Ovarian cancer (OVCA) is the leading cause of death among women due to a gynecologic malignancy, resulting in 15,500 deaths in 2012 in the United States. The major problems of ovarian cancer are late diagnosis and poor response to chemotherapeutic agents leading to poor five-year survival rate (18-35%). Ovarian canceris characterized by spreading along the peritoneal cavity. The cancer cells start to shed from ovarian carcinoma into the ascitic fluid as single cells and/or multicellular aggregates (MCAs) or spheroids. It has been found that MCAs/spheroids are highly resistant to chemotherapy. Resistance to chemotherapy has been linked to small signaling G-protein (small GTPases), such as Rac1 (Ras-Related C3 botulium toxin substrate 1) and Cdc42 (Cell division protein 42).
Their downstream effectors may play a role in OVCA metastasis as well as chemotherapeutic resistance. Ras GTPase-activating-like protein (IQGAP1) or 189-kDa scaffolding protein is an effector for Rac1 and Cdc42 that accumulates at cell-cell contact sites in the cytoplasm where E-cadherin, β-catenin, and α-catenin interact. IQGAP1 contains multiple binding sites for several molecules including Rac1/Cdc42, E-cadherin, actin, β-catenin, calmodulin, components of the mitogen-activated protein kinase pathway, and others, which are involved in cancer. Through interactions with these proteins, IQGAP1 plays a pivotal role in the regulation of cell-cell adhesion, migration, and signal transduction. IQGAP1 interacts with the β-catenin binding domainas a negative regulator of E-cadherin-mediated cell-cell adhesion. It dissociates the α-catenin from the β-catenin/cadherin complex resulting in weak cell-cell adhesion. It is expected that this interaction plays a role in disruption of MCAs/spheroids in OVCA.
Table of Contents
Abstract
Dedication
Acknowledgments
List of tables
List of figures
List of abbreviations
1. Chapter one: Introduction
1.1 Ovarian cancer
1.1.1 Incidence, etiology, risk factors, and screening
1.1.2 Types of ovarian cancer
1.1.3 Progression and subtypes of EOC
1.1.4 Stages of EOC
1.1.5 Treatment of EOC
1.1.6 Influence of OVCA progression on chemotherapy
1.1.7 Drug resistance in EOC
1.1.8 Mechanisms of drug resistance
1.1.9 MCAs and their resistance to therapy
1.2 The role of small GTPases in cancer progression
1.2.1 The Rho small GTPase family and their members Rac1 and Cdc42
1.2.2 Cellular functions and role of Rac1 and Cdc42 in cancer progression
1.3 Types of cell-cell adhesion
1.3.1 Classical cadherins mediated cell-cell adhesions in ovarian cancer
1.4 The relation between Rac1, Cdc42, and their effector IQGAP1 with cadherins
1.5 Inhibitors of Rac1 and Cdc
2.Chapter two: Rationale and goals
3. Research design/methodology
3.1 Specific Aim
3.1.1 Rationale for specific Aim
3.1.2 Research design for specific Aim
3.1.3 Methods for specific Aim
3.2 Specific Aim
3.2.1 Rationale for Aim
3.2.2 Research design for Aim
3.2.3 Methods for specific Aim
4.Summary and future directions
5. References
ABSTRACT
Ovarian cancer (OVCA) is the leading cause of death among women due to a gynecologic malignancy, resulting in 15,500 deaths in 2012 in the United States. The major problems of ovarian cancer are late diagnosis and poor response to chemotherapeutic agents leading to poor five-year survival rate (18-35%). Ovarian canceris characterized by spreading along the peritoneal cavity. The cancer cells start to shed from ovarian carcinoma into the ascitic fluid as single cells and/or multicellular aggregates (MCAs) or spheroids. It has been found that MCAs/spheroids are highly resistant to chemotherapy. Resistance to chemotherapy has been linked to small signaling G-protein (small GTPases), such as Rac1 (Ras- Related C3 botulium toxin substrate 1) and Cdc42 (Cell division protein 42). Their downstream effectors may play a role in OVCA metastasis as well as chemotherapeutic resistance. Ras GTPase-activating-like protein (IQGAP1) or 189- kDa scaffolding protein is an effector for Rac1 and Cdc42 that accumulates at cell- cell contact sites in the cytoplasm where E-cadherin, β-catenin, and α-catenin interact. IQGAP1 contains multiple binding sites for several molecules including Rac1/Cdc42, E-cadherin, actin, β-catenin, calmodulin, components of the mitogen- activated protein kinase pathway, and others, which are involved in cancer. Through interactions with these proteins, IQGAP1 plays a pivotal role in the regulation of cell-cell adhesion, migration, and signal transduction. IQGAP1 interacts with the β- catenin binding domainas a negative regulator of E-cadherin-mediated cell-cell adhesion. It dissociates the α-catenin from the β-catenin/cadherin complex resulting in weak cell-cell adhesion. It is expected that this interaction plays a role in disruption of MCAs/spheroids in OVCA.
