Effect of Akt1 Phosphorylation on Rhoc Gtpase Activity

EFFECT OF AKT1 PHOSPHORYLATION ON RHOC GTPASE ACTIVITY

IN INFLAMMATORY BREAST CANCER

Weam Othman Elbezanti

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences

Spring 2012

EFFECT OF AKT1 PHOSPHORYLATION ON RHOC GTPASE ACTIVITY

IN INFLAMMATORY BREAST CANCER

Weam Othman Elbezanti

Approved: ______Kenneth L. van Golen, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee

Approved: ______Randall L. Duncan, Ph.D. Chair of the Department of Biological Sciences

Approved: ______George Watson, Ph.D. Dean of the College of Arts and Sciences

Approved: ______Charles G. Riordan, Ph.D. Vice Provost for Graduate and Professional Education

ACKNOWLEDGMENTS

In the name of God the Beneficent, the Merciful. “…and say: My Lord! Increase me in knowledge” All the praises and thanks be to Allah, the Lord, whom gave me the faith and strength to do this research. Nothing from me except with the help of Allah. I would like to thank deeply professor Dr. Kenneth van Golen for taking me as a graduate student at the University of Delaware and giving me the opportunity to work in his laboratory under his mentorship; appreciating his enlightening guidance, extraordinary encouragement, and advice in helping me throughout the research, and whom gave me a wonderful research project that not only sparked in me a love for working on inflammatory breast cancer but also based on his humanitarian deep merciful feeling and worry for women infected with this horrible disease in different ages in the world particularly in North Africa of the highest infection in respect. My gratitude is also extended to my committee members whom did their outmost to support me. I sincerely thank Dr. Melinda Duncan for her helpful guidance to see “the big picture” and for her constructive criticisms that helped me become a stronger and more resilient researcher. I would like to thank Dr. William Cain for his valuable comments and support. I also would like to thank all my professors; especially, Dr. Randall Duncan for his support and kindness. I also appreciate the support of Drs. Jia Song,

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Erica Selva and Robert Sikes. I am also so thankful for my professors in Libya who encouraged me to study abroad-especially for Drs. Farouk Al-shridi, Mustafa Gebril and Muftafa Abugila. I would also like to thank all my lab members, particularly Dr. Heather Lehman for her support, help and constructive input throughout my research. I also would like to thank everyone who supported me in the department, including students and employees, especially, Jomnarong Lertsuwan, Rachel Addo, Kornkamon Nopmonkul, Lisa Gurski, Padma Srinivasan, Serge Ongaga, Senem Kurtoglu, Dr. mary Boggs, Linda Sequeira, and Ms. Betty Cowgill. There are not enough words to thank my beloved parents for their backing and continued support over the years of my study in Libya and here in the U.S.A. Without their encouragement and support, I would not have been able to study in the United States. I am also so thankful to my father-in-law and mother-in-law for their extraordinary support and prayers. I would like to thank my sisters, my sisters-in-law, my brothers and my brothers-in-law for their kindness and support. The presence of my faithful husband, Hesham, together with my two children Abd-Alwahab and Mohammed who have been beside me here in the states and have been a great support and happiness to me that enabled me to fulfill my theses work. Without their sincere love, I could not have continued in my education. Finally, my gratitude is extended to both educational authorities in Libya and U.S.A for the wonderful opportunity granted to me to complete my valuable study here in the states in this field of science.

TABLE OF CONTENTS

LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF ABBREVIATIONS ...... ix ABSTRACT ...... xii

Chapter

1 INTRODUCTION ...... 1

1.1 Breast Anatomy and Development ...... 1 1.2 Inflammatory Breast Cancer ...... 6 1.3 IBC Staging and Differences ...... 8

1.3.1 American Joint Committee on Cancer (AJCC) Case Definition of IBC: ...... 12 1.3.2 Population-based case definitions for IBC ...... 12

1.4 IBC in North Africa vs. the United States ...... 13 1.5 Risk Factors and Epidemiology ...... 15 1.6 Molecular Characteristics of IBC ...... 18 1.7 Rho GTPase ...... 20

1.7.1 Characteristics of Rho GTPase...... 21 1.7.2 Post translational Modifications ...... 25 1.7.3 Regulation of Rho GTPases by Upstream Regulatory Proteins: ...... 27

1.7.3.1 Upstream Rho Effector Proteins: The Rho GDIs ...... 30 1.7.3.2 Upstream Rho Effector Proteins: The RhoGEFs ...... 32 1.7.3.3 Upstream Rho Effector Proteins: The RhoGAPs ...... 32

1.7.4 RhoC GTPase ...... 33 1.7.5 Regulation of RhoC GTPase by Phosphorylation ...... 35

1.8 PI3K/Akt ...... 39

2 MATERIALS AND METHODS ...... 42

2.1 Cell Culture ...... 42 2.2 in vitro Transfection Experiments ...... 42 2.3 RhoC Activation Assay ...... 43 2.4 Western Blot Analysis ...... 45 2.5 RhoGAP Assay ...... 46 2.6 Immunoprecipitation ...... 46 2.7 GEF Fluorescence Assay ...... 47 2.8 2.8 Statistical Analysis ...... 48

3 RESULTS ...... 50

3.1 Phosphorylation of RhoC Does not Affect its Activation ...... 50 3.2 Inhibition of RhoC Phosphorylation by Akt1 Does not Affect GTP Hydrolysis ...... 53 3.3 Interaction of RhoC with GDI Is not Affected by RhoC Phosphorylation ...... 55 3.4 Phosphorylation of RhoC GTPase by Akt1 Does not Affect RhoC/GEF Interaction ...... 56

4 DISCUSSION, CONCLUSION AND FUTURE DIRECTIONS ...... 60

4.1 Discussion ...... 60 4.2 Conclusions ...... 66 4.3 Future Direction...... 67

REFERENCES ...... 70

Appendix

PERMISSION LETTERS ...... 79

LIST OF TABLES

Table1.1 The TNM staging system in breast cancer ...... 11

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LIST OF FIGURES

Figure 1.1 Composition of the mammary glands of the breast...... 5

Figure 1.2 Inflammatory breast cancer presentation...... 7

Figure 1.3 Human Rho GTPases...... 21

Figure 1.4 Structural domains in a typical Rho GTPases...... 24

Figure 1.5 Post-translational modifications of Rho GTPase...... 27

Figure 1.6 The GTPase cycle...... 29

Figure 1.7 Putative Akt phosphorylation consensus sequence in various members of the Rho GTPase subfamily...... 36

Figure 1.8 Akt1 phosphorylation of RhoC GTPase...... 38

Figure 1.9 Model of Akt1 phosphorylation of RhoC ...... 39

Figure 3.1 Diagram shows RhoC activation assay...... 51

Figure 3.2 RhoC GTPase ativity is not affected by inhibition of its phosphorylation by Akt1 in SUM149c cells...... 52

Figure 3.3 Inhibition of Akt1 phosphorylation of RhoC GTPases does not significantly alter hydrolysis activity...... 54

Figure 3.4 Interaction of RhoC GTPase with Rho GDI is not affected by the inhibition of Akt1 phosphorylation in SUM149 cells...... 56

Figure 3.5 Akt1 phosphorylation of RhoC GTPase dose not affect its interaction with RhoGEF...... 58

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LIST OF ABBREVIATIONS

AJCC American Joint Committee on Cancer BMI Body mass index BSA Bovine serum albumin CFP Cyan fluorescent protein DCIS Ductal carcinoma in situ ER Endoplasmic reticulum FBS Fetal bovine serum FMNL-2 Formin-like protein 2 DH Dbl-homology DOCK Dedicator of cytokinesis EGFR Epidermal growth factor receptor EMT Epithelial to mesenchymal transition ER Estrogen receptor F Farnesylated FRET Fluorescence resonance energy transfer FSH Follicle stimulating hormone FTase Farnesyltransferase GAP GTPase activating protein GDI Guanine dissociation inhibitor GEF Guanosine nucleotide exchange factor GDP Guanosine diphosphate GG Geranylgeranylated GGPP Geranylgeranyl pyrophosphate

GGTase Geranylgeranyltransferase GRF GDI-releasing factor GTP Guanosine triphosphate GTPase Guanosine triphosphatase iAkt Pharmacologic Akt inhibitor IBC Inflammatory breast cancer ICMT Isoprenylcysteine carboxymethyltransferase IGF Insulin-like growth factor LABC Locally advanced breast cancer LCIS Lobular carcinoma in situ Leu Leucine mTORC Mammalian target of rapamycin complex nIBC Non-inflammatory breast cancer PBS Phosphate buffered saline PDGFR Platelet-derived growth factor receptor PDK1 Phosphoinositide-dependent kinase-1 PH Pleckstrin homology PI Propidium iodide PI3K Phosphoinositide 3-kinase PIP Phosphatidylinositide phosphate PKB Protein kinase B PKC Protein kinase C PR Progesterone receptor PTEN Phosphatase and Tensin Homolog Ras RAt sarcoma RBD Rho binding domain RCE1 Ras-converting enzyme 1 RFP Red fluorescent protein

Rho Ras homologous ROCK Rho-associated protein kinase SEER Surveillance, Epidemiology and End Results program siAkt Small inhibitor ribonucleic acid to Akt siCtrl Small inhibitor RNA scrambled control siRNA Small inhibitor ribonucleic acid Ser Serine S73A RhoC phosphorylation mutant S73D RhoC phosphomimetic mutant Thr Threonine TNM Tumor node metastasis VEGF Vascular endothelial growth factor YFP Yellow fluorescence protein

ABSTRACT

Inflammatory breast cancer (IBC) is the most highly aggressive and lethal form of breast cancer. It is characterized by primary skin changes that include redness of skin, edema and peau d’ aurange [skin of an orange]. IBC is clinically distinguished by the rapid progression of these symptoms and it has a distinct pathology with tumor emboli that invade and block the dermal lymphatics of the skin over the breast. In the United States, 10-year disease-free survival rates are only 20% due to the aggressiveness and rapid metastasis of IBC. Therefore, it is important to understand the mechanism of metastasis of IBC. A key player that is an important contributor to the IBC metastatic phenotype is RhoC GTPase. RhoC GTPase is a member of Ras- superfamily, which is over-expressed in inflammatory breast cancer and is essential for its metastasis. It acts as a molecular switch, which cycles between a GTP-bound (“on”) state and GDP-bound (“off”) state. Regulation of RhoC GTPase activity occurs through its` interactions with GTPase-activation proteins (GAPs), GDP-dissociation inhibitors (GDIs), and guanine nucleotide exchange factors (GEFs). Previously, our laboratory demonstrated that RhoC is a substrate for Akt1 and its phosphorylation is required for the IBC metastatic phenotype. In addition to the regulation with upstream regulatory proteins, our laboratory has shown that phosphorylation of RhoC by Akt1 affects cell invasion. This project examines whether phosphorylation of RhoC by Akt1 alters the activation state of RhoC or its interaction with regulators of the GTPase cycle.

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Chapter 1

INTRODUCTION

1.1 Breast Anatomy and Development Mammary glands, or breasts are a distinguishing characteristic of mammals in humans. Their development starts in both sexes in the fifth week during embryonic development in which a pair of milk bands of ectoderm develops from axilla to the groin. By week 7 to 8 of gestation, a thickening takes place in the milk line known as “milk hill stage”. Then, the “disk stage” and the “globular stage” occur during which an invagination into the mesenchyme and tridimensional growth take place, respectively (Harris, Lippman, Morrow, & Osborne, 2008; Tavassoli, 1999). At week 10 to week 14 of fetal development, a flattening of the- ridge “disc stage” occur due to the additional invasion of the chest wall mesenchyme. Between week 12 and week 16 of gestation, the formation of the smooth muscle of nipple and areola starts as a result of a differentiation of the mesenchymal cells in the chest wall. The “budding stage” and “branching stage” occurs at 16 weeks’ gestation during which epithelial buds appear and form 15 to 25 strips, which will be the future secretory alveoli, respectively (Harris et al., 2008; Tavassoli, 1999). During the third trimester, placental hormones enter the fetal circulation and initiate the canalization of the epithelial strips. This stage known as “canalization stage” and continue to occur between week 20 to week 32 of gestation. Between the 32nd and 40th week, a fourfold increase in mammary gland mass due to the

