The Pennsylvania State University The Graduate School Department of Pharmacology

IDENTIFICATION OF NOVEL PHARMACOLOGICAL APPROACHES TO INHIBIT

NEUROTENSIN RECEPTOR-1 MITOGENIC SIGNALING IN BREAST CANCER

CELLS

A Dissertation in Pharmacology by Yasser Heakal

© 2009 Yasser Heakal

Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

December 2009 ii

The dissertation of Yasser Heakal was reviewed and approved* by the following:

Mark Kester Distinguished Professor of Pharmacology G. Thomas Passananti Professor of Pharmacology Chair of Committee Dissertation Advisor

Lisa Shantz Associate Professor of Cellular and Molecular Physiology

Christopher Herzog Assistant Professor of Pharmacology

Jong K. Yun Associate Professor of Pharmacology Director, Pharmacology Graduate Program

Melvin L. Billingsley Professor of Pharmacology, Biotechnology and Entrepreneurship

*Signatures are on file in the Graduate School. iii

ABSTRACT

G protein coupled receptors (GPCRs) represent the largest family of cell surface receptors and serve as the primary pharmacological targets for more than thirty percent of approved drugs on the market. In addition to targeting GPCRs to manage cardiovascular and central nervous system associated disease conditions, a growing body of literature suggests that many GPCRs are involved in cancer development, progression and metastasis.

Neurotensin receptor 1 (NTSR1) is a GPCR that has been recently identified as a mediator of tumorigenicity and metastasis. NTSR1, as well as its endogenous ligand, neurotensin (NTS), are coexpressed in several types of cancer including lung, pancreas, prostate, colon, head and neck as well as breast cancer cell lines and breast cancer tumor samples. We have previously reported that ceramide mimetics could inhibit breast cancer cell growth in vitro and in vivo . The direct effect of C6 ceramide on

GPCR-mediated cancer progression has not been characterized. Thus, understanding the biochemical and biophysical regulation of NTSR1 by ceramide can help further define NTSR1 as a novel target in breast cancer. Our results show that nanoliposomal formulations of C6 ceramide inhibit NTSR1-mediated MDA-MB-231 breast cancer progression (mitogenesis, migration, and matrix metalloproteinase-9 activity). In addition, liposomal C6 ceramide inhibited NTSR1-mediated, but not phorbol 12- myristate 13-acetate–mediated, activation of the mitogen-activated protein kinase pathway. Mechanistically, nanoliposomal short-chain ceramide reduces NTSR1 interaction with G αq/11 subunits within structured membrane microdomains (SMDs), iv consistent with diminished NTS-induced translocation of NTSR1 into membrane microdomains.

We next hypothesized that agonist-induced receptor palmitoylation mediates dynamic receptor translocation and signaling within SMDs. We have identified that endogenously expressed NTSR-1 in MDA-MB-231 breast adenocarcinomas as well as exogenously expressed NTSR-1 in HEK293T cells that do not normally express NTSR-

1 is palmitoylated at Cys 381 and Cys383. Inhibition of NTSR-1 palmitoylation in MDA-

MB-231 cells as well as NTSR-1 expressing HEK293T cells diminished NTS-mediated

ERK 1/2 phosphorylation. Additionally, NTSR1 mutated at Cys381 and/or Cys383 to serine showed diminished ERK1/2 stimulation and reduced ability to protect HEK293T cells against apoptosis induced by serum starvation. Mechanistically, C381,383S-

NTSR-1 showed reduced ability to interact with G αq/11 and diminished localization to

SMDs, where G αq/11 preferentially resides.

Based on these observations, we next hypothesized that ceramide effects on NTSR-

1 localization and signaling could be in part mediated through interference with NTSR-1 palmitoylation/depalmitoylation cycle. Our results suggest that ceramide could either inhibit NTSR-1 palmitoylation through direct biochemical or biophysical mechanism(s) or through acceleration of NTSR-1 deplamitoylation.

Collectively, our findings suggest that liposomal short-chain C6 ceramide can be utilized to inhibit NTS-dependent breast cancer cell growth. Our data also establish palmitoylation as a novel pharmacological target to disrupt NTSR-1 mitogenic signaling in breast cancer. v

TABLE OF CONTENTS

LIST OF FIGURES ...... ix

ACKNOWLEDGEMENTS ...... xi

Chapter 1. Introduction ...... 1

1.1 G-Protein coupled receptors role in cancer ...... 2

1.1.1 GPCRs Role in Cancer Development and Progression ...... 4

1.2 Neurotensin/Neurotensin Receptors ...... 7

1.2.1 Neurotensin Receptors ...... 8

1.2.2 Neurotensin Receptor-1 ...... 8

1.2.3 Neurotensin Receptor-2 ...... 12

1.2.4 Neurotensin Receptor-3 ...... 12

1.3 Structured Membrane Microdomains (SMDs) ...... 13

1.3.1 SMDs and Signal Transduction ...... 15

1.3.2 Role of SMDs in GPCRs Signaling ...... 19

1.3.3 Protein Palmitoylation and Localization to SMDs ...... 22

1.3.4 Sphingolipids and SMDs ...... 25

1.3.5 Modulation of SMDs as a Therapeutic Approach to Alter Signaling in Cancer ...... 29

1.4 Conclusions ...... 34

1.5 References .………….……………………………………………………..36

Chapter 2. Nanoliposomal Short-Chain Ceramide Inhibits Agonist- Dependent Translocation of Neurotensin Receptor-1 to Structured Membrane Microdomains In Breast cancer Cells ...... 55

2.1 Abstract ...... 56

2.2 Introduction ...... 57 vi

2.3 Materials and Methods ...... 58

2.4 Results ...... 67

2.4.1 Exogenous Short Chain Ceramide Inhibits NTSR-1-Mediated Cellular Proliferation in MDA-MB-231 Cells ...... 67

2.4.2 Exogenous Short Chain Ceramide Inhibits NTSR-1-Mediated Cellular Migration in MDA-MB-231 Cells ...... 70

2.4.3 Exogenous Short Chain Ceramide Inhibits NTSR-1-Mediated MMP-9 Expression/Activation in MDA-MB-231 Cells ...... 70

2.4.4 Exogenous Short Chain Ceramide Inhibits NTSR-1-Mediated, but not PMA-Mediated Activation of the MAP Kinase Pathway in MDA-MB-231 Cells ...... 72

2.4.5 NTSR-1 Signaling Requires Intact Structured Membrane Microdomains ...... 73

2.4.6 Exogenous Short Chain Ceramide Inhibits Agonist-Dependent Translocation of NTSR-1 into Structured Membrane Microdomains ...... 75

2.4.7 NTSR-1 Stimulation does not Alter G αq/11 Localization within SMDs ...... 78

2.4.8 Exogenous C6 Ceramide Inhibits NTSR-1Interaction with Gαq/11 within SMDs ...... 79

2.5 Discussion ...... 82

2.6 References ...... 86

Chapter 3 Neurotensin Receptor-1 Inducible Palmitoylation is Required for Efficient Receptor-Mediated Mitogenic Signaling within Structured Membrane Microdomains ...... 89

3.1 Abstract ...... 90

3.2 Introduction ...... 91

3.3 Materials and Methods ...... 93

3.4 Results ...... 98 vii

3.4.1 NTSR-1 is Palmitoylated in MDA-MB-231 Breast Cancer Cells ...... 98

3.4.2 Pharmacological Inhibition of NTSR-1Palmitoylation in MDA-MB-231 Cells Inhibits Receptor Mediated Mitogenic Signaling ...... 98

3.4.3 Generation and Expression of Wild Type and C381,383S- mutated NTSR-1 in HEK293T Cells ...... 100

3.4.4 NTS Treatment Induces Palmitoylation of WT-NTSR-1 but not C381,383S NTSR-1Expressed in HEK293T Cells ...... 102

3.4.5 NTS Treatment Promotes Viability of HEK293T Cells Expressing WT-NTSR-1 but not C381,383S NTSR-1 ...... 104

3.4.6 NTS Treatment Protects HEK293T Cells Expressing WT-NTSR-1 but not C381,383S NTSR-1from Apoptosis Induced by Serum Starvation ...... 104

3.4.7 WT-NTSR-1 but not C381,383S-NTSR-1Mutant Efficiently Mediates NTS-Induced ERK 1/2 Phsophorylation ...... 106

3.4.8 2-Bromopalmitate Inhibits WT-NTSR-1 Mediated but not PMA -Mediated Activation of MAP Kinase Pathway ...... 106

3.4.9 Palmitoylation of NTSR-1 is Required for Receptor Interaction with G αq/11 ...... 108

3.4.10 Palmitoylation of NTSR-1 is Required for Efficient Receptor Localization to SMDs ...... 108

3.4.11 NTSR-1 Palmitoylation Pattern may regulate NTSR-1 Stability ...... 111

3.4.12 NTSR-1 Double Palmitoylation is Required for Efficient Receptor Mitogenic Signaling ...... 112

3.4.13 Short Chain C6 Ceramide Inhabits NTS-induced NTSR-1 Palmitoylation ...... 113

viii

3.5 Discussion ...... 114

3.6 References ...... 119

Chapter 4 Conclusions, Therapeutic Potential and Future Directions ... 122

4.1 Therapeutic Potential of Short Chain Ceramides as SMDs Modulators ...... 123

4.1.1 Modulation of SMDs as a Therapeutic Approach for AIDs Treatment ...... 127

4.2 Therapeutic Potential of GPCRS-Palmitoylation Inhibitors ...... 129

4.2.1 Identification of NTSR-1 Palmitoylating Enzymes ...... 130

4.2.2 Potential Role of of NTSR-1 Palmitoylation in Receptor Stability ...... 133

4.2.3 Possible Cross Talk between of NTSR-1 Palmitoylation and Glycosylation ...... 135

4.2.4 Regulation of NTSR-1 Palmitoylation by Sphingolipids ...... 136

4.3 Summary ...... 138

4.4 References………………………………………………………………….140

ix

List of Figures

Chapter 1

Figure 1: NTSR-1 primary signaling pathway ...... 10

Figure 2: Ceramide raft formation ...... 17

Figure 3: Basic sphingolipid structures ...... 26

Figure 4: Ceramide metabolic pathways associated with drug resistance ...... 31

Figure 5: Sterol biosynthetic pathway ...... 33

Chapter 2

Figure 1: Anti-NTSR-1 antibody recognizes NTSR-1 in human cell lines ...... 61

Figure 2: Exogenous short ceramide inhibits NTSR1-mediated cellular proliferation ...... 69

Figure 3: Exogenous short chain ceramide inhibits NTSR1-mediated MMP9-dependent cellular invasion ...... 71

Figure 4: Exogenous short chain ceramide inhibits NTSR1 mediated ERK phosphorylation within SMDs ...... 74

Figure 5: Exogenous short chain ceramide inhibits NTS-dependent translocation of NTSR1 into SMDs ...... 78

Figure 6: NTSR1 Translocates to SMDs and Interacts with G αq/11 ...... 81

Chapter 3

Figure 1: Endogenously expressed NTSR-1 in breast cancer cells is palmitoylated ...... 99

Figure 2: NTSR-1 proteins can be overexpressed and glycosylated in HEK293T cells...... 101

Figure 3: Cysteine 381, 383 in NTSR-1 are required for optimal palmitoylation...... 103

x

Figure 4: NTSR-1 palmitoylation is required for NTS-mediated pro-survival activity ...... 105

Figure 5: NTSR-1 palmitoylation is required for NTS-mediated MAPK mitogenic signaling...... 107

Figure 6: NTSR-1 palmitoylation is required for optimal interaction with G αq/11A within SMDs...... 110

Figure 7: Mutant NTSR-1 receptors can be overexpressed and glycosylated in HEK293T cells ...... 112

Figure 8: NTSR-1 palmitoylation is required for efficient NTS -mediated MAPK mitogenic signaling...... 113

Figure 9: Effect of C6 ceramide on NTSR-1 palmitoylation ...... 114

Chapter 4

Figure 1: C6 ceramide disrupts NTSR-1 mitogenic signaling...... 124

Figure 2: NTSR-1 palmitoylation serves as coding signal...... 134

Figure 3: Possible mechanisms for ceramide mediated inhibition of NTSR-1 Palmitoylation ...... 137

Figure 4: Possible effect of C6 ceramide on NTSR-1 Palmitoylation/depalmitoylation cycle...... 138

xi

Acknowledgments

It would be impossible for me to spend 7 years in continuous pursuit of graduate studies, 6000 miles away from my homeland, Egypt, without continuous support and encouragement from my parents, my sister and her family. The friends I met over the years made this journey pleasant and joyful. My desire to pursue a career in science was inspired by many people who I have met including Paul Erhardt, my MS mentor, Xufei Huang, my Organic Chemistry mentor and Ahmad Zewail who I have admired and considered a role model throughout my journey in the United States. His book, Voyage through Time, has been always an inspiration.

Of course, without the support and encouragement from my PhD advisor, Mark Kester, this piece of work would not have been accomplished. I also appreciate the time of my committee members, Drs Yun, Herzog, Shantz and Billingsley who showed enthusiasm to discuss my research and provided me with valuable guidance when needed.

I would like to thank many people at PSU including Elaine Neidigh and Vicki for their help with many administrative issues whenever needed. They were generous and courteous all the time.

I would like to thank my friend and collaborator Matt Woll. Despite that our interaction lasted for brief period of time, we had joy and excitement discussing the possible impact of our collaborative research.

Chapter 1

Introduction

2

Cancer is a complex disease with multiple underlying causes ranging from genetics, environment to individual life style. Many signaling pathways and cellular deregulations were found to be associated with cancer initiation, development and progression. Each type of cancer is unique and requires different treatment regimens. Accumulating body of literature implicates G protein coupled receptors (GPCRs) in different stages of malignancy (1). Neurotensin receptor-1 (NTSR-1) which belong to the GPCR family has been associated with many types of cancers including pancreas, prostate, breast, colon and head and neck (2). The unique overexpression pattern of NTSR-1 in cancerous but not normal tissues provides an opportunity for selective pharmacological interventions.

In the next sections, GPCRs role in cancer and their potential regulation by cell membranes will be discussed in details in the following sections.

1.1 G-Protein Coupled Receptors (GPCRs)

GPCRs are the largest known family of cell surface receptors and represent the primary targets of about 30% of drugs currently on the market (3). GPCRs are characterized by 7-transmembrane α-helices with an extracellular N- terminus, an intracellular C-terminus and three loops on each side of the cell membrane. Based on sequence homology, GPCRs are divided into three distinct classes (4). Class A, which is the largest, includes receptors such as rhodopsin, adrenaline as well the olfactory receptors, transduce wide variety of signals ranging from photons, organic molecules, amines, proteins to lipids. The natural ligands of this class have diverse chemical structures. Class B contains only 25 members including the gastrointestinal peptide 3 hormone family such as glucagon and vasoactive intestinal peptide and parathyroid hormone receptors and structurally characterized by large extracellular ligand-binding domain. The natural ligands of members of this class are all endocrine peptides composed of >27 amino acids. Finally, class C which is the smallest and includes the glutamate receptor family (GABAa), calcium sensing receptors as well as some taste receptors. Members of this class are also characterized by a large extracellular amino terminus (3).

Agonist stimulation of GPCRs typically induces a conformational change in the spatial arrangement of certain helices forming an active receptor conformation which then interacts with heterotrimeric G protein subunits causing dissociation of the heterodimer into α and βɣ dimers which in turn regulate downstream effectors. The G α subunits hydrolyze GTP to GDP, an event known to be regulated by RGS (regulators of

G-protein signaling), followed by re-association of the heterotrimer and termination of the signaling cycle. To date, at least 16 α, 5 β and 12 ɣ subunits have been cloned.

GPCRs-mediated signaling is finely regulated by specific desensitization mechanisms to optimally turn-off signaling when necessary (3).

Two major molecules are involved in receptor desensitization, β-arrestins and G

Receptor Kinases (GRKs). Upon receptor stimulation, GRKs phosphorylate certain residues in the C-terminus forming docking sites for β-arrestins which turn off receptor- mediated signaling mainly by steric exclusion (5). Interestingly, β-arrestins are essential for transduction of signals that eventually lead to MAPK activation (6-8). Angiotensin type 1A receptor for example stimulates ERK 1/2 phosphorylation through both G protein mediated and β-arrestins mediated mechanisms (5). β-arrestins also can 4 function as scaffolding protein for Endothelin-A –dependent activation of β-cetenin (9).

β1 adrenergic receptor is another example in which activation of MAPK pathway is mediated through β-arrestin-dependent transactivation of epidermal growth factor receptor (EGFR) (10). Regulators of G-protein signaling (RGS) proteins are the newest family of proteins which regulate GPCRs downstream signaling events (11). RGS are also known as GTPase –activating proteins as they can influence G α subunits by enhancing their GTPase activity by up to 1000 folds resulting in G protein deactivation.

They also act as effectors antagonist by binding and sequestering activated G protein subunits. Based on RGS domain homology, they are divided into 8 subfamilies. RGS have been recently reviewed in details by Hurst and Hooks (12).

The evolution of GPCRs signaling pathways was accompanied by concurrent evolution of regulatory pathways that are essential for turning off signaling events physiologically. It is now well established that GPCRs are multi-tasking receptors, capable of activating multiple pathways simultaneously. This led to the notion that there are more complex mechanisms that regulate GPCRs signaling implicating cell membrane as a key player in this regulation process.

1.1.1 GPCRs Role in Cancer Development and Progression

A growing body of literature demonstrates that many GPCRs are overexpressed in tumor and adjacent tumor-microenvironment but not in normal tissues (1). Additionally, many types of tumors secrete GPCRs-endogenous ligands which form an autocrine, paracrine signaling loops and assist tumors to gain growth independence and 5 resistance to conventional therapeutic approaches (11). For example, overexpression of protease-activated receptor-1 in melanoma was found to be involved in metastasis via up-regulation of connexin 43 expression, a known mediator of tumor cell diapedesis

(outward movement of cells through intact walls of blood vessels) and attachment to endothelial cells (13). Many chemokine receptors which belong to the GPCR family also have been implicated in the progression of several types of cancers (14). CXCR1 and

CXCR2 expression in melanoma for instance, is associated with chemokine CXCL-8- dependent angiogenesis, tumor growth and metastasis (15-19). Similarly, CXCR3 expression in breast cancer has been linked to poor prognosis and found to promote metastasis in murine animal models (20-22). Antagonists of CXCR4, which is overexpressed in more than 23 types of cancers, are currently in the early stages of clinical development as they proved to be invaluable inhibitors of CXCR4-mediated metastasis in pre-clinical cancer models (23). Finally, CXCR6 expression in prostate cancer was also identified as a key mediator of metastasis and invasion through stimulation of mammalian target of rapamycin signaling pathway (24-26).

Additionally, GPCR- downstream regulators are also key players in GPCR-mitogenic signaling. For example, Endothelin-A receptor contributes to ovarian cancer metastasis through β-arrestin mediated activation of β-catenin signaling pathways (9). Interestingly, cellular transformation of normal to malignant cells is accompanied by distinct changes in RGS expression levels which could implicate these proteins in early stages of cancer development. In addition, RGS can serve as a predisposing factor for cancer development as single nucleotide polymorphisms have been detected in RGS 6 expressed in lung cancer. Hurst and Hooks recently reviewed the role of RGS in cancer

(10).

In addition to cancer progression, GPCRs contribute to the early stages of tumor development. In a recent study by Mills and co-workers, transgenic mice expressing lysophosphatidic acid (LPA) receptor and autotoxin, a secreted enzyme which mediates the production of extracellular (LPA) by hydrolyzing lysophosphatidylcholine, in mammary epithelium developed high frequency of late-onset, estrogen receptor positive, invasive and metastatic mammary cancer in the absence of any other oncogenic stresses (27). This study strongly implicates GPCRs as potential oncogenes and possibly valuable targets for cancer prevention and early detection. In addition, pharmacological modulation of GPCR signaling can also provide novel approaches to deliver large doses of chemotherapy to pancreatic cancer in vivo . A paradigm shifting study by Tuveson and co-workers revealed that pancreatic tumors are poorly perfused and vascularized which form an obstacle to efficiently deliver chemotherapeutic drugs to pancreatic cancerous tissue (28). Through pharmacological inhibition of hedgehog signaling pathway which is typically activated by a GPCR, tumor stromal tissue was depleted leading to enhanced tumor valscularization and drug intra-tumoral accumulation (28).

In conclusion, identification of GPCRs signaling pathways involved in cancer continues to yield novel approaches and unique targets to be considered for the development selective more efficient strategies for cancer therapy.

7

1.2 Neurotensin/Neurotensin Receptors

Neurotensin (NTS) is a 13 amino acid peptide that was first isolated in 1973 from the bovine hypothalamus by Carraway and Leeman (29). NTS plays important physiological functions in the central nervous system (CNS) and the periphery. In the CNS, NTS is involved in the modulation of dopaminergic transmission, hypothermia and pain (30,

31). NTS deficiency in the CNS was linked to the etiology of schizophrenia justifying the possibility of using NTS analogues as a therapeutic approach to alleviate schizophrenic symptoms (32). NTS is also expressed in the gastrointestinal (GI) tract where it regulates intestinal motility, billiary and pancreatic secretions and enhances fatty acid translocation from the intestinal lumen (33). In addition, NTS regulates certain cardiovascular functions and stimulates the growth of adrenal gland, fibroblasts and hepatocytes (34).

