srivastava, 2016) 4T1-luc intracardiac 90% reduction in brain metastasis model, fluorescence from (Ranjan and penfluridol 10 qd female Balb/c luciferase reporter Srivastava, 2016) ~33% reduction in brain 4T1 intracranial fluorescence from (Ranjan and penfluridol 10 qd xenograft luciferase reporter Srivastava, 2016)

BxPC-3 xenografts (subcutaneous) in ~50% reduction in tumor (Ranjan and penfluridol 10 qd athymic nude mice volume at day 27 Srivastava, 2016) ~33% reduction in hTALL2 cells in NSG luciferase (Gutierrez et al., perphenazine 10 qd mice bioluminescence 2014) 44.3% reduction in (Dolma et al., PNU 96415E 20 qd G362 xenograft, flank tumor mass 2016) ~66% reduction in (Hoeppner et al., quinpirole 10 qd LLC1 xenograft bioluminescence 2015) agonist OVCAR-3 xenograft, 26% tumor growth (Choi et al., thioridazine 10 qdX5 nude mice suppression 2008) slight, insignificant pretreatment reductions in tumor size pretreatment with 5 µM thioridazine of cells NCI-N87 xenograft or survival (Mu et al., 2014) thioridazine

300 Ocl-Ly10 xenograft, (Nagel et al., thioridazine μg/animal qd nude mice 2012) >50% reduction in tumor size and volume, 2774 xenografts, nude significantly reduced (Park et al., thioridazine 25 qd mice Ki67 staining 2014) Oral delivery 4T1 xenograft in 55% reduction in tumor thioridazine 32 qd BALB/c volume (Yin et al., 2015) pretreatment ~50% reduction in tumor thioridazine of cells NCI-H1299 size at day 47 (Yue et al., 2016) 2 MDA-MB-231 ~50% reduction in tumor (Park et al., trifluoperazine 10 x/week xenografts, nude mice volume at day 33 2016) significant reduction in CL97 tail vein injection bioluminescence/ tumor trifluoperazine 5 qd NOD/SCID size (Yeh et al., 2012) OVCAR-3 xenograft, 46% tumor growth (Choi et al., trifluperazine 10 qdX5 nude mice suppression 2008)

Figures

Figure 1. Organization of the dopamine receptor family.

Dopamine receptors are G protein coupled receptors which are divided into the D1 and D2 like families. Some tissues of interest where these receptors are expressed are included here.

Cancer Risk Ratios for PD and SCZ

34 33 32 31 30 29 28 27 26 25 24 23 22 21

20 #

D I

18 y

d 17 u t 16 S 15 14 13 12 11 10 9 8 7 6 5 4 3 PD 2 SCZ 1 0.0625 0.125 0.25 0.5 1 2 4 8 16 32 Risk Ratio (95% CI)

Figure 2. Forest plot of risk ratios from Table 2, by ID number.

Bars represent 95% confidence intervals. Studies of PD patients are shown in blue, while studies of SCZ patients are in red.

Figure 3. D2 antagonists affect vital metabolic processes in cancer.

Cancer stem cell-like activities, survival signaling, and proliferation, are reduced by treatment. However, intracellular calcium levels, autophagy, and apoptosis are increased.

Additionally, lipid synthesis and trafficking are disrupted. The direct mechanisms by which these alterations occur is not currently known, but these compounds may ultimately lead to cell death through these or other pathways.

CHAPTER 3. DOPAMINE D2 RECEPTOR ANTAGONISTS IN

GLIOBLASTOMA

Preface

Given the wide variety of responses to D2 receptor antagonist treatment which are discussed in Chapter 2, it is quite possible that the anticancer activity of D2 antagonists is through a D2R-independent mechanism. In Chapter 3, we present experiments which detail the role of the D2R in compound treatment response, but it also explores the possibility of another potential mechanism of action through which D2 antagonists may exert their cytotoxic activity. Namely, this chapter explores the role of calcium signaling in D2R antagonist response.

Calcium signaling is known to be dysregulated in cancer (Stewart et al., 2015;

Cardenas et al., 2016), and may present a potential target for anticancer therapy (Cardenas et al., 2016; Monteith et al., 2017). Proteins involved in calcium signaling present attractive targets for a number of reasons. First, due to the role of calcium ions as second messengers, calcium signaling is integral to many cellular and metabolic processes in response to extracellular stimuli (Monteith et al., 2017). Second, these ions also play an integral role in intraorganellar communication, such as that between the endoplasmic reticulum (ER) and the mitochondria (Cardenas et al., 2016). Critically, cancer cells appear to have an

“addiction” to low level calcium flow from the ER to the mitochondria through the inositol-

3-phosphate receptor (IP3R) and the mitochondrial calcium uniporter (MCU) complex

(Cardenas et al., 2016), though they are still sensitive to calcium overload. While all cells require that calcium levels within the cell be maintained with tight control within

biologically acceptable ranges, cancer cells may thus be more sensitive to changes in ion concentration than normal cells.

In this chapter, we describe how D2R antagonists of varying chemotypes have anticancer activity in GBM cell lines, rapidly and significantly reducing metabolic activity.

Although these cell lines express functional D2R, we find that response to D2R antagonists, such as thioridazine, is independent of D2R expression. Rather, cytotoxic effects correlated with calcium flux, loss of mitochondrial membrane potential, and release of cytochrome C from the mitochondria. This expands upon previously-published work discussed in Chapter

2 by providing verification of receptor presence and function in GBM cell lines and extending our understanding of D2R antagonist-induced calcium flux within the context of cancer cell monolayer culture.

Abstract

Dopamine D2-like receptors and their modulators have recently been implicated as a potential target for anticancer therapeutics (Sachlos et al., 2012; Dolma et al., 2016;

Ranjan et al., 2016). Numerous dopamine D2 receptor (D2R) antagonists, originally approved for use in psychiatry (e.g., for schizophrenia) have anticancer efficacy and a reasonable margin of safety (Li et al., 2014; Jandaghi et al., 2016). While these studies show anticancer effects for dopamine D2-like receptor antagonists, ligand concentrations required for such effects greatly exceed those pharmacologically predicted for a selective

D2 receptor effect. Thus, although the D2 receptor has been the postulated target, which of its signaling mechanisms and whether it is a direct effect (e.g., Akt signaling, lipid modulation, autophagy, etc.) remains unclear. To better understand the interrelationship between D2R antagonists and observed anticancer activity, we interrogated a panel of glioblastoma multiforme (GBM) cell lines and D2 antagonists of varying chemotype in regards to receptor pharmacology. Here, we show that while the D2R is present and functional in these cell lines, D2R antagonist cytotoxicity is dependent on calcium signaling in a manner that is independent of known D2R signaling effects.

Methods

Cell culture and reagents

U87MG, U251MG, A-172, Hs683, and LN-18 cell lines were obtained from ATCC

(American Type Culture Collection, Manassas, VA). U373MG (Uppsala) cells were from

Sigma Aldrich (St. Louis, MO), and SF-295 cells were obtained from Addex Bio (San

Diego, CA). U87MG, U251MG, and U373MG cells were grown in minimum essential media (MEM), while A-172, Hs683, and LN-18 cells were grown in Dulbecco’s MEM

(DMEM), and SF-295 cells were maintained in Roswell Park Memorial Institute

1640(RPMI 1640). All medias were obtained from Gibco (Waltham, MA). Cell lines were maintained in media with 10% fetal bovine serum (FBS) added (Hyclone, from

ThermoFisher Scientific, Waltham, MA) at 37ᵒC and 5% CO2 and used within 20 passages of receipt.

D2 antagonists haloperidol (McNeill Pharmaceuticals, Raritan, NJ), pimozide

(Sigma Aldrich), thioridazine (Sandoz Pharmaceuticals, Holzkirchen, Germany), chlorpromazine (Sigma Aldrich), triflupromazine, clozapine (Sandoz Pharmaceuticals), and remoxipride hydrochloride (AstraZeneca, Cambridge, UK) were diluted to 10 mM in dimethylsulfoxide (DMSO). D2 agonists PHNO (4-propyl-9-hydroxynaphthoxazine), ropinirole and sumanirole (Sigma Aldrich, St. Louis, MO) were kept at 100 mM in DMSO.

Thapsigargin (Sigma Aldrich), fendiline hydrochloride (Santa Cruz Biotechnology, Santa

Cruz, CA), and forskolin (Tocris Bioscience, Bristol, UK) were maintained at 10 µM. All drug stocks were stored at -20ᵒC except 10 mM ionomycin (Sigma Aldrich), which was stored at -80ᵒC.

Overexpression and knockdown

Stable cell lines expressing DRD2 shRNA or stably overexpressing DRD2 were used in these studies to determine the role of the receptor in compound response. To generate these lines, DRD2 GIPZ-shRNA viral particles (Dharmacon, Lafayette, CO) or myc-DDK tagged DRD2 lentiORF particles (Origene Technologies, Rockville, MD) were used according to manufacturer’s recommendations, with puromycin selection. Transient overexpression with GFP-DRD2 (Addgene #24099, (Jeanneteau et al., 2004)) was achieved with Lipofectamine 3000 according to manufacturer’s recommendations, using the higher recommended concentrations of lipofectamine. Cells were incubated with 1 µg

(6 well plate, for Western blotting) or 200 ng (96 well plates, for MTTs) DNA for 4 hours before a media change was carried out. At 24 h after initial transfection, cells were used for their respective assays.

Cyclic AMP quantification

Cyclic adenosine monophosphate (cAMP) was quantified by ELISA (enzyme- linked immunosorbent assay) following the supplier’s instructions (EMD Millipore,

Burlington, MA). Briefly, cells were plated at 10,000 cells/well in 24 well plates overnight, then treated for two hours with 10 µM forskolin and treated with varying concentrations of

D2 agonists before harvest in 250 µL 0.1 N hydrochloric acid and tested according to protocol. Data was compared to a standard curve for quantification.

Flow cytometry

Annexin V/7-AAD flow cytometry was carried out at given time points according to the manufacturer’s protocol with R-phycoethrin conjugated Annexin V (Annexin V-PE),

7-amino-actinomycin D (7-AAD), and Annexin binding buffer (BD Biosciences, Franklin

Lakes, NJ). Samples were tested on the PE (564-606 nm) and Cy-5 (>670 nM) channels on a FACSCanto 10 flow cytometer(BD Bioscience, San Jose, CA) in the Penn State

Hershey Flow Cytometry Core within an hour of staining and analyzed with FlowJo v10.

Sample sizes of at least 5000 cells were used per replicate, and all experiments were repeated 2-3 times.

JC-1 (5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide) staining was carried out with a MitoProbe JC-1 Assay Kit (Thermo Fisher

Scientific). Cells were harvested, resuspended to 10^6 cells/mL in warm PBS, and treated for 15 minutes before a 30 min incubation with JC-1 at a final concentration of 2 µM. Cells were then washed with PBS and resuspended in 500 µL of PBS before reading fluorescence in the Cy5 (>670 nm) and FITC (515-545 nm) channels on a FACSCanto 10 flow cytometer (BD Bioscience) in the Penn State Hershey Flow Cytometry Core and analyzed with FlowJo v10.