development of mammary lobuloalveolar structure. Moreover, the development and the pigmentation of the nipple-areolar complex takes place during this period. After birth, nipple of the neonate secrets colostral milk, known as “witches milk”, for 4-7 days. Prolactin is also secreted which in turn stimulate breast secretion. These secretions decline after 3-4 weeks due to withdrawal of placental hormones (Harris et al., 2008; Tavassoli, 1999). During puberty in females, the breasts start to develop again through the effect of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are secreted by the anterior pituitary gland as the result of the surging effect of gonadotropin-releasing hormone (GnRH)- that is secreted by the hypothalamus (Guyton & Hall, 2006). FSH in females stimulate the maturation of ovarian follicles into Graafian follicles, which secret estrogen (Harris et al., 2008). Similarly, one of the functions of the LH in the female is stimulation of estrogen and progesterone synthesis in the ovaries (Guyton & Hall, 2006). Once estrogen is stimulated, the growth of mammary gland and fat deposition in the breast will be stimulated. Estrogen is secreted by ovaries as well as by the adrenal cortices in nonpregnant female in very small amount (Guyton & Hall, 2006). It enhances the longitudinal growth and branching of ductal epithelium as well as the proliferation of periductal stroma, which will increase in elasticity, vascularity and fat deposition, which also produces estrogen. Full development of the mammary tissue needs synergestic effect of estrogen and progesterone (Harris et al., 2008; Tavassoli, 1999). During pregnancy, estrogen is also secreted in large amounts, 20- fold, by the placenta. The significantly higher estrogen level leads to further growth and

branching of the ductal system. Thus, the complete development of the glandular tissue will take place, which in turn leads to the production of milk. Growth hormone, prolactin, insulin and the adrenal glucocorticoids also contribute in the development of the breasts by playing role in protein metabolism (Guyton & Hall, 2006). The adult breast is located within the anterior thoracic wall and lies between the 2nd and 6th ribs vertically and horizontally between the sterna edge and the midaxillary line (Bland & Copeland, 2009; Harris et al., 2008). In general, breasts are composed of three major structures: a) skin, which is thin and includes hair follicles, sebaceous glands, and eccrine sweat glands. b) subcutaneous tissue, which contains connective tissue, blood vessels, nerves and lymphatic vessels. c) breast tissue, which is composed of parenchyma and stroma (Harris et al., 2008). The stroma and subcutaneous tissue contain fat, connective tissue, blood vessels, nerve and lymphatic vessels. The parenchyma is composed of 15 to 20 segments or lobes of glandular tissue that are arranged radially around the nipple, which is surrounded by a circular area called areola (Guyton & Hall, 2006; Harris et al., 2008; Spence & Mason, 1987). Each lobe is made up of 20 to 40 lobules each of which is composed of 10 to 100 alveoli or tubulosacular secretory units (Harris et al., 2008). Each lobe is drained by a single lactiferous (collecting) duct. All collecting ducts converge on the nipple. Just before reaching the nipple, lactiferous ducts converge on the lactiferous sinus or ampulla that functions as a small milk reservoir (Spence & Mason, 1987; Tavassoli, 1999) [Figure 1.1]. There are various types of muscle tissue that support the breast. Among these muscles are the pectoralis major and minor, serratus anterior, and latissimus dorsi muscles. The breast lies between two fascial layers: the superficial pectoral

fascia, which envelops the breast and the deep pectoral fascia. Fibrous bands known as suspensory or Cooper’s ligaments connect these two fascial layers and support the breast (Harris et al., 2008).

Figure 1.1 Composition of the mammary glands of the breast. (A)The breast that is composed of mammary gland, which consists of secretory lobules, alveoli, and lactiferous ducts (milk ducts) surrounded by adipose tissue. (B)The enlargements show a lobule. (C) Milk secreting cells of an alveolus. (This image was published in the Textbook of Medical Physiology, 11th edition, Guyton and Hall, pregnancy and lactation, 1039, copyright Elsevier, 2006).

1.2 Inflammatory Breast Cancer Inflammatory breast cancer (IBC) is the most aggressive and lethal form of locally advanced breast cancer. It tends to affect patients at younger ages as compared to non IBC and it is typically characterized by the rapid progression of its symptoms within three to six months of onset, so it is often classified as stage IIIB or IV at the time of diagnosis (Anderson, Schairer, Chen, Hance, & Levine, 2005; K. L. van Golen et al., 2002). It has unique molecular and epidemiologic characteristics as well as phenotype which suggest that IBC is a distinct biological entity (Robertson et al., 2010). The symptoms of IBC are quite distinct from non-IBC. The clinical symptoms of IBC include erythema, edema, “peau d’ orange”, thickening of the skin, and nipple retraction, but a palpable mass is usually absent (Figure 1.2) (Cristofanilli, Buzdar, & Hortobagyi, 2003). Pathologically, it is lympho-angioinvasive and distinguished by tumor emboli that invade and block the dermal lymphatics (Van der Auwera et al., 2005; Vermeulen, van Golen, & Dirix, 2010).

Figure 1.2 Inflammatory breast cancer presentation. (A) Common clinical symptoms of IBC including peau d’ aurange, erythema, and edema, nipple retraction (Image adapted from Anne's Helpful, Uncommon breast cancers part two: Inflammatory breast cancer. Mammonewsline Techtalk. 2010). http://mammonewsline.blogspot.com/2010/08/uncommon-breast-cancers-part- two_09.html). (B) Radiologic images of an IBC patient. The red arrow on the left image points out the distinct thickening of the skin of the breast that is characteristic of inflammatory breast cancer. The right image is the unaffected breast. (Image adapted from Levine PH, et al. What is inflammatory breast cancer? Revisiting the case definition. Cancers. 2010; 2(1): 143-152).

IBC is usually diagnosed by its clinical symptoms and by the presence of malignancy in the dermal lymphatics of skin biopsies (Kleer, van Golen, & Merajver, 2000). In addition to core biopsy and skin punch biopsy, IBC patients are recommended to undergo a diagnostic mammogram as well as ultrasound and MRI (Dawood et al., 2011). Because of the rapid progression and fast metastatic nature of IBC, patients need to be undergo aggressive treatment once IBC is diagnosed (Dawood et al., 2011; Kleer et al., 2000) The treatment method that is usually preferred is starting with systemic neoadjuvant chemotherapy followed by a mastectomy and radiation therapy (Cristofanilli et al., 2003; Dawood et al., 2011).

1.3 IBC Staging and Differences Inflammatory breast cancer (IBC) is a form of locally advanced breast cancer that is very aggressive and lethal. It is characterized by primary skin changes that include redness of skin, edema and peau d’ aurange [skin of an orange]. The appearance of edema and the redness of the skin is not a true physiologic inflammatory response. The reason for this appearance is the tumor emboli that invade and block the dermal lymphatics of the skin overlaying the breast (Anderson et al., 2005). The first scientist who described IBC was Sir Charles Bell in 1814. During that time period, IBC was thought to be a number of different diseases and was referred to by a variety of names, examples include, acute mammary carcinoma, mastitis carcinomatosa, and the cancer of pregnant and lactating women. These diseases were unified under the term inflammatory breast cancer (IBC), which was first proposed by Lee and Tannebaum in 1924 (Anderson et al., 2005; Hance, Anderson, Devesa, Young, & Levine, 2005). Variations in the definition of IBC over time and place lead to a huge difference and inaccuracy in the data being collected from cancer registries. Some suggest that there may be three subtypes of IBC according to clinical features and/or pathological features (Anderson et al., 2005):

 “Clin-only” or the clinical inflammation [clinical symptoms] without the pathologic evidence of plugging of the dermal lymphatics.

 “Path-only” or pathological plugging of dermal lymphatics without involvement of clinical inflammation.

 “Clin-path” or IBC with both clinical inflammation accompanied with pathological involvement of plugging of dermal lymphatics. In order to understand each definition, the reader should have general idea about different classifications of breast cancer around the world and which type of each classification is considered an inflammatory breast cancer. The most notable is the classification of breast cancer according to Poussee Evolutive system (Anderson et al., 2005): This is a French classification system which classified breast cancer into four groups:

 PEV 0: There is no observable increase in the size during past three months and there is no symptoms of inflammation

 PEV 1: There is an increase of the size of the tumor during the past three months, but no signs of inflammation.

 PEV 2: There is tumor with inflammatory signs that appear on less than half of the breast surface.

 PEV 3: There is tumor with inflammatory signs that involve more than half of the breast surface.

 PEV 2 and PEV3 were only considered IBC in the U.S.A.

The most common current staging of breast cancer in the United States and Europe is known as the TNM classification system, which takes into account to tumor size (T), lymph node involvement (N), and whether the cancer has metastasized (M) (Harris et al., 2008; Singletary & Connolly, 2006).

 Stage I: T (less than 2 cm), N (no lymph node involved), and M (no metastasis)

 Stage II A: T (any size), N (no lymph node involved), and M (no metastasis)

 Stage IIB: T (less than 5 cm), N ( 1-3 nodes involved), and M (no metastasis)

 Stage IIIA: T (larger than 5 cm), N ( 1 or more nodes involved), M (no metastasis).

 Stage IIIB: T (less than 5 cm), N ( 4 or more nodes involved), and M (no metastasis)

 Stage IIIC : It involves the skin of the breast or it is inflammatory

 Stage IV: T (any size), N (any nodes), and M (distant metastasis)

Moreover, there are sub classifications for T, N, and M to describe the breast cancer in more details. The letter T followed by a number from 0 to 4 describes the tumor's size and spread to the skin or to the chest wall under the breast. Higher T numbers mean a larger tumor and/or wider spread to tissues near the breast. The letter N followed by a number from 0 to 3 indicates whether the cancer has spread to lymph nodes near the breast and, if so, how many lymph nodes are affected. The letter M followed by a 0 or 1 indicates whether the cancer has spread to distant organs -- for example, the lungs or bones (Harris et al., 2008; Singletary & Connolly, 2006). T4, which refers to the largest tumor and/or wider spread to tissues near the breast, has 4 subsets: T4a, if a tumor invades the chest wall, T4b, if the tumor invade the skin, T4c, if the tumor invades both, and T4d, if it is “inflammatory, with diffuse, brawny induration of the skin and an erysipeloid edge”. Inflammatory breast cancer is classified as T4d, which has the worst prognosis (El-Tamer et al., 2002).

Table1.1 The TNM staging system in breast cancer

Stage 0 Tis (in situ) N0 M0

Stage I T1 N0 M0

Stage IIA T0 N1 M0

T1 N1 M0

T2 N0 M0

Stage IIB T2 N1 M0

T3 N0 M0

Stage IIIA T0 N2 M0

T1 N2 M0

T2 N2 M0

T3 N1 M0

T3 N2 M0

Stage IIIB T4 N0 M0

T4 N1 M0

T4 N2 M0

Stage IIIC Any T N3 M0

Stage IV Any T Any N M1

1.3.1 American Joint Committee on Cancer (AJCC) Case Definition of IBC: The AJCC definition is the most widely used in the United States. AJCC classified IBC according to TNM as T4d and stage IIIB or IV. It defines IBC as “clinicopathologic entity characterized clinically by diffuse edema (peau d'orange) and erythema of the breast, over the majority of the breast and often without an underlying mass. The reason for the clinical appearance is the pathologic plugging of the dermal lymphatics of the breast” (Anderson et al., 2005; Hance et al., 2005).

1.3.2 Population-based case definitions for IBC There is a variation of the definitions over time. In 1973, the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute coded IBC according to the International Classification of Diseases for Oncology 8530 designation. The National Association of Central Cancer Registries (NAACCR) also has identified IBC cases by ICD-O 8530. This definition required pathologic plugging of the dermal lymphatics with tumor emboli. However, Clinical inflammation is not considered for ICD-O8530. This definition was the most conservative IBC designation and it probably underestimates the true incidence of

IBC. Chang et al. noted very low IBC incidence rates of (0.5% in white women and 0.7% in black women by using this definition. However, using other codes, which may include LABC, from SEER, Levine et al. reported much higher incidence rates of 6% and 10% in white and black women respectively (Anderson et al., 2005; Hance et al., 2005). In 1988, SEER linked extent of disease (EOD) codes to AJCC designations SEER's EOD-E 70 corresponded to the AJCC T4d designation, i.e., “inflammatory carcinoma, including diffuse (beyond that directly overlying the tumor)

dermal lymphatic permeation or infiltration.” The tumor size code, EOD-S 998 was defined as “diffuse, widespread: 3/4's or more of the breast; inflammatory breast cancer.” (Anderson et al., 2005; Hance et al., 2005).