Neurotensin is produced from the cleavage of its precursor, pre-pro-neurotensin, by the pro-hormone convertase PC5 which also produces the related peptide, neuromedine (35). NST half-life in the blood stream is relatively short, about 2-6 minutes (36), due to its susceptibility for quick degradation via several ubiquitously expressed enzymes which include metalloendopeptidases 24.16 (EC 3.4.24.16) and

24.15 (EC 3.4.24.15), endopeptidase 24.11 (EC 3.4.24.11) and angiotensin-converting enzyme (ACE) (EC 3.4.15.1) (37). The susceptibility of NTS for degradation led to development of more stable synthetic analogs that can be used for experimental studies and targeted-therapeutic applications.

8

1.2.1 Neurotensin Receptors

NTS effects are mediated through specific neurotensin receptors. Three receptors have been identified and cloned to date. Two of these receptors, NTSR-1 and NTSR-2, are G protein coupled receptors (GPCR) while the third, NTSR-3, is a 100 kDa sortilin type receptor characterized by single transmembrane domain and plays a role in sorting molecules between the cell surface and intracellular organelles (discussed in 1.2.4) . A fourth receptor was recently isolated from the brain of bull frogs and had high affinity for endogenous NTS (36).

1.2.2 Neurotensin Receptor-1 (NTSR-1)

NTSR-1 mediates most of the known effects of NTS. In many studied tissues and cell systems, NTSR-1 stimulation leads to activation of phospholipase C through coupling to G αq (38) (Fig. 1.1). However, NTSR-1 could also activate diverse array of signaling pathways based on the expressing tissue and specific cell type. Several reports suggest that NTSR-1 can stimulate adenylyl cyclase through coupling to G αs subunits (39). In the colon epithelial cells, NTSR-1 stimulation leads to activation of IL-8 expression through activation of Rho-GTPase mediated NFkB-dependent pathways

(40).

NTSR-1 and NTS are co-expressed in several types of cancer (41). In pancreatic cancer for example, tumor but not normal tissues express NTSR-1 (42). Clinically, 75%-

90% of resected pancreatic adenocarcinoma expresses NTSR-1 (41). Despite high level of expression, reliance on NTSR-1 expression as a biomarker for pancreatic 9 cancer is not feasible due to comparable elevated expression levels in chronic pancreatitis (43). Guha et al. demonstrated that NTSR-1-mediated mitogenesis in pancreatic cancer is mainly transduced through potent stimulation of C-Raf-MEK-ERK

1/2 pathway (44, 45). A synergistic cross talk between NTSR-1 and insulin was also reported recently to occur through mTOR pathway which was found to be inhibited by metformin which is the most prescribed drug for the treatment of type-2 diabetes (46).

Metformin mainly abrogates the synergistic effects of insulin and NTS on cell proliferation. Metformin treatment inhibited the insulin-mediated potentiation of the mitogenic effect of NTS but had no effect on the effects mediated by NTS alone. This could be through reversal of insulin-mediated enhancement of intracellular calcium induced by GPCRs including NTSR-1.These findings suggest that metformin can be used in pancreatic cancer treatment regimens. More epidemiological studies are needed to determine if metformin use has any chemopreventive effects. Recently,

Olszweski and Hamilton demonstrated that NTSR-1 stimulation results in metastatic pancreatic cancer phenotype through induction of IL-8 expression In vitro (47, 48). In addition, NTSR-1 is also overexpressed in prostate cancer (49) and contributes to the development of bicalutamide-resistant phenotype (50). The mitogenic effect of NTS in prostate cancer is mainly mediated through MMP9-dependent transactivation of EGFR leading to stimulation of c-Src/Stat5b mitogenic pathway (51). 10

Fig.1.1. NTSR-1 primary signaling pathway.

The first evidence for possible involvement of NTSR-1 expression in the early stages of cancer development was reported by Souaze et al. (51) where they showed that

Wnt /APC signaling pathway activation is responsible for NTSR-1 promoter activation and receptor expression in pre-cancerous tissues of the colon (52). NTSR-1 expression in colon cancer increases during progression phase (53) leading to enhanced expression of other mitogenic pathways such as EGFR and early growth response gene-1 (54). Similar mitogenic effects and induction of resistance were observed in small cell lung carcinoma (55) and were found to be mediated mainly through coupling to G αq/11 and PLC stimulation (56). The U-growth hormone secretagogue receptor

1b/neurotensin receptor oncogenic signaling was recently validated as a valuable therapeutic target for lung cancer treatment (57). 11

In more recent studies, Souase et al. reported the co-expression of NTS and NTSR-

1 in breast cancer samples obtained from patients (58), which support the clinical relevance of their earlier finding that NTS counteract apoptosis in breast cancer cells through stimulation of BCl-2 expression (59). They also demonstrated the potential role

NTSR-1 plays in breast cancer progression through enhancing tumor growth and MMP-

9-mediated invasion (57). The high expression of NTSR-1 correlates with breast cancer

Scarff-Bloom-Richardson (SBR) grade, tumor size and number of metastatic lymph nodes (60). Stimulation of normal breast epithelial cells by estradiol induces the expression of NTS in the tissues adjacent to the tumor leading to a paracrine signaling within the tumor microenvironment (60). The molecular mechanism which links estrogen to NTSR-1/NTS expression is not well understood. It is possible that estrogen regulation of NTSR-1/NTS expression is mediated through Wnt/beta-catenin pathway; however, such possibility remains to be tested experimentally. Further studies may validate

NTSR-1/NTS as valuable primary target for breast cancer chemoprevention by anti- estrogen drugs such as Rolixifene (61).

Collectively, based on these studies, NTSR-1/NTS over expression in cancer tissues provides an opportunity to develop selective therapeutic and targeting strategies with possibly minimal serious side effects. This prompted several groups to design stable radio labeled neurotensin analogues for diagnostic and targeted-therapeutic applications (62). Additionally, further studies are needed to fully understand and identify the unique features of NTS signaling circuits in cancer as this would yield novel approaches for drug development.

12

1.2.3 Neurotensin Receptor-2 (NTSR-2)

NTSR-2, a GPCR, was first discovered in the rat brain when it was observed that the histamine H1 antagonist, levocabastine, selectively inhibited the binding of NTS to a low affinity binding site (63, 64). This observation was followed by the molecular cloning of

NTSR-2 in 1996 (65). Structurally, NTSR-2 differs from NTSR-1 mainly in the absence of N-glycosylation sites at its N-terminus and the absence of aspartate residue in the second transmembrane domain (66). The exact signaling events triggered by NTSR-2 are not yet fully characterized due to the variability in the experimental systems being employed among different groups; however, several reports demonstrated possible activation of MAPK pathway (67, 68). In addition, NTSR-2 may play a role in the regulation of NTSR-1 trafficking when both receptors are co-transfected in COS-7 cells

(69) however, the physiological significance of such interaction needs to be investigated in an endogenously expression clinically relevant system. Independent from NTSR-1, several reports suggest that NTSR-2 mediates several physiological functions including analgesia, hypothermia and pain control (70-72). The possible role of NTSR-2 in cancer remains to be established.

1.2.4 Neurotensin Receptor-3 (NTSR-3)

NTSR-3, a non-GPCR, was first cloned in 1998 after its affinity purification from the human brain (73). This 100 kDs receptor is 100% homologous to gp95/sortilin which is a sorting protein originally identified as an interacting protein with receptor associated protein (RAP) (72). The sortilin receptor family is characterized by a cysteine-rich 13 extracellular domain, single intracellular domain and a short intracellular tail that carries internalization signals (74). NTSR-3 is mainly expressed in the brain, more specifically in the hippocampus, dentate gyrus and cerebral cortex (75). The main functional roles of NTSR-3 are not well understood mainly due to its ability to interact with structurally diverse endogenous ligands such as nerve growth factor-β in addition to NTS (73).

NTSR-3 can possibly regulate NTS physiological functions through acting as an NTS- scavenger (76). The structure of sortilin ectodomain in complex with NTS was recently solved at two angstrom resolution revealing a distinct ten-bladed β-propeller domain which binds the C-terminal part of NTS (77). It has been suggested that heterodimerization of NTSR-1 and NTSR-3 is required for NTS signaling (78), however, such observation remain to be confirmed and evaluated in a more clinically relevant experimental model system. To establish the physiological significance, it would be important to detect such interaction in an in vivo model system or human-derived samples. The potential role of NTSR-3 in cancer remains to be established.

1.3 Structured Membrane Microdomains (SMDs)

The observation of the dynamic nature of antigens within biological membranes which was described in 1970 by Frye and Edidin (79) led to a paradigm shift in our understanding of the potential role of cell membranes in regulating cellular events.

Structured membrane microdomains (SMDs), also known as lipid rafts, a well studied type of membrane domains, were first described more than 20 years ago by Van Meer and co-workers who first reported the lateral segregation of membrane lipids into 14 distinct microdomains causing polarization of epithelial cells (80). Using confocal scanning laser flurescence microscope, they observed that florescent labeled sphingolipids are segregated into microdomains. The absolute proof of SMDs existence imposed a significant challenge for membrane biologist and biophysicist due to lack of reliable techniques useful for live visualization of these domains (81). To date, the rafts physical parameters such as size and shape remain not precisely determined. It is estimated that SMDs have a putative size of 5-200 nm in diameter, which is below the resolution limit of current optical microscopy techniques (80). Electron microscopy methods are insufficient due to inability to visualize these domains in live systems.

Other microscopic techniques such as atomic force microscopy, near-field optical microscopy and Forster-resonance energy transfer can be also utilized with equal limitations (82). Eggeling et al. (81) recently reported the first direct observation of lipid nano-scale dynamics (81). Through employing a novel stimulated emission-depletion

(STED) far-field fluorescence nanoscopy, they demonstrated that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins transiently (~10-20 ms) exist in cholesterol-mediated molecular complexes within < 20 nm diameter areas in living cells. These data represent that first microscopic evidence for lipid raft existence derived by observing living system.

It remains unclear whether raft dynamics are controlled by protein or lipid derived processes (81). Although sufficient evidence implicates cytoskeleton components such as actin filaments and microtubules in the regulation of raft formation and protein trafficking within membrane domains (83), it is possible that stimuli-induced changes in the levels of sphingolipid metabolites and cholesterol are the main driving force 15 controlling raft dynamics. Additionally, alternative novel mechanism recently suggested by Aydar and co-workers demonstrating that the GPCR sigma-1 receptor could play a key role in lipid raft re-modeling through direct cholesterol physical binding and sequestration (84).

The emerging role of cell membrane in regulation of signal transduction molecules and signaling events is based on SMDs ability to exclude and include specific proteins within regulated brief time intervals. The molecular mechanisms that target and regulate protein localization within specific membrane domains have been under extensive investigation as they might reveal novel pharmacological targets to control signal transduction in many disease conditions.

1.3.1 SMDs and Signal Transduction

Signaling molecules normally exist in cellular milieu in relatively low abundance.

Thus, the mechanics of the initiation and propagation of precise signals in the right cellular location at the right time remains to be a dilemma (85). Attempts to generate models based on theories such as random collision, scaffolding proteins or pre-coupled signaling circuits remain to be controversial (86). Thus, the identification of membrane domains as signaling hot spots provided a new paradigm that could explain the efficient timely engagement and segregation of signaling machinery components.

The role of SMDs in signal transduction was first suggested by Field et al. who demonstrated that SMDs function as platforms for Fc εRI receptor clustering and signaling (87, 88). In addition Kolesnick and co-workers reported that stimulation of 16 acid-sphingomyelinase which preferentially resides within SMDs results in hydrolysis of sphingomyelin, generating ceramide (89, 90) which forms distinct clustered rigid ordered domains that can include or exclude certain proteins leading to activation of specific signaling pathways (91) (Fig.1.2). Ira and Johnston used atomic force microscopy to show that acid-sphingomyelinase-mediated ceramide generation leads to clustering of nanoscale domains that could form signaling platforms (92, 93). The biophysical nature of SMDs will be discussed in another section of this document

(1.3.4). Ceramide-enriched SMDs are required for clustering of wide variety of receptors such as CD95, CD40 and several of their downstream effectors forming compartmentalized signalosome (90). In addition, ceramide platforms can regulate function of many proteins including kinase suppressor of Ras (94), PLA2 (95), cathepsin

D (96) and others. Recently, TLR4 signaling complex formation was also found to be highly dependent on acid sphingomyleinase mediated ceramide production and domain restructuring which allows for phosphorylation of PKCzeta and activation of MAPK downstream pathways (97). These results support an earlier finding by Olsson and

Sundler which implicates rafts in recruitment of CD-14 and MAP kinases upon LPS- mediated macrophage stimulation (98, 99). Earlier reports by Kester and co-workers

(100, 101) as well as Hundal and co-workers (102) also showed that ceramide recruits and activates PKCzeta within SMDs leading to Akt inactivation in vascular smooth muscle cells. 17

Fig 1.2. Ceramide-Enriched SMDs Formation.

In addition to the role of SMDs in regulating GPCR signaling (discussed in details in

1.3.2), many receptor tyrosine kinases (RTKs) are also regulated by SMDs (103). For example, Mineo et al. reported that epidermal growth factor receptor (EGFR) resides within SMDs and upon stimulation it migrates to non-SMD compartments where signal is terminated (104). In support of these results, Liu et al. conducted experiments using confocal microscopy techniques to show that EGFR co-localizes with GM-1 positive rafts in breast cancer cells (105). They also demonstrated that disruption of rafts by methyl-β-cyclodextrin (MCD) inhibited EGF-mediated chemotaxis of breast cancer cells in part through inhibition of Akt phosphorylation and PKCzeta translocation (104). On the contrary, using immune-electron microscopy, Rinegerike et al. reported that only 7% of EGFR reside within SMDs regardless of stimulation status (106). Conversely, Resh 18 and co-workers showed that cholesterol depletion leads to ligand independent activation of EGFR which they proposed to occur possibly via MCD-mediated receptor dimerization (107). This significant contradiction in the raft literature is very common and is mainly due to variability in experimental techniques and model systems among different groups. Therefore, it is prudent to evaluate raft research reports based on case-to-case basis rather than attempting to generalize certain findings.

In addition to regulation of specific signaling pathways, SMDs also act as a platform for cross talk between different signaling modalities. For example, CXCL 12 /CXCR 4, A

GPCR pathway, mediated transactivation of HER takes place within SMDs in prostate cancer cells promoting mitogenesis and invasion (108). Angiotensin receptor II also transactivates EGFR within SMDs through interaction with caveolin as a scaffolding protein (109). Other RTKs which associate with SMDs include PDGFR, IGF-1 R. A recent review by Arcaro and co-worker (110) provides an in-depth discussion of growth factor receptor signaling within SMDs.

In addition to signaling proteins, rafts regulate many ion channels such as TRPC

(111), BK channels (112), potassium channels (113, 114), funny f-channels (115) and others. For example, Kv1.3, a channel which is known to control differentiation, apoptosis and proliferation of lymphocytes, preferentially localizes to ceramide-enriched

SMDs (116). Rafts regulate the function of the channel through segregation from channel regulators such as Src-like tyrosine kinases which inhibit the activity of the channel.

Collectively, SMDs seem to be integral in functional regulation of a diverse array of proteins. Understanding the biophysical and biochemical basis of these regulatory 19 mechanisms may lead to identification of novel therapeutic approaches for many disease conditions.

1.3.2 Role of SMDs in GPCR Signaling

GPCRs-mediated signaling is highly regulated process. The initial signal is triggered upon GPCR coupling to a specific heterotrimeric G protein after agonist stimulation.

This protein-protein interaction requires both partners to co-localize to certain domains within brief time intervals. Thus, it has been hypothesized that membrane microdomains serve the role of a platform, concentrating signaling molecules and allowing for rapid engagement of effectors. GPCRs localization patterns within membranes is highly variable and cannot be predicted based on specific recognizable sequences. Several

GPCRs such as Kinin B1 receptor (117), luteinizing hormone receptor (118) and m2 mAchr (119) translocate to SMDs upon agonist stimulation where they interact with hetertrimeric G protein subunits which reside within SMDs. The neurokinin receptor-1 on the other hand preferentially localizes to SMDs under resting conditions and its activation is dependent on SMDs integrity (120).

Recent studies by Zheng et al. reported an important novel role for membrane microdomains in regulating agonist-selective signaling (121). They demonstrated that µ-

Opioid receptor is capable of stimulating two distinct pools of ERK leading to different transcriptional regulation and pharmacological effects based on the receptor membrane micro-localization (120). In the absence of an agonist, µ-Opioid localizes to SMDs, however, upon etorphine treatment but not morphine, µ-Opioid receptor translocates to 20 non-SMDs domains stimulating ERK phosphorylation that is dependent on forming a complex with β-arrestin. Interestingly, morphine treatment can also lead to ERK phosphorylation that is dependent on forming a complex with G αi2 within SMDs (120).

This finding could shed light on a mechanism than can explain an emerging novel concept in GPCR pharmacology known as functional selectivity, in which certain

GPCRs were found capable of mediating multiple opposing pharmacological effects

(122). Other opioid receptor isoforms including delta (123) and kappa (124) opioid receptors, also localize to SMDs where their protein-protein interactions and signaling pathways are regulated. Raft disruption can also result in switching in the G protein coupling mechanism. For example, CCR5 which normally couple to G αi within SMDs switches to pertussis toxin independent G protein mechanism upon raft disruption (125).

Dynamic localization of proteins within SMDs supports the hypothesis of compartmentalized signalplex formation and emphasizes the role of rafts in strategically segregating signaling molecules as a signaling restraining mechanism under resting conditions. Using a combination of biochemical and bioluminescence resonance energy transfer assay (BRET), Ponteir et al. demonstrated that G αs and adenylyl cyclase (AC) are strategically sequestered separately from β2 adrenergic ( β2AR) receptor. Disruption of raft integrity by cholesterol depletion resulted in a significant increase in β2AR- mediated cAMP production (126). Similar effect was reported earlier by Diaz et al. where they showed that disruption of rafts by MCD leads to phospholipase D (PLD) activation through exclusion from the raft domains(127, 128). PLD is the key enzyme involved in the production of phosphatidic acid containing bioactive lipids such as lysophosphatidic which signals through GPCRs and has mitogenic effects (129). 21

Alternatively, Lei et al. recently reported that SMDs can also restrain α1a adrenergic receptor activity by locking the receptor in an inactive conformation (130). Raft-mediated restraining of biological activity was also observed for other proteins such as NADPH oxidase which have higher activity outside raft domains (131) and TRPM8 channel which respond to menthol and cold mediated effectors only when channel-raft association is disrupted (132). Although several GPCRs undergo caveolin-dependent internalization (133), it is possible that they only transmit their signals within SMDs and then undergo clathrin dependent internalization within non-SMDs compartments. For example, using a combination of confocal microscopy analysis and biochemical assays,

Morris et al. found that α1a adrenergic receptor mediated signaling and desensitization occur mainly within SMDs before receptor-translocation to non-raft microdomains where it internalize through clathrin-coated pits (134). On the other hand, Cannabinoid receptor 1 interestingly undergoes both lipid raft and clathrin-coated-pits-mediated internalization (135). GPCR desensitization by GRKs can be also regulated by SMDs.

Villar et al. recently reported that dopamine D3 receptor phosphorylation by GRK4 occurs within SMDs (136).

In conclusion, SMDs play diverse regulatory roles in GPCRs signal transduction.

Understanding the molecular mechanisms that control the localization of GPCRs and their effectors and regulators such as RGS and GRKs to specific membrane compartments could provide clues toward the discovery of novel targets and therapeutic approaches that could be more pharmacologically tolerable than traditional receptor antagonism.

22

1.3.3 Protein palmitoylation and Localization to SMDs

Multiple factors are believed to be involved in protein localization to SMDs, however, the interactions among them are not fully understood (137). Microlocalization signal could be the amino acid sequences of the transmembrane domain(s), protein tendency to undergo homo- and hetero- dimerization, post-translational modifications and ability to interact with another protein that preferentially reside within SMDs such as caveolin and certain heterotrimeric G proteins (135).

Protein palmitoylation is a fatty acyl- post-translational modification that has been linked to protein sub-cellular localization and trafficking. It is the reversible addition of

16-carbon atom acyl chain to cysteine through a thio-ester linkage (138). Proteins can be palmitoylated at various domains in close proximity to the N-terminus, C-terminus or in the middle. In contrast to other lipidation-based post-translational modifications, there is no shared consensus, easily recognizable palmitoylation sites (139). The dynamic palmitoylation of proteins such as Ras facilitates their intracellular trafficking between cellular organelles and microlocalization within membranes (140). Inhibition of H-Ras palmitoylation for instance prevents its translocation to Golgi (138). Recently, palmitoylation has been also implicated in protein stability. Palmitoylation deficient proteins such as the yeast SNARE protein Tlg1P is more prone to ubiquitylation by the ubiquitin ligase Tul1p (141). Similarly, non-palmitoylated LRP6 is retained in the ER where it gets ubiquitylated (142). This finding implicates palmitotylation as an ER retention signal. Tanimura et al. also reported that the adapter protein, linker for activation of T cells (LAT) is palmitoylated (143). The palmitoylation deficient LAT mutant failed to reach the plasma membrane and was susceptible for degradation by 23 the proteosome pathway which suggests that lipidation is essential for protein stability

(141). Another study by Hundt et al. reported similar findings; however, they demonstrated that LAT plamitoylation does not play a role in raft localization as they reported that deficient mutants are absent from plasma membranes and only restricted to Golgi (144). However, a study by Brown and co-workers suggested that a combination of palmitoylation and other factors including protein-protein interactions are responsible for LAT raft localization (145).

For some membrane proteins, palmitoylation is sufficient for SMDs microlocalization.