Cytochrome C flow cytometry was carried out as previously reported (Waterhouse and Trapani, 2003). Briefly, cells were plated in 10 cm dishes and allowed to attach overnight before treatment as noted. Cells were harvested with 0.05% trypsin and washed with phosphate buffer saline (PBS) then permeabilized with digitonin permeabilization buffer (100 mM KCl, 50 µg/mL) for 5 min on ice. Cells were then fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature then washed three times with

PBS. After blocking for 1 h in 3% bovine serum albumin (BSA) + 0.05% saponin in phosphate buffered saline (PBS), cells were incubated overnight at 4ᵒC in a 1:50 dilution of rabbit anti-cytochrome C antibody (136F3, 1:50 dilution, Cell Signaling Technology).

After three PBS washes, cells were incubated with 1:500 AlexaFluor 488 conjugated

F(ab')2 goat anti-rabbit IgG (H+L) secondary antibody (ThermoFisher Scientific,

Waltham, MA) for 30 min at room temperature then rinsed two more times before reading fluorescence on the FITC (515-545 nm) channel of a FACSCanto 10 flow cytometer (BD

Bioscience). Data represents 2-3 experiments in triplicate with at least 5000 events per replicate.

Immunoblotting

Western blot analysis was carried out as previously described using the ScanLater

(Molecular Devices, San Jose, CA) system (Weissenrieder et al., 2018). Membranes were incubated with primary antibodies for D2R(1:1000 dilution, Millipore Sigma, Burlington,

MA), and vinculin (1:1000 dilution, Cell Signaling Technology, Danvers, MA) overnight at 4ᵒC after blocking. Eu-conjugated secondary antibodies (Molecular Devices) were applied for one hour at room temperature before rinsing, drying, and reading membranes on a SpectraMax i3x (Molecular Devices). Densitometry was quantified and normalized to the loading control, vinculin, with ImageStudio Lite (LI-COR, Lincoln, NE).

Calcium imaging

To measure single cell cytosolic Ca2+, U87MG cells were cultured on 30-mm glass coverslips for 24 hours in complete media (Grynkiewicz et al., 1985). Coverslips were then mounted to Teflon chambers and incubated for 30 minutes at 37C in complete media containing 2 M of Fura-2 AM. Using HEPES-buffered saline solution (HBSS; 140 mM

NaCl, 1.13 mM MgCl2, 4.7 mM KCl, +/-2 mM CaCl2, 10 mM D-glucose, and 10 mM

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); pH 7.4), cells were washed

4 times and kept in this solution for 10 minutes at room temperature prior to imaging. A digital fluorescence imaging system (InCytIm2; Intracellular Imaging, Cincinnati, OH,

USA) was used to measure fluorescence from single cells. The dye was alternately excited at 340 and 380 nm and emitted fluorescence was recorded at 510 nm. The emission ratio of 340/380 nm of each pixel was used to represent Ca2+ signal. Treatments were suspended to listed concentrations in HBSS. Ca2+ traces represent averages from many cells per coverslip and multiple independent experiments.

Similarly, to measure maximal Ca2+ responses to thioridazine, cells were cultured in 96 well plates for 24 hours in complete media. 96 well plates were then incubated for 30 minutes at 37C in complete media containing 2 M of Fura-2 AM. Cells were then washed

4 times with HEPES buffered saline solution and kept in this solution for 10 minutes at room temperature. Plates were loaded into FlexStation 3 and alternately excited at 340 and

380 nm and emitted fluorescence was recorded at 510 nm. Maximal Ca2+ was calculated at peak fluorescent signals and normalized to fluorescence at time zero.

For measurements of mitochondrial and ER Ca2+, U877MG cells were cotransfected with 1 g of R-CEPIA1er and G-CEPIA2mt plasmids (CEPIA; calcium- measuring organelle-entrapped protein indicators) and seeded on 30-mm glass coverslips for 24 hours in complete media (Suzuki et al., 2014). Coverslips were then mounted to

Teflon chambers and submerged with HBSS. To measure fluorescence from R-CEPIA1er and G-CEPIA2mt, a TCS SP8 (Leica, Wetzlar, Germany) equipped with a 63x objective captured images at the 552 nm/560–800nm and 488 nm/500–550 nm excitation/emission wavelengths, respectively. Leica X software (Wetzlar, Germany) was used to process and analyze images. Ca2+ signal was represented by normalizing each measurement by fluorescence recorded at time zero.

Quantification and statistical analysis

Data are shown as mean ±SEM, and experiments were repeated 2-3 times in triplicate or more. Statistical analysis was carried out with one or two way ANOVA followed by multiple comparisons with Dunnett’s posthoc to compare against controls or

Sidak’s posthoc to compare within treatment groups.

Results

We first studied the presence and signaling of D2R in a panel of glioma cell lines, including U87MG, U251MG, U373MG-Uppsala, Hs683, SF-295, A172, and LN-18.

While the D2R is present at both the protein and message level in all of the cell lines, expression levels varied and tended to be rather low (Figure 4, A-B). Some cell lines had more mature, glycosylated D2R (A172), while others had lower overall levels of D2R or predominantly unglycosylated protein (SF-295), which is less likely to signal properly.

D2R agonists sumanirole, PHNO (naxagolide), and ropinirole significantly reduced intracellular cyclic AMP levels in forskolin-stimulated GBM cells (p<0.01, one way

ANOVA with Sidak’s posthoc), suggesting that D2R were present and capable of signaling

(Figure 4C, 5A-C). Thus, D2R are present and functional in GBM cell lines, albeit at low levels.

Since D2R were present at varying levels and capable of signaling in these GBM cell lines, compound response was observed for any significant differences in drug response between cell lines. If the D2R is involved in the mechanism of action for these compounds, cell lines with varying expression levels would be expected to respond differently, and multiple D2R antagonists should have a cytotoxic effect. Varying chemotypes of D2 antagonist, including phenothiazines (thioridazine, chlorpromazine, triflupromazine, and metopimazine), phenylbutylpiperidines (haloperidol), diphenylbutylpiperidines (pimozide), substituted benzamides (remoxipride), and tricyclic benzodiazepines (clozapine) were represented in a 48 h MTT assay screening. As expected, most compounds reduced cell metabolic activity with IC50 values between 5-20 µM

(Figure 6A). Notably, these concentrations are much higher than expected given receptor

affinities (Figure 6B), though differences may be at least partially explained by differences in cancer cell culture vs. other biological systems. Cytotoxic compounds shared a characteristically steep Hill slope (Figure 6C, example) and rapid effect time (Figure 6D).

Cell lines A172, U251, and U87 all showed signs of apoptosis and necrosis, which occurred in a time and concentration dependent manner under treatment with thioridazine, pimozide, and haloperidol (Figure 6E-G, 7A-B). Cell proliferation, as measured by BrdU assays, was not negatively impacted until widespread, early-onset cell death was observed (Figure 6H).

However, remoxipride had no effect on cell metabolic activity, even at concentrations much higher than achievable in vivo (Figure 6I). This is notable, as remoxipride is highly selective compared to other tested compounds. Given this, the role of the D2R in D2 antagonist cytotoxicity was called into question.

Genetic and pharmacological means were then employed to further illuminate the role of the D2R in D2 antagonist cytotoxicity. MTT assays with agonist addbacks with D2R- saturating, noncytotoxic concentrations of ropinirole, sumanirole, and PHNO showed no changes in response (Figure 8A-C, 9A-C). To gain insight into possible off-target activity,

DRD2 was overexpressed and knocked down in U87MG (Figure 8D, 9D). Knockdown slightly reduced cell proliferation rates as measured by cell counts (Figure 8D-E, 9D-F, p<0.01 via two way ANOVA with Sidak’s posthoc), as previously published (Li et al.,

2014), but compound response was not affected by overexpression or knockdown (Figure

8D-F, 9G). As previously, remoxipride had no effect on cell proliferation regardless of cotreatment or genetic alterations (Figure 8F). Similar effects were seen with transient transfection of D2R, and similar results were seen with U251MG and A172 knockdown

(Figure 9B-C, 9E-F, 9H-I). Neither pharmacological competition nor genetic manipulation

of the D2R were able to rescue cells from the cytotoxicity induced by these compounds, suggesting a non-D2R mediated mechanism of action.

In the absence of strong data suggestive of a D2R mediated cytotoxicity, it appears likely that D2 antagonists operate through a nonselective target. As many of these compounds are known to affect ion flux within cells, we focused on the role of calcium signaling, which is critically altered in cancer cells (Cardenas et al., 2016; Monteith et al.,

2017). Cotreatment with 3 µM fendiline, a nonselective calcium channel blocker and calmodulin antagonist, sensitized U87MG and A172 cells to D2 antagonist thioridazine

(Figure 10A, 9B, p<0.05 via one way ANOVA with Sidak’s posthoc). This suggested that calcium signaling may be involved in the anticancer mechanism of action for these compounds, as has been previously suggested (Wolfe et al., 1999; Jandaghi et al., 2016;

Park et al., 2016). Indeed, treatment with these compounds, notably the phenothiazines, induced a rapid and sustained calcium response far beyond that induced by 2 µM of the

SERCA (sarcoendoplasmic reticulum calcium ATPase) blocker, thapsigargin (Figure

11A), both in the presence and absence of extracellular 2 mM calcium (Figure 10B-D).

These responses indicate that phenothiazines enhance cytosolic calcium by altering calcium transport across both the ER membrane and the plasma membrane. However, remoxipride had no such effect (Figure 10E), and genetic overexpression or knockdown of

D2R did not significantly alter calcium responses (Figure 10F, 11B), suggesting that D2R is not required for these alterations.

While it is clear that calcium signal is altered in glioma cell lines under treatment with these D2 antagonists, causality was uncertain. To determine if altered calcium signaling was required to induce cytotoxicity, BAPTA-AM was used to chelate cytosolic

calcium. Adequate BAPTA-AM preloading was determined via FLIPR (fluorescence imaging, plate reader) concentration response (Figure 10G). At a concentration which blocks thapsigargin-induced cytosolic calcium increase by ~50-70%, BAPTA-AM preloading significantly reduced cytotoxic responses to thioridazine in U87MG via both

MTT and cell count (Figure 10H-I, p<0.05 via two way ANOVA with Sidak’s posthoc).

To further elucidate the role of calcium in the anticancer mechanism of action for these compounds, mitochondria- and ER-targeted genetically-encoded calcium dyes CEPIA-mt2 and R-CEPIA-ER were transfected into cells and used to determine calcium levels specifically in mitochondria and ER of U87MG cells (Suzuki et al., 2014). This method allows for simultaneous imaging of calcium levels in the endoplasmic reticulum (ER) and the mitochondria (Figure 10J). Upon treatment with 10 µM thioridazine, calcium signals from the ER were reduced, indicating the emptying of ER stores (Figure 10K). The mitochondrial calcium signal, however, increased over this time, suggesting that the calcium released from the ER was taken up by the mitochondria. This is similar to the action of 2 µM thapsigargin (Figure 11C), which selectively inhibits SERCA pumps within the ER membrane and leads to calcium release into the cytoplasm. This cytosolic calcium is then buffered by mitochondria and was sufficient to disrupt the mitochondrial membrane potential within 45 min, as determined by JC-1 staining (Figure 10L). Cytochrome c was consequently released within 4 h (Figure 10M). Similar results were seen in A172 and

U251MG (Figure 11D-I). These results provide strong evidence for a calcium mediated mechanism for cytotoxicity induction by D2 antagonist thioridazine.