1.4 IBC in North Africa vs. the United States Inflammatory breast cancer is a highly aggressive and lethal disease no matter who it affects. However, as in the case of many diseases, IBC has a higher incidence in some geographic region and ethnic groups more than the others. According to data collected from Egypt, Tunisia and Algeria, North African countries (Maghreb region) have a higher frequency of IBC than the United States (Boussen et al., 2010). Because of the higher incidence rate in Tunisia and North Africa, inflammatory breast cancer is characterized by a peculiar geographic distribution. Several epidemiologic studies have been performed in Tunisia regarding IBC since the opening of the Salah Aziz Institute 1969. The first serious of publications were depending on Poussee Evolutive for classifying breast cancer cases. According to this classification, Poussee Evolutive 2 and 3 was considered an IBC, which was named at that time in those articles as rapidly progressing breast cancer (Boussen et al., 2010). Since the 1990s, the American Joint committee on Cancer (AJCC) definition of IBC was used to evaluate the incidence of IBC in Tunisia. The AJCC was defining every breast cancer that corresponding to T4d disease or a Poussee Evolutive 3 lesion as an IBC. However, several investigator believe that the definition of AJCC represents only a portion of IBC cases (Boussen et al., 2010). In the 1970s, IBC in Tunisia accounted for 30% to 55% of all breast cancer cases. Recently however, IBC in Tunisia was reported to be lower (5% -7%)

than in the 1970s. This could be due to the early diagnosis, awareness, and improvement of the socioeconomic level of Tunisian women. Moreover, using more stringent diagnostic criteria (pousess Evolutive 3/T4d) probably contributed to a more refined clinical diagnosis. Various studies show a higher incidence rate in Tunisia than in the United States. According to the largest cohort study from 1969 to 1974, which showed that 48.7% of 581 cases were Poussee Evolutive 3 (IBC), Mourali’s publication noted that Tunisia has a high incidence of rapidly progressing breast cancer (Mourali et al., 1980). A study by Lee et al. (from 1986 to 1987) reported 41% IBC cases, 29% of them has T4d and 12% with more localized disease (Le et al., 2005). From 1990 to 1996, Ben Ahmed et al. noted about a study on 729 breast cancer cases, 33.4% having T4 stage, including 14% with T4d (Ben Ahmed et al., 2002). According to a Tunisian national survey completed in 2004, Maalej et al. reported IBC as 6.2% (Maalej et al., 2008). As in the case in Tunisia, Egypt has a higher frequency of IBC than the U.S.A. Studies in Egypt proposed that IBC accounts for 10% of all breast patients. It affects younger women, progresses as rapidly, and tends to be more advanced at the time of diagnosis. The mean age of women with IBC in the US at the time of diagnosis is 57 and 59. However, Egyptian women has median age at the diagnosis of 42 years old (Robertson et al., 2010). A comparison between IBC in Egypt and the United States showed that erythema, edema and peau d’orange were found in 77% of Egyptian vs. 29% of US patients. Moreover, the number of tumor emboli was significantly higher in tumors from Egypt than from tumors in the United States. The same study has found that

there is high level of RhoC in 87% of IBC samples from Egypt and 14% of IBC samples in the USA. They concluded that IBC is more aggressive in Egypt than in the U.S. (Lo et al., 2008).(Soliman et al., 2009) Another study in the population-based cancer registry of Gharbiah, Egypt, which had 659 cases, grouped cases for three groups. The first group referred to as most likely IBC, who exhibited all the three features: erythema, edema, and peau d’ orange. The second group named possible IBC cases [patients who had any two of the three symptoms or had peau d’ orange only]. The third one called non-IBC for those who had edema only, erythema only, or none of the three symptoms. Most likely IBC accounted for 0.6%. However, most likely IBC cases and possible IBC cases together represent 11.1% (Soliman et al., 2009).

1.5 Risk Factors and Epidemiology IBC is the most dreadful form of locally advanced breast cancer with unknown etiology. Epidemiology of IBC is linked with its biology. Therefore, understanding the predisposing factors of IBC will inform us about the biology of IBC and how to avoid and treat it (Levine & Veneroso, 2008; Merajver, 2007). The most predominant risk factors that are associated with IBC: high body mass index (BMI ≥ 30) and younger age at diagnosis. In addition, IBC has higher incidence in some ethnic groups. African American women have at least a 50% higher incidence than white women (Levine & Veneroso, 2008; Molckovsky, Fitzgerald, Freedman, Heisey, & Clemons, 2009). Moreover, studies show that 40% of patients in a registry have a family history of some type of breast cancer (Robertson et al., 2010). Geographically, IBC has a higher frequency in North African countries. Even though BMI was highly suggested to be risk factor for IBC, a study in Algeria showed that there is no

significant association between BMI and IBC. However, it has been shown that postmenopausal obese women have worse prognosis compared to other IBC cases (Chaher et al., 2012). Data analysis shows that the mean age of Tunisian women that are affected with IBC is 43 years. 22% of them were younger than 35 years. In addition, the mean age of Egyptian women is 46.9 years old with range of 28-70 years old. In the United states, however, the mean age of women with IBC in the US at the time of diagnosis is 57.3 years old with range of 36 -75 years old. There are some difference in the median age between Tunisian, Egyptian and Algerian IBC. The median age for Tunisian and Egyptian IBC was approximately 43 year-old. Algerian women have relatively higher median age (48.5 years) with a majority of younger than 50. However, the median age for IBC women in the United States is 46 (Boussen et al., 2010; Chaher et al., 2012; Lo et al., 2008). Environmental factors are more likely to contribute to the higher incidence of IBC in North Africa than in the U.S.A. According to Levine, living in a rural region in Tunisia was strongly associated with IBC (T4d). Mourali et al. reported that approximately half of the premenopausal patients with Pousse´e E´ volutive-positive breast cancer, were living in rural areas (Boussen et al., 2010; Mourali et al., 1980). Rural area was associated with rapid progression breast cancer in both pre- and post-menopausal women in Tunisia (Anderson et al., 2005). The reason, which was suggested by some scientists, is that farming-related exposures and exogenous hormones may be implicated in IBC patients (Chaher et al., 2012). Another study in Egypt also supported that IBC patients typically live in rural areas. Moreover, it has been found in Tunisia that a higher percentage of PEV+ than PEV- cases had

blood type A (Anderson et al., 2005). Mourali et al. concluded from an analysis that “the most prominent parameters predicting high risk of diagnosis with Pousse´e E´ volutive cancer included being premenopausal (younger than 39 years), having a delay of diagnosis >6 months (associated with rural living), and having type A blood" (Boussen et al., 2010; Mourali et al., 1980). On the other hand, a study on Algerian women found that higher proportion of IBC patients was urban, premenopausal women in both low and medium income. It has been proposed that this is due to the difference in the socioeconomic status between these countries. Rural areas in Tunisia only have a private health care system that is not affordable for everybody. In the contrast, the health care system in Algeria is socialized and covers the entire population. In addition, the percentage of estrogen receptor (ER) positive IBC patients was similar between urban and rural patients (73% vs. 74% respectively). This probably indicates that both rural and urban patients are exposed to similar exogenous hormones. Thus, the farming-related exposure may not have the same implication in Algeria as opposed to the Tunisian and Egyptian patients (Lo et al., 2008). At the biomarker level, the percentage of Algerian IBC patients with ER positive (73%) was higher than IBC patients in Egypt (60%) and Tunisia (47%). Tunisian patients have higher Human Epidermal Growth Factor Receptor 2 ( HER2) and epidermal growth factor receptor (EGFR) (33% and 23% respectively) than in Algerian cases (15%). Interestingly, basal and HER2 tumor subtypes were more frequently found in Tunisian cases (33%), followed by luminal cases (29%). On the other hand, Algerian cases have predominant luminal A subtype (62%), followed by basal (18%), triple negative (17%), luminal B (10%) and HER2 (10%) subtypes. This

implies that there are molecular differences between these two neighboring countries and there is a unique molecular pattern of IBC in each region. Other reason that may explain the difference in results is technical variations, Immunohistochemistry assays “(sample collection, storage, fixing procedures and time, and antibodies employed), biomarker grading, as well as study design and sample size.” (Chaher et al., 2012).

1.6 Molecular Characteristics of IBC IBC is distinct and unique as compared to other forms of breast cancer because of its ability to invade the dermal lymphatic vessels. IBC was treated with the same treatments as locally advanced breast cancer (LABC) until the late 1990s. Because of its metastatic nature, however, surgery and irradiation were not enough to treat the IBC (Ellis, Bland, & Copeland, 1988; Maloisel et al., 1990). Even though, several studies suggested that IBC may be treated with chemotherapy with three cycles of 5-Flurouracil, Doxorubicin and cyclophosphamide in addition to radiation and/or radical mastectomy (Didolkar, Elias, Aisner, & Vachon, 1991; Maloisel et al., 1990), IBC was still dreadful and its disease free survival rate, according to the National Cancer Institute’s SEER program for the period 1975-1992, was significantly worse than non-IBC. Therefore, IBC should be considered as distinct entity. In order to fight this aggressive disease, it is important to understand this disease at molecular level and determine its specific driving molecules that lead to -its metastatic and invasive phenotype (Chang, Parker, Pham, Buzdar, & Hursting, 1998). Determination the status of estrogen receptor (ER) and progesterone receptor (PR) was the first step toward this goal. It has been shown by Paradise et al that ER+ and PR+ cases is lower in IBC compared to stage matched LABC (ER+, 44% versus 64% ; PR+, 30% versus 51%, respectively) (Dawood et al., 2011;

Delarue, May-Levin, Mouriesse, Contesso, & Sancho-Garnier, 1981; Paradiso et al., 1989). The higher frequency of negative ER and PR than non-IBC was associated with a worse prognosis and decreased overall survival rate among IBC patients (Dawood et al., 2011). Overexpression of Her2/neu, human epidermal growth factor receptor 2, oncogene and EGFR, epidermal growth factor receptor, in greater than 60% of inflammatory breast cancer cases was also found to be correlated with high degree of malignancy and shorter disease-free survival rate (Radunsky & van Golen, 2005). Several other molecular markers that may correlate with IBC have been evaluated. Among the most important molecular markers is the P53 tumor suppressor gene and E-cadherin. As in the case of many tumors, P53 was mutated in IBC patients. It has been shown that P53 in IBC patients either directly mutated or sequestered as wild type P53 protein in the cytoplasm. Even though, the loss of E- cadherin is a tumor marker for cancer, IBC was exception since it has high expression of E- cadherin. This suggests that IBC has a unique mechanism to metastasize (Kleer, van Golen, Braun, & Merajver, 2001; Moll, Riou, & Levine, 1992). All those studies that have investigated the mentioned molecular markers and other studies were essential toward understanding the prognostic factors that affect metastasis. However, none of them addressed the unique invasive nature of IBC. In order to establish a specific therapy that overcomes IBC, it was important to look for the molecular markers that are behind this invasive and metastatic phenotype. Our laboratory was one of the pioneers that starting looking at the specific genes that distinguish IBC from non-IBC.

An essential molecular marker that has been shown to be important for the metastasis of IBC is RhoC guanosine triphosphate (GTPase). It is a member of Rho family GTPase, which are involved in actin cytoskeleton rearrangements. Therefore, they are found to control cell motility, invasion and metastasis in cancer. RhoC GTPase is over expressed in greater than 90% of IBC tumors compared with 38% of non-IBC. It is correlated with high histologic grade, and poor prognostic outcome (K. L. van Golen et al., 1999). In addition to IBC, RhoC has been found to be essential for metastasis in other types of tumors such as pancreas, lung, and colon (Suwa et al., 1998).

1.7 Rho GTPase

Rho GTPase (Ras homology guanosine triphosphatases) subfamily belongs to the Ras superfamily. These low molecular weight proteins (21-28 kDa) act as molecular switches that switch on when bound to GTP and switch off after they hydrolyze the GTP , with the help of GTPase activating proteins (GAP), to GDP. Rho GTPases regulate various cell functions such as cell cycle, vesicular trafficking and cell polarity. The most predominant known function for this subfamily is regulation of the actin cytoskeleton (K. van Golen, 2009). Rho GTPases composed of 22 family members which are subdivided into eight groups according to the similarities in their sequence and structural similarity: Rho subfamily (RhoA, RhoB, RhoC), Rac subfamily (Rac1, Rac1b, Rac2, Rac3, RhoG), Cdc42 subfamily (Cdc42p, Cdc42b, TC10 and TCL), RhoD/RhoF, Rnd subfamily (Rnd1, Rnd2, Rnd3), RhoBTB subfamily (RhoBTB1 and RhoBTB2), Wrch1/ChP and RhoH subfamilies [Figure 1.3] (K. van Golen, 2009).

Figure 1.3 Human Rho GTPases. Amino acid sequences of the G-domains (corresponding to amino acids 5–173 of Rac1) of human Rho GTPase family members were aligned using the ClustalX and the dendritic-tree was made with TreeViewX. The 20 members of the Rho GTPases, including two splice variants, can be subdivided into eight major branches: the Rho, Cdc42, Rac, Rnd, RhoD/F, RhoU/V, RhoH, and RhoBTB subfamilies Human Rho GTPases (Figure adapted with kind permission from Springer science+Business Media: The Rho GTPases in Cancer, Chapter 1, Overview of Rho GTPase History, 2010, 11, E.V. Stevens and C.J. Der, Figure 1.1).