For example, the localization of the palmitoylation-deficient 5-HT1A receptor to SMDs is significantly reduced relative to wild type receptor implicating palmitoylation as SMDs targeting signal. In addition, palmitoylation deficient 5-HT1A interaction with G αi was also hindered leading to reduced signaling efficiency (146). Similarly, P2X7 receptor, an

ATP gated cationic channel, mainly localizes to SMDs. Inhibition of the receptor palmitoylation using 2-Bromo Palmitate (2-BP) or via mutagenesis resulted in significant reduction in SMD localization and an increase in proteolytic degradation due to retention in the endoplasmic reticulum (147). Interestingly, regulators of G-protein signaling

(RGS) 16 was found to be regulated by palmitoylation. Palmiotylation deficient RGS16 lost its ability to regulate G αq and G αi within SMDs (148, 149). In addition, palmitoylation of the small GTPase, Rap2b as well as (150) is also critical for ligand dependent translocation, activation and aggregation within SMDs (151-153). These examples demonstrate that palmitoylation could regulate GPCRs function indirectly through their effectors. 24

Despite the previous examples, palmitoylation of membrane proteins cannot be considered an absolute rule that reflect protein association with SMDs (135). Some proteins such as caveolin-1 which is palmitoylated at three cysteine residues always localizes to SMDs even when the palmitoylated cysteines are mutated (154). On the other hand, there are many palmitoylated proteins that do not localize to SMDs such as transferrin receptor which is usually used as a marker for non-raft domains. However, palmitoylation-deficient transferrin receptor undergoes accelerated endocytosis (155,

156). Interestingly, protein palmitoylation can serve as a negative regulator for SMD- localization. For example, the palmitoylated anthrax toxin receptors, TEM8, CMG2 and

LRP6 localize to SMDs only upon inhibition of palmitoylation (157). Unlike Death receptor 4 (DR4), a member of the TNFR family, which localizes to raft mainly due to palmitoylation (158), raft localization of death receptor 6 (DR6) which is also palmitoylated is uniquely mediated by N-glycosylation and not palmitoylation (159). It is more likely that palmitoylation is necessary for fine tuning the protein conformation by increasing hydrophibicity of specific segments, increasing the length of hydrophobic domains or modulation of the physico-chemical properties of certain residues that for example have preference for disordered membrane domains such amino acids that has aromatic groups and therefore allow better association with SMDs. Thus, it is very difficult to precisely predict the role of palmitoylation without taking into account other factors associated with unique structural features of the protein of interest.

Other lipidation events which have been linked to membrane targeting and SMDs localization include N-myristoylation and prenylation (161). Some proteins are subject to more than one lipidation events in order to achieve optimal function. H-Ras and N-Ras 25 for example require both palmitoylation and farnesylation in order to reach and bind plasma membrane (161). Prostacyclin receptor, a GPCR, is uniquely modified by both palmitoylation as well as prenylation and this combination of modification is required for coupling to G proteins (160). Combinations of multiple post-trasnlational modifications provide more flexibility to control protein sub-cellular localization and trafficking.

Taken together, protein localization to SMDs is a complex process and requires multiple factors to cooperate and synergize. Precise protein localization and trafficking is crucial for optimal function and efficient signaling as it provides multidimensional regulatory mechanism which encompasses in addition to conformational structure, location and time.

1.3.4 Sphingolipids and SMDs

Sphingolipids represent a class of bioactive lipids that constitute a major component of cellular membranes (161, 162). Due to their unique physico-chemical properties, sphingolipids tend to form distinct ordered membrane compartments and domains such as SMDs. Sphingoid base, 1,3-dihydroxy-2-aminoalken, forms the backbone structure of all known sphingolipids (163). Variability in the length of the alkyl chain and position and number of the double bonds provide a diverse array of structurally related sphingolipids. For instance, the attachment of fatty acid to sphingosine via amide ester bond generates the second messenger molecule, ceramide, which by itself constitutes the backbone of many complex sphingolipids such as sphingomyelin, cerebrosides, ganglosides and others (Fig.1.3). The advances in analytical LC/MS/MS techniques led 26 to the identification of many complex sphingolipids and several endogenous ceramide species of different fatty acid chain length ranging from two to 28 carbons. However,

C16 to C24 ceramide species remain to be the most abundant forms (164).

Fig.1.3. Basic Sphingolipid Structures.

Several reports have described the formation of ceramide-enriched microdomains that are heterogeneous relative to less ordered membrane compartments (165). The first experimental evidence for the formation of these microdomains was described by

Huang et al. where they demonstrated in an in vitro system using 2H-NMR technique that C16 ceramide clustering correlates with activation of phospholipase A2 (166) and modulation of PKC activity (167). Subsequently, Huang and co-workers reported that ceramide can displace cholesterol from lipid bilayers in 1:1 ratio (168). Consistent with these findings, London and co-workers also reported that ceramide selectively displaces cholesterol suggesting that both ceramide and cholesterol compete for limited space that accommodate lipids with small head groups (169). Similar observation was reported by Dobrowsky and co-workers (170) showing that elevation in endogenous ceramide levels using bacterial sphingomyelinase leads to depletion in cholesterol 27 levels within SMDs accompanied by changes in protein contents of the caveolin- enriched membranes (170). These findings suggest that combination of ceramide and cholesterol in different ratios forms diverse and distinct SMDs with variable range of physico-chemical properties.

Despite those short chain ceramide analogues, C2 and C6 are widely used experimentally due to their desirable physico-chemical characteristics, London and co- workers and other groups reported that there are critical differences in the biophysical behavior of these species relative to endogenous long chain ceramides (171). These differences are reflected in exhibiting biological effects that are not mediated by natural ceramides. Based on data generated by fluorescent quenching assays, raft stability is highly dependent on ceramide N-acyl chain length increases in the order

C8:0~C6:0

C2 and C6 ceramide analogues as experimental probes to study raft function in certain signaling systems. These findings were further validated by an elegant biophysical study by Schwille and co-workers where they used a combination of atomic force microscopy and fluorescence correlation spectroscopy to demonstrate that only long chain ceramides such as C16 and C18 at physiological concentrations tend to segregate from liquid ordered phase and form distinct gel-phase ceramide enriched ordered domains (173, 174). On the contrary, they showed that short chain ceramide 28 analogues are not capable of forming separate phases and perturb the physical structure of ordered-liquid domains resulting in reduced stability, viscosity and lipid packing (175). This growing body of literature could account for the differences in physiological effects that are frequently seen when using long versus short chain ceramides in experimental applications. Due to these unique properties, short chain ceramides can be used for many therapeutic applications through utilizing advanced delivery systems (176, 177).

To further understand the biophysical bases of interaction between ceramide and cholesterol within raft domains, Prieto and co-workers conducted an experiment using an in vitro large unilamellar vesicle system and reported that cholesterol-rich fluid membranes solubilize ceramide while solubility of cholesterol in ceramide domains is low (178). Despite the non-physiologic nature of these interesting experiments, the results hold up the notion that rafts are very dynamic structures and can be modulated by subtle changes in levels of cholesterol and sphingolipid metabolites. In support of these findings, Prieto and co-workers also reported that in biological membranes, cholesterol and raft domains can modulate the activity of sphingomyelinase, an enzyme responsible for ceramide production within plasma membranes (179). This finding would raise the possibility that cholesterol enriched rafts are in competition and/or balance with ceramide enriched rafts. Along this line, Kolesnick and co-workers and other groups established that acid-sphingomyelinase activity within rafts remodels the plasma membrane architecture via ceramide production (180). Another possible mechanism of interaction between ceramide and cholesterol was revealed by Matsuo and co-workers as they demonstrated that sphingomyelin, a precursor for ceramide, could modulate 29 cholesterol efflux through ABCG1 which is a half-type ATP binding cassette protein.

Reduction of SM levels resulted in reduced ABCG1-mediated cholesterol efflux (181).

Taken together, diverse array of microscopic, biophysical and biochemical data provide ample of evidence in support of SMDs or lipid rafts existence. These findings implicate cholesterol, ceramide and other sphingolipids as regulators of SMDs dynamics which provide an avenue to modulate rafts through pharmacological manipulation of enzymes involved in sphingolipid and/or cholesterol biosynthesis.

1.3.5 Modulation of SMDs as a Therapeutic Approach to Alter Signaling in

Cancer

The involvement of SMDs in regulation of many important signaling events resulted in an interest to control signal transduction pathways associated with disease conditions such as cancer through pharmacological manipulation of SMDs integrity. In cancer, several chemotherapeutic agents such as daunorobicin, doxorubicin, edelfosine, resveratrol, cisplatin and 1-beta-arabinofuranosylcytosine and others exert their cytotoxic effects in part through alteration of cell membrane architecture (182). Cisplatin, a classic widely used chemotherapeutic agent, requires intact SMDs to induce cellular cytotoxicity (183). Its early effects involve induction of CD95 clustering through activation of acid-sphingomyelinase-mediated ceramide production which triggers the redistribution of CD95 to SMDs (184). Recently, Cisplatin was also found to cause a rapid transient increase in membrane fluidity and inhibition of Na+/H+ membrane exchanger-1 causing intracellular acidification and subsequent cell death which is independent of DNA adducts formation (178). Earlier studies by Kolesnick and co- 30 workers and other groups also demonstrated the central role of acid-sphingomyelinase- mediated clustering of SMDs into ceramide-enriched microdomains as a signaling mechanism for many surface receptors and environmental stresses including ionizing radiation which is widely used approach for cancer treatment (185-187). Another interesting example is the effect of synthetic alkyl-lysophospholipids (ALPs) edelfosine and perifosine on multiple myeloma (MM), an incurable B cell malignancy (188). ALPs have unique structures where aliphatic side chains, a long and a short one, are linked to glycerol backbone through an ether linkage (189). These compounds induce selective apoptosis only in MM cell lines and patient MM cells but not in normal B and T lymphocytes or endothelial cells. The main mechanism of action of ALPs involves the recruitment of Fas/CD95 death receptor and pro-caspase-8 to SMDs. Interestingly, lipid raft disruption prevented ALPs-mediated death-inducing signaling complex formation within rafts and subsequent apoptosis. Recent studies suggest that edelfosine alters raft structure and organization by direct incorporation (190). Kester and co-workers also reported that ceramide mediated apoptosis is selective for malignant cells. Their studies demonstrated the safety and efficacy of administering liposomal short chain ceramide in multiple in vivo models including breast cancer and melanoma (191, 192). These findings may suggest that induction of ceramide formation could be an early step in

ALPs mediated apoptosis. In addition, malignant cells could have higher levels of cholesterol which facilitate rafts formation and death receptor clustering faster than normal cells. 31

Fig. 1.4. Ceramide Metabolic Pathways Associated with Drug Resistance. SMase: Sphingomyelinase, GCS: Glucosylceramide synthase, CRS: Cerebrosidase, CDase: Ceramidase, CS: Ceramide Synthase, CK: Ceramide Kinase, C1PP: Ceramide-1-phosphate phosphatase, SK: Sphingosine kinase, S1PP: Sphingosine-1-phosphate phosphatase.

A growing body of literature suggest that accelerated ceramide metabolism (Fig. 1.4) confers resistance to many chemotherapeutic modalities (193-196), emphasizing the central role of ceramide mediated formation of ordered domains in cell death. More specifically, up regulation of glucosyl ceramide synthase (GCS) and acid ceramidase

(197, 198) results in resistance towards many chemotherapeutics and ionizing radiation approaches (199). Inhibition of certain ceramide metabolizing enzymes that are unregulated in certain malignancies proved to be a promising strategy to increase 32 ceramide cellular levels and reverse resistance. As an example, Amiji and co-workers reported that systemic co-delivery of paclitaxel along with tamoxifen which inhibits GCS lowered the IC50 of paclitaxel by >10 folds in ovarian cancer experimental models

(200). Similarly, inhibition of acid ceramidase in prostate cancer sensitizes cell to ionizing radiation (197).

In addition to modulation of rafts via altering sphingolipid metabolism, it is possible to achieve the same goal through targeting cholesterol biosynthetic pathways (Fig 1.5). A pioneering study by Freeman and co-workers established the utility of cholesterol inhibition using simvastatin, an HMG-CoA reductase inhibitor, to suppress prostate cancer growth (201). Lowering cholesterol raft content inhibited Akt1serine-threonine kinase/Akt pathway and induced apoptosis in caveolin- and PTEN-negative LNCaP PCa cells. Interestingly, normal prostate epithelial cells were resistant to simvastatin- mediated apoptosis (193). On the other hand, high serum cholesterol levels increases tumor aggressiveness and growth (202, 203). Freeman and co-workers later reported that a subpopulation of androgen receptor localizes to SMDs in LNCap cells and androgen interaction with Akt1 occurs preferentially within SMDs (195). In addition to statins, ezetimibe, a cholesterol uptake blocking drug, also found to inhibit prostate cancer angiogenesis through lowering circulating cholesterol levels (204). Another novel approach to alter rafts is to target squalene synthase, the enzyme that controls the switch towards sterol biosynthesis (Fig1.5) (205). Squalene synthase expression in prostate cancer was found to be enhanced by androgens which causes a shift in the metabolism away from mevalonate/isoprenoid towards cholesterol leading to enhanced 33 de novo cholesterol synthesis that mainly affects SMDs cholesterol content while non- raft cholesterol remain unaffected (197). Consistent with Freeman observations, knockdown of squalene synthase expression as well as pharmacologic modulation using zaragozic acid suppresses proliferation and induces prostate cancer cell death

These data validate squalene synthase as a potential novel target for selective raft- based chemotherapeutic interventions.

Fig.1.5. Sterol Biosynthetic Pathway.

There are new emerging classes of compounds that can act as raft modulators in cancer. Omega-3 polyunsaturated fatty acids (PUFA) for instance down modulate 34

CXCR4 expression and function in MDA-MB-231 breast cancer cells via disruption of

SMDs in a manner similar to methyl-β-cyclodextrin causing partial exclusion of CXCR4 from SMDs (206). In addition, EGFR was also found to be excluded out of the rafts following PUFA treatments. The immunomodulatory effects of PUFA are also believed to be mediated through raft alteration and protein displacement (207, 208, 209). In addition, EGCG ( (–)-epigallocatechin-3-gallate) which is found in green tea also exerts its colon cancer inhibitory effects in part via modulation of SMDs (210). Patra et al. proposed that incorporation of EGCG into SMDs is sufficient to disrupt growth factor mediated signaling and results in growth inhibition of cancer but not normal cells due to an enhanced reliance of cancer cells on SMDs compartmentalized of mitogenic signaling pathways (211).

Although targeting SMDs might seem an appealing approach, it remains selectively challenging. SMDs exist in every single cell in the human body and potential toxicity cannot be underestimated. In addition, targeting a subpopulation of rafts within a cell is currently not feasible. It is possible in the near future with the advancement in analytical tools and methodologies such as LC/MS/MS to identify unique lipid markers for SMDs in diseases such as cancer which might open new avenues towards safe modulation of specific subsets of SMDs in cancerous but not healthy tissues. Future clinical use of lipidomics as a strategy to identify sphingolipid-based resistance in individual patients may hold a promise in personalized cancer chemotherapy.

1.4 Conclusions

The emerging role of cell membranes as regulators of signal transduction opens new avenues for novel pharmacological interventions. SMDs dynamics can be altered 35 by modulating sphingolipids metabolism, cholesterol metabolism or directly through physical disruption. These approaches provide an opportunity to manipulate GPCRs signaling in wide variety of disease conditions. Regulation or NTSR-1 by cell membrane has not been investigated. Thus, one of the main aims of our studies was to reveal the role of SMDs in NTSR-1 signaling. We also characterized the effect of nanoliposomal ceramide NTSR-1 mitogenic signaling through altering SMDs integrity. Based on these studies and due to lack of knowledge regarding NTSR-1 post-translational modifications, we conducted experiments to determine if there is a link between NTSR-

1 palmitoylation and receptor dynamic membrane localization which is regulated by

NTS. Our data led to the possibility of potential regulation of protein palmitoylation by sphingolipid homeostasis. Despite our exciting findings, several novel avenues remain to be explored including potential cross talk between multiple post-translational events and the potential central role of SMDs in dynamic protein palmitoylation. These studies would continue to reveal new strategies to modulate signal transduction for wide variety of therapeutic applications.

36

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55

Chapter 2

Nano-Liposomal Short-Chain Ceramide Inhibits Agonist-Dependent Translocation of Neurotensin Receptor-1 to Structured Membrane Microdomains in Breast Cancer Cells

56

2.1 Abstract

Neurotensin receptor-1 (NTSR1) is a G-protein coupled receptor that has been recently identified as a mediator of tumorigenicity and metastasis. NTSR1, as well as its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples but not in normal breast tissue. We have previously published that ceramide mimetics could inhibit breast cancer growth in vitro and in vivo. Thus, understanding the biochemical and biophysical regulation of NTSR1 by ceramide can help further define NTSR1 as a novel target in breast cancer. Our results demonstrate that nano-liposomal formulations of ceramide inhibit NTSR1- mediated MDA-MB-231 breast cancer progression (mitogenesis, migration and MMP-9 activity). In addition, liposomal ceramide inhibited NTSR1-mediated, but not PMA- mediated activation of the MAP kinase pathway. Mechanistically, nano-liposomal short chain ceramide reduces NTSR1 interaction with G αq/11 subunits within structured membrane microdomains (SMDs), consistent with diminished NTS-induced translocation of NTSR1 into SMDs. Collectively, our findings suggest that exogenous short chain ceramide has the potential to be used as an adjuvant therapy to inhibit NTS- dependent breast cancer progression.

57

2.2 Introduction

Neurotensin (NTS), originally characterized as a tridecapeptide neurotransmitter (1) or gastrointestinal hormone and mitogen (2, 3), has recently been demonstrated to be expressed in metastatic and drug resistant cancers (4, 5). The G-protein coupled neurotensin receptor, NTSR1, is also overexpressed in many types of cancer (6), including breast cancer (7). 91% of invasive ductal breast tumors were positive for

NTSR1 (7). In fact, NTSR1 and NTS were coexpressed in 30% of these ductal breast tumors (7). In normal mammary tissue, NTSR1 and NTS are not expressed (7). NTSR1 activation has been observed in both ER-positive and ER- negative breast cancer cells, stimulating BCl-2 (8) and matrix metalloprotease-9, respectively (7). In addition, siRNA knockdown of NTSR1 in xenografted MDA-MB-231 cells resulted in a 70% fold decrease in tumor growth compared with wild type cells (7). Taken together, these studies identify NTSR1 as a key target in breast cancer.

Structured membrane microdomains (SMDs), also known as lipid rafts, are regions in the membrane enriched in cholesterol, glycolipids and sphingomyelin (9). Enrichment of SMDs with polyunsaturated fatty acids (PUFA) (10), sphingolipid or glycosphingolipid metabolites, such as ceramide (11, 12) alter the lipid microenvironment in a way that may interfere with receptor-mediated signaling within microdomains. In fact, the structure of SMDs is highly unstable as these lipid metabolites are dynamically and temporarily formed and degraded (13). Often, these structured membrane microdomains contain scaffolding elements, such as caveolin-1, which interacts with promitogenic signaling elements including glycophosphatidyl-anchored proteins, acylated proteins, G protein-coupled receptors (GPCRs), trimeric and small G-proteins 58 and their effectors (14). We, and others, have previously demonstrated that short chain ceramide localizes to SMDs and causes an increase in the phosphorylation and localization of PKCzeta within caveolin-enriched microdomains to inactivate promitogenic and prosurvival Akt (15, 16). As nano-scale ceramide formulations have previously been shown to selectively inhibit breast and ovarian cancer growth in vitro and in vivo (17-19), we now investigate if exogenous nanosized formulations of liposomal C6 ceramide alters NTSR1-induced prosurvival, promitogenic signal transduction cascades in breast cancer cells.

2.3 Materials and Methods

Materials and Cell Culture. Dioleoyl phosphatidylethanolamine (DOPE), dioleoyl

phosphatidylcholine (DOPC), D-erythro -hexanoyl-sphingosine (C 6-ceramide), polyethyleneglycol-450-C8-ceramide (PEG-C8) were purchased from Avanti Polar

Lipids (Alabaster, AL). Di-hydro-erythro -hexanoyl-sphingosine (DHC 6) was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Polyacrylamide gel electrophoresis gradient gels and gelatin containing gels were purchased from

Invitrogen (Carlsbad, CA), and enhanced chemiluminescence reagent was purchased from Thermo Fisher Scientific Inc. Human MDA-MB-231 breast adenocarcinoma cells and HEK-293T were obtained from American Type Culture Collection (Manassas, VA) and grown at 37°C in RPMI 1640 medium and DMEM, respectively, supplemented with

10% fetal bovine serum (FBS). The MDA-MB-231 cell line is a highly aggressive metastatic, estrogen receptor-negative, EGFR positive and NTSR1 positive model of human breast cancer. BrdU cell proliferation ELISA colorimetric kit was obtained from 59

Roche (Indiana). NTS was purchased from Sigma-Aldrich and was dissolved in 0.05% acetic acid just before use in experiments. JMV-449, a synthetic, stable NTS analogue was purchased from NeoMPS (Strasbourg, France). All antibodies were purchased from

Santa Cruz Corp, CA. Peptide-N-glycosidase F (PNGaseF) was obtained from Sigma,

Saint Louis, Missouri. Phorbol ester (PMA) was obtained from Promega Inc (Madison,

WI). Human NTSR1 and HA-tagged cDNA was purchased from Missouri S&T cDNA

Resource Center, Rolla, MO (Catalogue Number: NTSR100000). Unless it is specified in the figure, “control” indicates untreated cells.