Conclusions

To explain these results, we propose a model where D2 antagonists exert cytotoxicity in glioblastoma cell lines through a calcium dependent mechanism which is largely independent of D2R. Our data contributes to the rich literature available which suggests anticancer activity for these compounds, but redirects it significantly by observing a proximal mechanism and noting that D2R signaling is not required for such effects. These findings are significant because they suggest this class of FDA approved drugs may be quickly repurposed for the treatment of cancer and may cooperate with calcium-modulating compounds, such as fendiline. We have also underscored the viability of targeting calcium signaling as a therapeutic strategy in GBM. Future research beyond the scope of this work may reveal a protein target for these antagonists, perhaps a calcium pump, or a physical effect on the membrane which causes these calcium signaling responses in cells.

Respective Contributions

JSW designed and carried out all experiments and wrote the manuscript. Martin

T. Johnson and Mohamed Trebak provided guidance for calcium imaging experiments.

Jeffrey D. Neighbors, Richard B. Mailman, and Raymond J. Hohl provided insight into experimental planning and edited the manuscript.

Figures

Figure 4. The dopamine D2 receptor is present and signals in GBM cell lines.

A) Western blotting indicates presence of D2R in both mature, glycosylated states (~80 kDa) and immature, lower molecular weight species (~50 kDa) in untreated glioma cell lines (quantified in Figure S1). B) DRD2 transcripts are present in untreated glioma cell lines at varying, often low levels. Standard deviations for GAPDH expression were less than 0.085. C) The D2 agonist sumanirole signals through Gi/o and reduce cytoplasmic cAMP levels in a concentration-dependent manner in U87MG, with a IC50 of 250 nM, compared to previously reported EC50 values of 17-75 nM for cell based assays in D2R expressing CHO cells (McCall et al., 2005). Data is representative of 2-3 independent experiments in triplicate. *, p<0.05 via two way ANOVA with Sidak’s post-hoc.

Figure 5. Cyclic AMP levels in GBM cells are reduced by D2 agonists.

Sumanirole, ropinirole, and PHNO concentration-dependently reduced cyclic AMP levels in A172 and U251MG cells over the course of 2 h. Competitive ELISAs were carried out as for Figure 1C, and data represents two independent experiments in triplicate.

Figure 6. D2 antagonists induce cytotoxicity in GBM cell lines.

A) 48 h MTT IC50s (µM) are presented as a heat map. B) expected receptor/ligand affinities for tested ligands(Besnard et al., 2012). Cytotoxic activity occurs only at nonselective concentrations. C) representative 48 h MTT concentration response curve for thioridazine in U87MG cells. D2 antagonist MTT curves have a characteristic sharp Hill slope starting between 1 and 30 µM. D) 4 h MTT dose response curve. Cytotoxic responses to these compounds occur quickly. E) cell counts for A172 cells under treatment with 10

µM D2 antagonists. F) 24 h annexin V/7-AAD flow cytometry indicates the induction of apoptosis in U251MG cells. Apoptosis is induced in a time (G) and concentration (H) dependent manner in U87MG. I) 24 h BrdU incorporation suggests that proliferation is not significantly affected by D2 antagonist treatment until widespread cell death is seen. J) remoxipride does not significantly affect U87MG response via 48 h MTT. K) remoxipride does not significantly affect the induction of apoptosis via 24 h annexin V/7-AAD flow cytometry. Data is representative of three or more independent experiments in triplicate for

MTT and BrdU experiments, while flow cytometry experiments are representative of three independent experiments of 5000-10000 cells. Two way ANOVA with Sidak’s post-hoc was used to analyze significance. *, p<0.05. **, p<0.01.

Figure 7. Thioridazine induces apoptosis in GBM cell lines.

Thioridazine increases signs of apoptosis and necrosis in a time and concentration dependent manner in GBM. A) apoptosis, necrosis, and cell death are induced by treatment with 10 µM thioridazine in U251MG cells in a time dependent manner. B) at 24 h, thioridazine increases apoptotic, necrotic, and dead cell populations in a concentration dependent manner in both A172 and U251MG cell lines.

Figure 8. D2R is not involved in cytotoxic responses to D2 antagonists.

A) addition of 100 µM ropinirole, a D2 agonist, was not able to reduce thioridazine’s toxic effect in U87MG cells in a 48 h MTT assay. B) sumanirole (30 µM) does not reduce the induction of apoptosis as seen by 24 h annexin V/7-AAD flow cytometry. C) 30 µM PHNO does not affect pimozide response for U87 cells in a 48 h MTT assay. D) quantification of lentiviral D2R knockdown and overexpression via immunoblot. E) knockdown of DRD2 in A172 cells via stable lentiviral transduction reduces cell proliferation slightly, but does not significantly affect response to 10 µM thioridazine. F) knockdown and overexpression of DRD2 in U87MG cells does not affect thioridazine response via 48 h MTT. G) regardless of genetic manipulation, cells do not have a cytotoxic response to remoxipride. Data is representative of two independent experiments carried out in triplicate and is analyzed by two way ANOVA with Sidak’s post-hoc. *, p<0.05.

Figure 9. D2R antagonist cytotoxicity is independent of D2R.

A) Ropinirole does not rescue the concentration dependent cytotoxic response in U87 cells, as measured by annexin V/7-AAD flow cytometry. B) 30 µM sumanirole fails to significantly rescue cytotoxicity in A172 cells treated with 30 µM pimozide, while 3 µM fendiline (non-cytotoxic concentration, data not shown) increases cytotoxicity. C) 50 µM ropinirole failed to significantly rescue cytotoxicity of U251MG cells treated with 1 and

10 µM ropinirole. D) Protein expression levels of GBM cell lines stably transduced with

DRD2 GIPZ-shRNA (Dharmacon, Lafayette, CO) or myc-DDK tagged DRD2 lentiORF

particles (Origene Technologies, Rockville, MD). Cell counts of A172 cells (E) or

U251MG cells (F) treated with 10 µM thioridazine over the course of 72 h show that thioridazine response remains the same, regardless of DRD2 knockdown. Stable expression of the shDRD2 vector reduced maximal cell number slightly in both cell lines, which could be due to transfected cells being less prone to close growth behaviors. 4 h MTTs of U87

(G), A172 (H), and U251MG (I) cells transfected with DRD2 show no significant alterations in cytotoxicity.

Figure 10. D2 antagonists except remoxipride alter calcium signaling in GBM.

A) 3 µM fendiline, a nonselective calcium channel blocker and calmodulin antagonist, sensitizes U87MG cells to thioridazine via 48 h MTT. B) 10 µM thioridazine induces a strong and sustained increase in cytosolic calcium release as measured by single cell imaging with Fura-2 AM. This increase is due to both ER calcium release (as shown by the increase in the absence of extracellular calcium) and calcium influx from the

extracellular space. Unlike the positive control, 2 µM thapsigargin (Figure 11A), the cell is unable to clear this signal for a significant amount of time. C) thioridazine increases cytosolic calcium signal via Fura-2 AM in a concentration dependent manner as measured by FLIPR assay. D), maximal calcium responses of selected D2 antagonists as measured by Fura-2 AM FLIPR assay. E) remoxipride does not affect cytoplasmic calcium levels as measured by single cell imaging with Fura-2 AM. F) maximal calcium responses are not significantly altered by overexpression or knockdown of DRD2. A is representative of three experiments in triplicate, while single cell imaging experiments are representative of three independent experiments with at least two cover slips and 40 cells. FLIPR assays are representative of at least two independent experiments carried out in quadruplicate. G)

BAPTA-AM can concentration-dependently reduce cytoplasmic calcium signal from 2 µM thapsigargin exposure in U87 cells via FLIPR assay. H) BAPTA-AM concentration- dependently reduces cytotoxic response in U87MG cells via 4 h MTT. I) BAPTA-AM treatment reduces the induction of apoptosis by 10 µM thioridazine in U87 cells via 4 h flow cytometry. J) representative images from G-CEPIA2mt and R-CEPIA1er stained

U87MG cells treated with 10 µM thioridazine. K) Quantification of CEPIA staining in

U87MG under treatment with 10 µM thioridazine. Similar results were seen with 2 µM thapsigargin (Figure 11). L) JC-1 staining for mitochondrial potential indicates loss of mitochondrial potential within 15 min under treatment with thioridazine. M) Release of cytochrome c is induced by treatment with 10 µM thioridazine for 4 h. Experiments were analyzed via two way ANOVA with Dunnett’s post-hoc. *, p<0.05. **, p<0.01. ***, p<0.005. ****, p<0.0001.

Figure 11. Calcium signaling is altered in D2 antagonist-treated GBM cells.

A) Fura-2 AM calcium trace of U87 cells treated with 2 µM thapsigargin in the absence of calcium. Intracellular calcium levels rise, indicating a release of calcium from the endoplasmic reticulum, then drop slowly as calcium is removed from the cell. By 400 seconds, the intracellular signal has reached baseline. BAPTA-AM preloading for 30 min chelates calcium signal and increases cell viability of A172 (B) and U251MG (C) cells cotreated with 3 µM thioridazine for 4 h. Significances are provided for BAPTA-treated samples vs controls. D) knockdown of DRD2 with shRNA did not significantly alter

calcium response in U251MG or A172 cells. E) CEPIA trace of U87 cells treated with 2

µM thapsigargin. Trace is representative of 12 cells gathered from three cover slips on three separate days. JC-1 staining after 45 min of treatment indicates a rapid depolarization of A172 (F) and U251MG (G) mitochondria. A172 (H) and U251MG (I) cells showed cytochrome C release after 4 hours of treatment. Release was not significantly different between 30 and 10 µM treatments.

CHAPTER 4. DOPAMINE D2 RECEPTOR MODULATION IN

SPHEROIDS

Preface

As mentioned in Chapter 2, D2R and its antagonists have been tied to cancer stem cell maintenance and activity (Sachlos et al., 2012; Yeh et al., 2012). This association with stem-like behavior could provide another layer of potential for anticancer therapeutics which may be missed in the context of the standard monolayer cell culture studies which were carried out in Chapter 3, as selective activity at stem cells could reduce tumor growth, spread, and chemoresistance (Wang et al., 2015a; Kulsum et al., 2017). Chapter 4 thus explores the role of D2R modulation in spheroid cultures of GBM cells, which may be used to model stem-like populations (Lee et al., 2006). This model has increased in use since its development both due to the enrichment of stemness markers, such as Oct4, Nanog, and

Nestin, and the higher phenotypic resemblance to patient tumors (Lee et al., 2006; Yeh et al., 2012; Wang et al., 2015a; Zhang et al., 2016). Spheroid formation and survival are dependent upon a number of factors aside from stemness, including EGFR and FGFR signaling cascades, metabolic factors promoting anchorage independent growth, and cell- cell adhesion mechanisms (Johnson et al., 2013; Wang et al., 2015a). These experiments show that D2R activity appears to modulate a spheroid-formation effect which is independent of stemness markers Nestin and SRY box 2 (SOX2) and may involve one or more of these other factors that contribute to the phenotype.

Abstract

GBM is a common, and lethal, central nervous system cancer. This cancer is difficult to treat because most anti-cancer therapeutics do not readily penetrate into the brain due to the tight regulation of solute transport at the cerebro-vascular barrier.