1.7.1 Characteristics of Rho GTPase Rho GTPases are generally small proteins, consisting of approximately 200 amino acids. They were originally identified by their similarity to the Ras proteins

by sharing with them approximately 30% amino acid identity particularly in sequences important for GDP/GTP binding and regulation, effector binding, and posttranslational lipid modifications. Similar to Ras, Rho GTPases contain a set of consensus GDP/ GTP-binding sequence elements, an effector interaction domain, and C-terminal CAAX tetrapeptide motifs. They are distinguished from Ras by an extra sequence known as the insert region (Der, 2011; Manser, 2005; K. van Golen, 2009). The general structure of Rho GTPases as shown in Figure.1.4 below composed of an N- terminal G-domain which is involved in GTP/GDP binding, hydrolysis, and effector binding, as well as a C-terminal membrane targeting domain (K. van Golen, 2009). The amino terminal G domain is characterized by consensus GDP/GTP- binding motifs shared with other GTP-binding proteins. Rho family proteins possess high affinity binding for both guanine nucleotides (GDP and GTP) through the consensus GDP/GTP binding motif. Their biological functions are controlled by cycling between active GTP-bound and inactive GDP-bound states. The exchange between the GDP and the GTP-bound states is accompanied by the conformational changes in Rho in two amino terminal regions switch I (which corresponds to Ras residues 30–38) and switch II (Ras residues 60–76). These switch regions are critical for proper interaction with upstream regulators such as GEFs and GDIs and downstream effector molecules, hence termed the “effector region.” In addition, Rho family proteins possess a unique Rho GTPases short sequence [13 amino acids] positioned between residues analogous to Ras residues 122 and 123 called the insert sequence. This sequence may also be involved in effector interaction (Der, 2011; K. van Golen, 2009).

The carboxyl terminal CAAX (C = cysteine, A = aliphatic amino acid, X = terminal amino acid) tetrapeptide sequence and the hypervariable domain are membrane targeting signals. The CAAX motif signals three posttranslational modification steps: the addition of either a farnesyl or geranylgeranyl isoprenoid lipid group to the cysteine of the CAAX motif, proteolytic removal of the AAX residues, and carboxylmethylation of the prenylated cysteine residue. These modifications promote subcellular localization of Rho GTPases to the plasma membrane and other endomembranes. The hypervariable domain, which is upstream to CAAX, exhibits the greatest sequence divergence between highly related isoforms (Der, 2011; K. van Golen, 2009).

Figure 1.4 Structural domains in a typical Rho GTPases. Highlighted residues are important for GTPase activity: core effector domain (purple), GTP-binding motifs (white), Rho insert domain (blue), hypervariable domain (stripes), and the CAAX domain (spots). The exchange between the GDPand the GTP-bound states is accompanied by the conformational changes in Rho in two amino terminal regions switch I (which corresponds to Ras residues 30–38) and switch II (Ras residues 60– 76). These switch regions are critical for proper interaction with upstream regulators such as GEFs and GDIs and downstream effector molecules, hence termed “effector region.” Another region involved in effector-mediated signaling is an alpha-helical “insert region,” positioned between residues corresponding to Ras residues 122 and 123, that is present in Rho GTPases, but not other Ras superfamily members. The hypervariable domain (which exhibits the greatest sequence divergence between highly related isoforms) and the CAAX motif (cysteine-aliphatic-aliphaticterminal amino acid) are membrane targeting signals. The CAAX motif signals for a series of posttranslational modifications to promote subcellular localization of Rho GTPases to the plasma membrane and other endomembranes (Figure adapted with kind permission from Springer science+Business Media: The Rho GTPases in Cancer, Chapter 1, Overview of Rho GTPase History, 2010, 11, E.V. Stevens and C.J. Der, Figure 1.3).

1.7.2 Post translational Modifications Rho GTPases are synthesized as cytosolic proteins that need to go through a series post translational modifications in order protect them from degradation, regulate protein-protein interaction, facilitate their interaction to the membrane and determine their subcellular localization (Der, 2011; Symons, 2004). As previously mentioned, the C-terminal domain contains the CAAX motif, which is important for posttranslational modifications. The CAAX motif signals for isoprenylation [addition of isoprenyl group to the cysteine residue of the CAAX] through prenyltransferase. The isoprenyl lipid group that is added could be one of the following (Symons, 2004) [figure 1.5]:

a) Covalent attachment of a 15 carbon isoprenoid which is known as farnesyl pyrophosphate (FPP) through the catalysis of farnesyltransferase. Farnesylation occurs when the X residue in the CAAX motif is neither leucine (Leu) nor phenylalanine (Phe.) (Roberts et al., 2008; K. van Golen, 2009).

b) Addition of a 20 carbon geranylgeranyl pyrophosphate through the catalysis of geranylgranyl transfrase-1. Geranylation occurs when the X residue in the CAAX motif is either leucine or phenylalanine (Roberts et al., 2008; K. van Golen, 2009).

Geranylgeranylation is more predominant than farnesylation; however, most Ras and Rho proteins are farnesylated. RhoB GTPase can either be farnesylated or geranylgeranylated. RhoA and RhoC GTPase, however, are only geranylgeranylated (Roberts et al., 2008; K. van Golen, 2009).

The isoprenyl group targets the Rho GTPases protein to the endoplasmic reticulum where the –AAX terminal amino acid residues are cleaved by protease

known as RAS converting enzyme 1 (RCE 1). Then the carboxyl group of the terminal cysteine is methylated by isoprenylcysteinecarboxymethyltransferase (ICMT). The subcellular localization of the small GTPases at this point depends on other targeting signals located adjacent to the CAAX motif [upstream to it]. It consists of either cysteines that are sites of palmitoylation, or a polybasic region which consists of six or more positively charged residues [e.g. RhoC] (Symons, 2004). After being fully processed, Rho GTPases are typically sequestered in the cytosol by GDI (Guanidine dissociation inhibitor) [will be discussed later] until other events occur that lead to the dissociation of RhoGDI and translocation of Rho proteins to the plasma membrane, Golgi apparatus, endoplasmic reticulum, or endosomes. This depends on the targeting signal [palmitoylated or polybasic region] (Konstantinopoulos, Karamouzis, & Papavassiliou, 2007; Symons, 2004).

Figure 1.5 Post-translational modifications of Rho GTPase. Rho GTPases are prenylated, Farnesylated (F) or geranylgeranylated (GG). Then, they go under proteolytic removal of AAX by Ras converting enzyme 1 (RCE1) and carboxymethylation by isoprenylcysteine carboxymethyltransferase (ICMT). Thus, they bind to Rho guanine nucleotide dissociation inhibitors ( Image reprinted by permission from Macmillian publishers Ltd: Nature Reviews Drug Discovery, Konstantinopoulos te al., 2007).

1.7.3 Regulation of Rho GTPases by Upstream Regulatory Proteins: In order to be effective, Rho GTPases need to transiently cycle between inactive form (GDP-bound) to active form (GTP-bound) and return to its inactive form to go through the cycle again, as shown in figure 1.6. The GTPase cycle of Rho family proteins is controlled by three different functional classes of regulatory proteins. The first class of regulators is Rho guanine nucleotide dissociation inhibitors (GDIs), which sequester the GTPases proteins in the cytosol by binding with the isoprenyl moiety of the GTPase through their C-terminal domain. In addition, GDI binds the switch regions of the GTPases through its N-terminal regulatory arm, thus

preventing the exchange of GDP for GTP, the hydrolysis of GTP, and binding of an effector. GDI protein keeps Rho GTPases as soluble cytosolic proteins until it is released by GDI-releasing factor (GRF) through an unknown mechanism (Der, 2011; K. van Golen, 2009). The second class of regulators is Guanine nucleotide exchange factors (GEFs), which activate Rho GTPases through stimulating their intrinsic exchange activity to promote an exchang of GDP for GTP, thus become active. RhoGEFs , also known as DbI family proteins, activate GTPases through their DbI homology (DH) catalytic domain, which stimulate the exchange of GDP for GTP and their pleckstrin homology (PH) domain, which is believed to regulate DH domain as well as promote association of GEF with the plasma membrane. After being activated, Rho GTPases bind to specific effector protein leading to downstream signals and a cellular response (Der, 2011; K. van Golen, 2009). The third class of regulators of Rho GTPase proteins are GTPase activating proteins (GAPs), which act as negative regulators by stimulating the weak intrinsic GTP hydrolysis activity of Rho GTPases to cause the formation of inactive GDP-bound form. GAPs enhance GTP hydrolysis through their conserved Rho GAPs domain (Der, 2011; K. van Golen, 2009).

Figure 1.6. The GTPase cycle. GTPases act as molecular switches that cycle between a GDP-bound “inactive” and GTP-bound “active” conformation. This process is tightly regulated by various proteins. GDIs sequester GDP-bound Rho GTPases in the cytosol by binding to their prenyl group. The GRF will liberate the GDI/GTPase complex, allowing the GTPase to translocate to the inner leaflet of the plasma membrane and insert its prenylation site into the membrane. GEFs at the membrane will stimulate the exchange of GDP for GTP, allowing the GTPase to become active and interact with effector molecules. The intrinsic rate of GTP hydrolysis of the Rho GTPases is quite slow. The GAP proteins help to greatly increase the rate of hydrolysis, hydrolyzing GTP to GDP, and thus allowing a GDI to once again sequester the GTPase in the cytosol until further activation (Figure adapted with kind permission from Springer Science+Business Media: The Rho GTPase in Cancer, Chapter 1, Overview of Rho GTPase History, 2010, 11, E. V. Stevens and CJ. Der).

1.7.3.1 Upstream Rho Effector Proteins: The Rho GDIs Rho GDIs are one of the regulators of Rho and Rab families of the Ras superfamily (Leonard et al., 1992). There are three genes encoding Rho GDI:

 Rho-GDI ( also known as RhoGDI-1, and RhoGDIα), which is the most abundant and ubiquitously expressed. It interacts with various members of Rho family including RhoC GTPase (Fukumoto et al., 1990; Ueda, Kikuchi, Ohga, Yamamoto, & Takai, 1990).

 RhoGDI-2 (also known as RhoGDIβ, LyGDI, and D4GDI). It is mainly expressed in haematopoietic cells, but it is also expressed in other tissues. It interacts with several types of Rho GTPase in vitro (Lelias et al., 1993; Scherle, Behrens, & Staudt, 1993), but with much lower affinity than Rho GDI-1 (Platko et al., 1995). Most of these interactions are not detected yet in vivo (Gorvel, Chang, Boretto, Azuma, & Chavrier, 1998).

 RhoGDI-3 ( also known as RhoGDI-γ and GDIγ). It is specific for RhoB and RhoG (Brunet, Morin, & Olofsson, 2002; Gorvel et al., 1998). It is different from the cytosolic GDIs by being membrane associated (Zalcman et al., 1996) and can be targeted to the Golgi complex and to other cellular membrane by a unique amino-terminal extension (Brunet et al., 2002). It is expressed at low level and preferentially expressed in brain and pancreas (Adra et al., 1997).

Structure of Rho GDIs:

Rho GDIs consist of two main domains:

1. The C-terminal domain [an immunoglobulin like fold], which comprises of 140 residues [amino acids 74-204) contains hydrophobic residues that is considered as the binding pocket of the isoprenyl group group (Gosser et al., 1997; Keep et al.; Lian et al., 2000). There is also negative charge amino acids [acidic patch] which is important for the extraction of Rho proteins from the membrane

(Hoffman, Nassar, & Cerione, 2000; Longenecker et al., 1999).

2. The N-terminal (amino acids 5-55), which is the region of the “regulatory arm”. This domain binds to switch I and switch II region of Rho protein preventing the exchange and the hydrolysis.

It has been shown that Rho GDIs can interact with the same affinity both states: the GTP bound and the GDP bound. Interestingly, however, when the Cdc42 Rho GTPase is attaching to the membrane, the GDI has higher affinity to extract the inactive form. A study which shows how GDIs extract Rho proteins from the membrane suggests a two steps for the interaction (Garcia-Mata, Boulter, & Burridge; Symons, 2004):

 The first stage occurs when the regulatory arm of the GDI binds to the switch I and switch II domains of the Rho proteins. This association permits the carbonyl of the threonine 35, which is located in switch I region and is conserved in all Rho GTPase, to coordinate with Mg+2 that is required for nucleotide stabilization by compensating the negative charge of the phosphate groups (Symons, 2004).

 The second stage is slower and leads to the movement of the isoprenyl group from the membrane to the hydrophobic pocket of the GDI. The acidic patch on the GDI helps in this process by competing with the acidic phospholipid of the lipid bilayer for the polybasic tail of the Rho GTPpase proteins (Symons, 2004).

The structure of the RhoGDIs directly contributes to their function. Generally, the Rho GDIs were considered as negative regulators of Rho GTPase, however, it is shown that they have more complicated function (Boulter et al.).

1.7.3.2 Upstream Rho Effector Proteins: The RhoGEFs Rho guanine exchange factors (Rho GEFs), as mentioned before, are a large family of GDP/GTP exchange proteins that activate the Rho GTPase proteins by catalyzing the physical exchange of GDP for GTP when Rho GTPases are attached to the plasma membrane. They play a major role in cancer by their direct contribution to Rho GTPase hyperactivation (K. van Golen, 2009). These proteins contain an ~ 150 amino acid DBI-homology domain (DH) and approximately 100 amino acid pleckstrin homology (PH) domain. The DH domain is responsible for the catalytic activity of the GEFs, while the PH domain likely binds to phosphoinositide second messengers to recruit GEFs to the plasma membrane. It is also thought that PH domain may activate the DH catalytic domain and contribute to its correct folding (Symons, 2004). There are other domains that are specific for each individual GEF, which may influence its activation and localization. Two types of RhoGEFs that deserve mention because of their role in diseases such as cancer are the Vav and DOCK proteins. It has been shown that Rho GTPases can be activated by the Vav family of GEFs, which can be stimulated by protein tyrosin kinase receptors. A family of GEF known as a dedicator of cytokinesis (DOCK) proteins that interact also with other proteins than Rho GTpases and have domains that can affect the activation of downstream molecules (Symons, 2004; K. van Golen, 2009).