HEK-293T Transfection. Cells were grown in 10 cm plates until 50% confluency.

Transfection was performed using Lipofectamine 2000 (Invitrogen) transfection agent according to the manufacturer recommendations. After 48 hours, the cells were harvested using RIPA buffer (100 mM Tris pH 8.0, 300 mM NaCl, 1 % v/v Nonidet

P40,1% sodium deoxycholate, 0.2% SDS) and NTSR1 expression was assayed using western blot analysis.

Validation of NTSR1 Antibody.

To ensure specificity and reliability of a goat polyclonal IgG Anti-NTSR1 (sc25042,

Santa Cruz, Inc.), we have utilized several experimental approaches. First, we purchased a positive control from Santa Cruz, IMR-32 cell lysate: sc-2409, which is a human neuroblastoma cell line. The antibody recognized a band in human IMR-32 cell lysate at an analogous molecular weight to human MDA-MB-231 breast cancer cells

(Fig. 2.1A), a molecular weight consistent with previous reports (20). To further confirm specificity, we overexpressed NTSR1 in HEK-293T cells which normally do not express this receptor (this was confirmed using RT-PCR, data not shown) using a non-HA- 60 tagged construct. Western blot analysis of the lysates obtained from tranfected cells recognized bands at comparable molecular weight relative to MDA-MB-231 cells as well as IMR-32 cells, while no bands were detected in the non transfected cells (Fig. 2.1B).

Additionally, we repeated the same overexpression experiment using an HA-tagged

NTSR1 construct (Fig. 2.1C). Cell lyasates containing equal amount of total protein were immunoprecipitated using Anti-HA antibody. Band corresponding to molecular weight ~52-54 kDa was observed when the blots were probed with both Anti-HA and anti-NTSR1 antibodies. The selectivity of the NTSR1 antibody to the epitope that was used to generate the antibody was confirmed by a competition study using a blocking peptide, where the bands completely disappeared (data not shown). In all supplemental and manuscript data, we used a 1:500 dilution along with the SuperSignal West Femto

Chemiluminescent Kit from Pierce for optimal signal detection. Therefore, together, the data confirm that the antibody was able to selectively and specifically detect NTSR1 of human origin. To avoid the possibility of false positive bands in the immunoprecipitation experiments due to IgG detection at molecular weight around 55kDa, we utilized two different anti-HA antibodies from two different species. IP was done using a mouse monoclonal anti-HA and IB was done using a rabbit anti-HA.

61

Fig. 2.1. Anti-NTSR1 antibody recognizes NTSR1 in human cell lines. A. Anti-NTSR1 goat polyclonal antibody recognizes NTSR1 in human MDA-MB-231 breast cancer cell lysates and human IMR32 neuroblastoma cell lysates. An apparent 52 kDa band is observed in both lysates, while an additional 54 kDa band is observed in the MDA-MB-231 cell line. B. Anti- NTSR1 antibody recognizes NTSR1 ( 52-54 kDa) in HEK293T cells transiently transfected with NTSR1 construct. No corresponding bands were visualized in untrasfected HEK293T control cells. Equal protein loading was confirmed by detecting GAPDH expression level. C. Anti- NTSR1 antibody recognizes NTSR1 ( 52-54 kDa) in HEK293T cells transiently transfected with HA-tagged NTSR1 construct; the same band was detected using Anti-HA tag antibody. No corresponding bands were visualized in untrasfected HEK293T control cells. The 51 kDa legend corresponds to the position of Glutamic Dehydrogenase (SeeBlue Plus2 Pre Stained Standard, Invitrogen).

Liposome Formulation and Extrusion. Lipids, dissolved in chloroform (CHCl 3), were combined according to the following molar ratios: 1,2-Dioleolyl-sn-Glycero-3-

Phosphoethanolamine (DOPE): 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):

C8 mPEG 750 Ceramide: 1,2-Distearoyl-sn -glycero-3-phosphoethanolamine-N- 62

[methoxy(polyethylene glycol)-2000] (Ammonium salt): C6-Ceramide or Dihydro-C6- ceramide (dC6-ceramide) (1.75: 3.75: 0.75: 0.75: 3). This formulation is highly stable and can be successfully used in in vivo experiments due to pegylation. These liposomes are inert pharmacologically within clinically relevant concentrations. The biologically inactive dihydro-C6-ceramide was used as a negative control under the described experimental conditions. Ceramide-free liposomes (ghost) containing similar concentrations and ratios of the other lipid components were also used as a negative lipid control. Lipid mixtures were dried under a stream of nitrogen above lipid transition temperatures, and hydrated with sterile phosphate-buffered saline (PBS). The resulting solution underwent sonication for 10 minutes followed by extrusion through 100-nm polycarbonate membranes. The amount of total lipids in C6 ceramide, dihydroceramide, and ghost liposomal formulations remained constant in all experiments. In all experiments, C6 ceramide was delivered in a non-toxic nano-liposomal formulation as previously described by us (18) as well as previously characterized by the National

Cancer Institute Nanotechnology Characterization Laboratory

(http://ncl.cancer.gov/MK_022207_073007.pdf ). For short term signaling and sucrose gradient experiments, we used 10 M liposomal C6 ceramide to maximize biochemical and biophysical effects. For “endpoint” analysis (migration, mitogenesis), we utilized

1M liposomal C6 ceramide to rule out any long term potential non-specific toxic effects of ceramide. Previous studies have demonstrated that the IC 50 value for liposomal C6 ceramide is 5 M (18) .

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Cell Proliferation. Highly invasive estrogen negative MDA-MB-231 cells were seeded at 2.0 x 10 5 cells/well in 96-well plates and grown overnight prior to 24-h serum starvation. Cells were treated with the indicated concentration of JMV-449 alone or in the presence of 1µM C6 ceramide liposomes, di-hydro-C6-ceramide liposomes or ghost liposomes. After 48 hours, cells were labeled with BrdU (20 l/well) for 5 hours then

BrdU incorporation into DNA was measured according to the manufacturer’s protocol

(Roche Applied Biosciences fluorescent ELISA kit).

Western Blot Analysis. MDA-MB-231 cells were seeded at 4.0 x 10 5 cells/well in 60- mm plates and grown overnight prior to 24-h serum starvation, In some experiments, it was necessary to starve cells for up to 3 days to downregulate basal ERK

phosphorylation. Cells were then treated with liposomal C 6 for 1 hour prior to 100 nM

NT or PMA stimulation for 15 minutes. Cells were washed once with cold PBS followed by the addition of 150 µl of cold lysis buffer (1% Triton X-100, 20 mM Tris, 150 mM

NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na 4P2O7, 1 mM glycerolphosphate, 1 mM

Na 3VO 4, and 1 µg/ml leupeptin in ddH 2O, pH 7.5) on ice. Cells were lysed for 15 min on ice, and cell lysate was harvested and centrifuged at 15,000 g for 15 min. Thirty-five micrograms of protein were loaded in 4 to 12% precasted SDS-polyacrylamide gel electrophoresis gradient gels and probed for pErk 1/2. Blots were stripped and re- probed for Erk 2 to demonstrate equal loading. Protein bands were visualized using enhanced chemiluminescence and quantified using Image-J software.

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Gelatin Zymography. MDA-MB-231 cells were seeded at 4.0 x 10 5 cells/well in 100- mm plates and allowed to grow till 80% confluency. Cells were then treated with 100 nM

NTS in the presence or absence of 1 µM C6 Ceramide, di-hydro-C6- Ceramide or ghost liposomes for 48 hours. Conditioned media were then collected and concentrated using

Centriprep concentrators (YM-30). Aliquots of the concentrated media containing equal amounts of total proteins were then subjected to non-reducing SDS-gel elctrophoresis using gels that contain 1% gelatin (Invitrogen). The gels were then developed according to the manufacturer protocol.

Wound Healing Assay. MDA-MB-231 cells were seeded at 4.0 x 10 5 cells/well in 6 well plates marked with a line grid and allowed to grow until confluency was reached. The confluent cell layer were scratched using 200 l pipette tip and the cells were treated with 100 nM NTS in the presence or absence of 1 µM C6 ceramide in 0.1 % serum containing media. Photomicrographes were taken using a digital camera (Diagnostic instruments, inc.) installed on a microscope (Olympus), 10x magnification power.

Image-J software was used to measure the wound width.

PNGase F Deglycosylation. MDA-MB-231 cells were seeded at 4.0 x 10 5 cells/well in

100-mm plates and allowed to grow till 80% confluency. Cells were lysed using modified lysis buffer (50mM Tris-HCl, pH 7.5; 150 mM NaCl; 1mM EGTA; 10 mM MgCl 2; 0.5 %

Triton X-100; protease inhibitor cocktail). NTSR1 was immuno-precipitated as previously described (16). Immuno precipitated protein was subjected to deglycosylation 65 using peptide-N-glycosydase (PNGase F) (Sigma) according to the manufacturer recommended protocol. The samples were then analysed using SDS-PAGE electrophoresis

Detergent-free Fractionation of Caveolin Rich Domains (16, 21) . We chose a detergent-free protocol based upon high reproducibility and lack of potential experimental artifacts. MDA-MB-231 cells were seeded at 4.0 x 10 5 cells/well in 100-mm plates and allowed to grow till 80% confluency. Cells were then treated with 10 µM C6 ceramide nano-liposomes or Ghost nano-liposomes (1hour) followed by 100 nM NTS

(15 minutes) and then washed with ice-cold PBS twice. Sucrose gradient fractionation was performed according to Song et al. protocol (21). Due to the low concentration of

NTSR1 receptor protein in the fractions, trichloroacetic (TCA) acid-acetone protein precipitation was required in order to be able to analyze the fractions using western blot analysis. In brief, 250 l of freshly prepared TCAstock (100% w/v) was added to 1 ml sucrose gradient fraction and incubated on ice for at least 30 minutes. The samples were then microcentrifuged at 14K rpm for 5 minutes. The supernatants were then removed and the pellet washed twice with ice-cold acetone. After drying the pellets, the pellets were dissolved in 20 l PBS and then 10 l 3X SDS sample buffer was added followed by analysis using SDS-PAGE electrophoreses as described in western blot analysis.

Detergent-based fractionation of Caveolin-rich Domains suitable for Co-IP. We chose this procedure, which is performed at pH 7, in order to preserve protein-protein 66 interactions. Within physiological pH ranges, proteins maintain their tertiary structure and therefore preserve their ability to interact with other proteins. The high pH required for detergent-free fractionation that we have utilized previously could disrupt such interactions. This procedure was performed as described in the previous detergent-free fractionation method except that the cells were lysed using 1% Triton-X in MBS buffer

(25 mM MES, pH 6.5, and 150 mM NaCl). Fractions 4, 5 and 6 that represent the caveolin-enriched fractions were separated and subjected to immuno-precipitation using anti G αq/11 antibody. The immuno-precipitates were separated using SDS-PAGE electrophoresis and the blots were probed using anti-NTSR1 antibody. The blots were then developed as described previously in the western blot section.

Cholesterol Depletion-Repletion. MDA-MB-231 cells were grown in 6-well plates until

80% confluency and then starved for 24 -72 hours. To remove cholesterol, serum- starved cells were incubated in RPMI containing the indicated concentration of methyl-

β-cyclodextrin (M βCD) (Sigma) for 1 h at 37°C. For cholesterol repletion, depleted cells were incubated with a solution containing 0.2 mM of preformed M βCD/cholesterol (10:1 mol/mol) complexes, similar to a previously described procedure (16). The cells were then stimulated with 1 M NTS or PMA for 15 minutes and then lysed using NP-40 lysis buffer. Total proteins were separated on SDS-PAGE and the immunoblots were probed for Phospho-ERK and ERK-2. Kester and co-workers have demonstrated that M βCD treatment under these experimental conditions is sufficient to disrupt SMDs integrity

(16). 67

Statistical Analysis. Differences among treatment groups were statistically analyzed using a two-tailed Student's t test for statistical analyses. Where appropriate, One-way analysis of variance with Bonferroni multiple comparison post-hoc test and t tests analysis were performed using GraphPad Prism 4.0 software. A statistically significant difference was reported if p < 0.05 or less. Data are reported as mean ± S.E. from at least n = 3 separate experiments.

2.4 Results

2.4.1 Exogenous Short Chain Ceramide Inhibits NTSR1-Mediated Cellular

Proliferation in MDA-MB-231 Cells.

Neurotensin (NTS) has been shown to induce cellular proliferation of several cancer cell lines including prostate cancer PC3 cells and breast cancer (4). We utilized a BrdU incorporation assay to initially assess the effect of NTS on DNA synthesis. Treatment of cells with increasing doses of JMV-449, a synthetic stable analogue of NTS, for 48 hours after a 24-hour serum starvation resulted in a dose-dependent increase in DNA synthesis (Fig. 2.2A). The observed increase in DNA synthesis after treatment with 1 and 10 M NTS analogue was not statistically significant compared to 100 nM treatment indicating a level of saturation. Using the lowest saturating dose for NTS, 100 nM, we evaluated the effect of liposomal ceramide on NTS-mediated mitogenesis. Cells were serum-starved for 24 hours before being stimulated with 100 nM JMV-449 in the presence or absence of 1 µM liposomal C6 ceramide (Fig. 2.2B), a dose that was previously shown to inhibit breast cancer proliferation. NTS-induced BrdU incorporation was significantly reduced in the presence of liposomal C6 ceramide, but not liposomal 68 di-hydro C6 ceramide. It has been suggested that the lack of allelic double bond in di- hydro C6 ceramide prevents the formation of hydrogen bonding which is responsible for the bulky rigid head group structures in the case of C6 ceramide (172). In addition, these liposomal formulations did not have any effect on basal level DNA synthesis under these experimental conditions (Fig. 2.2C). 10 % fetal bovine serum was used as a positive control and produced an effect consistent with that observed with NTS treatment.

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Fig.2.2. Exogenous short ceramide inhibits NTSR1-mediated cellular proliferation. A. MDA-MB-231 cells starved for 16 hours were treated with the indicated doses of the NTS1 mimetic, JMV-449, for 48 hours and then were labeled with BrdU (20 l/well) for 5 hours. Confirmatory data demonstrate that NTS itself also stimulated BrdU incorporation with saturation at doses higher than 10uM (data not shown). B. MDA-MB-231 cells were treated with 100 nM JMV-449 in the presence or absence of 1 M C6 ceramide, dihydro C6 ceramide or ghost control liposomes. Fluorescence was measured using a fluorescence plate reader according the manufacturers protocol. C. MDA-MB-231 cells were treated with 10% serum as positive control or C6 ceramide, dC6 ceramide or ghost liposomes under the same experimental conditions used in A and B. Mean + S.E., n = 3 separate experiments.* P < 0.05. 70

2.4.2 Exogenous Short Chain Ceramide Inhibits NTSR1 Mediated Cellular

Migration in MDA-MB-231 Cells.

It has been shown previously that NTS induces migration of breast cancer cells (7).

Using a cellular scratch assay, 100 nM NTS significantly enhanced cellular migration compared to untreated cells (Fig 2.3A). Pretreatment of cells with liposomal C6 ceramide for 1 hour mitigated NTS-induced breast cancer migration compared with ceramide-free liposomes. Ceramide-free liposomal formulations (ghost liposomes) had no effect on cellular migration.

2.4.3 Exogenous Short Chain Ceramide Inhibits NTSR1-Mediated MMP-9

Expression/Activation in MDA-MB-231 Cells.

Previous studies have shown that NTSR1 stimulation induces the expression and activity of matrix metalloproteinase-9 (MMP-9), which has been implicated in tumor invasion of blood vessels (7). We used gelatin zymography to study the effect of C6- ceramide on MMP-9 activity. Treatment of cells with NTS enhanced MMP-9 gelatinolytic activity compared to untreated cells (Fig. 2.3B). Pretreatment of cells with liposomal C6- ceramide significantly reduced NTSR1 mediated activation of MMP-9 while liposomal di- hydro C6 ceramide (Fig. 2.3B) and ceramide-free liposomes (data not shown) had only a minimal non-significant effect. As a positive MMP-9 standard control, conditioned media from MCF7 cells showed a band at the same molecular weight (~92 kDa) as our zymographed samples as well as a recombinant standard (data not shown).

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Fig.2.3. Exogenous short chain ceramide inhibits NTSR1- mediated MMP9-dependent cellular invasion. A. Confluent MDA-MB-231 cells were scratched using a 200 l pipette tip and then treated with 100 nM NTS in the presence or absence of M C6 ceramide or ghost liposomes. Pictures were taken at time 0 and at 22 hours using digital camera installed on Olympus Microscope at 10X magnification power. The width of the wounded area was measured using ImageJ software. Mean + S.E., n = 3 separate experiments. * P < 0.05. B. MDA- MB-231 cells were treated with 100 nM NTS in the presence or absence of 1 M C6 ceramide or dihydro C6 ceramide liposomes. After 48 hours, conditioned media were collected, concentrated and analyzed using gelatin zymography. The negative image was used for clarity. Mean + S.E., n = 3 separate experiments. * P < 0.05.

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2.4.4 Exogenous Short Chain Ceramide Inhibits NTSR1-Mediated, but not PMA-

Mediated, Activation of the MAP Kinase Pathway in MDA-MB-231 Cells.

To assess the effect of NTS on the promitogenic MAP Kinase pathway, we performed a time response study and measured the increase in ERK1/2 phosphorylation using western blot analysis. After serum starvation for 24 hours, MDA-MB-231 cells were treated with 100 nM NTS for the indicated periods of time (Fig. 2.4A). Phospho ERK 1/2 was transiently activated reaching maximal activation after 15 minutes treatment. Based on this study, we chose 15 minutes as a time point to study the effect of liposomal C6 ceramide on NTS-mediated ERK 1/2 activation. Pretreatment of cells with 10 uM liposomal C6 ceramide inhibited NTS-induced ERK 1/2 activation (Fig. 2.4B). As an additional control, we also showed that the MEK inhibitor U0124 also reduced NTS- stimulated ERK stimulation. These results suggest that NTS effect on breast cancer cells is mediated, in part; through the activation of the mitogenic ERK 1/2 pathway and ceramide can inhibit this biochemical signaling cascade.

In order to determine the specificity of C6 ceramide-induced inhibition of NTSR1- mediated MAP Kinase activation, we tested the ability of C6 ceramide to inhibit PMA- induced activation of ERK phosphorylation. PMA was chosen in these studies due to its ability to directly activate downstream PKC isozymes, bypassing NTSR1, as well as its direct effectors (G αq/11 , and phospholipase C). MDA-MB-231 cells were serum starved before a 1 hour pretreatment with 10 M C6 ceramide. Cells were then stimulated with 1

M PMA for 15 minutes and lysates were assayed using western blot analysis. C6 ceramide did not alter basal ERK phosphorylation as well as PMA-induced ERK phosphorylation (Fig. 2.4C). This suggests that C6 ceramide treatment selectively 73 disrupts NTSR1 interaction with signaling intermediates that lie upstream of PKC isozyme such as heterotrimeric G protein subunits.

2.4.5 NTSR1 Signaling Requires Intact Structured Membrane Microdomains

(SMDs)

In order to examine the importance of SMDs for NTSR1-mediated mitogenic signaling, we used Methyl-β-cyclodextrin (MCD) to deplete cholesterol, a major constituent of

SMDs. After 24 hour serum starvation, cells were pretreated with Methyl-β-cyclodextrin in the presence or absence of cholesterol for 30 minutes, before being stimulated with

100 nM NTS or 1 M PMA. MCD treatment resulted in inhibition of ERK1/2 activation, while cholesterol repletion restored NTSR1 activity (Fig. 2.4.D). These results suggest that NTSR1 requires intact SMDs to signal properly. In contrast, MCD did not disrupt

PMA- induced ERK activation, alluding to the specificity of receptor-mediated, but not non-receptor-mediated signaling within intact SMDs.

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Fig.2.4. Exogenous short chain ceramide inhibits NTSR1 mediated ERK phosphorylation. A. MDA-MB- 231 cells were starved for 24 hours and then stimulated with 100 nM NTS for the indicated periods of time. Cell lysates were analyzed using western blot. B. MDA-MB-231 cells were starved for 24 hours and then stimulated with 100 nM NTS in the presence or absence of C6 ceramide liposomes or U0124, MEK inhibitor. C. MDA-MB-231 cells were starved for 24 hours and then treated with 1 M PMA in the presence or absence of 10 uM nano-liposomal C6 ceramide. D. (P.75) MDA-MB-231 cells were starved for 24 hours before stimulation with 1nM NTS or 1 M PMA for 15 minutes in the presence or absence of 10 µM M βCD. As a control, choles terol was replenished before NTS stimulation in selected treatments. Representative blots from at least 3 separate experiments. Mean + S.E., n = 3 separate experiments. * P < 0.05.

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2.4.6 Exogenous C6-Ceramide Inhibits Agonist-Dependent Translocation of

NTSR1 into Structured Membrane Microdomains

Given that NTSR1-signaling responses require functional SMDs, we next analyzed if there was a biophysical component to ceramide inhibited NTSR1 signaling. We initially 76 examined if exogenous ceramide, which localizes within SMDs (16), could alter localization of NTSR1 within SMDs. MDA-MB-231 cells were serum starved for 24 hours and then treated with 10 M C6 ceramide for 1 hour before 1 µM NTS stimulation for 15 minutes. Due to the low sensitivity of sucrose gradient fractionation experiments in systems where endogenously expressed low abundant protein membrane localization is under study, we have utilized a higher NTS treatment concentration to ensure a detectable response. The lysates were subjected to detergent-free sucrose gradient fractionation and the isolated fractions were subsequently analyzed by western blotting.