Hypothesis: The inhibition of Rac1 and Cdc42 in ovarian cancer will lead to the disruption of MCAs/spheroids.
Aim1: To investigate if small inhibitors of Rac1 and/or Cdc42 disrupt in vitro formation of MCAs/spheroids. Aim2: To confirm decreased activity of Rac1 and Cdc42 as well as their effector IQGAP1 after inhibiting Rac1 and Cdc42.
Significance of study: Inhibiting Rac1 and Cdc42 is expected to result in dispersing MCAs/spheroids, thus making them responsive to chemotherapy. This will help overcome resistance in ovarian cancer that is attributed to MCAs/spheroids aggregation.
Dedication
This thesis is dedicated to my wife, Lujain Ashoor, for her endless love, support and encouragement throughout our marriage and particularly during my thesis process; and to my parents and brothers for constant love and a solid foundation upon which I continue to build to this day.
Acknowledgements
In the name of Allah the most gracious, most merciful. All praise and glory to Allah the almighty who alone made it possible for this small work to be accomplished and I am asking Him to accept my efforts.
Last but not least, my deep gratitude to my parents, my brothers and my precious wife, the source of my success, for her solid support and generosity.
List of Tables
Table I. Stages of ovarian cancer, characteristics of the disease at every stage, and relative five-year survival rate
Table II. Drugs used as first-line treatment for ovarian cancer
Table III. Drugs used, or that are under investigation for use, as second line treatment for ovarian cancer
Table IV.Common upstream effectors of Rac1 an Cdc
Table V. Common downstream effectors for Rac1 and Cdc42 and their Biological functions
List of Figures
Figure 1. Schematic of ovarian cancer metastasis
Figure2. Role of Rho small GTPase family in cell-cell adhesion
Figure 3. Chemical Structures of Rac1 and Cdc42 inhibitors and a Structural control
Figure 4. Disruption of MCAs by small inhibitors
Figure 5. A sketch of proposed Aim1 & Aim
Figure 6. A sketch of Aim 1 research design
Figure7. Steps of the hang drop assay
Figure 8. Steps of the liquid overlay assay
Figure 9. The Lactate dehydrogenase (LDH) assay protocol
Figure 10. Steps of the G-LISA assay
Figure 11. Evaluating the expression of phosphorylated and total IQGAP1 by Western blot
Figure 12. Examining the cellular localization of adherens junctions’ molecules
List of Abbreviation
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1.Chapter One : Introduction
1.1. Ovarian Cancer
Ovarian carcinoma (OVCA) is a neoplastic transformation that affects ovarian tissue. It originates either in the ovaries or the fallopian tubes. Ovarian carcinoma remains a significant health problem with the highest fatality rate of any gynecologic malignancy in the United States (1,2).