1.7.3.3 Upstream Rho Effector Proteins: The RhoGAPs Rho GTPase activating proteins (RhoGAPs) activate the weak intrinsic GTP hydrolysis activity of Rho family proteins to form the inactive GDP-bound protein. Presently, there are approximately 70 human Rho GAPs identified (Lin & van Golen, 2004). They are multi domain proteins with different lipid and protein

interactive domains that participate in protein-protein interactions, membrane targeting, and cellular localization. RhoGAPs may contain from one to as many as nine domains. Examples of these functional domains are: a conserved GAP domain, SH2, and SH3 domains. The conserved GAP domain, which is approximately 140 amino acids, defines the Rho GAP family. Although the sequence of Rho GAP domain differs from those of other classes of GAPs, such as RasGAPs, the basic GTPase-activating mechanism of RhoGAP domain appears to be similar to that of RhoGAP. The RhoGAP domain is composed of nine helices and a highly conserved arginine residue located in a loop structure (Gamblin & Smerdon, 1998). During the GAP reaction, conformational changes place the catalytic arginine residue of RhoGAP into the active site of Rho GTPase and stabilize charges that occur during the formation of the transitional state. The first RhoGAP protein that was purified, p50RhoGAP, which was found to act on Cdc42, Rac and Rho in vitro (Barfod et al., 1993; Lancaster et al., 1994).

1.7.4 RhoC GTPase RhoC GTPase is a member of the Rho family of Ras superfamily GTPases that acts as molecular switches which cycles between GDP-bound and GTP-bound states [70]. It is a small protein (~22 kDa) which consists of 193 amino acids. Like other Rho family, RhoC composed of:

 N-terminal G-domain which is GTPase and effector binding domain (K. van Golen, 2009), as well as switch I, and switch II domains. Interestingly, X-ray crystallographic structure of RhoC showed that there are two activated states for RhoC: partialy active and fully active state (Wheeler & Ridley, 2004).

 The C-terminal domain is the targeting domain that is responsible for the localization (K. van Golen, 2009). RhoC is

only geranylgeranylated which is mostly localized at the membrane. The C-terminal domain contains the hypervariable region in which the greatest difference occurs between RhoA, RhoB and RhoC (Wheeler & Ridley, 2004).

Rho GTPases interact with several downstream effector proteins, but a few of them have been identified such as Formin-like 2 (FMNL2) (Kitzing, Wang, Pertz, Copeland, & Grosse), Rho-associated protein kinase (ROCK), mDia1 and 2, Rhotekin, Rhophilin, and Citron Kinase (Wheeler & Ridley, 2004). RhoC is able to interact with these downstream effectors in vitro, however whether these downstream proteins interact with RhoC in situ remains to be shown. Likely, the interaction of RhoC with certain effectors is cell-type dependent. Additionally, evidence from our laboratory may suggest that regulation outside of the GTPase cycle, namely phosphorylation (the main subject of this thesis) could affect RhoC-effector protein interactions. What makes RhoC GTPase an important protein to study is its overexpression in several types of highly aggressive and metastatic cancer phenotype. It has been shown by the van Golen group that RhoC GTPase is overexpressed 2- 8 fold in 90% of IBC tumors compared with stage matched non-IBC specimens (K. L. van Golen et al., 1999; K. L. van Golen, Z. F. Wu, X. T. Qiao, L. Bao, & S. D. Merajver, 2000a; K. L. van Golen, Z. F. Wu, X. T. Qiao, L. W. Bao, & S. D. Merajver, 2000b). The van Golen laboratory subsequently demonstrated RhoC as a prognostic marker for detecting invasive carcinomas with small tumors (less than 1 cm) that have the potential to metastasize (Kleer et al., 2002). RhoC GTPase is involved in the regulation of the actin cytoskeleton and the formation of focal adhesions that are required for polarization and movement (Chardin, 1999). Overexpression of RhoC GTPase correlates with the increase of

vascularization, aggressiveness, invasiveness, and metastatic phenotype of inflammatory breast cancer (K. L. van Golen et al., 2000a). It is overexpressed in various types of the invasive cancer phenotype including colorectal, gastric, ovarian and bladder cancer. Since its expression is restricted to metastatic cancer, targeting RhoC GTPase holds significant hope as a new therapeutic approach to treat inflammatory breast cancer as well as other types of cancer. Thus, it is important to understand how RhoC GTPase is regulated and find a way to manipulate its expression (K. van Golen, 2009). As other Rho GTPases, RhoC is regulated by the same upstream regulatory proteins which are GAPs, GEFs, and GDIs during the GTPase cycle. Moreover, Dr. van Golen’s laboratory has shown that RhoC GTPase is a substrate for Akt1 and its phosphorylation is required for the metastatic phenotype of IBC. It seems that in addition to the upstream regulatory proteins, phosphorylation of RhoC GTPase by Akt1 may affect its activation and its interaction with upstream regulatory proteins.

1.7.5 Regulation of RhoC GTPase by Phosphorylation It has been suggested that phosphorylation by protein kinases, which play an important role in the regulation of intracellular signaling pathways, can affect activity and/or cellular localization of many members of the Rho protein superfamily (Petsko & Ringe, 2004; K. van Golen, 2009). Phosphorylation of RhoA by protein kinase A, for example, on serine 188 negatively inhibits its function by addition the negative charge phosphate group that leads to increase the binding of RhoA to RhoGDI, which sequester it in the cytoplasm (Ellerbroek, Wennerberg, & Burridge). A survey of the protein sequence of RhoA, RhoB, RhoC, Rac, Cdc42, and RhoG shows a putative Akt (protein kinase B) phosphorylation site ( xxRxRxxS/Txx),

as shown in Figure1.7 (Kwon, Kwon, Chun, Kim, & Kang, 2000; K. van Golen, 2009). Interestingly, serine 73 in RhoC lies within the switch II region, which is important for effector binding. X-Ray crystal structures propose that RhoC GTPase undergoes two conformational changes upon GTP binding. Changes of RhoC conformation may occur when this serine is phosphorylated affecting the ability of RhoC to bind with its effector proteins (Dias & Cerione, 2007).

Figure 1.7. Putative Akt phosphorylation consensus sequence in various members of the Rho GTPase subfamily. Several members of the Rho subfamily contain a putative site for phosphorylation. This site lies within the GTPase switch region, potentially effecting GTPase activation or interactions with downstream effectors (Figure adapted with kind permission from Springer Science+Business Media: The Rho GTPases in Cancer, Chapter 10, Regulation of Rho GTPase Activity Through Phosphorylation Events: A Brief Overview, 2010, Heather Unger and Kenneth van Golen, Figure 10.1)

The van Golen lab has shown that Akt1/PKBα is active in both: the patient-derived IBC cell line, SUM149, and patient samples. This increase in activity of Akt1/PKBα in SUM149, leads to high levels of serine phosphorylated RhoC GTPase [Figure 1.8. a] (Lehman, 2011). RhoC GTPase is overexpressed in IBC and it correlates with worse prognosis and increased metastasis and invasive phenotype of

IBC (K. L. van Golen et al., 2000a). Our laboratory has found that the RhoC-mediated invasion in the SUM149 IBC cells can be significantly decreased when Akt1/PKBα is inhibited either by pharmacologic inhibitors or through the transfection with siRNA against Akt1 or reintroduction of phosphatase and tensin homolog (PTEN) [Figure.1.8.c] (Lehman, 2011). PTEN is a gene that is lost in IBC and acts as a tumor suppressor gene through its phosphatase effect [PTEN regulatory functions in tumor suppression and cell biology (K. van Golen, 2009). Moreover, the invasive capability of the IBC cells was significantly decreased when RhoCS73A mutant, which cannot be phosphorylated by Akt, was introduced into the IBC cell line [Figure1.8.d]. Likewise, the invasive capability that is inhibited through the knockdown of siAkt1was rescued when RhoC S73D, phosphomimetic mutant, was introduced to the SUM149 [Figure1.8.b] (Lehman, 2011).

Figure 1.8 Akt1 phosphorylation of RhoC GTPase. (a) Immunoprecipitation of RhoC GTPase followed by an immunoblot with a phospho-serine specific antibody. Serine phosphorylation of RhoC in the SUM149 IBC cells is confirmed. (b) Transfection of SUM149 IBC cells with a RhoC GTPase phosphomimetic mutant (RhoCS73D) and RhoA GTPase phosphomimetic mutant (RhoAS73D). In these mutants the Akt phosphorylation at serine 73 of RhoC was changed to an aspartate to mimic a phosphorylated state. Inhibition of Akt1 in the cells containing RhoAS73D resulted in decreased invasion of the IBC cells, while little effect was seen when inhibiting Akt1 in the cells containing RhoCS73D.(c) Akt phosphorylation of RhoC GTPase is required for IBC cell invasion. Matrigel invasion assay demonstrating a significant decrease in invasion of the SUM149 IBC cells after transfection with an Akt phosphorylation mutant (RhoCS73A), where the putative Akt phosphorylation site in the RhoC sequence could not be phosphorylated (Figures adapted with kind permission from University of Delaware: Molecular Mechanisms of Farnesyl Transferase Inhibitor Treatment On Inflammatory Breast Cancer, Chapter 4, Heather Lehman 2011)

These findings suggest that Akt1 phosphorylation of RhoC GTPase mediates the invasion of inflammatory breast cancer. It is unknown how RhoC GTPase mediate invasion and metastasis in IBC and how phosphorylation affects RhoC GTPase. Knowing the impact of phosphorylation on RhoC GTPase holds considerable hope for IBC patients by allowing us create specific therapy against RhoC GTPase. In this research we will examine our hypothesis, which is that Akt1 phosphorylation of RhoC GTPase influences its interactions with upstream regulatory proteins.

Figure 1.9. Model of Akt1 phosphorylation of RhoC (Figure derived with kind permission from University of Delaware: Molecular Mechanisms of Farnesyl Transferase Inhibitor Treatment on Inflammatory Breast Cancer, Chapter 6, Heather Lehman 2011)

1.8 PI3K/Akt The phosphoinositide 3-kinase (PI3K)/Akt (Protein kinase B) signaling pathway is an important signaling pathway that control cell survival, growth,

apoptosis, and metabolism. Moreover, this signaling pathway is involved in cellular motility, invasion, and metastasis. Aberrant activation of this pathway has shown to be involved in cancer, thus it is an attractive therapeutic target that can benefit cancer patients (Manning & Cantley, 2007; Testa & Bellacosa, 2001). PI3Ks are lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring in inositol phospholipid to produce the second messenger phosphatidylinositol-3,4,5 triphosphate (PIP3). Akt/PKB, which is a signal transducer of PI3K pathway, is a serine/threonine kinase that belongs to the AGC kinase superfamily. It is composed of three domains: PH domain (Pleckstrin homology domain), a kinase domain, and a C-terminal domain (Song, Ouyang, & Bao). Activation of receptor tyrosine kinases by specific signaling molecules such as insulin-like growth factor-1 (IGF-1) leads to the activation of PI3K, thus synthesizing PIP3. PIP3 works as a docking site and recruits two protein kinases that contain PH domains to the plasma membrane: Akt and PDK1 (phosphoinositide- dependent protein kinase 1) enabling PDK1 to phosphorylate Akt on Thr308, which is highly conserved and lies within the kinase domain. To become fully activated, Akt needs another phosphorylation on Ser473, which lies in the hydrophobic motif. This allows Akt to dissociate from the membrane and translocate to the cytoplasm, nucleus, mitochondria and other organelles to transduce signals by phophorylating a number of substrates such as BAD, caspase 9, GSk3β, and Rac1 (Wong, Engelman, & Cantley, 2010). There are three highly similar isoforms of Akt: Akt1(PKBα), Akt2(PKBβ), and Akt3(PKBγ). Although these isoforms are activated through similar mechanisms, they differ in their tissue specificity and functional roles. Akt1 is the

most ubiquitous in tissues and Akt1-null mice show developmental defects and growth retardation. Akt2 is also abundant, but is specifically ubiquitous in insulin-responsive tissues such as skeletal muscles and brown fat. Akt2-null mice displayed defects in glucose homeostasis and developed insulin-resistant diabetes. Akt3 is expressed at lower levels and it can be found in neuronal tissues. Akt3-null mice had a decrease in brain size and brain developmental defects (Lehman, 2011). The three isoforms plays totally distinct roles in cancer. Akt1has been shown in recent studies to be involved in breast, ovarian, and colorectal carcinoma through a mutation in the lipid-binding pocket of Akt1 that activates it by localization to the plasma membrane, thus activating downstream signals. Akt1 decreases mammary epithelial cell migration in non-inflammatory breast cancers. On the other hand, inhibition of Akt1in inflammatory breast cancer cell lines significantly decreases their invasive capabilities, which indicates the important role of Akt1 in IBC invasion (Lehman, 2011). Akt2 is also involved in ovarian cancer (Cheng et al., 1992). In contrast to Akt1, Akt2 plays a pro-invasive role in non-inflammatory breast cancer, promoting adhesion and migration of breast cancer cells (Irie et al., 2005; Manning & Cantley, 2007) and its inhibition significantly decreases non- IBC cell invasion. On the other hand, studies suggest that Akt3 contributes to the aggressiveness of steroid hormone- insensitive cancers. Akt3 mRNA is upregulated and has increased activity in estrogen receptor (ER)-negative breast tumors. In addition, it has an increased activity in androgen-insensitive prostate cancer cell lines (PCa) (Nakatani et al., 1999). Akt3, but not Akt1, has an important effect on IBC cell survival. It’s inhibition leads to a significant decrease in apoptosis (Lehman, 2011).