Fractions 4 and 5 (Fig. 2.5A) are enriched with caveolin, a protein that is known to mainly localize within SMDs and is extensively used in the literature as a marker for these domains (22). Fractions 8 to 12 contain very low level of caveolin and represent the buoyant non-SMD fractions. Caveolin content in the SMD fractions, 4 &5, does not change upon any treatment (Fig. 2.5A). After western blot analysis, we found that both the non-N-glycosylated and N-glycosylated isoforms of NTSR1 (characterized as shown in figure 2.5B) localize mainly to fraction 8-12 outside the caveolin-enriched microdomains (Fig. 2.5A). Upon receptor stimulation, mature NTSR1 isoform translocates into caveolin-enriched SMDs (Fig. 2.5A). This movement is transient as the receptor either internalizes or moves out of the rafts after 20 minutes treatment (data not shown). Most strikingly, we observed an approximate 2 kDa increase in the receptor molecular weight upon entrance into SMD, fractions 4 and 5. The identity of the band of higher molecular weight (~54 kDa) was confirmed to be the N-glycosylated isoform as shown in Fig. 2.5B, where treatment with PNGase F casued a shift of the band to a level similar to that of the lower molecular weight band (~52 kDa). It is apparent that the 77

NTSR1 antibody identified the more glycosylated NTSR1 isoform under these immunoprecipitation conditions. This may reflect that the protein is not heat-denatured, in contrast to regular western blot analysis where the protein is heat denatured. It is most likely that this change in the mobility of the protein upon enrichment into SMDs is due to receptor hyperglycosylation. Pretreatment of cells with liposomal C6 ceramide before NTS stimulation resulted in inhibition of NTS-dependent NTSR1 translocation into SMDs, while treatment of cells with C6 ceramide alone does not affect the receptor localization (Fig. 2.5A). The percentage of NTSR1 receptor that translocates into the

SMDs fraction was quantitated relative to total NTSR1 in all fractions using ImageJ software and was found to be about 30% (Fig.2.5A, 2.6B). These results suggest that accumulation of C6 ceramide within structure membrane microdomains prevents NTS- induced translocation of NTSR1 into these caveolin-rich microdomains.

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Fig.2.5. Exogenous short chain ceramide inhibits NTS-dependent translocation of NTSR1 into SMDs. A. MDA-MB-231 cells were starved for 24 hours before stimulation with 1 µM NTS or control (untreated cells) for 15 minutes in the presence or absence of 10 µM liposomal C6 ceramide (pretreatment for 1 hour). Cell lysates were then subjected to detergent-free sucrose gradient fractionation as described in the methods section. The inserted bar graph represents densitometric quantification of the percentage of NTSR1 in fractions 4 and 5 relative to the total NTSR1 in all fractions. Representative blots from 3 separate experiments. * P < 0.05.. B. NTSR1 was immunoprecipitated from MDA-MB-231 lysates. The immunoprecipitates were then subjected to N-deglycosylation using PNGase F and then analyzed using SDS-PAGE electrophoresis. It is important to note that the immunoprecipitation conditions isolate the N- glycosylated isoform(s). Representative blots from 3 separate experiments. * P < 0.05.

2.4.7 NTSR1 Stimulation does not Alter G αααq/11 Localization within SMDs.

Several reports suggest that NTSR1 can couple to multiple G proteins including G αq and G αs (23). Additionally, Souaze et al. suggested the participation of G αq/G βγ and 79

PKC signaling pathways in NTS-dependent breast cancer progression (7). Therefore, we examined if NTS-induced NTSR1 translocation to SMDs regulates NTSR1/G αq/11 interaction. We again performed detergent-free sucrose gradient fractionation and observed an approximate 37% increase in NTSR1 localization to SMD fractions upon

NTS receptor stimulation, which was abrogated by ceramide pretreatment (Fig. 2.6A).

Gαq/11 mainly localize to SMDs and receptor stimulation with 1 µM NTS did not alter membrane localization (Fig. 2.6A). We also observed that ERK-2 mainly localizes to the non-caveolar-rich domains, however, a fraction of ERK-2 exists within SMDs and could be sufficient for signal transduction upon NTSR1 stimulation. Thus, it is suggested that

NTS-induced NTSR1 translocation within SMD may lead to enhanced interaction of

NTSR1 with G αq/11 .

2.4.8 Exogenous C6-Ceramide Inhibits NTSR1 Interaction with G αq/11 within SMDs.

To further confirm the mechanistic importance of NTSR1 translocation into SMDs for

NTS mitogenic signaling, we designed an experimental protocol that combines both detergent-based sucrose fractionation followed by co-immunoprecipitation of SMDs fractions. It was necessary to perform detergent-based sucrose gradient fractionation in order to maintain protein-protein interactions; the alkaline pH necessary for detergent- free fractionation can disrupt protein-protein interaction. The SMD fractions were separated and subjected to immuno-precipitation using anti-Gαq/11 antibody, followed by immuno-blotting using anti-NTSR1 antibody (Fig. 2.6B). The results show that G αq/11 interacts with NTSR1 within SMDs only upon its stimulation with 1 M NTS for 15 minutes. It is important to note that the higher molecular weight, hyperglycosylated, 80

NTSR1 is the major receptor population that interacts with G αq/11 . Pretreatment with 10

µM nano-liposomal ceramide for 1 hour before NTS stimulation disrupted this critical interaction (Fig. 2.6B). This result suggests that the receptor membrane translocation is essential for NTS coupling to G αq/11 within SMDs.

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Fig.2.6. NTSR1 Translocates to SMDs and Interacts with G αααq/11 . A. MDA-MB-231 cells were starved for 24 hours before stimulation with 1 µM NTS for 15 minutes or control in the presence or absence of 10 µM liposomal C6 ceramide (pretreatment for 1 hour). Cell lysates were then subjected to detergent-free sucrose gradient fractionation followed by western blot analysis of the isolated fractions as described in the methods section. B. Quantification of the western blots in panel A (based on n=3 independent experiments) showing the percentage of NTSR1 that localizes within SMDs relative to total NTSR1 C. (P.82) MDA-MB-231 cells were seeded in 10 cm plate and then starved for 24 hours before being stimulated with 1 µM NTS for 15 minutes in the presence or absence of 10 µM C6 liposomal ceramide. Cell lysates were then subjected to TritonX based detergent- based sucrose gradient fractionation method followed by co-immunoprecipitation of the

SMD-containing fractions using anti-Gαq/11 . The immunoprecipitates were resolved on SDS-PAGE electrophoresis and the blots were probed with anti-NTSR1. Representative blots from n=3 separate experiments.

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2.5 Discussion

G protein-coupled receptors (GPCRs) are very attractive targets for drug development (24). Recently, many GPCRs; such as neurotensin, endothelin, chemokine and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers (24). NTSR1 is overexpressed in several types of cancer and correlate with tumor metastasis and acquired resistance to conventional chemotherapeutic regimens (3). SMDs and caveolae have been shown to be involved in the regulation of various essential cell functions including fine-tuning of components of the cell signaling machinery (9). It is not well understood how GPCRs localize within

SMDs (14). Some GPCRs localize exclusively into SMDs such as the gonadotrophin- releasing hormones receptor (GnRH) (25) and alpha1a-adrenergic receptor (26) while others are restricted from SMDs such as the oxytocin receptor (22, 25, 27). Several 83

GPCRs such as the angiotensin II type 1 receptor translocate into SMDs only upon agonist stimulation (28). On the other hand, CB1 cannabinoid receptor translocates outside SMDs upon agonist binding (14). Such receptor translocations might be essential for the formation of an active receptor-signaling complex. Recently, reports from several labs, including ours (15, 16) have shown that ceramide tends to localize within SMDs, which serves to assemble large scale membrane domains and/or trigger signaling cascades (15, 16). Mehga and London showed that ceramide selectively displaces cholesterol from ordered lipid domains to form ceramide-rich SMDs (11). Yu et al. also supported this finding and additionally showed that such displacement of cholesterol can alter multiple proteins restricted within the SMDs (29). Such findings are supported by Gidwani at al. (12) who showed that incorporation of short-chain ceramides into SMDs correlates with inhibition of phospholipase D and downstream signaling by Fc εRI (12).

These findings prompted us to investigate the effect of short-chain ceramide on

NTSR1 signaling in breast cancer. The results of the functional studies including cellular proliferation, migration and MMP-9 expression suggested that liposomal short-chain ceramide antagonized NTSR1-mediated functions associated with breast cancer cell tumorigenesis. Previous studies showed that the MAP kinase pathway might, in part, mediate NTSR1 mitogenesis and/or oncogenesis (7). We found that liposomal C6 ceramide reduce NTSR1-dependent MAP kinase signaling (ERK 1/2), correlating with diminished breast cancer progression. The inability of C6 ceramide to disrupt PMA- induced ERK phosphorylation suggests that its effect on NTSR1 signaling is receptor specific. Consistent with this observation, cholesterol depletion using MCD reduced 84

NTSR1-dependent ERK phosphorylation but had no significant effect on PMA- induced

ERK phosphorylation, indicating that MDA-MB-231 cells are still responsive to non- receptor mediated signaling even in the presence of C6 ceramide. Taken together, these results suggest that C6 ceramide altered SMDs disrupts receptor-mediated, but not non-receptor-mediated, signaling pathways.

We envisioned that the biophysical mechanism by which ceramide leads to a decrease in NTSR1 signaling is mainly due to the modulation of lipid microenvironment within SMDs. We found that upon NTSR1 stimulation, the receptor translocates into

SMDs and this localization could be reversed with exogenous C6 ceramide. We then went further and examined the effect of NTS stimulation on NTSR1 membrane localization, and interaction with G proteins that are candidate transducers of NTSR1- dependent MAP kinase activation. Previous reports show that NTSR1 can couple to

Gαq/11 and G αs (23). However, Souaze et al. suggested that G αq signaling pathway is more likely to be responsible for NTSR1 mitogenic signaling in breast cancer (7).

Using sucrose gradient fractionation, we found that G αq is preferentially localized to

SMDs, in agreement with Oh and Schnitzer (30), suggesting that G αq interaction with

NTSR1 may occur within SMDs. We confirmed this interaction by analyzing the SMD fractions using co-immuno-precipitation and noted that C6 ceramide disrupts

NTSR1/G αq/11 interactions. Based on these data, we believe that the intact SMDs are essential for the formation of the receptor signalplex and disruption of these domains, via C6 ceramide, can prevent the signal initiation. Our previously published data show that exogenous ceramide accumulates into caveolin-rich micordomains (16). We believe that this causes a change in the lipid microenvironment and prevents the movement of 85

NTSR1 into SMDs, thus decreasing interactions with G protein dependent signaling cascades. Although, the data suggest that the G protein coupling for NTSR1 in breast cancer occurs mainly SMDs, we cannot role out the possibility that SMD-localization may also regulate receptor desensitization, trafficking and/or recycling of NTSR1/G αq complex. Inhibition of NTSR1 localization by ceramide could also disrupt mitogenic signaling in breast cancer due to the reduction in available and responsive NTSR1.

However, it is important to note that C6 ceramide treatment did not have any significant effect on caveolin-1 (Fig 2.5A, 2.6B), or NTSR1 expression level (data not shown).

In conclusion, our finding suggests a novel pharmacological approach to target

NTSR1 in breast cancers. We demonstrate that nano-liposomal short chain ceramide inhibits NTSR1 induced breast cancer progression via biochemical (ERK), and biophysical (SMDs) mechanisms, findings consistent with our previous in vitro and in vivo work (17). We also provide a mechanism by which modulating SMD-dependent

NTSR1 signaling inhibits the receptor ability to interact with G αq subunits. Collectively, our studies implicate nanoliposomal C6 ceramide as a potential systemic adjuvant for breast cancer therapy. In support of our conclusion that short chain ceramide nano- formulations may have broad valuable clinical applications, we have recently reported that short chain ceramide incorporated into calcium phosphate nanocomposite particles diminish growth of MCF7 cells, a highly differentiated NTSR1 overexpressing and responsive breast cancer cellular model (31).

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2.6 References

1 Uhl,G.R. and Snyder,S.H. Neurotensin receptor binding, regional and subcellular distributions favor transmitter role, Eur.J.Pharmacol., 41: 89-91, 1977.

2 Kitabgi,P. and Freychet,P. Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release, Eur.J.Pharmacol., 56: 403-406, 1979.

3 Carraway,R.E. and Plona,A.M. Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools, Peptides, 27: 2445- 2460, 2006.

4 Vias,M., Burtt,G., Culig,Z., Veerakumarasivam,A., Neal,D.E. and Mills,I.G. A role for neurotensin in bicalutamide resistant prostate cancer cells, Prostate, 67: 190- 202, 2007.

5 Seufferlein,T. and Rozengurt,E. Galanin, neurotensin, and phorbol esters rapidly stimulate activation of mitogen-activated protein kinase in small cell lung cancer cells, Cancer Res., 56: 5758-5764, 1996.

6 Evers,B.M. Neurotensin and growth of normal and neoplastic tissues, Peptides, 27: 2424-2433, 2006.

7 Souaze,F., Dupouy,S., Viardot-Foucault,V., Bruyneel,E., Attoub,S., Gespach,C., Gompel,A. and Forgez,P. Expression of neurotensin and NT1 receptor in human breast cancer: a potential role in tumor progression, Cancer Res., 66: 6243-6249, 2006.

8 Somai,S., Gompel,A., Rostene,W. and Forgez,P. Neurotensin counteracts apoptosis in breast cancer cells, Biochem.Biophys.Res.Commun., 295: 482-488, 2002.

9 Quest,A.F., Leyton,L. and Parraga,M. Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease, Biochem.Cell Biol., 82: 129-144, 2004.

10 Schley,P.D., Brindley,D.N. and Field,C.J. (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells, J.Nutr., 137: 548-553, 2007. 87

11 Megha and London,E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function, J.Biol.Chem., 279: 9997-10004, 2004.

12 Gidwani,A., Brown,H.A., Holowka,D. and Baird,B. Disruption of lipid order by short- chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI, J.Cell Sci., 116: 3177-3187, 2003.

13 Jacobson,K., Mouritsen,O.G. and Anderson,R.G. Lipid rafts: at a crossroad between cell biology and physics, Nat.Cell Biol., 9: 7-14, 2007.

14 Barnett-Norris,J., Lynch,D. and Reggio,P.H. Lipids, lipid rafts and caveolae: their importance for GPCR signaling and their centrality to the endocannabinoid system, Life Sci., 77: 1625-1639, 2005.

15 Hajduch,E., Turban,S., Le,L., X, Le,L.S., Lipina,C., Dimopoulos,N., Dugail,I. and Hundal,H.S. Targeting of PKCzeta and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide, Biochem.J., 410: 369-379, 2008.

16 Fox,T.E., Houck,K.L., O'Neill,S.M., Nagarajan,M., Stover,T.C., Pomianowski,P.T., Unal,O., Yun,J.K., Naides,S.J. and Kester,M. Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains, J.Biol.Chem., 282: 12450-12457, 2007.

17 Stover,T.C., Sharma,A., Robertson,G.P. and Kester,M. Systemic delivery of liposomal short-chain ceramide limits solid tumor growth in murine models of breast adenocarcinoma, Clin.Cancer Res., 11: 3465-3474, 2005.

18 Stover,T. and Kester,M. Liposomal delivery enhances short-chain ceramide- induced apoptosis of breast cancer cells, J.Pharmacol.Exp.Ther., 307: 468-475, 2003.

19 Devalapally,H., Duan,Z., Seiden,M.V. and Amiji,M.M. Paclitaxel and ceramide co- administration in biodegradable polymeric nanoparticulate delivery system to overcome drug resistance in ovarian cancer, Int.J.Cancer, 121: 1830-1838, 2007.

20 Boudin,H., Lazaroff,B., Bachelet,C.M., Pelaprat,D., Rostene,W. and Beaudet,A. Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain, J.Comp Neurol., 425: 45-57, 2000.

21 Song,K.S., Li,S., Okamoto,T., Quilliam,L.A., Sargiacomo,M. and Lisanti,M.P. Co- purification and direct interaction of Ras with caveolin, an integral membrane 88

protein of caveolae microdomains. Detergent-free purification of caveolae microdomains, J.Biol.Chem., 271: 9690-9697, 1996.

22 Insel,P.A., Head,B.P., Patel,H.H., Roth,D.M., Bundey,R.A. and Swaney,J.S. Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae, Biochem.Soc.Trans., 33: 1131-1134, 2005.

23 Pelaprat,D. Interactions between neurotensin receptors and G proteins, Peptides, 27: 2476-2487, 2006.

24 Dorsam,R.T. and Gutkind,J.S. G-protein-coupled receptors and cancer, Nat.Rev.Cancer, 7: 79-94, 2007.

25 McArdle,C.A., Franklin,J., Green,L. and Hislop,J.N. The gonadotrophin-releasing hormone receptor: signalling, cycling and desensitisation, Arch.Physiol Biochem., 110: 113-122, 2002.

26 Morris,D.P., Lei,B., Wu,Y.X., Michelotti,G.A. and Schwinn,D.A. The alpha1a- adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits, J.Biol.Chem., 283: 2973-2985, 2008.

27 Xu,W., Yoon,S.I., Huang,P., Wang,Y., Chen,C., Chong,P.L. and Liu-Chen,L.Y. Localization of the kappa opioid receptor in lipid rafts, J.Pharmacol.Exp.Ther., 317: 1295-1306, 2006.

28 Ishizaka,N., Griendling,K.K., Lassegue,B. and Alexander,R.W. Angiotensin II type 1 receptor: relationship with caveolae and caveolin after initial agonist stimulation, Hypertension, 32: 459-466, 1998.

29 Yu,C., Alterman,M. and Dobrowsky,R.T. Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1, J.Lipid Res., 46: 1678-1691, 2005.

30 Oh,P. and Schnitzer,J.E. Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default, Mol.Biol.Cell, 12: 685-698, 2001.

31 Kester,M., Heakal,Y., Fox,T., Sharma,A., Robertson,G.P., Morgan,T.T., Altinoglu,E.I., Tabakovic,A., Parette,M.R., Rouse,S., Ruiz-Velasco,V. and Adair,J.H. Calcium Phosphate Nanocomposite Particles for In Vitro Imaging and Encapsulated Chemotherapeutic Drug Delivery to Cancer Cells, Nano.Lett., 2008.

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Chapter 3

Neurotensin Receptor -1 Inducible Palmitoylation is Required for Efficient Receptor-Mediated Mitogenic-Signaling within Structured Membrane Microdomains

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3.1 Abstract

Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer development and progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for receptor localization and signaling within SMDs upon

NTS stimulation. In this study, we identified that endogenously expressed NTSR-1 in

MDA-MB-231 breast adenocarcinomas, and exogenously expressed NTSR-1 in

HEK293T cells that do not normally express NTSR-1 is palmitoylated at Cys 381 and

Cys383. Inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1 expressing HEK293T cells diminished NTS-mediated ERK 1/2 phosphorylation.

Additionally, NTSR1 mutated at Cys381 and/or Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HEK293T cells against apoptosis induced by serum starvation. Mechanistically, C381,383S-NTSR-1 showed reduced ability to interact with G αq/11 and diminished localization to SMDs, where G αq/11 preferentially resides. In addition, our results also suggest that ceramide effects on NTSR-1 localization and signaling could be, in part, mediated through interference with NTSR-1- palmitoylation/depalmitoylation cycle. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer.

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3.2 Introduction

The overexpression of the G-protein coupled neurotensin receptor, NTSR-1, and its endogenous ligand, NTS, plays a key role in the development and progression of several types of tumors including breast (1), pancreas (2), prostate (3), colon (4), and lung (5) cancers. In invasive ductal breast carcinomas, Dupouy et al. recently demonstrated that NTS expression is regulated by estrogen (1). Moreover, NTSR-1 expression level in breast cancer is correlated with the Scarff-Bloom-Richardson (SBR) grade, tumor size, and tendency to metastasize to lymph nodes (1). Additionally, patients with low NTSR-1 expression had better prognosis relative to those with high expression levels (96.5% ten years survival rate versus 66.2%) (1). Therefore, identification of novel pharmacological approaches to inhibit NTSR-1 mitogenic signaling is needed.

Palmitoylation is the post-translational addition of a 16-carbon fatty acid, palmitate, to a cysteine residue of a protein via a thioester or amide bond (6). Unlike myristoylation and isoprenylation, palmitoylation is a dynamic, reversible process, which allows for regulation of protein lipophilicity during signal transmission (6). Rhodopsin receptor was the first GPCR to be identified as a target for palmitoylation (7). GPCR palmitoylation alters receptor conformation and thereby, regulates the interactions of the receptor with specific downstream effectors (8). A palmitoylation/depalmitoylation cycle upon stimulation was observed for several GPCRs, such as the D1 dopamine (9-11), β2- adrenergic (12) and α2A-adrenergic receptors (13). Palmitoylation of chemokine (C-C motif) receptor 5 CCR5 (14) and A1 adenosine receptors (15) was found to be 92 necessary for receptor delivery to the plasma membrane, while non-palmitoylated receptors are degraded. In addition to receptor palmitoylation, downstream effectors such as heterotrimeric G protein subunits are also subject to several posttranslational modifications through lipidation. For example, G αq/11 and G αs palmitoylation is required for membrane anchorage and interaction with GPCRs (16).

To date, over 20 putative mammalian palmitoyl acyl transferases (PAT) characterized by the presence of DHHC-cysteine-rich domain have been identified.