Numerous studies have suggested that dopamine D2 receptor antagonists, such as first generation antipsychotics, may have anticancer efficacy in vivo and in vitro. The role of the D2R itself in the anticancer effects is unclear, but there is evidence suggesting that D2R activation promotes stem-like behavior in GBM. We aimed to observe the role of the dopamine D2 receptor (D2R) and its modulators (at selective concentrations) in spheroid formation and stemness of the GBM cell line, U87MG, to clarify the validity of the D2R as a therapeutic target for cancer therapy. Spheroid formation assays and western blotting of the glioblastoma cell line, U87MG, were used to observe responses to treatment with the D2R agonists sumanirole, ropinirole, and PHNO and the D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride. Stable lentiviral transduction of DRD2 or shDRD2 were used to validate the role of the D2R in assay behaviors.

D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride decrease spheroid formation behaviors at a selective 100 nM concentration, while D2R agonists

PHNO, sumanirole and ropinirole increase the formation of spheroids. These results were recapitulated with genetic overexpression and knockdown of the D2R, and combination experiments indicate that the D2R is required for the effects of the pharmacological modulators. Furthermore, spheroid proliferation and invasive capacity increased under treatment with 100 nM sumanirole and decreased under treatment with 100 nM thioridazine. Expression levels of the stemness markers Nestin and Sox2, as well as those

of differentiation marker glial fibrillary acidic protein (GFAP), were not altered by 100 nM thioridazine or sumanirole for 72 h or continuous treatment with these compounds for 7 d during a spheroid formation assay. In conclusion, signaling activity of the dopamine D2 receptor may be involved in the spheroid formation phenotype in the context of the U87MG cell line. However, this modulation may not be due to alterations in stemness marker expression, but due to other factors that may contribute to spheroid formation, such as cell- cell adhesion or EGFR signaling modulation by the D2R.

Introduction

A common central nervous system cancer, GBM has a remarkably poor prognosis compared to many other cancers (Thakkar et al., 2014). Less than ten percent of patients will survive for five years after diagnosis (Batash et al., 2017). Even among patients who engage in clinical trials, only about a quarter of patients will survive for two years (Batash et al., 2017). Additionally, patients often suffer from debilitating symptoms such as headaches, memory loss, seizures, and mood swings depending on tumor location

(Alexander and Cloughesy, 2017). There are many complications of GBM treatment, including limitations on resection (due to tumor location or diffuseness) and exclusion of potential therapeutics from the tumor by the cerebro-vascular or blood-brain barrier (BBB)

(Alexander and Cloughesy, 2017; Batash et al., 2017). Thus, current surgical options, chemotherapeutics, and radiotherapy can only marginally improve patient survival and not cure GBM (Thakkar et al., 2014; Alexander and Cloughesy, 2017; Batash et al., 2017). It is clear that new options are needed for GBM patients.

One potential treatment modality for GBM, as well as for other treatment-resistant cancers, is to target cancer stem cells (CSCs), a purported group of slow-cycling, undifferentiated cells capable of continual self-renewal (Wang et al., 2015a). CSCs are linked to increased tumor metastasis and chemoresistance, and putative populations of

CSC-like cells have been identified in numerous cancers, including GBM (Chen et al.,

2017; Kulsum et al., 2017). GBM cells grown in spheroid-inducing, serum-free media are enriched in normal embryonic stemness markers and are thought to provide a better model for in vivo behaviors of GBM, as they mimic the highly proliferative, undifferentiated state of most GBMs (Lee et al., 2006). Spheroid formation and spheroid culture in this type of

system have become a common method of studying GBM and CSCs due to enhanced behavioral similarities to in vivo tumors, expression of putative stemness markers, and stem-like self-renewal behaviors (Lee et al., 2006). Treatments that are able to cross the

BBB and are selective for CSCs over nonmalignant cells may offer a high-efficacy, low- adverse effect option for patients with GBM that could potentially extend survival or improve quality of life.

Interestingly, dopamine D2-like receptor (D2R) targeting compounds were identified as a potential selective CSC treatments in acute myeloid leukemia, lung cancer, and GBM through pharmacological screens (Lee et al., 2006). These receptors are Gαi/o- coupled GPCRs that are found throughout the nervous system, though they are also present elsewhere in the body (Boyd and Mailman, 2012a). High expression of D2R has been tied to poor prognosis in numerous cancers (Li et al., 2006; Hoeppner et al., 2015; Kanakis et al., 2015; Mao et al., 2015; Cherubini et al., 2016b), and D2R knockdown impairs the growth of the GBM cell line, U87MG (Li et al., 2014). Critically, D2R antagonists such as those used in the above studies can cross the BBB and readily access CNS tumors. Many of these compounds are also FDA-approved treatments for conditions such as schizophrenia with well-characterized adverse effect profiles, pharmacokinetics, and pharmacodynamic profiles (Mailman and Huang, 2007; Boyd and Mailman, 2012a) . If the

D2R is involved in stemness, treatment with already-available, relatively safe compounds could extend survival for GBM patients.

Although the D2R and its antagonists have been identified in numerous screens, no study has yet clearly shown a direct relationship between D2R antagonist cytotoxicity and

D2R receptor activity in order to elucidate the role of the receptor in cancer cell growth.

Moreover, these studies were carried out with relatively nonselective concentrations

(typically micromolar, when D2R receptor affinities are typically in the low nanomolar range for these compounds) of D2R antagonists, a concentration at which binding to other receptors and off target enzymes is likely. In this work, we further characterized the role of D2R in GBM spheroid formation and stem-like behaviors in the context of the commonly used GBM cell line, U87MG. To that end, we employ a combination of genetic modulation and the application of varying chemotypes of D2R modulators at concentrations selective for D2R binding to reduce the likelihood of off target effects interfering in the study.

Materials/Methods

Reagents and cell culture

U87MG cells were obtained from ATCC (Manassas, GA), and all experiments were performed within 20 passages of receipt. U87MG-shDRD2 and U87MG-OE-DRD2 were generated according to manufacturer’s protocols with lentiviruses, respectively DRD2

GIPZ-shRNA viral particles (Dharmacon, Lafayette, CO) or myc-DDK tagged DRD2 lentiORF particles (Origene Technologies, Rockville, MD). For monolayer cultures, cells were maintained in MEM (Gibco, Waltham, MA) + 10% FBS (Hyclone, Logan, UT).

Spheroids were generated and maintained in spheroid media [DMEM/F12 (1:1) + 20 ng/mL epidermal growth factor (Sigma Aldrich, St. Louis, MO), 5 µg/mL insulin (Sigma

Aldrich), 10 ng/mL basic fibroblast growth factor (Sigma Aldrich) and 0.4% bovine serum albumin (Research Products International, Mount Prospect, IL). Media was mixed fresh for each experiment, and 1x B-27 growth supplement (Gibco) was added to spheroid media immediately before use. All cultures were incubated at 37ᵒC and 5% CO2.

Spheroid forming assays

Sphere-forming assays were carried out as previously described (Johnson et al.,

2013). Briefly, cells were plated at 200 cells/well in given concentrations of compounds in spheroid media in low adhesion 96 well plates (Corning Costar, Corning, NY) and incubated for 7 d before quantification. PBS was used in all outside wells to reduce evaporation. For counting, spheroids were defined as rounded aggregates of cells with a smooth surface and poor cell to cell definition. Loose cellular aggregates of well-defined cells were not included in spheroid counts.

Immunoblotting

Western blotting was performed as previously described (Weissenrieder et al.,

2018) using the ScanLater (Molecular Devices, San Jose, CA) visualization system. 4-12% bis/tris gels (Life Technologies, Carlsbad, CA) were loaded with even amounts of protein

(8-12 µg/well) as determined by Pierce BCA assay (Thermo Fisher Scientific, Waltham,

MA) and run at 175 V for 60-90 min in MOPS (3-(N-morpholino)propanesulfonic acid) buffer (Life Technologies). Transfer was carried out on ice at 20 V for 120 min. Primary antibodies (Cell Signaling Technology, Danvers, MA) included: Nestin (10C2 mouse mAb, #33475, 1:1000 dilution), GFAP (D1F4Q rabbit mAb, #12389, 1:500 dilution),

SOX2 (D6D9 rabbit mAb, #3579, 1:500 dilution), and vinculin (E1E9V rabbit mAb,

#13901, 1:1000 dilution). ScanLater Eu-conjugated secondary antibodies (Molecular

Devices) were used for visualization with a SpectraMax i3x multimode plate reader

(Molecular Devices). Images were quantified with ImageStudio Lite software (Licor,

Lincoln, NE).

Invasion assays

Invasion assays were carried out with Corning BioCoat Matrigel Invasion Chamber

8.0 micron 24 well plates (Corning, NY) according to manufacturer’s instructions. Briefly,

U87MG spheroid cells were plated in rehydrated transwell inserts at 15 x10^4 cells/well in DMEM/F12 not supplemented with growth factors, with complete sphere media in the plate well as a chemoattractant. Invasion assays were carried out for 48 h before insert removal and cell counting. Inserts were washed, then stained with DiffQuik (Siemens,

Munich, Germany). Photos were taken of the inserts and quantified with ImageJ.

Statistics and analysis

All data shown are representative of 2-3 independent experiments. Spheroid counts were carried out in quadruplicate or more for each experiment, whereas cell counts and invasion assays were carried out in triplicate. Time course data was analyzed with two way

ANOVA followed by a Dunnett’s posthoc, whereas spheroid count data and immunoblot signals were analyzed with one way ANOVA plus Dunnett’s posthoc. For spheroid forming assays, at least eight replicate wells were counted; results are presented as box plots, with the whiskers representing minimum and maximum values and the box representing quartiles. All other data points shown are presented as mean ± SEM.

Results

Selective concentrations of D2R modulators alter sphere formation of U87MG.

First, we investigated the function of D2R modulators in a common model of stemness by measuring spheroid-forming capability of the U87MG cell line. For this assay, we used a standard 100 nM concentration for all compounds, which is closer to expected receptor affinities and thus should provide higher selectivity for D2R than concentrations

of these compounds that have historically been used (Strobl et al., 1990; Gil-Ad et al.,

2004; Sanchez-Wandelmer et al., 2010; Cheng et al., 2015; Mao et al., 2015; Yin et al.,

2015; Weissenrieder, 2019). Over the course of 7 d, 100 nM of D2 antagonists thioridazine, pimozide, haloperidol, and remoxipride reduced spheroid formation, whereas 100 nM of agonists sumanirole, ropinirole, and PHNO increased the number of spheroids formed

(Figure 12A). At these concentrations, there is no significant effect on MTT measurements of cell metabolic activity in either monolayer or spheroid cultures (Figure 12B-C). Thus, at non-cytotoxic, yet D2R saturating, concentrations, multiple chemotypes of D2 antagonist reduce sphere formation while multiple chemotypes of agonist increase it in the context of

U87MG.

Genetic modulation of D2R alters sphere formation of U87MG.

While such pharmacological evidence is strongly supportive of a D2R-mediated reduction in spheroid-forming capacity, we also manipulated DRD2 gene expression to further elucidate the role of the D2R in spheroid-forming phenotypes. Stable, lentivirally- transduced cell lines were used due to the 7 d time period of these assays. Stable transduction of sh-RNA against DRD2 were able to reduce protein expression by about half, whereas stable overexpression of DRD2 increased expression nearly fourfold over that of the parental line (Figure 13A, p<0.01 by one way ANOVA with Dunnett’s posthoc).