1.1.1. Incidence, Etiology, Risk Factors, and Screening
In the United States, OVCA is the eighth most common type of cancer and the fifth leading cause of cancer death due to a gynecologic malignancy (2). In 2012, it expected to result in 15,500 deaths and 22,280 women will be diagnosed with OVCA (1). This cancer mainly develops in older women (60 years or older) and it is more common in white women than African-American women (2,3). OVCA confined to the ovary has a 5-year survival of 92% (1). However, most women with OVCA are diagnosed with advanced stage disease, which has a 5-year survival of only 30%- 40% (1,3). The pathogenesis and etiology of OVCA remain unclear but several risk factors have been identified, such as reproductive factors, exogenous hormones, gynecology-related conditions, environmental, and genetic risk factors (4). Approximately 10% of OVCA are hereditary with 90% having mutated genes such as breast cancer Type 1 and 2 susceptibility protein (BRCA1/2) (4,5).
Currently, only two tests used to screen for OVCA are administered to women with familial OVCA (6). Transvaginal sonography is a test that places a small ultrasound probe in the vagina; it can help image the neoplasm in the ovary. The other test detects cancer antigen 125 (CA-125), which is a protein in the blood that is at elevated levels in many women with OVCA. Currently, there are no routinely used OVCA screening methods in the USA.
1.1.2. Types of Ovarian Cancer
There are three basic classes of OVCA, depending on the type of cells in which it occurs; they are germ cell tumors, stromal tumors, and epithelial tumors (7). Germ cell tumors start from the germ cells that produce the ova (eggs). They are common in young females with a high survival rate. Stromal cell tumors occur in the connective tissue. Epithelial tumors arise from cells that cover the surface of the ovary or epithelial ovarian carcinoma (EOC); and eighty to ninety percent of ovarian malignancies are of this type, which is also the most lethal OVCA neoplasm. Due to this, OVCA research is primarily aimed at EOC diagnosis and treatment (7).
1.1.3. Progression and Subtypes of EOC
Epithelial ovarian cancers are the most common type of OVCA. They arise from the ovarian surface epithelium (OSE) or from the fimbria of the fallopian tubes and account for approximately 90% of ovarian malignancies (7). The OSE undergoes a multistep process during carcinogenesis. This process is characterized by a progression of changes at the cellular and genetic level that ultimately reprogram a cell to transform from a normal to a fully malignant phenotype (7). There are three stages of carcinogenesis: initiation, promotion, and progression. During initiation, carcinogenic factors directly damage the DNA. Promotion, or clonal expansion, starts after the initiation and involves the gradual accumulation of genetic alterations in proliferating premalignant cells. Progression occurs due to additional genetic alterations that facilitate the final transformation to cancer.
Recent studies have established two models that categorize EOC into Type I and II, based on clinical, morphological, and molecular genetics (8-10). Type I tumors (originate from OSE) are characterized clinically by stepwise development and histologically low- grade serous carcinoma associated with frequent mutations, such as BRAF, KRAS, beta- catenin and PTEN mutations. Type II tumors (originate in the fimbria of the fallopian tubes) exhibit high-grade serious carcinomas which are undifferentiated, aggressive tumors with frequent mutations in the p53 gene (< 80% of cases) or have CCNE1 (encoding cyclin E1) amplifications. The Type II tumors are genetically highly unstable with an approximate 30 % five-year survival rate. The two models may be used to draw attention to the molecular genetic events that play a role in ovarian tumor progression and can shed light on new approaches to early detection, prevention, and treatment (9).
1.1.4. Stages of EOC
The categorization of EOC into different stages is based on either tumor invasion or the spread of cancer. According to the International Federation of Gynecology and Obstetrics (FIGO), there are four stages of EOC (stage I, stage II, stage III, and stage IV) (7,11,12). Each stage of EOC has a subgroup system (subgroup A, B, and C), except stage IV (Table I). Stage I is characterized by a localization of cancer in one and/or both ovaries, which does not spread outside the ovary. In stage II, EOC spreads from one or both ovaries to other organs within the pelvic extension such as uterus, bladder, and fallopian tubes. In stage III in addition to the spread of stage II, it extends beyond the pelvic region into the lining of the abdomen and metastasis to lymph nodes. Stage IV is the most advanced stage of EOC with neoplastic metastasis which extends to other organs outside of the peritoneal cavity. Buys and co-workers used CA-125 and transvaginal ultrasound to investigate the effect of screening for OVCA on mortality (6). This study has shown that 76% of EOC patients are initially diagnosed with disseminated intra-abdominal disease at stage III and/or stage IV with an expected five-year survival rate of 34% and 18%, respectively (6).