Recent studies suggest that targeting PATs could lead to the development of novel anti- neoplastic chemotherapeutic agents (17). Although, characterization of the specific

PATs that are responsible for GPCR palmitoylation are still not well defined, there are currently several lipid-based and small molecule based-experimental PAT inhibitors that can serve as lead compounds for the development of more specific drug-like inhibitors

(18). We previously published that intact structured membrane micordomains (SMDs) are required for NTSR-1 signaling and interaction with G αq/11 (19) . We now hypothesize that NTSR-1 is palmitoylated and this post-translational modification is required for optimal receptor signaling within SMDs. In this study, we identify NTSR-1 as a target for

PAT and we investigate the functional and mechanistic roles of NTSR-1 palmitoylation in receptor-mediated pro-survival signaling. We validate the potential of utilizing PAT inhibitors to selectively disrupt NTSR-1 mitogenic signaling in breast cancer.

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3.3 Materials and Methods

Materials and Cell Culture. Polyacrylamide gel electrophoresis gradient gels were purchased from Invitrogen (Carlsbad, CA), and enhanced chemiluminescence reagent was purchased from Thermo Fisher Scientific Inc. Human MDA-MB-231 breast adenocarcinoma cells and HEK293T were obtained from American Type Culture

Collection (Manassas, VA) and grown at 37°C in RPMI 1640 medium and DMEM, respectively, supplemented with 10% fetal bovine serum (FBS). The MDA-MB-231 cell line is a highly aggressive metastatic, estrogen receptor-negative, epidermal growth factor receptor (EGFR) positive and NTSR-1 positive model of human breast cancer.

NTS was purchased from Sigma-Aldrich and was dissolved in 0.05% acetic acid just before use in experiments. All antibodies were purchased from Santa Cruz Corp, CA.

Peptide-N-glycosidase F (PNGaseF) was obtained from Sigma, Saint Louis, Missouri.

Phorbol ester (PMA) was obtained from Promega Inc (Madison, WI). Human NTSR1 and HA-tagged cDNA was purchased from Missouri S&T cDNA Resource Center, Rolla,

MO (Catalogue Number: NTSR100000). MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and caspase 3/7 apoptosis assay reagents were purchased from Promega. PNGase F and O-glycosidase were obtained from Sigma, Saint Louis, MO. [9,10 -3H]palmitate (American Radiolabeled

Chemicals).

Site Directed Mutagenesis. The coding region for human NTSR-1 was subcloned into the EcoRI and XbaI restriction sites of the CMV promoter expression plasmids 94 pCDNA3.1(+) and N-terminal HA-tagged pCDNA3.1(+). Cysteine residues 381 and 383 of NTSR-1 were mutated individually and simultaneously to serine residues by site directed PCR mutagenesis to generate C381S, C383S and C381,383S-NTSR-1 mutant.

All sequences were verified by DNA sequencing (Molecular Genetics Core Facility,

Pennsylvania State University, Hershey, PA).

PNGase F and O-glycosidase Deglycosylation . HEK293T cells were seeded in 10- mm plates and allowed to grow until 70% confluency and then transfected with 1µg

DNA using lipofectamine 2000 according to manufacturer recommendation. After 48 hours, Cells were lysed using modified lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EGTA, 10 mmol/L MgCl 2, 0.5% Triton X-100, protease inhibitor cocktail]. NTSR-1 was immunoprecipitated as previously described (16).

Immunoprecipitated protein was subjected to deglycosylation using PNGase F or O- glycosidase according to the manufacturer's recommended protocol. The samples were then analyzed using SDS-PAGE electrophoresis.

Western Blot Analysis. Transfected HEK293T cells were seeded at 4.0 x 105 cells/well in 6 well plates and grown overnight prior to 24-h serum starvation. Cells were then stimulated with 1 µM NTS or phorbol ester phorbol 12-myristate 13-acetate PMA for 15 minutes in the presence or absence of 2-bromopalmitate (2-BP). Cells were washed once with cold PBS followed by the addition of 60 l of cold lysis buffer (1%

Triton X-100, 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na 4P2O7, 1 95

mM glycerolphosphate, 1 mM Na 3VO 4, and 1 g/ml leupeptin in ddH 2O, pH 7.5) on ice.

Cells were lysed for 15 min on ice, and cell lysate was harvested and centrifuged at

15,000 g for 15 min. 35 µg of protein were loaded in 4 to 12% precasted SDS- polyacrylamide gel electrophoresis gradient gels and probed for P-ERK 1/2. Blots were stripped and re-probed for Erk 2 to demonstrate equal loading. Protein bands were visualized using enhanced chemiluminescence and quantified using Image-J software.

Metabolic Radiolabeling. HEK293T cells transiently transfected with HA-tagged WT-

NTSR-1 and C381,383S-NTSR-1 constructs were grown in 10 cm plates till they reached 80% confluence then starved over night. 50 µCi/ml [ 3H] Plamitate was added to the cells for 4 hours before stimulation with 1 µM NTS for 15 minutes. Following stimulation, cells were lysed using Radioimmunoprecipitation (RIPA) buffer and NTSR-1 was immunoprecipitated using anti-HA antibody. Immunoprecipitated samples were then split into two parts. One part was analyzed by SDS-gel electrophoreses and the blots were probed with anti-HA antibody to ensure equal loading while the remaining portions were analyzed using auto-radiography where the blots were sprayed with

ENHANCE (PerkinElmer, Waltham MA) and the film was exposed for 6-8 weeks at -81

οC.

MTS Cell Viability Assay. HEK293T cells transfected with WT-NTSR-1 or C381,383S-

NTSR-1 were seeded in 96 well plates at a density of 4000 cell/well. After 48 hours, the cells were starved for 24 hours and then treated with 200 nM NTS or 5% FBS for additional 24 hours. Two hundred nanomolar NTS was added again for another 24 96 hours before the addition of MTS reagent, in the presence of phenazine methosulfate

(PMS)). Cell viability was quantified according to the manufacturer protocol where absorbance was measured at 495 nm using a plate reader.

Caspase 3/7 Assay Apoptosis Assay. HEK293T cells transfected with WT-NTSR-1 or

C381,383S-NTSR-1 were seeded in 96 well plates at a density of 4000 cell/well. After

48 hours, the cells were starved in the presence or absence of 200 nM NTS for additional 48 hours. ApoOne caspase 3/7 assay (Promega, WI) was used to measure caspase 3/7 activity according to the manufacturer instructions.

Acyl Exchange Palmitoylation Assay (20). MDA-MB-231 cells were grown in 10 cm plates till they reached 70-80% confluence then lysed using modified lysis buffer (50mM

Tris-HCl, pH 7.5; 150 mM NaCl; 1mM EGTA; 10 mM MgCl 2; 0.5 % Triton X-100; protease inhibitor cocktail). After measuring protein concentration, cell lysates were solubilized overnight at 4 οC. Cell lysates were then centrifuged at 4 οC at 15000g for 10 min to remove the insoluble material. NTSR-1 was immunoprecipitated over night using anti-NTSR-1 antibody. Gamma bind beads (GE, NJ) were added to the immunoprecipitate mixture and rotated over night at 4 οC. The immunoprecipitates were washed with PBS for 15 minutes, lysis buffer for 1 hour then 2 times with PBS for 15 minutes. 50 µl N-ethylmaleimide (NEM) (30 mg/ml) added to 700 µl PBS and the samples were rotated at 4 οC overnight. The beads were then washed 4-5 times using

PBS at a required pH of 7-7.5. After splitting each sample into 2, 800 µl 1M NH 2OH 97

(~pH 7) was added to one sample of each pair while the other served as an internal negative control. The samples were then incubated rotating at 37 οC for 3-5 hours.

The beads were then washed with PBS 2-3 times. 1-biotinamido-4-[4-

(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC) was added to each samples followed by incubation on a rotator overnight at room temperature. The beads were then washed twice with PBS and 1X loading dye was added. After heating at 37 οC for 30 minutes, the samples were analyzed by SDS gel electrophoreses. The blots were probed with avidin antibody and visualized using enhanced chemiluminescence.

Detergent-free Fractionation of Caveolin Rich Domains (17, 21) . We chose a detergent-free protocol based upon high reproducibility and lack of potential experimental artifacts. Transfected HEK293T cells were seeded at 4.0 x 10 5 cells/well in

100-mm plates and allowed to grow till 80% confluency. Cells were then treated with 1

µM NTS (15 minutes) and then washed with ice-cold PBS twice. Sucrose gradient fractionation was performed according to Song et al. protocol (21). The samples were then analyzed using SDS-PAGE electrophoreses as described in western blot analysis.

Statistical Analysis. Differences among treatment groups were statistically analyzed using a two-tailed Student's t test for statistical analyses. Where appropriate, one-way analysis of variance with Bonferroni multiple comparison post-hoc test and t tests analysis were performed using GraphPad Prism 4.0 software. A statistically significant 98 difference was reported if p < 0.05 or less. Data are reported as mean ± S.E. from at least n = 3 separate experiments.

3.4 Results:

3.4.1 NTSR-1 is Palmitoylated in MDA-MB-231 Breast Cancer Cells.

Based upon NTSR-1 sequence, we suggest the presence of two putative palmitoylation sites within close proximity to the C-terminus. We identify Cys 381 and

Cys 383 as putative palmitoylation sites using published sequences. We initially tested the hypothesis that the endogenously expressed NTSR-1 in MDA-MB-231 breast cancer cells is palmitoylated, using a non-radioactive fatty acyl exchange labeling assay. This assay assesses palmitoylation via cleavage of palmitoyl moieties with hydroxyl amine (NH 2OH) after blocking all exposed free cysteine residues with NEM.

The NH 2OH exposed cysteine(s) are visualized by conjugation with Biotin (Btn-BMCC)

(Fig 3.1A upper panel). As an internal negative control, minimal signal is observed for the immunoprecipitated NTSR-1 in the absence of NH 2OH. This assay identified basal- palmitoylation of an immunoprecipitated ~54 kDa protein (Fig. 1A upper panel), that co- blotted with NTSR-1 antibody (Fig 3.1A lower panel). Collectively, these data clearly demonstrate that NTSR-1 is palmitoylated under basal conditions.

3.4.2 Pharmacological Inhibition of NTSR-1 Palmitoylation in MDA-MB-231 Cells

Inhibits Receptor-Mediated Mitogenic Signaling.

To further define the role of NTSR-1- palmitoylation in a non-overexpression cell system, we examined the pharmacological effect of 2-BP on NTSR-1-mediated- 99 enhancement of the constitutively active MAP kinase signaling pathway in MDA-MB-231 breast cancer cells that endogenously express NTSR-1.

Fig.3.1. Endogenously expressed NTSR-1 in breast cancer cells is palmitoylated . A. MDA-MB-231 cells were seeded in 10 cm plates followed by receptor immunoprecipitation. After receptor immunoprecipitation, free cysteine residues were protected using N-ethylmaleimide (NEM) followed by NH 2OH-mediated cleavage of palmitate groups. The free cysteine residues were then labeled with biotin using biotin-conjugated 1-biotinamido- 4-[4- (maleimidomethyl)cyclohexanec arboxamido]butane (BMCC) and immunoprecipitates were analyzed using SDS electrophoresis using an avidin antibody to detect the biotin- labeled palmitoylated residues. B. MDA-MB-231 breast cancer cells were seeded in 6 well plates and serum starved for 48 hours before 1 µM NTS stimulation for 15 minutes. The cells were lysed using RIPA buffer and equal total protein samples were analyzed using SDS gel electrophoresis. The blots were probed with P-ERK1/2 antibody and quantified using Image-J software. Representative plots from 3 separate experiments. Mean + S.E., * P < 0.05.

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Data in figure 3.1B demonstrate that 2-BP-pretreatment abrogated both basal and

NTS-strengthened P-ERK activity. This result further confirm that the endogenously expressed receptor is indeed palmitoylated and also demonstrate the potential of exploiting PATs inhibitors as a novel pharmacological approach to ameliorate NTSR-1- mediated mitogenic signaling in breast cancer. These treatment conditions as observed by microscopic examination of cell viability (data not shown) are not toxic as demonstrated in the experiment described in Figure 3.5B where PMA-mediated stimulation of ERK is not inhibited by 2-BP.

3.4.3 Generation and Expression of Wild Type and C381, 383S-mutated NTSR-1 in

HEK293T Cells

In order to further confirm that NTSR-1 is palmitoylated, we conducted site direct mutagenesis studies. Cys 381 and Cys 383 were mutated to serine, generating double mutant NTSR-1 constructs. HEK293T cells, which don’t normally express the receptor, were transfected with the constructs and grown for 48 hours before western blot analysis was performed to measure protein expression. The expression levels

C381,383S NTSR-1 and WT-NTSR-1 constructs were comparable as shown in Fig.

3.2A. No expression was observed with untransfected control lysates. Consistent with our previously published studies (21), fully mature glycosylated receptor isoforms were detected at molecular weight ~ 52 kDa, while non-mature isoforms were detected at molecular weight ~42 kDa. To confirm and validate the specificity of glycosylated NTSR-

1 species, we assessed N- and O-type glycosylation via PNGaseF enzyme and O- glycosidase, respectively. (Fig.3.2B). We observed a shift in the high molecular weight isoforms of NTSR-1 for PNGaseF but not O-glycosidase treatment. Taken together, 101 these data suggest that wild type as well as putative palmitoylation deficient mutant

NTSR-1 constructs can be expressed and N-glycosylated in HEK293T cells.

Fig.3.2. NTSR-1 proteins can be overexpressed and glycosylated in HEK293T cells. A. HEK293T cells were seeded in 10 cm plates and allowed to attach before transfection with NTSR-1 constructs using Lipofectamine 2000. After 48 hours, the cells were lysed using RIPA buffer and samples containing equal total protein were analyzed using SDS gel electrophoresis. The blots were probed with anti-NTSR-1 antibody and quantified using Image-J software. B. Transfected HEK293T cells were lysed using Ripa lysis buffer and NTSR-1 was immunoprecipitated using anti-NTSR-1 antibody. The protein was subjected to PGNaseF or O- deglycosydase and samples were analyzed using SDS-gel elecrophoresis. Representative plots from 3 separate experiments. Mean + S.E., * P < 0.05. 102

3.4.4 NTS Treatment Induces Palmitoylation of WT-NTSR-1, but not C381,383S-

NTSR-1 Expressed in HEK293T Cells.

To further confirm if NTSR-1 is palmitoylated at specific cysteine residues, we utilized a metabolic radiolabeling technique using [ 3H] palmitate. As shown in Fig. 3.3,

WT-NTSR-1, but not C381,383S-NTSR-1, is palmitoylated under basal conditions.

Additionally, stimulation of the receptor with 1µM NTS for 15 minutes resulted in an approximate 50% increase in WT-NTSR-1, but not C381,383S-NTSR-1 receptor palmitoylation, suggesting that this post-translational modification serves as a regulatory mechanism for NTSR-1.

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Figure 3.3 Cysteine 381, 383 in NTSR-1 are required for optimal palmitoylation. HEK293T cells transfected with HA tagged WT-NTSR-1 and C381,383S NTSR-1 were starved for 24 hours before 4 hours [ 3H] palmitate labeling. Cell lysates were subjected to immunoprecipitation using anti-HA antibody. One portion of the samples were analyzed using SDS-gel electrophoresis to determine equal expression level of NTSR-1 constructs. The other portions were analyzed using fluororadiography. The blots were sprayed with ENHANCE and exposed to film at -80 οC for 4-6 weeks. Representative plots from 3 separate experiments. In this particular experiment, we used HA tagged construct to increase the efficiency of receptor immunoprecipitation relative to using anti-NTSR-1. Mean + S.E., * P < 0.05.

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3.4.5 NTS Treatment Promotes Viability of HEK293T Cells Expressing WT-NTSR-1 but not C381, 383S-NTSR-1

The physiological significance of NTSR-1 mutation at putative palmitoylation sites was evaluated using MTS cell survival assay. Serum starved HEK293T expressing WT-

NTSR-1 that were treated with 200 nM NTS exhibited improved cell viability relative to untransfected HEK293T cells and HEK293T expressing C381,383S-NTSR-1 (Fig.3 4A).

10% FBS treatment was used as a positive control in these experiments. These data suggest that these two palmitoylation sites, C381 and C383, play a critical role in receptor pro-survival signaling.

3.4.6 NTS Treatment Protects HEK293T Cells Expressing WT-NTSR-1 but not

C381, 383S-NTSR-1 from Apoptosis Induced by Serum Starvation.

To further confirm the importance of NTSR-1 palmitoylation for NTS-mediated pro-survival function, we analyzed casapse 3/7 activity after 48 hours serum starvation

(Fig.3.4B). HEK293T cells transfected with WT-NTSR-1 and treated with 200 nM NTS showed less apoptosis relative to untreated cells as well as cells transfected with

C381,383S-NTSR-1. As a negative control, 10% FBS treated cells had minimal caspase

3/7 activity. Collectively, these data suggest that palmitoylation of NTSR-1 at C381 and/or C383 is necessary to protect against stress-triggered (serum starvation) apoptosis.

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Fig.3.4. NTSR -1 palmitoylation is required for NTS-mediated pro- survival activity. A. Transfected HEK 293T cells were seeded in 96 well plates at density of 4000 cells/ well. Cells were serum-starved for 24 hours before stimulation with 5% FBS, or 200 nM NTS every 24 hours for 2 days. Cell viability was determines using MTS assay where absorbance was measured using a plate reader at 495nm. B. Transfected HEK 293T cells were seeded in 96 well plates at density of 4000 cells/ well. Cells were serum-starved for 24 hours before stimulation with 5% FBS, or 200 nM NTS. We used 200 nm in these longer term experiments to avoid rapid receptor desensitization. Caspase 3/7 activity was measured using a fluorescence plate reader (560 Ex /590 Em ). Results obtained from n=3 separate experiments. Mean + S.E., * P < 0.05. (Reproducible equal transfection efficiency was optimized before carrying out the assay).

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3.4.7 WT-NTSR-1, but not C381,383S-NTSR-1 Mutant, Efficiently Mediates NTS- induced ERK 1/2 Phosphorylation.

As NTS-induced MAP kinase activity is associated with proliferation and/or survival of breast cancer cells, we next decided to study the effect of palmitoylation deficiency on NTSR-1 stimulation of the MAP kinase pathway. After transfecting HEK293T cell with WT-NTSR-1 and C381,383S-NTSR-1, we stimulated the cells using 1µM NTS for

15 minutes. As shown in Fig.3.5A, only WT-NTSR-1 efficiently stimulated ERK phosphorylation compared to an approximate 50% reduction for mutant receptor. These results further support the biochemical importance of NTSR-1 palmitoylation for efficient pro-mitogenic/prosurvival receptor signaling.

3.4.8 2-Bromopalmitate Inhibits WT-NTSR-1-Mediated, but not PMA-Mediated,

Activation of MAP Kinase Pathway

To confirm the previous site-directed mutagenesis experimental strategies, we next utilized a pharmacological approach to target palmitoylation-dependent NTSR-1 mitogenic signaling. We utilized 2-BP, which has been shown to be an inhibitor of palmitoyl acyl transferases (22-24). Pretreatment of HEK293T cells transfected with

WT-NTSR-1 with 10 µM 2-BP for 30 minutes significantly reduced NTS-mediated ERK phosphorylation (Fig.3.5B). In order to ensure the specificity of this effect, we tested 2-

BP upon PMA-mediated ERK phosphorylation. 2-BP did not inhibit PMA-induced ERK phosphorylation, suggesting that the 2-BP effect is at the receptor level and not due to non-specific disruption of downstream signaling pathways. 107

Fig.3.5. NTSR -1 palmitoylation is required for NTS-mediated MAPK mitogenic signaling. A. HEK293T cells were seeded in 6 well plates were transfected with NTSR-1 constructs the next day. After 24 hours, the cells were then serum-starved for another 24 hours before 1 µM NTS stimulation for 15 minutes. The cell lysates were then analyzed using SDS-gel electrophoresis and blots were probed using P-ERK1/2 antibody. B. HEK293T cells were seeded in 6 well plates and were transfected with NTSR-1 constructs the next day. After 24 hours, the cells were then serum-starved for another 24 hours before 10 µM 2-BP treatment followed by 15 minute-1 µM NTS stimulation. The cell lysates were then analyzed using SDS-gel electrophoresis and blots were probed using P-ERK1/2 antibody. Representative plots from 3 separate experiments. Mean + S.E., * P < 0.05.

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3.4.9 Palmitoylation of NTSR-1 is Required for Receptor Interaction with G αq/11.

To further identify the biochemical mechanism responsible for regulated NTSR-1 palmitoylation to stimulate the MAP kinase pathway, we next investigated NTSR-1 /

Gαq/11 protein-protein interaction, which we have shown previously to be essential for efficient induction of ERK phosphorylation by NTS (22). Fig.3.6A reveals that the palmitoylation-deficient C381,383S-NTSR-1 mutant failed to interact with G αq/11 following 15 minutes stimulation with 1 µM NTS. These co-IP data suggest that palmitoylation is essential for efficient NTSR-1/ G αq/11 coupling. In the next series of experiments, we investigated the biophysical mechanism by which non-palmitoylated

NTSR-1 mutants may have impaired ability to co-localize within SMDs, where G αq/11 mainly resides (22).