Chapter 2

MATERIALS AND METHODS

2.1 Cell Culture The inflammatory breast cancer line SUM149 which is derived from a primary IBC was maintained at 37ºC in a 90% : 10% air to CO2 in Ham’s F12 medium ((Mediatech, Inc., Manassas, VA, USA, , #10-080 ) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA, #FP-0500-A), 1% L-Glutamine (Mediatech, , Inc., #25-005), 1% Penicillin/Streptomycin (Mediatech, ,Inc., #30-001), 1% Antibiotic/Antimycotic (Mediatech, , Inc., #30-004), 1% Insulin/Transferrin/Selenium cocktail (Mediatech, #25-800), and 1μg/ml Hydrocortisone (Sigma-Aldrich, #H0888). Hydrocortisone (Invitrogen, Carlsbad, CA, USA), and an insulin/transferring/selenium cocktail (Gibco, Carlsbad, CA).

2.2 in vitro Transfection Experiments Using FuGene HD transfection reagent (Roche, Branchburg, NJ), SUM149 cells were transiently transfected when they are 60-80% confluent with siRNA directed against Akt1, a scrambled siRNA control (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), wildtype RhoC 3xHA, RhoCS73A, RhoCS73D. Transfected cells were incubated at 37ºC in 5% CO2 for 48 hours. The SUM149 cell line was also treated with 10 µM of a pharmacological Akt inhibitor (Akt Inhibitor II from

Calbiochem, Gibbstown, NJ) directly applied to the cells and incubated for 24 hours at 37 ºC in 5% CO2.

2.3 RhoC Activation Assay At harvesting time, media was removed and cells are washed with PBS and put on ice. Cells were then lysed using GST-fish buffer (10% glycerol, 50 mM

Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 2mM MgCl2) in addition to 5μl/ml protease inhibitor cocktail (Calbiochem, Gibstown, NJ)) and 10μl/ml phosphatase inhibitor (Thermo Scientific, #78420) and were kept on ice for 5 minutes. Cells were then harvested using a flat scraper and transferred to a microcentrifuge tube and sonicated for 30 seconds. Then the cell lysate was centrifuged for 20,000 rpm for 5 minutes at 4 ºC. Supernatant was then transferred to a fresh centrifuge tube and snap frozen in liquid nitrogen and stored at -80 ºC or used immediately after determining protein concentration. A BCA Protein Assay kit (Thermo Scientific, #23225) was used to determine protein concentration at an absorbance wavelength of 562 nm. A total 1000µg protein from each sample is needed to do the RhoC activation assay. The fusion protein needs to be fresh for each experiment. It is prepared from the glycerol stocks of E. coli which express C21, the Rho binding domain of RhoC’s effector protein Rhotekin. This C21 is fused to a GST protein in the bacteria. Approximately 5µl of glycerol stock was then added to 50 ml LB (Lauria-Bertani) broth containing ampicillin (100 µg/ml). Cells were then grown overnight at 37ºC with shaking. Following incubation, the starter culture was then diluted 1:10 in 500 ml of ampicillin (100 µg/ml) containing LB and incubated for 1h at 37ºC with shaking. After one hour, 0,1 mM IPTG (isopropyl β-D1 thio galactopyranoside) was added to

induce GST fusion protein production. Cells were left to continue growing for 2 hours at 37ºC with shaking. Bacterial cells were then harvested by centrifugation with Sorvall GSA-3 rotator at 500 rpm for 20 minutes at 4ºC. Following centrifugation the supernatant was removed and cells were resuspended in 10 ml bacterial lysis buffer (20% sucrose, 10% glycerol, 50 mM Tris-HCl pH 8.0, 0.2 mM Na2S2O5, 2 mM MgCl2, 2 mM DDT, fresh protease inhibitor). Then cells were sonicated in lysis buffer for two to three minutes with a fisher Sonic Dismembrator Model 500 set to mark 6. The cell lysate was then centrifuged at 10,000 rpm for twenty minutes at 4ºC using Sorvall SS-34 rotor. The supernatant was then removed to a new tube, and one ml of fifty percent glutathione-Sepharose 4B bead/lysis buffer slurry was added to it. This suspension was rotated for thirty minutes at 4ºC. After rotation, the beads were retrived by centrifugation. Then the beads were washed with 5 ml lysis buffer and spun down at 10.000 rpm for 5 minutes at 4ºC using Sorvall SS-34 rotor. This washing processes was repeated for three times. The final bead was then resuspended in one ml of GST-Fish buffer. For each sample 1000 µg of protein was added to 100 µl of GST-RBD/glutathione-Sepharose bead slurry. The mixture was then put in the tube rotator in the cold room to rotate overnight. After the samples have rotated overnight, they were spun down in the centrifuge for 10 minutes at 10,000 rpm at 4ºC. The supernatant is then discarded. Then, pellets were washed with 1 ml GST-Fish buffer and spun down for 30-45 seconds at 15,000 rpm at room temperature. The supernatant was discarded. This washing processes was repeated for three times. Then, beads were resuspended in 30

microliters of GST-Fish buffer. The samples are ready to be used for western blot, or can be frozen at -80ºC.

2.4 Western Blot Analysis The bead, which was prepared in the activation assay and was resuspended in 30 microliters of GST-Fish buffer, of each sample as well as 40 µg for total protein of each sample were mixed with 2X Laemeli buffer (Sigma-Aldrich, #S3401). Then samples were boiled to be denatured for 3-5 minutes in boiling water, separated by SDS-PAGE on Criterion pre-cast 4-20% Tris-HCl gels (BioRad, Hercules, CA), and transferred to nitrocellulose at 110V for 1 hour. Non-specific binding was blocked by overnight incubation with 3% powdered milk (Nestle Carnation) in 1X PBST (phosphate-buffered saline) (Thermo Scientific, Waltham, MA) with 0.05% Tween-20 (Sigma Chemical Co., St. Louis, MO) for one hour at room temperature. Then primary antibody, which is IgY chicken antibody made in our lab specifically against RhoC, was added to the membrane at a concentration of 1: 250 and incubated overnight at 4°C, while rocking. After incubation, membranes were washed for four times with PBST for 3 minutes each and once in PBS for 3 minutes. After incubation, the secondary antibody, donkey anti-chicken-horseradish peroxidase (HRP), donkey anti-chicken IgY agarose (Gallus Immunotech, Inc., Cary, NC) was added to the membrane at a concentration of 1:5000 and incubated for one hour at room temprature. After incubation with the secondary antibody, membrane was washed for four times with PBST for 3 minutes each and once in PBS for 3 minutes. Following washing, protein bands were visualized with the use of ECL (Millipore, Co., Billerica, MA) and exposed to Hyperfilm (Amersham, Piscataway, NJ). The

membranes were then stripped and reprobed for beta-Actin. β-actin (Cell Signalling, #4967L).

2.5 RhoGAP Assay SUM149 IBC cells were plated in 6-well tissue culture dishes and allowed to reach 75% confluency. Cells were transfected using FugeneHD transfection reagent with a scrambled control siRNA, Akt1-specific siRNA, RhoCS73A, or RhoCS73D plasmid. The Akt pharmacologic inhibitor (iAkt) was added directly to the cells. The untreated cells were used as a control. Treatments were allowed to incubate for 24-48 hours and protein was harvested. Protein was harvested using RIPA buffer as previously described. The entire biochemical reaction was carried out in a 96-well tissue culture plate at room temperature. Twenty-five micrograms of each protein sample was added to 1X RhoGAP assay reaction buffer (Cytoskeleton, Inc.). An 800 µM GTP solution was added to each reaction mixture, the plate was shaken for five seconds, and the reaction was incubated at room temperature for 20 minutes. Immediately after the incubation, CytoPhos reagent (Cytoskeleton, Inc.) was added into each well and a green color was allowed to develop for approximately 6 minutes, indicating inorganic phosphate production. The absorbance of the plate was read at a wavelength of 650 nm. In addition to the protein samples of interest, control reactions included His-tagged RhoC GTPase protein only (Cytoskeleton, Inc.), p50 RhoGAP, and reaction buffer only.

2.6 Immunoprecipitation For immunprecipitation assays, proteins were harvested using RIPA buffer (1X PBS, 150mM NaCl, 50mM Tris-HCl pH7.4, 2mM EDTA, 1% NP-40,

0.1% SDS), 10μl/ml phosphatase inhibitor (Thermo Scientific, #78420), and 5μl/ml protease inhibitor cocktail (Calbiochem, Gibstown, NJ)) was used to harvest protein from each cell line. A BCA Protein Assay kit (Thermo Scientific, #23225) was used to determine protein concentration at an absorbance wavelength of 562 nm. Whole cell lysates (250 μg) were incubated overnight at 4ºC with primary antibodies specific to RhoC GTPase (developed in our lab), or GDI mouse monoclonal IgG (Santa Cruz Biotechnology, #13120 ) Antibody-bound proteins were incubated with donkey anti- chicken IgY agarose (Gallus Immunotech, Inc., Cary, NC) or Protein A/G PLUS- Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4ºC with gentle rocking for three hours. Samples were washed five times with cell lysis buffer and 1X PBS. Immunoprecipitates were then resuspended in Laemmli buffer and boiled for five minutes. Samples were separated by SDS-PAGE on a 4-12% Tris-HCl Criterion precast gel (BioRad, Hercules, CA) and transferred to a nitrocellulose membrane. The membrane was blocked by overnight incubation with 4% BSA in phosphate-buffered saline with 0.05% Tween-20 or 3% powdered milk (Nestle Carnation) in phosphate- buffered saline with 0.05% Tween-20 (Sigma Chemical Co., St. Louis, MO). Anti- RhoC immunoprecipitate blots were incubated with GDI mouse monoclonal IgG antibody. Anti GDI imunoprecipitate blots were incubated with RhoC GTPase antibody overnight. After washing stips, secondary antibody was used as described previously. Protein bands were then visualized with the use of ECL (Millipore Co., Billerica, MA).

2.7 GEF Fluorescence Assay SUM149 IBC cells were plated in 6-well tissue culture dishes and allowedto reach 75% confluency. Cells were transfected as described above. 10 µM of

Akt pharmacologic inhibitor (iAkt) was added directly to the cells. The untreated cells were used as a control. Treatments were allowed to incubate for 24-48 hours and protein was harvested. Protein was harvested using RIPA buffer as previously described. The entire biochemical reaction was carried out in a black flat-bottom 96- well half plate (Corning plate Cat# 3686) at room temperature. Twenty-five micrograms of each protein sample was added to 2x exchange assay reaction buffer (Cytoskeleton, Inc.), which contain 1.5 µM mant-GTP (the florescent nucleotide analog N-methylanthraniloyl-GTP). Distilled water was added to adjust the volume to 90 µl in each well. The fluorescence of the reaction mixture immediately was read (ex: 450nm, em: 460 nm) for 150 seconds [five readings]. After five readings 10µl of either GEF, hDbs protein (8µM) or distilled water (intrinsic control) was added to in respective wells and immediately after pipette up and down twice, fluorescence was read (ex: 450nm , em: 460 nm) for 30 minutes [60 readings]. In addition to the protein samples of interest, control reactions included His-tagged RhoC GTPase protein only (Cytoskeleton, Inc.), hDbs- His protein, and reaction buffer only.