Overexpression of DRD2 slightly increased cell proliferation, while knockdown slightly decreased it over the course of 96 h as measured by cell counts (Figure 13B, p<0.01 by two way ANOVA with Dunnett’s posthoc). However, the differences in number of spheres formed is more pronounced. Knockdown reduced spheroid formation by nearly half, and overexpression doubled sphere formation (Figure 13C, p<0.001 by one way ANOVA with

Dunnett’s posthoc). While it is possible that this effect is due to the proliferative changes in the monolayer culture, the vast majority of cells placed in the spheroid growth paradigm would be dead by the time that proliferation rates significantly vary. Differences in magnitude of effect between monolayer culture growth rates and spheroid formation assays also make it more likely that the presence of D2R more greatly facilitates stemness or survival in the more restrictive environment of spheroid media.

Effects on the sphere-forming phenotype are responsive to D2R expression levels.

To further control for the possibility of off-target pharmacological effects, we assayed the role of the receptor further with a combination of pharmacological competition and genetic manipulation. First, we ascertained the effects of cotreatment with D2R antagonists and agonists. In these studies, we found that cotreatment with 100 nM agonists,

PHNO, sumanirole, or ropinirole, was able to at least partially block the reduction in spheroid formation caused by treatment with 100 nM antagonists, thioridazine, pimozide, and haloperidol (Figure 14A). We then compared the behaviors of wild type (Figure 12A),

DRD2 overexpressed (Figure 14B) and DRD2 knockdown (Figure 14C) U87MG cell lines under treatment with D2R modulators. When DRD2 was overexpressed (Figure 14B), the ability of antagonists to reduce sphere formation was impaired (p<0.05 for all four antagonists by one way ANOVA with Dunnett’s posthoc). Of the agonists, only 100 nM sumanirole significantly increased spheroid formation (p<0.05). It is possible that overexpression led to the near-saturation of signal through the D2R at a basal level, thus ablating any further effects from agonist treatment. When DRD2 was knocked down, none of the compounds, either agonist or antagonist, were able to alter the spheroid-formation phenotype of the cells (Figure 14C, not significantly different from control by one way

ANOVA and Dunnett’s posthoc). This suggests that the observed effect requires the D2R, and is thus not off-target.

Invasive and proliferative behaviors are affected by D2R modulators.

The combination of results from pharmacological and genetic modulation in this assay suggested that there was a clear D2R effect on the spheroid-forming phenotype, but we wished to ascertain if this phenotype was due to increased stemness. It is possible that increased sphere formation is related to increases in adhesion proteins or overall proliferation rate more than to stemness markers. We chose to study the glial differentiation factor, glial fibrillary acidic protein (GFAP), and two common stemness markers, Nestin and SOX2 (sex determining region Y-box 2) in both preformed spheres treated for 72h

(Figure 15A,C) and spheres formed over 7 d in media containing the D2R modulators

(Figure 15B,C). As elsewhere, we used the more selective 100 nM concentration for all treatments. Interestingly, we saw no changes in any of these markers under these conditions. However, changes were observed in crystal violet staining, which was used as a surrogate marker of spheroid proliferation (Figure 15D). Sumanirole was able to increase total cell membrane staining at 96 h and 168 h, but thioridazine significantly decreased it

(p<0.01 for both compounds at 96 and 168 h). Spheroids were also more able to invade in a 48 h transwell assay when treated with sumanirole than thioridazine or remoxipride

(Figure 15E, p<0.001 by two way ANOVA with Dunnett’s posthoc). Taken together, these data suggest that there is an effect on cellular proliferation and invasive behavior with D2R modulation, but this effect is independent of the regulation of stemness markers Nestin and

SOX2.

Conclusions/Discussion

In this work, we have observed a clear spheroid-formation phenotype tied to D2R activity in U87MG cells. Pharmacological and genetic manipulation are able to modulate spheroid formation in this cell line in a manner which can be ablated with the addition of antagonistic factors. It is worth noting that overexpression and knockdown of this receptor was sufficient to ablate both agonist and antagonist effects. In the case of overexpression, it is possible that the effects of agonism were overwhelmed by increased low-level constitutive signaling from the D2R. It appears that these receptors are engaging in appreciable signaling in the absence of supplied D2R ligand, though it is possible that the cells are producing dopamine on their own or a factor present in the growth media is providing some level of agonism. Similarly, heightened receptor availability negated the effects of pharmacological antagonism.

These findings modify our understanding of D2R in a common model of CSCs, as we have seen clear spheroid-formation effects at selective concentrations of D2R modulators, but we were unable to observe alterations in stemness markers Nestin or SOX2 or the differentiation marker, GFAP. This suggests that while there are changes in sphere formation, they may not be attributable to increases in stemness. In the absence of increased stemness, it appears that cell viability and spheroid growth in the context of serum-free, suspension growth must be due to other factors. These could include, but are not limited to, increased cell-cell adhesion, reduced susceptibility to death signals, or an enhanced reliance on other growth signaling systems which are favored in this media, such as EGFR signaling.

Respective Contributions

JSW conceived, designed, carried out, and analyzed all experiments with the aid of Jessie L. Reed and Emily J. Koubek. JSW wrote the manuscript. Jeffrey D. Neighbors,

Richard B. Mailman, and Raymond J. Hohl contributed to experimental design and analysis and edited the manuscript.

Figures

Figure 12. Spheroid formation is altered by D2 modulators.

A) 100 nM of D2R antagonists thioridazine, pimozide, haloperidol, and remoxipride significantly reduce spheroid formation of U87MG cells in a 7d assay. 100 nM of D2R agonists PHNO, sumanirole, and ropinirole significantly increased the number of spheroids formed. B) 100 nM concentrations of D2 antagonists thioridazine, haloperidol, pimozide, and remoxipride are nontoxic to monolayer cultures of U87MG. C) 100 nM concentrations of D2 antagonists are nontoxic to U87MG cells cultured as spheroids. Spheroid formation assays were analyzed by one-way ANOVA with Dunnett’s posthoc. **, p<0.01; ***, p<

0.001; ****, p<0.0001.

Figure 13.Genetic modulation of D2R alters spheroid formation capacity..

A) Stable transduction with lentiviral vectors produced significant alterations in D2R protein expression level. Transduction with shDRD2 reduced protein expression by half

(knockdown, KD on blot), whereas stable overexpression of DRD2 increased protein expression by ~3.5 fold (overexpression, OE on blot). B) Overexpression of DRD2 slightly, but significantly, increases cell proliferation rates as measured by cell counts, while knockdown significantly decreased it over the course of four days. C) Expression levels of

D2R correlate with spheroid formation capacity. Knockdown reduced spheroid formation by half, while overexpression significantly increased sphere formation. Spheroid formation assays and western blot data were analyzed by one-way ANOVA with Dunnett’s posthoc, while cell counts were analyzed with two-way ANOVA with Dunnett’s posthoc. **, p<0.01; ***, p< 0.001; ****, p<0.0001.

Figure 14. The D2R is involved in observed spheroid formation phenotypes..

A) cotreatment with 100 nM D2R agonists PHNO, sumanirole, and ropinirole partially ablated the reduction in spheroid number induced by 100 nM of D2 antagonists thioridazine, pimozide, and haloperidol over the course of 7 d. B-C) modulation of D2R

expression impacted compound response in a 7d spheroid formation assay. Wild type

(Figure 1A) responses to antagonists were reduced by overexpression of D2R (B), while knockdown blocked all compound responses (C). Spheroid formation assays were analyzed by one-way ANOVA with Dunnett’s posthoc. *, p<0.05;**, p<0.01; ***, p<

0.001; ****, p<0.0001.

Figure 15. D2R modulators alter spheroid behaviors but not stemness markers.

Pharmacological modulation of D2R activity alters malignant behaviors of spheroids, but do not change stemness marker expression levels. A) U87MG spheroids were treated with vehicle, 100 nM sumanirole, or 100 nM thioridazine for 72 h. Expression of stemness markers Nestin and SOX2 and glial differentiation marker GFAP were not significantly altered. B) U87 MG spheroids were formed over the course of 7d in the presence of vehicle,

100 nM sumanirole, or 100 nM thioridazine. Expression of markers was unchanged by treatment. C) Quantification of A and B. Densitometry values were normalized to loading control, vinculin. D) Spheroid proliferation was measured by crystal violet staining over

7d under treatment with vehicle, 100 nM sumanirole, or 100 nM thioridazine. D2R agonist sumanirole increased spheroid proliferation at 96 and 168 h, whereas 100 nM thioridazine significantly reduced it. E) Invasion of cells from U87MG spheroids was measured via transwell assay after 48 h under treatment with 0, 100, and 1000 nM concentrations of

sumanirole, thioridazine, and remoxipride. Sumanirole significantly increased invasion at

100 and 1000 nM, while thioridazine and remoxipride inhibited it in a concentration dependent manner. Western blots were analyzed via one way ANOVA, while two-way

ANOVA was used for cell proliferation and invasion assays. Dunnett’s posthoc was used for each. *, p<0.05;**, p<0.01; ***, p< 0.001; ****, p<0.0001.

CHAPTER 5. DOPAMINE D2 RECEPTOR ANTAGONISTS AND THE

ISOPRENOID PATHWAY

Preface

As discussed in the previous chapters, D2 antagonists have shown efficacy in both cell culture and animal models of cancer (Chapters 2-4). Additionally, case reports of patients suggest that these compounds may have anticancer efficacy in humans (Csatary,

1972; Nagel et al., 2012). These findings suggest that D2 antagonists may have therapeutic utility in humans, but there are some indications that they may have a narrow therapeutic window (Choi et al., 2008). In this case, D2 antagonists may best serve patients in combination treatment paradigms, where they may cooperate with other chemotherapeutics to have a synergistic or additive effect (Rho et al., 2011; Sachlos et al., 2012). Because these compounds are known to have effects on lipid metabolism and trafficking (Masson et al., 1992; Ferno et al., 2006; Vik-Mo et al., 2009; Sanchez-Wandelmer et al., 2010), it is possible that lipid-targeting medications may be used to sensitize cells to their effects. In

Chapter 5, we report on the potential for interplay between thioridazine treatment and the isoprenoid biosynthetic pathway, which is required for cholesterol synthesis and isoprenylation of small GTPases to examine if this may be a viable therapeutic strategy.

This work was published on BioRXIV as “Thioridazine as an anticancer therapeutic: interplay with the isoprenoid biosynthetic pathway”(Weissenrieder et al., 2019).

Abstract

Glioblastoma multiforme is a form of cancer with poor survival prognosis and few treatment options. The cerebrovascular barrier complicates the delivery of chemotherapeutic agents and contributes to poor treatment response in patients with this disease. Recently, dopamine D2 receptor antagonizing compounds, including the FDA- approved phenothiazine, thioridazine, were identified as potential anticancer therapeutics, but their mechanism of action is as yet poorly understood. We investigated the hypothesis that the cytotoxicity of thioridazine may be tied to disruption of lipid metabolism, specifically the synthesis of isoprenoids and cholesterol by the isoprenoid biosynthetic pathway. We show that, while pathway inhibitors lovastatin and zoledronate can sensitize

U87MG and U251MG cells to thioridazine treatment, the addition of pathway intermediates cannot prevent thioridazine’s cytotoxic effects. Treatment with methyl- schweinfurthin G, which is known to disrupt lipid trafficking, is able to sensitize these cell lines as well, suggesting that cholesterol availability or localization may be involved in these effects. However, all measured effects were of very small, biologically insignificant magnitude and thus findings are of limited utility.