3.4.10 Palmitoylation of NTSR-1 is Required for Efficient Receptor Localization to

SMDs

To test the hypothesis that the reduced NTSR-1/ G αq/11 interaction is due to the diminished ability of the receptor to reside within SMDs for extended intervals, we studied NTSR-1-membrane localization using detergent-free sucrose gradient fractionation. Data in Fig.3.6B and 3.6C demonstrate that palmitoylation-deficient

NTSR-1 localization within caveolin-enriched SMDs is diminished upon NTS stimulation, relative to WT-NTSR-1. In particular, we show reduction in mutated but not wild type

NTSR-1 localization within SMDs utilizing both HA-tagged (Fig.3.6B) and un-tagged

(Fig.3.6C) constructs. This reduction of glycosylated, but palmitoylation deficient NTSR-

1 within the caveolin enriched domains (fractions 4 and 5) quantified in n=3 separate 109 experiments and levels were reduced from 25+4 to 4 +4 %. Equal loading of the gels for both experiments was confirmed by comparable caveolin levels. Moreover, the data clearly show that only the glycosylated NTSR-1 isoforms localize within SMD fractions 4

& 5. The non-mature, non-glycosylated NSTR-1 isoforms preferentially reside within the non-SMDs fractions 8-12. Taken together, these results suggest that both palmitoylation and glycosylation of NTSR-1 could be necessary for efficient SMDs localization and subsequent interaction with G αq/11 . In the absence of NTS treatment, we observed That

NTSR-1 localizes to SMDs to lesser extent in the case of mutant receptor relative to wild type (data not shown).

110

Fig.3.6. NTSR -1 palmitoylation is required for optimal interaction with G αq/11A within SMDs. A. HEK293T cells transfected with WT-NTSR-1 and C381,383S NTSR-1 were serum- starved for 24 hours before 15 minute stimulation with 1µM NTS. The cells were then lysed using TritonX-100 buffer and immunoprecipitated using anti-Gαq/11 antibody. The immunoprecipitates immunoblotted with anti-NTSR-1. B/C (P. 111). HEK293T cells transfected with HA tagged WT-NTSR-1 and HA tagged C381,383S NTSR-1 were serum- starved for 24 hours before 15 minute stimulation with 1µM NTS. The lysates were then subjected to detergent free-sucrose gradient fractionation and the fractions were immunoblotted using with either anti-HA (B) or anti-NTSR-1 (C). Representative plots from n= 3 separate experiments .

111

3.4.11 NTSR-1 Plamitoylation may Regulate NTSR-1 Stability

We have generated 3 mutant NTSR-1. C381S, C383S and C381,383S. All constructs can be transiently expressed in HEK293T cells. No expression was observed with untransfected control lysates. Consistent with our previously published studies (22), fully mature glycosylated receptor isoforms were detected at molecular weight ~ 52 kDa, while non-mature isoforms were detected at molecular weight ~42 kDa. Interestingly, we observed that only C383S-NTSR-1 mutant exhibited significantly lower expression levels in comparison to other mutants (Fig.3.7). These results suggest that specific palmitoyalation patterns may serve as a coding signal for receptor stability, retention and/or degradation.

112

Fig. 3.7. NTSR-1 receptors can be overexpressed and glycosylated in HEK293T cells. HEK293T cells were seeded in 10 cm plates and allowed to attach before transfection with NTSR-1 constructs using Lipofectamine 2000. After 48 hours, the cells were lysed using RIPA buffer and samples containing equal total protein were analyzed using SDS gel electrophoresis. The blots were probed with anti-NTSR-1 antibody and quantified using Image-J software. Representative plots from 3 separate experiments. Mean + S.E., * P < 0.05.

3.4.12 NTSR-1 Double Palmitoylation is required for Efficient Mitogenic Signaling

We next tested if palmitoylation of both cysteine residues, 381 and 383, is required for efficient NTSR-1 ERK 1/2 phosphorylation. Interestingly, stimulation HEK293T cells expression C381S, C383S and C381,383S NTSR-1 resulted in 50% reduced ERK 1/2 phosphorylation in comparison to cells expression WT-NTSR-1 (Fig. 3.8). These data suggest that palmitoylation of each residue plays different function and possibly occur within different cellular compartments. Again, we probed for NTSR-1 expression and observed the reduced expression level of C383S-NTSR-1 (Fig.3.8). These data support the observation that palmitoylation pattern may serve as a receptor stability signal.

113

Fig.3.8. NTSR-1 double palmitoylation is required for NTS-mediated MAPK mitogenic signaling. A. HEK293T cells were seeded in 6 well plates were transfected with NTSR-1 constructs the next day. After 24 hours, the cells were then serum-starved for another 24 hours before 1 µM NTS stimulation for 15 minutes. The cell lysates were then analyzed using SDS-gel electrophoresis and blots were probed using P-ERK1/2 antibody. Representative plots from 3 separate experiments. Mean + S.E., * P < 0.05.

3.4.13 Short Chain C6 Ceramide Inhibits NTS-induced NTSR-1 Palmiotylation

Based on our previously published observation that ceramide inhibits NTSR-1- mediated mitogenesis through biophysical disruption of receptor translocation to SMDs, and our recent findings that NTSR-1 palmitoylation is required for mitogenic signaling within SMDs, we decided to test the hypothesis if ceramide effects were in part mediated through inhibition of NTSR-1 induced palmitoylation. We conducted an acyl exchange based palmitoylation assay as described previously and found that 114 pretreatment of MDA-MB-231 breast cancer cells with 10 µM liposomal C6 ceramide inhibited NTS-induced NTSR-1 (Fig. 3.9). These data suggest that ceramide could modulate NTSR-1 functions through interference with receptor palmitoylation/depalmitoylation cycle.

Fig. 3.9. Effect of C6 ceramide on NTSR-1 Palmitoylation. MDA-MB-231 cells were seeded in 20 cm plates and allowed to grow overnight. After 24 hours serum starvation, cells were stimulated with 1 µM neurotensin in the presence or absence of C6 ceramide pretreatment for 30 minutes. Cell lysates were analyzed as described previously using biotin acyl exchange assay. Representative blots from 3 separate experiments.

3.5 Discussion

The need to identify novel pharmacological approaches to inhibit NTSR-1 mitogenic signaling in cancer is justified by recent studies demonstrating NTSR-1 overexpression correlating with tumor metastatic potential and resistance to anti-neoplastic chemotherapeutic agents. Our previous findings (22) revealed a unique biophysical mechanism that has the potential to yield a more selective targeting approach for 115

NTSR-1 mediated oncogenesis. We specifically demonstrated that NTS-stimulated

NTSR-1 preferentially localizes within SMDs, to interact with G αq/11 subunits. Thus, we hypothesized that agonist-inducible receptor palmitoylation is the primary signal that targets the receptor to these domains, increasing residence time and facilitating interaction with G αq/11 . Our data suggest that palmitoyltation is required for efficient receptor-mediated mitogenic-signaling within SMDs. Thus, our studies potentially argue for the use of PAT inhibitors as a new therapeutic modality for NTSR-1 expressing cancers. It is possible that NTS/NTSR-1 binding binding induces a conformational change in the receptor 3-D structure, exposing a putative interaction site with a PAT enzyme within the cell membrane. Receptor palmitoylation increases protein lipophilicity, resulting in enhanced localization to SMDs, which allows for optimal interaction with G αq/11. Alternatively, GPCR-palmitoylation results in the formation of an additional fourth intra-cellular loop that results in a conformational change, which could serve as a docking site for critical downstream protein/protein interaction (8).

We have combined both site-directed mutagenesis and pharmacological approaches to study NTSR-1 palmitoylation. Using a pharmacological approach (2-BP) and a molecular approach (C381,383S mutations), we demonstrated similar effects on

ERK phosphorylation in two different cell lines. We chose 2-BP, a lipid based inhibitor, due to its ability to irreversibly inhibit palmitoylacytransferase activity of all PATs (25). In order to exclude the possibility of off-target effects that would disrupt signal transduction at downstream levels, we showed that PMA-mediated MAP kinase signaling remains intact, suggesting a direct effect of PAT on NTSR-1 All of our mutants were equally expressed and glycosylated in HEK293T model system. Documentation of 116 functional, physiological dysfunction by both molecular and pharmacological strategies strongly suggests that NTSR-1 palmitoylation is a critical event for efficient NTS- mediated mitogenesis and survival.

Our data suggest that there is potential cross talk between palmitoylation and glycosylation post-translational modifications of NTSR-1. Specifically, we demonstrate that only glycosylated isoforms of NTSR-1 localize within SMDs by palmitoylation. Even though these data suggest that cysteine sites, 381 and 383 are critical for sub-cellular localization and interaction with G αq, it is of interest that loss of either one of these cysteines results in equal reduction of ERK 1/2 phosphorylation. Our studies further suggest that palmitoylation of glycosylated NTSR-1 can affect downstream G protein signaling via stability and/or membrane micro-localization of the receptor. Although our data suggest that NTSR-1 localization within SMDs is reduced when receptor palmitoylation is impaired, other mechanisms cannot be excluded. For example, palmitoylation-deficient A1 adenosine and CCR5 receptors as well as the palmitoylation-deficient Rous sarcoma virus glycoprotein are more susceptible for proteolysis (26-28). Thus, the diminished NTSR-1 protein level within caveolin-enriched

SMD fractions 4 and 5 could also be attributed to enhanced protein degradation.

Possible NTSR-1 ubiquitination could be detected using anti-ubiquitin antibody in order to assess NTSR-1 degradation mechanism. Manipulation of the ubiquitin pathway pharmacologically can be also utilized to study the potential crosstalk between palmitoylation and ubiquitination on NTSR-1 half life.

In addition, our observation that palmitoylation pattern could play a role in receptor stability provide potentially novel function for GPCRs-palmitoylation. Previous reports by 117 other groups implicated palmitoylation in protein expression, ER retention and degradation by ubiquitin pathway (29, 30). Regulation of NTSR-1 stability by dynamic palmitoylation could be a possible mechanism for receptor desensitization. More studies are required to further understand the relationship between NTSR-1palmitoylation pattern and receptor stability.

Palmitoylation/depalmitoylation cycle for many proteins involved in cell proliferation and survival is gaining increasing attention. In analogy to phosphorylation/dephosphorylation modifications, palmitoylation is tightly regulated and sphingolipids might play a role e in such regulation. Our data suggest that ceramide could inhibit NTSR-1 palmitoylation. However, the exact mechanism is not clear. It is possible that ceramide directly interfere with PAT or biophysically inhibitors NTSR-

1/PAT co-localization. Additionally, ceramide might exert its effect through enhancing

NTSR-1 depalmitoylation. Future studies are required to address such possibilities.

Pulse-chase experiments could be useful to examine NTSR-1 palmitoylation/deplamitoylaton over time. The effect of C6 ceramide on this cycle through either enhancement or retardation would generate a clue regarding the exact mechanism of action of C6 ceramide.

Enhanced protein palmitoylation is involved in the pathogenesis of many disease conditions such as cancer, Huntigton’s disease, immune diseases and various cardiovascular diseases (31). Therefore, identification of functional roles of protein palmitoylation at the molecular level can further validate palmitoyl acyltransferases as strategic targets for the development of novel pharmacological approaches for disease treatment. Accumulating evidence in the literature suggest that PATs may have 118 substrate specificity (32). For example, Singaraja et al. recently identified DHHC8 as the

PAT which regulates ATP-binding cassette transporter (ABC-A1) through palmitoylation

(33). They proposed that an increased DHHC8 protein levels could lead to an increase in ABCA-1 mediated lipid efflux. Similarly, our findings could enable selective targeting of the specific PAT(s) which regulate NTSR-1 functions using siRNA approach resulting in the desired selective pharmacological effects. In conclusion, we suggest that NTSR-1 palmitoylation is required for receptor localization and signaling within SMDs. We also provide for the first time a novel pharmacological approach to inhibit NTSR-1 mitogenic signaling in cancer through inhibition of receptor palmitoylation.

119

3.6 References

1 Dupouy,S., Viardot-Foucault,V., Alifano,M., Souaze,F., Plu-Bureau, Chaouat,M., Lavaur,A., Hugol,D., Gespach,C., Gompel,A. and Forgez,P. The neurotensin receptor-1 pathway contributes to human ductal breast cancer progression, PLoS.ONE., 4: e4223, 2009.

2 Wang,L., Friess,H., Zhu,Z., Graber,H., Zimmermann,A., Korc,M., Reubi,J.C. and Buchler,M.W. Neurotensin receptor-1 mRNA analysis in normal pancreas and pancreatic disease, Clin.Cancer Res., 6: 566-571, 2000.

3 Seethalakshmi,L., Mitra,S.P., Dobner,P.R., Menon,M. and Carraway,R.E. Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations, Prostate, 31: 183-192, 1997.

4 Gui,X., Guzman,G., Dobner,P.R. and Kadkol,S.S. Increased neurotensin receptor- 1 expression during progression of colonic adenocarcinoma, Peptides, 29: 1609- 1615, 2008.

5 Goedert,M., Reeve,J.G., Emson,P.C. and Bleehen,N.M. Neurotensin in human small cell lung carcinoma, Br.J.Cancer, 50: 179-183, 1984.

6 Resh,M.D. Palmitoylation of ligands, receptors, and intracellular signaling molecules, Sci.STKE., 2006: re14, 2006.

7 Karnik,S.S., Ridge,K.D., Bhattacharya,S. and Khorana,H.G. Palmitoylation of bovine opsin and its cysteine mutants in COS cells, Proc.Natl.Acad.Sci.U.S.A, 90: 40-44, 1993.

8 Chini,B. and Parenti,M. G-protein coupled receptors, cholesterol and palmitoylation: facts about fats, J.Mol.Endocrinol., 2009.

9 Jin,H., Zastawny,R., George,S.R. and O'Dowd,B.F. Elimination of palmitoylation sites in the human dopamine D1 receptor does not affect receptor-G protein interaction, Eur.J.Pharmacol., 324: 109-116, 1997.

10 Jin,H., Xie,Z., George,S.R. and O'Dowd,B.F. Palmitoylation occurs at cysteine 347 and cysteine 351 of the dopamine D(1) receptor, Eur.J.Pharmacol., 386: 305-312, 1999.

11 Ng,G.Y., Mouillac,B., George,S.R., Caron,M., Dennis,M., Bouvier,M. and O'Dowd,B.F. Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor, Eur.J.Pharmacol., 267: 7-19, 1994. 120

12 Loisel,T.P., Adam,L., Hebert,T.E. and Bouvier,M. Agonist stimulation increases the turnover rate of beta 2AR-bound palmitate and promotes receptor depalmitoylation, Biochemistry, 35: 15923-15932, 1996.

13 Kennedy,M.E. and Limbird,L.E. Palmitoylation of the alpha 2A-adrenergic receptor. Analysis of the sequence requirements for and the dynamic properties of alpha 2A- adrenergic receptor palmitoylation, J.Biol.Chem., 269: 31915-31922, 1994.

14 Blanpain,C., Wittamer,V., Vanderwinden,J.M., Boom,A., Renneboog,B., Lee,B., Le,P.E., El,A.L., Govaerts,C., Vassart,G., Doms,R.W. and Parmentier,M. Palmitoylation of CCR5 is critical for receptor trafficking and efficient activation of intracellular signaling pathways, J.Biol.Chem., 276: 23795-23804, 2001.

15 Gao,Z., Ni,Y., Szabo,G. and Linden,J. Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors, Biochem.J., 342 ( Pt 2): 387-395, 1999.

16 Wedegaertner,P.B., Chu,D.H., Wilson,P.T., Levis,M.J. and Bourne,H.R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha, J.Biol.Chem., 268: 25001-25008, 1993.

17 Ducker,C.E., Griffel,L.K., Smith,R.A., Keller,S.N., Zhuang,Y., Xia,Z., Diller,J.D. and Smith,C.D. Discovery and characterization of inhibitors of human palmitoyl acyltransferases, Mol.Cancer Ther., 5: 1647-1659, 2006.

18 Draper,J.M. and Smith,C.D. Palmitoyl acyltransferase assays and inhibitors (Review), Mol.Membr.Biol., 26: 5-13, 2009.

19 Heakal,Y. and Kester,M. Nanoliposomal short-chain ceramide inhibits agonist- dependent translocation of neurotensin receptor 1 to structured membrane microdomains in breast cancer cells, Mol.Cancer Res., 7: 724-734, 2009.

20 Drisdel,R.C., Alexander,J.K., Sayeed,A. and Green,W.N. Assays of protein palmitoylation, Methods, 40: 127-134, 2006.

21 Heakal,Y. and Kester,M. Nanoliposomal short-chain ceramide inhibits agonist- dependent translocation of neurotensin receptor 1 to structured membrane microdomains in breast cancer cells, Mol.Cancer Res., 7: 724-734, 2009.

22 Jennings,B.C., Nadolski,M.J., Ling,Y., Baker,M.B., Harrison,M.L., Deschenes,R.J. and Linder,M.E. 2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)- benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro, J.Lipid Res., 50: 233-242, 2009.

23 Mikic,I., Planey,S., Zhang,J., Ceballos,C., Seron,T., von,M.B., Watson,R., Callaway,S., McDonough,P.M., Price,J.H., Hunter,E. and Zacharias,D. A live cell, image-based approach to understanding the enzymology and pharmacology of 2- bromopalmitate and palmitoylation, Methods Enzymol., 414: 150-187, 2006. 121

24 Webb,Y., Hermida-Matsumoto,L. and Resh,M.D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids, J.Biol.Chem., 275: 261-270, 2000.

25 Jennings,B.C., Nadolski,M.J., Ling,Y., Baker,M.B., Harrison,M.L., Deschenes,R.J. and Linder,M.E. 2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)- benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro, J.Lipid Res., 50: 233-242, 2009.

26 Gao,Z., Ni,Y., Szabo,G. and Linden,J. Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors, Biochem.J., 342 ( Pt 2): 387-395, 1999.

27 Ochsenbauer-Jambor,C., Miller,D.C., Roberts,C.R., Rhee,S.S. and Hunter,E. Palmitoylation of the Rous sarcoma virus transmembrane glycoprotein is required for protein stability and virus infectivity, J.Virol., 75: 11544-11554, 2001.

28 Percherancier,Y., Planchenault,T., Valenzuela-Fernandez,A., Virelizier,J.L., renzana-Seisdedos,F. and Bachelerie,F. Palmitoylation-dependent control of degradation, life span, and membrane expression of the CCR5 receptor, J.Biol.Chem., 276: 31936-31944, 2001.

29 Abrami,L., Kunz,B., Iacovache,I. and Van Der Goot,F.G. Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum, Proc.Natl.Acad.Sci.U.S.A, 105: 5384-5389, 2008.

30 Abrami,L., Leppla,S.H. and Van Der Goot,F.G. Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis, J.Cell Biol., 172: 309-320, 2006.

31 Planey,S.L. and Zacharias,D.A. Palmitoyl acyltransferases, their substrates, and novel assays to connect them (Review), Mol.Membr.Biol., 26: 14-31, 2009.

32 Roth,A.F., Wan,J., Bailey,A.O., Sun,B., Kuchar,J.A., Green,W.N., Phinney,B.S., Yates,J.R., III and Davis,N.G. Global analysis of protein palmitoylation in yeast, Cell, 125: 1003-1013, 2006.

33 Singaraja,R.R., Kang,M.H., Vaid,K., Sanders,S., Vilas,G., Arstikaitis,P., Coutinho,J., Drisdel,R.C., El-Husseini,A.E., Green,W.N., Berthiaume,L. and Hayden,M.R. Palmitoylation of ATP-Binding Cassette Transporter A1 Is Essential for Its Trafficking and Function, Circ.Res., 2009.

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Chapter 4

Conclusions, Therapeutic Potential and Future Directions

123

The emerging role of cell membrane as a regulator of signal transduction is exciting, but equally challenging. Despite the advances made in our understanding of biological membranes, the mechanisms of domains formation, dissolution as well as protein microlocalization remain beyond our full comprehension. Reliable methodologies are still needed to distinguish, characterize and study various types of membrane domains in living systems. In addition to biochemical techniques, several microscopy techniques have been developed including electron microscopy, atomic force microscopy, near- field optical microscopy and Forster resonance energy transfer. Despite their high resolution, they are limited by inability the track fast lipid diffusion in live systems (1).

The controversy regarding raft hypotheses is mainly due to lack of standardized methodologies used in raft studies. It is very common in the raft literature to come across contradicting results, controversies and disagreements regarding membrane localization of specific proteins. Despite this, there is sufficient evidence supports the existence and function of plasma membrane domains as regulators of cellular signaling.

4.1 Therapeutic Potential of Short Chain Ceramides as SMDs modulators

Our work demonstrates that short chain ceramide disrupts NTSR-1 mitogenic signaling in breast cancer cells, in part, via selective exclusion from SMDs where

NTSR-1 interacts with G αq (Fig.4.1). As discussed previously, unlike endogenous ceramide species, short chain ceramides have distinct physical properties and tend to destabilize cholesterol-enriched domains and increase membrane fluidity. The novel approaches developed by Kester and co-workers to systemically deliver ceramide establish the clinical potential of this therapeutic approach in the treatment of cancer 124 and other diseases (2-5) where undesired NTS-mediated signaling contributes to disease pathogenesis . The use of liposomal ceramide proved a useful approach to inhibit cancer growth in several in vivo cancer animal models (6). Ceramide selectively induces apoptosis in malignant transformed cells while normal cells are spared (7, 8).

Short chain ceramide delivery using other nano-delivery vehicles including calcium phosphate nanoparticles (9) and thermoresponsive dendrimers provides alternative desirable characteristics for specific disease conditions (10). Targeted forms of nano delivery systems would allow selective manipulation of SMDs with minimal if any side effects. However, it is important to keep in mind that short chain ceramides have other biochemical effects in addition to biophysical SMDs modulation such as regulation of

PKC-zeta activity and localization which affects cell viability.