2.8 2.8 Statistical Analysis All experiments were performed on a minimum of three separate times with individual transfections or treatments and performed with no less than three replicates per experiment. Statistical analysis of the combined experiments was performed using GraphPad Prism. Significance was defined as a P value ≤0.001. Data is represented as mean ± standard deviation. For Western blot analysis, images ImageJ 1.45 software (http://rsbweb.nih.gov/ij/) was used and densitometry was performed according to recommendations outlined by Gassmann et al. (Gassmann, Grenacher, Rohde, &

Vogel). Western blot images were first converted to grayscale. The density profile of each band was determined by arbitrarily defining a rectangular box around the bands. This box was then used to measure all lanes and a profile plot was formed, with peaks of the plots representing the relative density of the contents of the rectangle over each lane. Each peak of interest was analyzed using the wand tool of ImageJ and labeled with a size, expressed as a percentage of the total size of all the peaks being analyzed. This was performed first for beta-actin loading controls followed by the proteins bands of interest. Relative density values were calculated for the loading controls and proteins of interest by choosing one standard and one sample as a control and dividing the percent values of each band by the control protein percent value. Finally, the relative densities of the proteins of interest were scaled by using the relative densities of the loading controls. The relative density of each protein of interest was divided by the relative density for the corresponding loading control to gain final values.

Chapter 3

RESULTS

3.1 Phosphorylation of RhoC Does not Affect its Activation It has been shown by our lab that Akt1 phosphorylation of RhoC GTPses is essential for IBC invasive phenotype. We would like to know the consequences of this phosphorylation. To be effective, RhoC GTPase needs to transiently cycle between GTP bound and GDP bound states. RhoC can bind to its effector proteins to lead to specific cellular response only in GTP bound form (K. van Golen, 2009). The first aim of this study is to determine if Akt1 phosphorylation of RhoC GTPase affects RhoC’s activation. For this aim, RhoC activation assays were performed to look at active levels of RhoC in SUM149 IBC cells. This experiment is based on pulling down active RhoC with its effector protein, Rhotekin. Figure 3.1 is a diagram of the main principles of my experimental design. The idea is that we start with a bacterial culture of E. coli which has expressed in it C21, which is the Rho binding domain of RhoC’s effector protein Rhotekin. This C21 is fused to a GST protein in the bacteria. The GST-C21 is incubated with glutathione sepharose beads, which bind to the GST portion to make the GST-fusion protein (Lucey, Unger, & van Golen, 2010). I then incubated SUM149 cell lysates, including the mentioned treatments in chapter 2, with the GST-fusion protein overnight. This allows the RhoC in the cells to bind to the C21 Rho binding domain. The SUM149 IBC cells were treated with the mentioned treatments. After washing and centrifugation steps the C21-RhoC bound

complex is eluted out, and then prepared for gel electrophoresis and western blotting. The blots are then probed for both total RhoC levels and active RhoC levels in all of the samples. As shown in figure 3.2a, there was no significant change in the active and total level of RhoC between all treatments. significance ( P of RhoC 3XHA vs RhoCS73A = 0.2356, P of RhoC 3XHA vs RhoCS73D =0.1742, P of UT vs iAkt = 0.4230, and P of siCtrl vs siAkt1 = 0.5003).

Figure 3.1 Diagram shows RhoC activation assay. (Figure adapted with kind permission from M Lucey, H Unger, E Dashner and KL van Golen, “RhoC GTPase Activation Assay.” JoVE, 2010 Aug 22. http://www.jove.com/index/details.stp?iid=2083, doi: 10.3791/2083)

Figure 3.2 RhoC GTPase ativity is not affected by inhibition of its phosphorylation by Akt1 in SUM149c cells. (a) Effectiveness of siRNA against AKT1 in SUM149 cells. Densetometry was performed and relative intensity of AKT1 represented as percentage. (b) Representative RhoC pulldown activation assay of untreated cells as control [UT] and cells that was transfected with RhoC 73A [mutant], RhoC S73D [phosphomimetic mutant], RhoC 3XHA [tagged RhoC], AKT inhibitor [iAKT], si AKT1 and si control [siCtrl]. Active and total RhoC were detected using antiRhoC IgY. (c) Densitometry using imageJ determined the relative expression of total and active RhoC that is normalized to B-Actin represented as arbitrary units (A.U). Data are represented as mean ± standard deviation using GraphPad Prism.

3.2 Inhibition of RhoC Phosphorylation by Akt1 Does not Affect GTP Hydrolysis The second aim of my project is to demonstrate the interaction of Rho GDI, GEFs, and GAPs with RhoC GTPase to test my hypothesis, which is is Akt1 phosphorylation of RhoC GTPase influences its interactions with upstream regulatory proteins. RhoGAP is one of the upstream regulatory proteins in the RhoC GTPase cycle. It is an essential negative regulator that enhances the intrinsic GTPase activity which hydrolyzes GTP to GDP. We would like to know the effect of inhibition of Akt1 phophorylation on the ability of RhoC to interact with GAP. We will determine that by looking for the difference in the ability in hydrolyzing GTP to GDP. We used the RhoGAP assay, which is an in vitro colorimetric assay that measures the production of inorganic phosphate that is produced during the hydrolysis of GTP to GDP (Tcherkezian & Lamarche-Vane, 2007). Each of protein samples with different treatments, as previousely mentioned, was mixed with the reaction buffer and 800 µM of GTP and with P50 RhoGAP domain. After 20 minutes incubation at room temprature, CytoPhos reagent (Cytoskeleton, Inc.) was added into each well and a green color was allowed to develop for approximately 6 minutes, indicating inorganic phosphate production. Absorbancce was read at wavelength 650nm.The results are shown in the figure 3.3, there is a slight decrease in GTPases capabilities of RhoC when Akt phosphorylation is inhibited either with the RhoCS73 mutant, siAkt1, or pharmacologic inhibitor of Akt (iAkt) in comparision to the control. However, this decrease is not significance ( P of UT vs RhoCS73A = 0.1900, P of UT vs RhoCS73D =0.7176, P of UT vs iAkt = 0.5978, and P of siCtrl vs siAkt1 = 0.3402).

Figure 3.3 Inhibition of Akt1 phosphorylation of RhoC GTPases does not significantly alter hydrolysis activity. An in vitro GAP assay measuring the levels of inorganic phosphate produced by cell during GTP hydrolysis when stimulated with GTP shows a slight decrease in GAP activity with inhibition of RhoC phosphorylation by Akt. Data are represented as mean ± SEM. SUM149 cells was used. Untreated cells was used as control [UT] and cells that was transfected with RhoC 73A [mutant], RhoC S73D [phosphomimatic mutant], RhoC 3XHA [taged RhoC], AKT inhibitor [iAKT], si AKT1 and si control [siCtrl]

3.3 Interaction of RhoC with GDI Is not Affected by RhoC Phosphorylation GDI is one of RhoC’s upstream regulatory proteins that sequester RhoC GTPases in the cytoplasm and inhibit the dissociation of the bound nucleotide (both GDP and GTP). It binds to RhoC through its flexible C-terminal domain that mask the isoprenyl moiety and through its regulatory arm that binds switch I and switch II regions. Serine 73 lies within switch II region and it has been shown that it is a putative Akt1 phosphorylation site that is required for metastasis of IBC. We would like to test the inhibition of this phosphorylation on the interaction between GDI and RhoC GTPase. To know this, I have done an immunoprecipitation assay for both GDI and RhoC followed by western bloting for RhoC and GDI respectively. As shown in figure 3.4 from the band and by using densitometry, there is no important difference between all treatments as compared to the control.

Figure 3.4 Interaction of RhoC GTPase with Rho GDI is not affected by the inhibition of Akt1 phosphorylation in SUM149 cells. (a) Immunoprecipitation of RhoC GTPase followed by an immunoblot with a RhoGDIα specific antibody. SUM 149 cells was used. Untreated cells was used as control [UT] and cells that was transfected with RhoC 73A [mutant], RhoC S73D [phosphomimetic mutant], RhoC 3XHA [tagged RhoC], AKT inhibitor [iAKT], si AKT1 and si control [siCtrl]. (b) Immunoprecipitation of RhoGDIα followed by an immunoblot with a RhoC specific antibody using the same treatments mentioned in (a)

3.4 Phosphorylation of RhoC GTPase by Akt1 Does not Affect RhoC/GEF Interaction GEF is a major class of RhoC’s GTPases upstream regulatory proteins that activate RhoC by exchanging GDP for GTP. The catalytic domain by which GEF promotes the activity of RhoC is the DbI (DH) domain (Schmidt & Hall, 2002). To

examine the third part of my second aim in which I would like to demonstrate the interaction of RhoGEFs with RhoC GTPase, I have performed RhoGEF exchange assay. This assay measures the uptake of the fluoresecent nucleotide analog N- methylanthraniloyl-GTP (mant-GTP) into GTPases. The uptake can be measured due to the spectroscopic difference between free and GTPase-bound mant-GTP. As mant- GTP gets bound in the nucleotide binding pocket of GTPase, its fluorescence (ex: 450 nm, em: 460nm) increases. Therfore, the enhancement of mant-GTP fluorescence intensity in the presence of small GTPase indicates nucleotide uptake (or exchange for already bound nucleotide) by the GTPase (Cytoskeleton inc.). In this assay, I used cell lysate to test each treatment. I mixed 25µg of with 2X exchange assay reaction buffer, which contain 1.5 µM mant-GTP and distilled water was added to adjust the volume. The fluorescence of the reaction mixture immediately was read (ex: 450nm, em: 460 nm) for 150 seconds [five readings]. After five readings 10µl of either GEF, hDbs protein (8µM) or distilled water (intrinsic control) was added. As shown in figure 3.5, there is only the effect of GEF on RhoC, but there is no difference between treatments as compared to the control ( P of RhoC 3XHA vs RhoCS73A = 0.9799, P of RhoC 3XHA vs RhoCS73D =0.9987, P of UT vs iAkt = 0.7600, and P of siCtrl vs siAkt1 = 0.8747).

Figure 3.5 Akt1 phosphorylation of RhoC GTPase dose not affect its interaction with RhoGEF. An in vitro RhoGEF exchange assay measuring nucleotide exchange by measuring the uptake of the fluorescent nucleotide analog , mant-GTP. The uptake

58 can be measured due to spectroscopic difference between free and GTPase bound mant-GTP. As mant-GTP gets bound in the nucleotide binding pocket of GTPase, its fluorescence (ex: 450 nm, em: 460nm) increases. Therefore, enhancement of fluorescent intensity in the presence of RhoC GTPase [treated and untreated SUM149 cells] and GEF will reflect the effect of inhibition of Akt1 phosphorylation on GEF activity. (a) Shows the Dbs exchange activity for SUM149 untreated cells. (b) Shows the Dbs exchange activity for SUM149 cells transfected with RhoC 3XHA. (c) Shows the Dbs exchange activity for SUM149 cells transfected with RhoC S73A.(d) Shows the Dbs exchange activity for SUM149 cells transfected with RhoC S73D. (e) Shows the Dbs exchange activity for SUM149 cells treated with AKT inhibitor. (f) Shows the Dbs exchange activity for SUM149 cells transfected with siAKT1. (g) Shows the Dbs exchange activity for SUM149 cells transfected with si control. (h). Student t test using JMP shows mean comparision ± SEM.

Chapter 4

DISCUSSION, CONCLUSION AND FUTURE DIRECTIONS

4.1 Discussion Inflammatory breast cancer is the most aggressive and atrocious form of locally advanced breast cancer with unknown etiology. This dreadful disease is characterized by the rapidity of its progression and it appears to be metastatic upon inception, which is the most lethal attribute of this cancer. Women affected with IBC have less than 45% and 20% 5- and 10-year disease free survival rate, respectively. After one year from diagnosis, 90% of women with IBC have gross metastasis. IBC patients may have better survival rates with the understanding of the molecules that drive invasion and metastasis of IBC (Hance et al., 2005). The incidence in the U.S. is 1- 6% of all newly diagnosed women with breast cancer each year. According to SEER program data that compares trends and pattern of breast cancer, between 1975-1977 and 1990-1992, the incidence of IBC increased from 0.3 to 0.7 cases per 100,000 person-years (Cristofanilli et al., 2003). In addition, IBC has a peculiar geographic distribution. North African countries appear to have a higher frequency of IBC (~10%), according to studies in Egypt and Tunisia. Previously, it was thought that the incidence of IBC in North Africa was as high as 30%. However, with stricter criteria of diagnosis and re- evaluation of historic cases, the incidence levels of in countries such as Tunisia, IBC

incidence is approximately one third of what was originally thought (Boussen et al., 2010; Lo et al., 2008). Regardless of geographical location or population affected, IBC is extremely deadly and conventional therapies are ineffective. IBC has notoriously been understudied. Prior to the year 1999, IBC was studied incidentally, that is individual IBC samples would be included in a larger breast cancer study. However, very little was learned about the molecular mechanisms underlying IBC. In 1999, our laboratory published the first study focused on understanding IBC. A comparison of gene expression in the SUM149 IBC cell line, two different human mammary epithelial cell (HMECs) lines and lymphocytes from the patient that the SUM149 cells were isolated from, was performed. A group of 17 differentially expressed genes were isolated and the expression levels of these genes blindly tested in IBC and Stage-matched non-IBC patient samples. Two genes, RhoC GTPase and Wisp3 were shown to be altered significantly in IBC patients (K. L. van Golen et al., 1999; K. L. van Golen et al., 2000b). Our laboratory has shown that RhoC GTPase is overexpressed in inflammatory breast cancer. Moreover, RhoC GTPases is required for IBC cell invasion and metastasis. RhoC GTPase has been indicated to be essential for metastasis in a large number of cancers. RhoC GTPase is overexpressed in 90% of IBC tumors compared to stage matched non-IBC tumors (K. L. van Golen et al., 2000a). RhoC GTPase act may as molecular switch to control many cellular aspects such as cytoskeleton reorganization (Ridley, 2001; Sahai & Marshall, 2002). RhoC is regulated by upstream regulatory proteins: GEF, GAPa and GDIs. In addition