Introduction

Glioblastoma multiforme (GBM) is one of the most common central nervous system malignancies (Brodbelt et al., 2015; Alexander and Cloughesy, 2017; Ostrom et al.,

2018). While metastasis of GBM is rare, survival rates are dismal, with the vast majority of patients surviving under two years after diagnosis (Koshy et al., 2012; Brodbelt et al.,

2015; Ostrom et al., 2018). The current standard of care, consisting of maximal surgical resection, radiotherapy, and chemotherapy with temozolomide (Alexander and Cloughesy,

2017). While many types of cancer have benefited strongly from the development of targeted therapeutics, many of these have shown limited efficacy in GBM due to the exclusion of therapeutics by the blood-brain barrier (BBB) and the immune privilege of the environment (Maxwell et al., 2017; Touat et al., 2017). Therapy is also complicated by inter- and intra-patient tumor heterogeneity (Sottoriva et al., 2013; Patel et al., 2014;

Morokoff et al., 2015) and diffuse tumor margins (Chaichana et al., 2014; Pessina et al.,

2017). A novel treatment method using alternating electric fields has shown limited anticancer efficacy, but no survival improvement over the current standard of care (Stupp et al., 2012). To improve patient outcomes, new treatment options are needed.

Lipid modulation shows promise as an anti-GBM therapeutic target. Glial cancers like GBM are known to be highly dependent on lipid metabolism, cholesterol levels, and liver X receptor (LXR) function, suggesting lipid-targeting therapeutics, especially those which modulate cholesterol synthesis or trafficking, could have potential in the treatment of GBM (Villa et al., 2016). Cholesterol is a product of the isoprenoid (or mevalonate) biosynthetic pathway (Figure 16), which may be pharmacologically inhibited by statins, such as lovastatin. Statins inhibit the rate limiting step of cholesterol synthesis catalyzed by HMGCoA reductase. Other enzymes within this pathway may also be modulated with chemical inhibitors, allowing the role of individual pathway intermediates in a given phenotype to be readily discerned by experimentation.

Recently, dopamine D2 receptor (D2R) antagonists have been identified as potential anticancer therapeutics (Rho et al., 2011; Sachlos et al., 2012; Yeh et al., 2012; Cheng et al., 2015; Huang et al., 2016). In the case of GBM, their ability to pass through the blood- brain barrier makes them particularly attractive compounds (Batash et al., 2017; Touat et

al., 2017; Harder et al., 2018). Numerous compounds of this class are approved by the Food and Drug Administration for treatment of mental illnesses, such as schizophrenia. A number of these D2R antagonists, especially those of the phenothiazine chemotype, have been shown to induce apoptosis (Gil-Ad et al., 2004), decrease survival signaling (Park et al., 2014), and reduce tumor size in xenograft models (Shin et al., 2013). All of these effects are desirable in an anticancer therapeutic.

Interestingly, D2R antagonists are also known to disrupt lipid synthesis and trafficking (Masson et al., 1992). It is not known if this dysregulation is required for their anticancer activity. By the early 1990s, it was evident that cytotoxic concentrations of the phenothiazine D2R antagonist, chlorpromazine, inhibited sphingomyelinase activity and induced accumulation of unesterified cholesterol in large droplets within the cell (Masson et al., 1992). Treatment with D2R antagonists such as haloperidol and clozapine increased sterol responsive element binding protein 1/2 (SREBP) protein levels and induced upregulation of sterol-responsive genes such as HMGCR (HMG-CoA reductase), APOE

(apolipoprotein E), ABCA1 (ATP binding cassette A1), LXR1/2 (liver X receptor 1/2), and others in the context of GaMg glioma cells (Ferno et al., 2006; Vik-Mo et al., 2009). This increase in mRNA may lead to a buildup of synthetic pathway intermediates, but appears to block cholesterol synthesis (Sanchez-Wandelmer et al., 2010; Canfran-Duque et al.,

2013).

Here, we more closely describe the role of the isoprenoid pathway in the mechanism of action for the phenothiazine D2R antagonist, thioridazine. Through a series of experiments involving supplementation with isoprenoid pathway intermediates and

cotreatment with isoprenoid pathway inhibitors, we show that the anticancer efficacy of thioridazine is not reliant upon isoprenoid pathway disruption.

Methods

Cell culture and reagents

Cell lines U87MG and U251MG were purchased from American Type Culture

Collection (ATCC, Manassas, VA). Both lines were cultured in MEM (Gibco, Waltham,

MA) plus 10% FBS (HyClone, Logan, Utah) at 37ᵒC and 5% CO2. Cell lines were used within 20 passages of receipt.

Thioridazine (Sandoz Pharmaceuticals, Holzkirchen, Germany) was maintained as a 10 mM stock in dimethyl sulfoxide at -20ᵒC. Farnesol, geranylgeraniol, and mevalonate

(1M stock) were obtained from Sigma Aldrich (St. Louis, MO). Lovastatin was activated as previously described (Dong et al., 2009). DGBP was synthesized as previously described

(Shull et al., 2006) and dissolved in water. Likewise, methyl schweinfurthin G was synthesized (Koubek et al., 2018) and protected from light at all times. All compound stocks were maintained at -20ᵒC at 10 mM in DMSO unless otherwise noted.

MTT assay

MTT assays were carried out as previously described (Weissenrieder et al., 2018).

Briefly, U87MG and U251MG cells were plated at 4000 cells/well in 96 well plates and allowed to seat overnight before treatment with 100 µL treatment media per well.

Treatments were mixed in phenol red free MEM (Gibco, Waltham, MA) + 10% FBS. After

44 h of incubation at 37ᵒC and 5% CO2, 10 µL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide, Life Technologies, Waltham, MA) was added to each well. At 48 h, 100 µL of stop solution (10% 1 N HCl, 10% Triton X–100, 80% 2-

propanol) was added and incubated at 37ᵒC overnight, then A570 (test) and A690

(reference) were read on a Spectramax i3x (Molecular Devices, Sunnyvale, CA).

Absorbances were normalized to vehicle controls.

Statistics

All data is representative of 2-3 independent experiments in triplicate.

Concentration response curves were generated via non-linear regression and compared with GraphPad Prism 8 (San Diego, CA). Flow cytometric data was analyzed via chi- square.

Results

Cotreatment with isoprenoid pathway inhibitor lovastatin sensitizes GBM cells to thioridazine treatment.

To observe if the cytotoxic effects of thioridazine were due to isoprenoid pathway alterations, we used 48 h MTT assays under concomitant treatment with pharmacological inhibitors of the pathway. For these experiments, we used 30 µM digeranyl bisphosphonate

(DGBP), 10 µM zoledronate, and 3 µM activated lovastatin. At these concentrations, these compounds are known to selectively inhibit stages of the isoprenoid pathway (Figure 16).

Lovastatin is a commonly used competitive inhibitor of 3-hydroxy-3- methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the isoprenoid pathway. Treatment with lovastatin reduces substrate synthesis and availability for all future pathway enzymes, decreasing both cholesterol synthesis and isoprenoid pyrophosphate availability. Zoledronate, a bisphosphonate, acts downstream of lovastatin to reduce farnesyl pyrophosphate (FPP) synthesis and modulate downstream enzymes, such as farnesyl transferase and squalene synthase (Dunford et al., 2001). This represents

a branch point in the isoprenoid pathway, where FPP may be used to isoprenylate proteins, synthesize geranylgeranyl pyrophosphate (GGPP), or begin the committed cholesterol biosynthetic process. Farnesyl transferase requires the farnesyl pyrophosphate (FPP) substrate generated by the isoprenoid pathway to post-translationally modify, and thus regulate, small GTPases. These proteins, including Ras family members, are known to contribute to cancer development and metastasis. Squalene synthase, however, requires

FPP for the first committed, rate-limiting step to synthesize cholesterol, itself a required component of cell membranes. Downstream of FPP synthesis, DGBP selectively inhibits the synthesis of GGPP by geranylgeranyl diphosphate synthase (GGDPS), reducing the substrate availability for the isoprenylation of small GTPases in the Rho and Rab families by their respective transferases (geranylgeranyl transferase 1 and 2, respectively) (Wiemer et al., 2007; Wiemer et al., 2011). This can disrupt cytoskeletal regulation, thus potentially contributing to dysregulation of metabolic processes and trafficking of required metabolic and structural substrates. These enzymes are later-stage enzymes in the isoprenoid pathway, subject to the regulation of upstream enzymes such as 3-hydroxy-3- methylglutaryl-CoA reductase (HMGCR).

Metabolic activity of both U87MG and U251MG GBM cell lines were examined with 48 h MTT assays. We observed a small, yet statistically significant, reduction in inhibitory concentration (IC50) for thioridazine for cells cotreated with lovastatin and zoledronate (p<0.0001), but not for cells cotreated with DGBP (p>0.05) (Figure 17A-B).

This suggests that the isoprenoid biosynthetic pathway may be involved in the mechanism of action for thioridazine, since an inhibitor of this pathway is able to sensitize cells to thioridazine treatment. However, the disruption of small GTPase regulation by

geranylgeranylation may not be involved in this process, indicating that the earlier steps in the biosynthetic pathway may be more critical for cell survival under treatment with thioridazine.

Exogenous supplementation of isoprenoid pathway intermediates does not protect

GBM cells from thioridazine treatment.

Having observed that isoprenoid biosynthetic pathway inhibition could sensitize

GBM cell lines to thioridazine treatment, we then examined whether the addition of pathway intermediates was sufficient to prevent the cytotoxic effects of thioridazine. This process is helpful to control for any non-selective effects of the pharmacological inhibitors in Figure 17. For FPP and GGPP, we supplied the alcohols of these pyrophosphates, which are more chemically stable and membrane permeable than the highly charged pyophosphates. When we treated cells with thioridazine and 5 mM mevalonate, 30 µM farnesol, or 20 µM geranylgeraniol, we saw no significant increases in IC50 (Figure 18A-

B). Indeed, supplementation with pathway intermediates farnesol and geranylgeraniol actually increased cytotoxicity (p<0.0001 for both). This may indicate that supplementation induced feedback inhibition on the isoprenoid pathway or that the protective effects of lovastatin and zoledronate are due to off-target interactions. However, the effects of mevalonate were more complex. Addback of mevalonate did not significantly sensitize U87MG cells to thioridazine, and it had a very slight, but statistically significant, protective effect on U251MG. This difference may be due to metabolic differences in the cell lines, but complicates findings.

Cotreatment with methyl-schweinfurthin G sensitizes cells to thioridazine treatment.