Fig.4.1. C6 ceramide disrupts NTSR-1 mitogenic signaling. 125

SMDs modulation through manipulation of sphingolipid metabolic pathways represents another clinically applicable approach. As discussed previously, several enzymes can serve as key pharmacological targets including acid-sphingomyelinase

(11), glucosyl ceramide synthase (12), sphingosine kinase (13-15), acid ceramidase

(16-18) and others. Inhibition of these enzymes proved valuable approach to sensitize cancer cells to different therapeutic approaches including chemotherapy and ionizing radiation. Cabot and co-workers also demonstrated the possibility to reverse multidrug resistance in cancer by targeting glucosyl ceramide synthase (19, 20). In addition, HIV-1 infectivity could be suppressed by targeting sphingomyelinase (21) and glucosylceramide synthase (22) (discussed in details in section 4.1.1). Additionally, cholesterol lowering drugs, statins, disrupt SMDs integrity via inhibition of HMG-CoA reductase, the first rate limiting step in cholesterol biosynthesis. Statins are widely used drugs and relatively safe as they proved efficacious in preventing cardiovascular diseases. Accumulating body of literature supports their potential benefit in preventing several types of malignancies including breast, prostate and colon cancers (23-28). In addition, preclinical studies suggest that statins could be also useful to suppress HIV-1 infectivity (29). However, it is important to appreciate that raft disruption might not be the main mechanism of action of statins as other alternative mechanisms could be more fundamental to their chemopreventive, anti-neoplastic or anti-HIV effects. For instance, inhibition of HMG-CoA reductase prevents the formation of downstream isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate which are required for isoprenylation of wide variety of proteins involved in cell survival such as Rho proteins (30-32). The inability of these proteins to undergo post-translational 126 modifications disrupts their normal trafficking and membrane anchoring mechanisms leading to cell death. Clinical use of statins possibly in combination with other drugs such as COX-2 inhibitors could be a promising tolerable pharmacological approach to prevent cancer (33-35).

Alternatively, squalene synthase can be targeted to selectively inhibit raft cholesterol biosynthesis (36). It is not obvious why squalene synthase inhibition selectively reduces raft cholesterol. The data reported by Brussleman’s et al. (32) imply the possibility that cholesterol biosynthesis is a localized process at the sub cellular level. Squalene synthase as a key pharmacological target for cancer treatment remains to be further validated in different cancer models as well in HIV-1 infection. Tissue targeting of squalene synthase could be another hurdle that could be solved using targeted drug delivery approaches.

Our work also demonstrates a direct effect of short chain ceramide on the G protein coupled receptor, NTSR-1, in breast cancer cells. It would be of interest to further test if similar ceramide effects can be observed in other GPCR signaling systems that require intact SMDs to activate certain signaling pathways. Utilizing short chain ceramide analogs may yield desired clinical effects relative to classical direct receptor antagonism causing possibly less side effects via selective inhibition of specific signaling routs triggered only within SMDs. Additionally It is of interest to determine the effect of endogenously generated ceramide species on GPCRs signaling and more specifically

NTSR-1. Such studies may help us better understand the pathogenesis of diseases associated with deregulation of sphingolipid metabolism and/or GPCR function. 127

In conclusion, several pharmacological therapeutic approaches are emerging as modulators of SMDs mediated signaling. Taking advantage of these modalities to alter

GPCRs signaling could lead to valuable clinical effects.

4.1.1 Modulation of SMDs as a Therapeutic Approach for AIDS Treatment

A growing body of literature implicates membrane rafts of mammalian cells as a key player in different stages of infections by a variety of pathogens including bacteria, viruses, prions and parasites (37, 38). Among these viruses are HIV, influenza virus,

Ebstein-Barr virus, Echovirus and Rhinovirus (39, 40). Viruses utilize host cell membranes for entry, assembly and budding. HIV-1 entry into susceptible mammalian cells is a complex multistep process that begins by binding of viral glycoprotein gp120 to

CD4 on T Cells (207). Other accessory proteins are also required for this step including

ICAM-3, macrophage mannose receptor and lymphocyte function antigen-1 (41). A subsequent conformational change in gp120 enables binding to accessory chemokine receptors, CCR5 and CXCR4 followed by second conformational change exposing the transmembrane glycoprotein gp42 which is inserted into the cell membrane leading to a complete fusion between the host and viral membranes (209). Despite there is strong evidence that HIV entry is dependent on membrane raft integrity, there is significant controversy in the literature. Both Kozak et al. (42) and Popik et al. (43, 44) reported that CD4 and CCR5 but not CXCR4 are associated with SMDs. In support of these findings, Del real et al. (45) demonstrated that a non raft associated CD4 mutant inhibited HIV infection. Conversely, Bachelerie and co-workers reported that CCR5 does not localize to raft domains and HIV viral entry takes place outside rafts (46). It was later proposed that CD4 localization to rafts could be required for virus post-binding 128 fusion steps but not initial entry process (47). Despite this literature inconsistency, disruption of rafts integrity in host cells by cholesterol depletion led to significant inhibition of HIV infection. In addition, Blumenthal and co-workers reported that raft modulation by increasing intracellular ceramide using fenretinide also inhibited viral infectivity (48). Sphingomyelinase was also found to inhibit viral entry through clustering of CD4 which prevents co-receptor engagement (49). Additionally, inhibition of glucosyl ceramide formation using 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) also suppressed HIV-1 entry and Env mediated fusion in several cell lines (50). HIV entry into macrophages was also found to be dependent on intact SMDs. (51). Carter et al. used four pharmacological agents, MCD, nystatin, flipin and lovastatin that disrupt rafts integrity through different mechanism and found that viral release from macrophages significantly decreased. The effects were reversed by the addition of water soluble cholesterol (218).

In addition to viral entry, SMDs have been also linked to viral replication, assembly and budding (52-55). Treatment of infected cells with cyclo dextrins or statins inhibits viral production and reduces the infectivity of the produced viral particles (56). Khanna et al. tested the feasibility of utilizing cholesterol depletion as a chemo-preventive strategy in humanized SCID animal model. Pre-treatment of the mouse vaginal mucosa with 2-hydroxypropyl cyclodextrin inhibited viral transmission from HIV-1 infected cells

(223). Similar effects were observed using statins, however, these results remain to be confirmed and reproduced in patients. A novel approach to inhibit viral production through targeting SMDs was recently described by Balogh et al. where they used a new cholesterol-specific monoclonal antibody which recognizes clustered membrane 129 cholesterol. Treatment of human T-cells and macrophages with this antibody resulted in remodeling and rearrangement of HIV-1 receptors and co-receptors leading to an inhibition of HIV-1 infection and production (57).

Taken together, the role of host SMDs in HIV-1 infection and progression is well established. Despite successful preclinical studies, confirming these finding based on patient data is still needed. One major future hurdle for this strategy is to selectively target SMDs domains in susceptible and infected cells selectively.

4.2 Therapeutic Potential of GPCRs Palmitoylation Inhibitors.

Over the years, the progress in studying protein palmitoylation has been relatively slow. The main impediment was the lack of reliable robust assays to determine palmitoylation status of proteins as well as due to the dynamic nature of this post- translational modification (58). Despite the accumulating body of literature, functional consequences of protein palmitoyaltion cannot be predicted with absolute certainty.

For many years, metabolic radiolableing, a costly, time consuming technique with limited sensitivity, has been extensively utilized to identify palmitoylated proteins (59).

Recently, Green and co-workers reported the development and use of fatty acyl exchange assay as a more robust sensitive method to detect palmitoylation of low abundant endogenously expressed proteins such as GPCRs and ion channels (60).

Simply, the palmitoylated protein of interest can be manipulated chemically by selective cleavage and labeling of palmitoylated cysteine residues using labeling tags such as biotin. Different assay variations can be employed for final detection and quantification such as the use of SDS gel electrophoresis where biotin can be detected using HRP- 130 avidin or even a more sensitive approach can be employed such as mass spectrometry or radiofluorography in case a robust radiolabeled tag is used. As we have shown earlier, despite the low abundance of endogenously expressed NTSR-1, we have successfully utilized this assay to detect the palmitoylation of endogenously expressed

NTSR-1 in human breast cancer cells.

It is well established that a family of enzymes known as palmitoyl acyl trasnferases

(PATs) is responsible for protein palmitoylation (61-63). Smith and co-workers reported the development of a robust high throughput assays that would enable identification of

PATs as well as small organic molecule PAT-inhibitors (64-67). In brief, fluorescent lipidated peptides can be used to characterize PAT activities in vitro and in intact cells where they can be separated and fluorescence is quantified. The fluorescent peptides are useful to identify subcellular localization of palmitoylated products.

The advances in the tools and assays currently available to study protein palmitoylation will have a tremendous impact on the development and progress of this field. Pharmacologic manipulation of palmitoylation cycle will lead to the discovery of new drugs and treatments for diseases such as cancer.

4.2.1 Identification of NTSR-1 Palmitoylating Enzymes.

In the past, it has been proposed that protein palmitoylaition is a non-enzymatic process mainly due to lack of powerful methodology. Until quite recently, studies on yeast led to the identification of several acyl transferase enzymes responsible for palmitoylation activities. Currently, there are more than 20 putative PATs that are 131 ubiquitously expressed and share a conserved Asp-His-His-Cys (DHHC) cytoplasmic motif (68). At the subcellular level, PATs localize to ER, Golgi and plasma membrane

(69). In addition, a recent interesting study by Noritake et al. demonstrated that PATs could be mobile and differentially regulated (70). They showed that DHHC2 undergo dynamic translocations based on substrate protein activity. These results add more complexity to understanding compartmentalized palmitoylation processes and its regulation mainly because of the possibility that a single PAT can be deployed to different cellular compartments based on certain signals. Targeting specific PATs could be an interesting pharmacological approach as it has been shown that individual PATs exhibit substrate specificity (71).

PATs localize mainly to plasma membranes, golgi, ER however their membrane microlocalization have not been determined (72). It would be of interest to study the effect of SMD disruption on protein palmitoylation and more specifically NTSR-1.

Despite recent studies which suggest that certain PATs are mobile (70), it is possible that SMDs serve as platforms for protein PAT co-localization and palmitoylation reactions. Such studies would provide additional approaches to modulate PATs activities.

In order to pursue our interesting findings further, it is necessary to identify the individual PAT(s) responsible for NTSR-1 palmitoylation. Our data suggest that the two cysteine residues can be palmitoylated separately and they may serve distinct functions. This suggests that the two palmitoylation events could occur within separate subcellular compartments by distinct PATs. Despite the complexity of our analysis, our 132 studies suggest the possibility of targeting NTSR-1 palmitoylation at two different levels which could be more useful to minimize potential side effects.

A possible experimental approach to identify NTSR-1-PATs is to conduct a yeast two hybrid (YTH) screen. There are several new technical modifications of YTH that can be adapted in order to identify possible protein-protein interactions which may take place within plasma membrane. Mammalian yeast two hybrid systems could be the method of choice to identify PATs that specifically process NTSR-1 (73). Another experimental approach is to utilize siRNA designed against known putative PATs and screen for the

NTSR-1 processing enzyme(s). The palmitoylation assay methods developed by Green and co-workers would make this approach feasible experimentally assuming that

NTSR-1-PAT enzyme(s) are members of currently known putative PATs. However, it is important to keep in mind that palmitoylation could be a redundant process and attempting to target specific PAT may not be sufficient therapeutically.

Following the identification of NTSR-1-PATs, target validation would be necessary.

In vitro and in vivo cancer models can be utilized to test if NTSR-1-PATs are valuable targets to inhibit NTS- mediated mitogenic signaling. One might argue that using a direct receptor antagonist which currently available could be sufficient. However, NTSR-

1-PATs could be preferentially expressed in cancer tissues rendering this pathway mitogenic only in malignant transformed cells. Thus, targeting these enzymes may lead to fewer side effects relative to classical direct antagonism.

133

4.2.2 Potential Role of NTSR-1 Palmitoylation in Receptor Stability.

GPCRs undergo several types of post-translational modifications including glycosylation, phosphorylation, isoprenylation and palmitoylation. The exact function of

GPCRS palmitoylation is not fully understood. Several GPCRs require palmitoylation in order to localize to SMDs. In addition, plamitoylation was found to be necessary for optimal coupling to heterotrimeric G proteins and signal initiation (74, 75). Recent data from two independent groups implicated palmitoylation in protein expression, ER retention and degradation by ubiquitin pathway. A pioneering study by Van der Goot and co-workers (76, 77) first identified that the Wnt signaling protein LRP6 protein is palmitoylated. Palmitoylation deficient LRP6 mutants were retained in the ER and found to be susceptible to mono- ubiquitination. A more recent study by Kanellopoulos and co- workers reported that palmitoylation deficient P2X7 receptors are not expressed on the cell surface and retained in the ER (78). The half life of the protein was significantly reduced due to enhance degradation through proteosomes relative to wild type P2X7 receptors.

Based on these findings, it is possible that the observed low expression level of

C383S NTSR-1 construct is attributed to lower protein stability and degradation which strongly suggests that both residues are required to serve dissimilar functions. The palmitoylation of specific residue but not the other could serve as a degradation code in analogy to the phosphorylation codes hypothesis proposed by Tobin et al. (79) where they proposed that different combination of phosphorylated sites at the C-terminal of a

GPCR leads to different signaling outcomes (79). Similarly, different palmitoylation patterns could also serve distinct functions (Fig.4.2). Simple experimental approaches 134 such as detection of palmitoylation using ubiquitin specific antibodies could be employed. Pharmacological inhibition of ubiquitination pathway could also provide further evidence for dependence of receptor degradation on functional ubiquitin pathway.

In addition, we have observed that deletion of only one palmitoylation site is sufficient to reduce the NTSR-1-mediated ERK 1/2 phosphorylation by about 50% which imply that double palmitoylation serve a very unique function (Fig 3.8).

Additional studies are required to establish these possible novel functional consequences of NTSR-1 palmitoylation. The half life of mutant NTSR-1 should be determined in order to fully assess the importance of palmitoylation for receptor stability.

The results will shed more light on the unique features of palmitoylation among other lipidation events.

Fig.4.2. NTSR-1 palmitoylation serves as coding signal. 135

4.2.3 Possible Cross Talk between NTSR-1 Palmitoylation and Glycosylation

Most membrane proteins including GPCRs are glycosylated at least one site near the N-terminus (80, 81). Glycosylation is a complex process in which oligosaccharides are linked to the amide nitrogen of asparagines (N-glycosylation) or occasionally through the hydroxyl of serine or threonine (O-glycosylation). GPCRs glycosylation is required for optimal receptor expression, folding, stability and normal function. In a recent report by Viana and co-workers (82, 83), glycosylation of TRPM8 channel was found to serve as a targeting signal for SMDs localization where the channel can function optimally. Glycosylation deficient TRPM8 exhibited reduced ability to localize to

SMDs and the channel function was significantly compromised.

NTSR-1 has at least three possible glycosylation sites near the N-terminus. Our sucrose fractionation data suggest that there is a possible cross talk between receptor palmitoylation and glycosylation. Palmitoylation-deficient mutants showed reduced ability to undergo glycosylation and to localize to SMDs (Fig. 3.6). In addition, sucrose fractionation data from MDA-MB-231 breast cancer cells also suggest that only the fully glycosylated isoform is capable to localize to SMDs (Fig.2.5). It is not clear if palmitoylation or glycosylation are pre-requisite post-translation modification for one another. Further experimentation is needed to establish this potentially novel relationship. Multiple experimental approaches can be pursued including pharmacological as well as mutagenesis. Reliance on pharmacological approach utilizing tunicamycin, a glycosylation inhibitor, for example would not be sufficient as these agents interfere with other cellular processes including palmitoylation. Therefore site directed mutagenesis of glycosylation sites would yield more informative results. 136

Utilizing siRNA designed against specific PATs can be also utilized to study the effect of palmitoylation inhibition on glycosylation in native systems. Such studies would lead to identification of novel pharmacological targets to control disease conditions.

4.2.4 Regulation of NTSR-1 Palmitoylation by Sphingolipids.

It is well established that palmitoylation/depalmitoylation cycle is a fundamental regulatory mechanism that control function and signaling properties of many proteins such as Ras, GPCRs, hetertrimeric G proteins, ion channels and many others (33, 37).

In analogy for protein phosphorylation/dephosphorylation, palmitoylation/depalmitoylation cycle is tightly regulated process. Sphingolipid functions as regulators of many key proteins such protein kinases could serve as good candidates as regulators of palmitoylation cycles either biochemically, biophysically or both. Interaction of sphingolipids with PATs could directly or indirectly alter palmitoylation via effects on compartmentalization and subcellular localization. These possible mechanisms along with our data prompted us to examine the direct effect of short chain ceramide on NTSR-1 palmitoylation. Our preliminary data suggest that ceramide could inhibit NTSR-1 palmitoylation, however, it is not clear if this is a direct biochemical inhibition via ceramide interaction with a PAT active binding site (Fig4.3A) or due to inhibition of NTSR-1 ability to co-localize with the processing PAT (Fig4.3B). In addition, we did not determine if this is indeed inhibition of palmitoylation or acceleration of depalmitoylation which is also an enzymatic but ill-characterized process (Fig.4.4). 137

Thus, more experiments are needed to address these critical questions as the possible clinical therapeutic implications could be invaluable.

A

B

Fig.4.3. Possible mechanisms for ceramide mediated inhibition of NTSR-1 palmitoylation.

138

Fig.4.4. Possible effect of ceramide on NTSR-1 palmitoylation/depalmitoylation cycle.

4.3 Summary

In conclusion, our findings establish a novel role for sphingolipids in regulating

GPCRs membrane microlocalization biophysically as well as biochemically. In addition we implicate sphingolipid homeostasis as putative regulators of palmitoylation/depalmitoylation cycle. Further studies are required to test if endogenously generated ceramide species causes similar effects on GPCRs signaling and protein palmitoylation. In addition, studies on other GPCRs are also needed to test 139 the possibility to generalize our findings beyond NTSR-1. Such studies would reveal novel pharmacological targets to managing many disease conditions including cancer.

140

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78 Gonnord,P., Delarasse,C., Auger,R., Benihoud,K., Prigent,M., Cuif,M.H., Lamaze,C. and Kanellopoulos,J.M. Palmitoylation of the P2X7 receptor, an ATP- gated channel, controls its expression and association with lipid rafts, FASEB J., 23: 795-805, 2009.

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80 Davis,D.P. and Segaloff,D.L. N-linked carbohydrates on G protein-coupled receptors: mapping sites of attachment and determining functional roles, Methods Enzymol., 343: 200-212, 2002.

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Vitae

Yasser Heakal

Education

1996-2001 B.Sc. in Pharmaceutical Sciences, Cairo University College of Pharmacy, Cairo, Egypt. 2002-2005 M.S. in Medicinal and Biological Chemistry, University of Toledo College of Pharmacy, Toledo, OH, USA. 2005-2009 Ph.D. in Pharmacology, The Pennsylvania State University College of Medicine, Hershey, PA, USA. 2007-2009 M.B.A. The Pennsylvania State University, Harrisburg, PA, USA.

Publications

Yasser Heakal . Homospermidine as a selective targeting vector for cancer diagnostic and therapeutic agents. M.S. Thesis in Medicinal Chemistry, 2005.

Banu S. Zolnik, Stephan T. Stern, James M. Kaiser, , Yasser Heakal, Jeffrey D. Clogston, Mark Kester, and Scott E. McNeil. Rapid Distribution of Liposomal Short-Chain Ceramide In Vitro and In Vivo. Drug Metabolism and Disposition . 2008; 36(8): pp1709-15.

Mark Kester, Yasser Heakal , A. Sharma, Gavin P. Robertson, Thomas T. Morgan, Erhan Altinoulu, Amra Tabokov, Mylisa R. Parette, Sarah Rouse, Victor Ruiz-Velasco, James Adair. The Use of Calcium Phosphate Nanoparticles (CPNP) for In Vitro Imaging and Drug Delivery, Nano Lett. , 2008, 8 (12), pp 4116–4121.

Yasser Heakal & Mark Kester. Nano-Liposomal Short-Chain Ceramide Inhibits Agonist- Dependent Translocation of Neurotensin Receptor-1 to Structured Membrane Microdomains in Breast Cancer Cells. Mol. Cancer Res , 7: 724-734, 2009.

Yasser Heakal , Matt Woll, Robert Levenson, Mark Kester. Neurotensin Receptor-1 Inducible Palmitoylation is Required for Receptor Efficient Mitogenic Signaling within Structured Membrane Microdomains. ( Submitted ).

Yasser Heakal, James Bortner, Karam ELbayoumy & Mark Kester. Intranasal Delivery of Nano- Liposomal Resveratrol for Lung Cancer Chemoprevention. (work in progress).

Meeting Presentations Yasser Heakal , Mark Kester. Exogenous Short Chain Ceramide Abrogates Neurotensin Mediated Breast Cancer Progression. Advances in Breast Cancer Research, AACR Meeting, 2007, San Diego, CA, USA. Yasser Heakal , Mark Kester. Exogenous Short-Chain Ceramide Inhibits Agonist-Dependent Translocation of Neurotensin Receptor-1 to Structured Membrane Microdomains in Metastatic Breast Cancer Cells. ACS National Meeting 2008, Philadelphia, PA, USA. Yasser Heakal , Mark Kester. Nano-Liposomal-Short Chain Ceramide Inhibits Neurotensin Receptro-1-Dependent Breast Cancer Progression. 5th International Charleston Ceramide Meeting 2009, Charleston, SC, USA (Invited Oral Presentation).