to its regulation with upstream regulatory protein, our laboratory found that RhoC is phosphorylated by Akt1 and this phosphorylation is needed for metastasis and invasion of the SUM 149 IBC cell line. How RhoC mediates invasion and how phosphorylation may affect this process is still mystery. Previous work from our lab showed that the PI3K/Akt signaling pathway is upregulated in IBC patients while there are also high levels of serine phosphorylation in the SUM149 inflammatory breast cancer cells. Inhibition of Akt1 significantly decreased the invasive capabilities of SUM149 cells. In addition, there is also a significant decrease in invasion of SUM149 cells after transfection with an Akt phosphorylation mutant (RhoCS73A), where the putative Akt phosphorylation site in the RhoC sequence is mutated. Our laboratory found that Akt1 is responsible for IBC cell motility and invasion through phosphorylation of RhoC GTPase (Lehman, 2011). RhoC has been shown to have a putative Akt phosphorylation site at serine 73 in its sequence and there is a high level of phosphoserine in the SUM 149 IBC cell line. Akt1 phosphorylation site, Serine73, lies within switch II region of RhoC GTPase. Interestingly, the crystal structure of RhoC proposed that there are two activated states for RhoC [partially and fully active] during its conversion from inactive GDP-bound to active GTP-bound states (Dias & Cerione, 2007). This phosphorylation site might play an important role in the conformational changes of RhoC and its ability to interact with upstream regulatory proteins and/or downstream effector proteins. In this research project, we investigated the effect of Akt1 phosphorylation on the activity of RhoC and on its interaction with upstream regulators.

Because previous work demonstrated that inhibition of Akt1 phosphorylation of RhoC GTPase leads to decreased IBC cell invasion resembled prior studies that specifically inhibited RhoC activity, we postulated that phosphorylation would have an effect on the activation state of the GTPase. As detailed above, activity can also be altered by interaction with upstream regulatory proteins such as RhoGDIs, RhoGEFs, and RhoGAPs. Total Rho activity is a snapshot in time. Therefore, although no changes in total activity may be readily apparent, changes in the interactions of the GTPase with upstream effectors may affect the frequency or longevity of activation. The activation assay showed that there is little to no change in the activity levels of RhoC upon inhibition of phosphorylation by Akt1. This implies that RhoC is still able to bind with GTP and interact with the Rho binding domain (RBD) of the downstream effector Rhotekin, even if we inhibit Akt1 phosphorylation. However, there are important issues that were not considered in this assay. First, since there are two activated states for RhoC (Dias & Cerione, 2007), we still need to investigate whether this GTP bound state is fully or partially active. That is to say the protein may not go through both conformational changes. To date, the significance of the conformational changes is unknown. The changes in conformational change may be studied by examining the crystal structure. The second issue is that we are pulling down active RhoC with only a domain of its effector protein, Rhotekin. To date, dozens of downstream Rho effector proteins have been identified, yet those specific to RhoC remain unknown (K. van Golen, 2009). This activation assay gives us an idea that RhoC is still active in all cases of inhibiting phosphorylation. However, this also does not give us the real picture about the effect of the negative charge of the

phosphate group on the interaction between RhoC and the whole Rhotekin protein. Because every protein has its unique conformation, phosphorylation of Rho may increase its binding affinity for some effector proteins or hinder its binding for others through the effect of its negative charge. The consequences of Rho protein phosphorylation remain debated. RhoA GTPase contains a conspicuous serine in the 188 position of the C-terminal hypervariable region. Studies are conflicting with some showing that phosphorylation is required for activation whereas others show that phosphorylation inhibits activation. One possibility is that the effect of RhoA phosphorylation is cell-type dependent (Ellerbroek et al.; Rolli-Derkinderen et al., 2005; K. van Golen, 2009). Currently, it is believed that phosphorylation affects cellular localization of RhoA through altered charge and/or interactions with RhoGDIs. Similar to RhoA, the phenotypic effect of phosphorylation of RhoC serine73 appears to be IBC-specific (K. van Golen, 2009). Since Akt1 phosphorylation of RhoC is important for metastasis and invasion of the SUM149 IBC cell line (Lehman, 2011), this phosphorylation probably affect the interaction of RhoC with its downstream effectors and/or cellular localization of RhoC GTPases. The Rho GAP assay also showed that slight decrease in the GTP hydrolysis rates of RhoC GTPase with inhibition of Akt phosphorylation site of RhoC. In this test, however, GTP hydrolysis cannot be attributed to the effect of GAP on RhoC only because there are other Rho GTPase proteins that may bind with GAP and hydrolyse GTP to GDP to produce the inorganic phosphate , which may interfere with our results. Thus this assay examines the GTP hydrolysis for all GTPases that are found in the cell and it is not specific for RhoC. For specific results, wild type RhoC

and mutated RhoC should be purified. Then, every purified type should be examined for its GTPase hydrolysis ability. Unfortunately, this is beyond the technical capacity of the assay. In the GAP assay, we wanted to examine the effect of Akt1 phosphorylation of RhoC on the interaction of RhoC with GAP. In this experiment, however, we used the GAP binding domain only, which cannot show us whether or how negative charge of a phosphate group can affect protein- protein interaction. Moreover, this phosphate group may affect RhoC localization and the interaction in vitro is not an indication for a real interaction in vivo. Finally, GAP is regulated by phosphorylation (Manser, 2005; Symons, 2004) and the inhibition of Akt1 phosphorylation in vivo may affect other signaling pathways, through cross talk, which in turn may regulate GAP function. Thus, to examine the real Interaction in vivo, we may use the fluorescence resonance energy transfer (FRET) technique as discussed in future directions. The immunoprecipitation of GDI with RhoC and vice versa showed little to no effect upon inhibition of Akt phosphorylation of RhoC. GDI binds Rho proteins through two regions. The isoprenyl binding pocket and the acidic patch, which lies within the c- terminal, and the regulatory arm, which is located within the N-terminal region and binds to switch I and switch II domain in a way that stabilizes nucleotide state through a coordination between threonine 35 and Mg+2 that compensates the negative charge of the phosphate group (Manser, 2005). Switch II region contains the serine 73 of RhoC (K. van Golen, 2009) that we are testing the effect of its phosphorylation in our hypothesis. It seems that this serine 73 phosphorylation is not essential for GDI binding. However, it may affect the localization of RhoGDI complex

and/or affect the regulation of GDI by phosphorylation, thus enhance or hinder the dissociation of GDI. In the GEF activity fluorescence assay, we can clearly see the effect of DbI domain on the activity of Rho. However, it appears that there is no effect of RhoC phosphorylation on its interaction with DbI binding domain. The same issue is applied in this experiment, which is using only the DbI domain that is responsible for catalytic activity to stimulate exchange of GDP for GTP (Der, 2011). To determine the effect of RhoC phosphorylation on its interaction with GEF, we should use whole protein and monitor the interaction by FRET technique.

4.2 Conclusions In this project we examined the most obvious regulators of Rho activity that would affect RhoC GTPase activation and as a consequence IBC cell invasion. Although the total activity or RhoC and none of the obvious upstream regulators or are affected by serine73 phosphorylation, this project has given us some definite conclusions expanding our knowledge of IBC and RhoC biology.

 Akt1 does not affect RhoC activation.  Phosphorylation of serine 73 has no effect on GEF stimulated release of GDP.  Phosphorylation of serine 73 has no effect on GAP activated hydrolysis.  Phosphorylation of serine 73 has no effect on RhoC/GDI interactions. .

4.3 Future Direction In this study we examined the consequence of Akt1 phosphorylation of RhoC GTPase. Although we did not detect changes in the activation state or RhoC or in the interaction of upstream effectors with the GTPase, this project has given us a definite direction for future studies. For future work, we would like to know first whether Akt1 phosphorylation affects RhoC localization. We can do this purpose by two methods: 1] By using fluorescence live cell microscopy: We could perform a transient transfection of RFP( Red fluorescent protein) -labeled wild type RhoC, RFP-labeled RhoC S73A, and RFP-labeled RhoC S73D,RFP labled RhoC in cells treated with a pharmacological inhibitor Akt, siRNA specific for Akt1, and a siRNA control]. We will be imaging cells by laser scanning microscopy and looking for changes in localization (ex. increased or decreased membrane binding) in response to mutation. 2] Cell fractionation: This experiment is based on the difference in solubility between the membrane part and the cytoplasmic part. To do this, we could harvest treated SUM149 cells [transfected with the constructs outlined above. Western blot analysis would allow us to see if there is a different compartmentalization of RhoC. Since phosphorylation could affect localization, we could find different mutant proteins localize to different compartments. We have suggested that the phosphorylation may affect RhoC’s interaction with its upstream regulatory protein. However, we have not looked at the downstream effector proteins. In the RhoC activation assay, I have used a part of an effector protein, C21, which is the RBD of RhoC’s effector protein Rhotekin, with

RhoC [using the previously mentioned treatments]. Then after pulling down active RhoC, I used western blot to see only the active levels RhoC which should have interacted with Rhotekin. The problem in this experiment is that using only a portion of protein does not give us real idea about protein-protein interaction. That is because we neglected the effect of the negatively charged phosphate group when it interacts with whole protein. The other problem in this experiment is that whole RhoC has been shown to interact with Rhotekin in vitro (K. van Golen, 2009), it is still not known if these two proteins meet each other and interact in vivo. The second question would be: if we find that these two proteins interact with each other in vitro and we previously assumed that phosphorylation may affect RhoC’s localization, Do these proteins still meet each other and interact within the cell? To answer this question, we can use two ways: using fluorescence resonance energy transfer (FRET) technique and immunoprecipitation as an alternative technique. The disadvantage of immunoprecipitation is that when we break the cell, some protein may interact with each other in vivo, but they may not have been interacted in vitro. On the other hand, by using FRET technique, we know if this interaction actually happens in the cell and we will see the interaction in an intact live cell. This technique is distance-dependent transfer of energy from a donor molecule to an acceptor molecule (Held, 2005).

 Express cyan fluorescent protein (CFP)- RhoC wild type [in addition using mutants and the other treatments]. This would be the donor dye chromopore that absorb energy.

 Express yellow fluorescent protein (YFP)-Rhotekin. Then, measure the FRET efficiency. This will be the acceptor to which the energy is transferred (Held, 2005).

We excite with blue light and see the color. If there is no interaction, the color will be the same and we will see blue light. But, if there is an interaction, we will see the yellow light (Held, 2005). Finally, to determine if phosphorylated RhoC interacts with different effector proteins, we could clone the RBD of a dozen or so known downstream Rho effectors. The RBDs could be expressed and interactions with wild type RhoC,

RhoCS73A and RhoCS73D loaded with nonhydrolyzable GTPS or GDP tested in vitro. This technique has been used successfully to identify RhoA effectors (K. van Golen, 2009). Thus, we need to answer some questions to be able to find way that helps targeting RhoC GTPase: does phosphorylation affects RhoC’s localization rather than activation? Does it affect both? Does the phosphorylation of RhoC affect its interaction of downstream effector proteins and does it determine which effector RhoC would bind?.

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From: Weam Elbezanti Date: Tue, May 1, 2012 at 3:19 AM Subject: I urgently need your response please [Not available on Science Direct.] To: [email protected] Cc: [email protected]

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Dear Weam Elbezanti

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From: Weam Elbezanti Date: Mon, May 7, 2012 at 7:26 PM Subject: permission To: Kenneth Van Golen

Dear Dr. van Golen

I would like to ask you for a permission please to use a figure, which is a diagram that shows RhoC activation assay.[ By Lucey M, Unger H, van Golen KLRhoC GTPase Activation Assay. J Vis Exp. 42 (2010)].

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From: Kenneth van Golen Date: Tue, May 8, 2012 at 8:17 AM Subject: Re: permission To: Weam Elbezanti

Weam-

With this email I give you permission to use the RhoC activation assay figure that you have put in your thesis.

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From: [email protected] To: [email protected]; [email protected] Subject: Time sensitive request Date: Thu, 26 Apr 2012 20:28:15 -0400 Dear Dr. Lehman

I am writing to inquire about permission to use some figures from your dissertation in my thesis. This is a time sensitive request, as I am hoping to obtain permission for use of this figure ASAP. The thesis will be submitted in a few days from the present time.

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I hope to hear from you soon. Sincerely,

Weam Elbezanti

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Dear Weam,

I give you full permission to use any figures you request from my dissertation. You can reference the figures as having permission from me for use.

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