We also ascertained the effects of cotreatment with a novel anticancer therapeutic from the schweinfurthin family, methyl-schweinfurthin G (MeSG). These compounds are known to have potent anticancer effects in the sub-micromolar range in models of central nervous system malignancies such as GBM (Beutler et al., 1998). While their anticancer mechanism of action is currently unknown, this class of compounds is known to disrupt lipid trafficking and metabolism in cell culture systems (Koubek et al., 2018). When we cotreated U87MG and U251MG cells with 100 nM MeSG, we saw a half-log order shift suggesting a more robust sensitization of these cell lines to thioridazine (Figure 19A-B).

This suggests that perhaps the localization and/or concentration of cholesterol, its metabolites, or its precursors may affect sensitivity of cell lines to thioridazine treatment.

Conclusions

Taken together, our results indicate that depletion of isoprenoid pathway intermediates via upstream inhibition may increase sensitivity of the GBM cell lines,

U87MG and U251MG, to the anticancer activity of thioridazine. However, treatment with these intermediates added back to the media is not protective. This suggests that, while this pathway may be perturbed to sensitize cells, its products are not sufficient to protect GBM cells from the cytotoxic effects of thioridazine. Such findings may be rationalized in a number of ways. For one, a fully functional isoprenoid pathway may provide some protection against thioridazine, but low level function without the addition of pathway intermediates may be sufficient for this effect. In this paradigm, only profound, near-total blockade of the pathway is sufficient to have an effect. Another option is that such effects are dependent upon cholesterol concentration, and cholesterol is not synthesized quickly

enough from intermediates to protect against cotreatment with thioridazine, which may have cytotoxic effects at much earlier time points. If so, addbacks could cause feedback inhibition which would effectively reduce pathway flux while thioridazine is inducing cytotoxicity.

Future work may further clarify the role of the isoprenoid biosynthetic pathway in thioridazine response. This work may include quantitation of cholesterol and isoprenoid pathway intermediates under treatment with thioridazine or related compounds. It may also involve carrying out experiments with lipid depleted medium, cholesterol extraction with methyl-β-cyclodextran, or cholesterol supplementation to more closely observe the role of the cholesterol synthesis branch of this pathway. It may also be instructive to observe the effects of isoprenoid pathway modulation and thioridazine treatment in the context of cancer stem cell enriched populations, as may be modeled by spheroid cultures. These cultures appear to better model patient responses to compounds and may provide a clearer picture of the in vivo effects of these treatments (Lee et al., 2006).

Importantly, it is imperative to note that while the effects of isoprenoid biosynthetic pathway inhibitors are statistically significant, the magnitude of the effect is negligible in all experiments except perhaps for the experiments involving MeSG, where the curves are separated by half a log order. These findings may thus assist researchers in understanding the cytotoxic cascade initiated by thioridazine, but they likely have no direct translational applicability. Current findings support a conclusion that thioridazine induces a metabolic crisis which may relate to lipid metabolism. However, these effects are not causative of cytotoxicity, but rather an effect of it.

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Respective Contributions

JSW conceived, designed, carried out, and analyzed all experiments with the aid of Jessie L. Reed. JSW wrote the manuscript. Jeffrey D. Neighbors and Raymond J. Hohl contributed to experimental design and analysis and edited the manuscript.

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Figures

Figure 16. The isoprenoid biosynthetic pathway.

The isoprenoid pathway is involved in the synthesis of cholesterol and isoprenyl groups. It may be inhibited at key steps. Notably, the statins, such as lovastatin, inhibit the rate limiting step near the beginning of this pathway, synthesis of mevalonate from HMG-CoA via HMG-CoA reductase (HMGCR). Zoledronate inhibits the conversion of isopentenyl pyrophosphate (IPP) to geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP) by farnesyl diphosphate synthase (FDPS). The pathway branches out at FPP, which can be used to isoprenylate proteins through the action of farnesyl transferases (FTases), as a substrate to synthesize geranylgeranyl pyrophosphate (GGPP), or as a substrate for the committed step of cholesterol synthesis by squalene synthase (SS). Digeranyl bisphosphonate (DGBP) inhibits the conversion of FPP to GGPP by geranylgeranyl

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diphosphate synthase (GGDPS), reducing substrate availability for geranylgeranyl transferases 1 and 2 (GGTase 1/2).

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Figure 17. Isoprenoid pathway inhibitors sensitize GBM cells to thioridazine.

In a 48 h MTT assay, 3 µM lovastatin and 10 µM zoledronate sensitize U87MG (A) and

U251MG (B) cells to thioridazine treatment (p<0.0001). However, 30 µM digeranyl bisphosphonate has no significant effect on the sensitivity of these cell lines.

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Figure 18. IBP intermediates do not strongly affect thioridazine sensitivity.

Exogenous supplementation of isoprenoid pathway intermediates does not have a pronounced effect on GBM cell sensitivity to thioridazine. Addback of geranylgeranyl and farnesyl moieties induced a low-magnitude, but highly significant (p<0.0001) sensitization of U87MG (A) and U251MG (B) cells to thioridazine treatment, according to 48 h MTT data. 5 mM mevalonate had no effect on U87MG, but was slightly protective for U251MG

(p<0.05).

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Figure 19. Methyl-schweinfurthin G sensitizes GBM cells to thioridazine.

Cotreatment with 100 nM methyl-schweinfurthin G sensitizes GBM cell lines to thioridazine. In a 48 h MTT, U87MG (A) and U251MG (B) cell lines were significantly sensitized to thioridazine by 100 nM concentrations of the anticancer candidate compound, methyl-schweinfurthin G, which induced a half log order increase in sensitivity

(p<0.0001).

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CHAPTER 6. FUTURE DIRECTIONS

Introduction

The studies presented within this dissertation provide insights about the role the

D2R and its pharmacological modulators may play in cancer cell proliferation and treatment. However, our current understanding of these receptors and compounds also raises new questions about mechanism, therapeutic viability, and other potential effects of these compounds on cancer sensitivity. In this chapter, potential lines of inquiry for future research will be discussed.

D2R antagonists and calcium signaling

While it is clear that D2R antagonists are capable of altering calcium flux within

GBM monolayer cultures, many questions remain about the mechanism by which the flux is induced. It is possible that increases in cytosolic calcium levels are due to selective modulation of proteins such as the plasma membrane calcium ATPase (PMCA) and the sarco/endoplasmic reticulum calcium ATPase (SERCA). It is also possible that calcium effects are due to direct inhibition of calmodulin. To observe the role of these proteins in compound response, it may be useful to carry out docking studies to observe the potential for compound binding or to carry out overexpression experiments to observe if protein overexpression ablates the cytotoxicity of D2R compounds. Such experiments would be complicated by the biological requirement for calcium modulating proteins that has led to functional redundancy within these systems. For instance, knockdown experiments are not expected to be useful in these studies, as knockdown of specific proteins is known to either be compensated for by homologous proteins or cytotoxic in itself due to dysregulation of intracellular calcium homeostasis (Bruce, 2018).

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However, the relatively high concentrations required for these effects, coupled with the chemical structures of cytotoxic D2R antagonists, suggests another possibility.

Chemically, these compounds are all cationic amphiphiles, with structures that contain both hydrophilic and hydrophobic moieties. Such compounds are capable of insertion into the membrane and may act as detergents. Indeed, numerous ionic amphiphiles are used as detergents, including the common laboratory detergent, sodium dodecyl sulfate (SDS, an anionic amphiphile). Concentrations of many D2R antagonists are much higher in fatty tissues, such as the brain, as compared to the plasma (Daniel et al., 1997). If sufficient concentration of these compounds is present to disrupt membrane integrity, it could cause rapid increases in cytosolic calcium levels. The potential for this could be examined in a cell-free system, through the measurement of membrane integrity and rigidity via biophysical techniques such as atomic force spectroscopy or electron spin resonance.

Membrane composition may be varied to observe the role of various phospholipids and cholesterol in sensitivity to membrane disruption.

If it is determined that the anticancer efficacy of these compounds is due to direct action at a calcium-regulating protein such as PMCA, SERCA, or calmodulin, other compounds targeting that protein could be developed or re-examined as potential anticancer therapeutics. This could lead to the development of a new treatment modality in the context of cancer in general. However, if these effects are due to loss of membrane integrity rather than action at a protein target, it would suggest that such compounds would have a limited therapeutic window and a high potential for adverse effects. This is due to intrinsic biological limitations on viable membrane compositions. While cancer cells do have altered plasma membrane lipid composition, it is likely not sufficiently different to

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selectively target with a detergent. On the other hand, the modulation of cancer cell membrane lipid profiles with membrane lipid therapies such as 2-hydroxyoleic acid has been proposed as a potential therapeutic strategy in glioma, suggesting that a selective alteration in membrane composition of cancer cells could reduce tumor growth (Llado et al., 2014). Thus, if a membrane altering effect were more selective, it may have some utility in anticancer treatment.

Immunological effects of D2R antagonists

As noted in Chapter 2, anti-tumor activity appears to occur in vivo at lower blood plasma concentrations of D2R antagonist than would be expected when compared to cytotoxic concentrations in cell culture experiments. This would suggest a systemic-level effect of the compounds that is not present in cell culture. The immune system is one body system which is of great interest in anticancer therapy, as it can actively combat tumor growth. In the context of many peripheral cancers, the use of immunomodulatory treatments such as immune checkpoint inhibitors and antibody-based therapies have greatly improved patient response to treatment and survival. While most animal studies investigating the antitumor activity of D2R antagonists are in the context of immunodeficient mice, it is tempting to consider the potential effects of these compounds on an intact immune system.

It is possible that D2R antagonist treatment could enhance immune response at the tumor site for a number of reasons. It is known that these compounds alter calcium flux and induce ER stress (Wolfe and Morris, 1999; Jandaghi et al., 2016; Park et al., 2016).

These effects may lead to the surface expression of calreticulin, which may serve as an “eat me” signal to attract immune cells (Galluzzi et al., 2017). This effect could be readily tested

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via flow cytometry by staining for surface expression of calreticulin. In vivo, effects could be observed in immunocompetent models of cancer, either genetic or orthologous xenograft. If the immune system contributes to compound response, tumor size will be reduced and the effect may be amplified under cotreatment with immune modulators, such as checkpoint inhibitor therapy.

D2R antagonists and spheroids

Although spheroid formation, a common surrogate readout for stemness, is altered by selective concentrations of D2R modulating compounds, these changes are not paralleled by alterations in stemness markers Nestin and SOX2 or differentiation marker

GFAP. Therefore, it is not known what induces the change in phenotype. A few possibilities emerge from these findings.

Firstly, it is possible that stemness is altered, but the particular markers used in this study are not informative within this particular experimental system. Potential markers for

CSCs are fiercely contested in the field, and their use is rather uncertain depending on experimental readouts since they are largely co-opted from studies of human pluripotent stem cells. Future studies could observe changes in other potential stemness markers via flow cytometry for CD133/CD44, or via fluorescence of Oct-4-GFP reporter cells to further define the role of stem-like properties in this phenotype under treatment with selective concentrations of D2R modulators.

Secondly, stemness may not be affected at all. In this event, alterations in cell-cell adhesion or metabolic processes favoring anchorage-independent growth may lead to an apparent increase in stem-like behavior in this experimental paradigm. Alterations in cell- cell adhesion could be controlled for by carrying out limiting-dilution spheroid formation

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assays to remove the possibility that cells may survive via aggregation. If cell-cell adhesion is involved, protein expression of adhesion molecules such as β1 integrin and intracellular adhesion molecules (ICAMs) may also increase under treatment with D2 agonists and decrease under treatment with antagonists. This could be measured by immunoblotting. It is also possible that observed effects in sphere formation are merely an artifact of the spheroid-forming system and may not be biologically relevant. The D2R is known to be involved in EGFR transactivation via an interaction with ADAM proteins (Yoon and Baik,

2013), and its presence or activation may be sufficient to promote EGF-dependent growth, which is favored in the spheroid-formation media since it is high in EGF. In this case, the effect may not be particularly relevant from a translational perspective. Immunoblotting for downstream effectors of EGFR or concurrent treatment with higher levels of EGFR or an EGF signaling inhibitor may shed light on this possibility.

Investigation into these prospective mechanisms by which selective concentrations of D2R modulators elicit changes in the spheroid formation phenotype will further clarify the role of the D2R in cancer cell stemness. It may also illustrate the limitations of the spheroid-formation model of cancer cell stemness, as it is possible that cell-cell adhesion or EGF sensitivity are more important to this phenotype in U87MG cells, and spheroid formation under these conditions is not representative of stem cell-like behavior. It is also possible that this cell line may perform differently than patient-derived spheroid cultures; in this case, repeating the same series of experiments in patient-derived spheroids may be more instructive.

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Anticancer efficacy of D2 antagonists in humans

D2R antagonists have already shown potential for anticancer activity in human patients in case studies, but this possibility has, as yet, not been tested in a clinical trial.

This would definitively determine whether or not such compounds have potential as anticancer therapeutics in human patients. This could be crucial for patients with CNS cancers which currently have dismal survival prognosis and limited treatment options, such as GBM, as it could provide another therapeutic option to patients who are not currently responding to their therapeutic regimen. Since the majority of the D2R compounds which have been studied in the context of cancer are FDA-approved for psychiatric indications, their safety profiles, pharmacokinetics, and pharmacodynamics are well known and it would be relatively easy to carry out a limited in-human study to determine safety and efficacy in patients. Many other studies could be carried out to further elucidate mechanism, but only such a study would determine efficacy and therapeutic window in humans to determine if a larger scale study is warranted.

Central nervous system modulation of anticancer effects

Finally, it has recently become clear that alterations within the central nervous system can have clear effects on immune system response and the efficacy of anticancer therapy (Chang et al., 2014). These effects are not well understood, but may have a very real impact on patient survival or treatment response. Since D2R antagonists are psychoactive and are known to modulate dopamine signaling throughout the brain, it is possible that these compounds could contribute to a sort of “psychomodulation” of anticancer activity. This idea is very interesting, but may prove difficult to explore experimentally. Typical treatment modalities and therapeutic readouts would not allow

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-/- discernment between CNS-modulated and peripheral effects. D2R , immunocompetent mouse models of cancer may allow the observation of the receptor’s central nervous system role in cancer development, though this would not account for the complex neuropharmacology of these compounds. Another option would be to correlate murine fMRI activity with anticancer activity under treatment with D2 antagonists, though this would be highly speculative and thus of limited utility.

Conclusions

Future research in the area of D2R and D2R antagonists in cancer may focus in a number of areas. The role of D2R in stemness could be elucidated further, as could the pharmacological effects of D2R antagonists on calcium flux. However, the most critical studies at this juncture would be those that focus on whole organism systems, such as murine models or in-human studies. These could further illuminate the role of the central nervous system and the immune system in anticancer treatment response, as well as clarify the anticancer efficacy of D2R antagonists in human patients. Such in-human studies would be necessary to determine if these compounds have any potential for use as anticancer therapeutics in the future.

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CHAPTER 7. CONCLUSIONS

Introduction

This body of work has contributed to our understanding of both dopamine D2 receptors and chemical modulators of this receptor in the context of cancer, specifically

GBM. This central nervous system malignancy has a very poor prognosis, and few treatment options are available to patients at this time. By developing more understanding of the anticancer activities of a group of compounds which are known to cross the cerebrovascular barrier, researchers can determine the viability of these compounds as chemotherapeutics in humans. This work has summarized the state of the literature on the subject while expanding our knowledge of the mechanism of action of D2R antagonists as anticancer agents in the context of both monolayer and spheroid cell culture using established GBM cell lines. The conclusions drawn from this research may be applied to future research in the area.

D2R antagonists elicit D2R-independent cytotoxicity in GBM cells.

The reported ability of D2R antagonists to induce cytotoxicity initiated this line of inquiry. While numerous sources have observed cytotoxicity, reduced proliferation, and reduced metabolic activity in cancer cells in the literature (reviewed in Chapter 2), we noted that concentrations required for this cytotoxicity were orders of magnitude higher than that which would be expected for a D2R mediated effect. Thus, we screened numerous chemotypes of D2R antagonists in a panel of GBM cell lines in order to further our understanding of this cytotoxicity. These findings suggested that multiple D2R antagonists of differing chemotype were able to reduce GBM cell metabolic activity with IC50 concentrations in the 1-50 micromolar range, as seen in Chapter 3. However, one D2R

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antagonist, remoxipride, was unable to elicit this response. This antagonist is highly selective for D2-like receptors, with modest selectivity for D2 over D3 or D4 receptors

(Seeman and Tallerico, 1998). This finding, coupled with the disparity between ligand affinity for the D2 family of receptors and the effective IC50 concentrations, led to the hypothesis that the anticancer attributes of this group of compounds may be independent of the D2R.

Although D2R are expressed in GBM cells (Chapter 3) and patient tumors in a number of cancers (Li et al., 2006; Mao et al., 2015; Cherubini et al., 2016a), we observed that D2R antagonist response was not associated with D2R expression in GBM cell monolayer culture. This is evident in cell lines with varying levels of expression, as well as in transiently transfected and stably transduced GBM cell lines. When coupled with the lack of cytotoxicity of a selective D2R antagonist and the high concentrations required for cytotoxic behavior, such results strongly indicate that a non-D2R-mediated mechanism is involved in cytotoxicity.

Indeed, we found evidence for a non-selective effect of cytotoxic D2 antagonists.

The cytotoxic activity of these compounds is linked to calcium signaling within the cell.

Calcium floods the cytoplasmic compartment within seconds of treatment with ~IC50 concentrations of D2 antagonists (Chapter 3). Such high concentrations are known to be cytotoxic to the cell, and we show that this calcium flux is tied to loss of mitochondrial membrane potential and cytochrome C release. This provides another possible mechanism of action by which these compounds could elicit direct cytotoxicity in cancer cells.

Despite the lack of effect of D2R expression on cancer cell cytotoxicity under treatment with D2 antagonists, it is still possible that D2R may contribute to tumor growth

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in some way. Previous research has suggested that D2R expression correlates with patient prognosis (Li et al., 2006), and knockdown with shRNA reduced cancer cell proliferation

(Li et al., 2014). We also observed a relationship between D2R expression and cell proliferation rate. While D2R expression was not correlated with D2R antagonist response in monolayer cultures, we were able to see significant alterations in spheroid formation phenotypes for U87MG cells treated with selective concentrations of these pharmacological modulators. While it is not clear whether this effect is due to alterations in stem-like behavior, cell-cell adhesion, or metabolic pathways, future work may further elaborate the role of this receptor in cancer cell growth, proliferation, and stemness.

Conclusions: Providing Context

As discussed in Chapter 1, GBM is a devastating central nervous system malignancy with dismal survival rates and few therapeutic options. Patients need new treatment options, and repurposing already-characterized and/or already FDA-approved drugs may provide much-needed alternatives to the current standard of care. Potential compounds to repurpose for anticancer therapy include D2R antagonists, of which numerous examples are approved by the Food and Drug Administration for use as antipsychotic compounds. The work presented here underscores the in vitro cytotoxicity of these compounds and extends our understanding of compound efficacy in GBM cell lines.

However, it also proposes a different anticancer mechanism of action, one which may involve calcium overload. While more research is needed to identify the target involved in the anticancer activity of these compounds, such work could lead to new therapeutic possibilities for patients. It is possible that current lead compounds may show anticancer efficacy in humans with acceptable adverse effect profiles. Alternatively, they may be

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reworked and derivatized into more effective, safer anticancer therapeutics given more understanding of the target which must be engaged for these effects. This is true whether the compound target is a dopamine receptor or a calcium transporter, such as a plasma membrane calcium ATPase (PMCA). Only time and more research will tell if D2 antagonists or their derivatives will prove useful as anticancer agents.

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VITA Jillian S. Weissenrieder

Education: 2015-2019 Pennsylvania State University College of Medicine PhD. in Biomedical Sciences Advisor: Raymond J. Hohl, MD/PhD. Dissertation Title: Dopamine D2 Receptor Antagonists as Potential Therapeutics for Glioblastoma Multiforme 2013-2015 Millersville University B.S. in Chemistry (Biochemistry track) Research Advisor: Edward Rajaseelan, PhD. 2010-2013 Harrisburg Area Community College

Selected Publications: • Weissenrieder, J.S., Reilly, J.E., Neighbors, J.D., Hohl, R.J. Inhibiting geranylgeranyl diphosphate synthesis reduces nuclear androgen receptor signaling and neuroendocrine differentiation in prostate cancer cell models. The Prostate. 2019 Jan;79(1):21-30. doi: 10.1002/pros.23707. • Koubek, E.J., Weissenrieder, J.S., Neighbors, J.D., and Hohl, R.J. Schweinfurthins: Lipid Modulators with Promising Anticancer Activity. Lipids. 2018 Aug;53(8):767-784. doi: 10.1002/lipd.12088. • Weissenrieder, J.S., Neighbors, J.D., Mailman, R.B., Hohl, R.J. Cancer and the dopamine D2 receptor: a pharmacological perspective. JPET. Submitted. • Weissenrieder, J.S., Moldovan, G.L., Johnson M. T., Trebak, M., Neighbors, J.D., Mailman, R.B., Hohl, R.J. D2 antagonist thioridazine elicited cytotoxicity in glioblastoma multiforme is calcium-dependent and independent of the D2-receptor. Nature. Submitted (letter). • Weissenrieder, J.S., Reed, J.L., Koubek, E.J., Moldovan, G.L., Neighbors, J.D., Hohl, R.J. The dopamine D2 receptor contributes to the spheroid formation behavior of U87MG glioblastoma cells. Pharmacology. Submitted. • Weissenrieder, J.S., Reed, J.L., Neighbors, J.D., Hohl, R.J. Thioridazine as an anticancer therapeutic: interplay with the isoprenoid biosynthetic pathway. bioRxiv. doi:10.1101/532788. Professional Service: 2017-2018 Penn State Hershey Graduate Student Association, Research Forum Chair 2017-2018 Instructor, Beginning Laboratory Statistics Workshop, Penn State College of Medicine 2017 Lecturer, CHEM 316 – The Professional Chemist, Penn State University Awards and Funding: 201 Graduate Alumni Endowed Scholarship 2017 College of Medicine Alumni Society Endowed Scholarship 2017 Predoctoral Training Grant, 2T32CA060395-21A1 (Viruses and Cancer), Pennsylvania State University Hershey Medical Center. Craig Meyers, PI 2015-2017 Pritchard Distinguished Fellowship 2016 American Chemical Society Award in Analytical Chemistry 2014-2015 - Sandra L. Turchi biochemistry endowment 2014-2015 – Richard Sasin Scholarship