1.1.5. Treatment of EOC
The treatment of EOC is standard and it involves surgery, platinum- and taxane- based therapy. Surgery has two main goals: staging EOC and removal of the tumor from the ovary/ovaries and other sites as much as possible (13). In early stages, surgery is able to cure the disease and the five-year survival rate for stage I and II epithelial ovarian carcinoma is around 90% (1,2,12). A study of two parallel-randomized phase III clinical trials in early stage EOC (IA or IB) compared platinum-based adjuvant chemotherapy with surgery. There was no evidence of significant differences between chemotherapy and surgery on survival in any pretreatment subcategory (14). For advanced stages, maximal surgical cytoreduction or debulking followed by platinum- and taxane- based chemotherapy (Table I) showed 10-30% five-year survivals for women with stage III and IV epithelial ovarian carcinoma (3,12,15).
Chemotherapy, the second important modality of treatment, uses several conventional drugs, such as platinum agents, taxanes, and anthracyclines, to kill the ovarian cancer cells (Tables II & III). The administration method for chemotherapy in ovarian cancer patients can be intravenous and/or intra-peritoneal (IP). The efficacy of IP administration chemotherapy either alone or in combination with an intravenous route, was demonstrated to improve patient survival (16-19).
In the United States, the first line chemotherapy treatment for EOC usually involves a combination of carboplatin and paclitaxel (Table II), which has response rates up to 80% (20,21). Chemotherapy is typically given in cycles: a period of chemotherapy treatment followed by a period of rest. Each cycle of chemotherapy is given over a period of three weeks. However, the recommendations for the number of cycles of treatment vary with the stage of the disease. For patients with advanced-stage disease (stages III- IV), 6 to 8 cycles of chemotherapy are recommended, whereas 3 to 6 cycles are recommended for earlier-stage disease (22). If OVCA recurs, liposomal doxorubicin, the only Food and Drug Administration (FDA) approved second line treatment for recurrent OVCA, is used (23-25). There are, however, several drugs still under clinical trials (Table III) for evaluation of their effectiveness and side-effects including: docetaxel (26), etoposide (27), gemcitabine (23,24), and topotecan (25,28).
Radiation therapy is the third type of treatment. It is rarely used in the initial treatment of EOC due to limited effectiveness in advanced stage patients. It may control cancer in selected patients with early stage EOC due to the localization of cancer in one or both the ovaries (29).
1.1.6. Influence of OVCA Progression on Chemotherapy
Ovarian carcinoma is the leading cause of death from gynecologic malignancy, resulting in approximately 22,280 estimated new cases in 2012 with an estimated 15,500 deaths in 2012 in the USA (1). Many patients suffer from recurrence of EOC that becomes resistant and incurable by chemotherapy (30). Approximately 75% of OVCA Table I. Stages of ovarian cancer, characteristics of the disease at every stage and relative five-year survival rate (11).
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Table II. Drugs used as first-line treatment for ovarian cancer (21).
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Table III. Drugs used, or that are under investigation for use, as second line treatment for ovarian cancer (23-28).
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patients are initially diagnosed with disseminated intra-abdominal disease (stage III-IV) (6) and because of this late diagnosis less than 40% of these patients survive for five years (1-3).
OVCA patients with advanced disease frequently develop malignant ascites (31,32). Cancer cells detach from ovarian carcinoma into the ascitic fluid as single cells (SCs) and/or multicellular aggregates (MCAs) and later adhere or interact with mesothelial cells that line the inner surface of the peritoneum and grow as secondary tumors (31,32) (Figure 1). The secondary tumors resulting from MCAs or spheroids dissemination are highly resistant to chemotherapy (31,32). The resistance to chemotherapy is accompanied by an increase in cell proliferation which is due to increased transition of G1 to S phase of the cell cycle and suppression of apoptosis (31,33-36).
1.1.7. Drug Resistance in EOC
In general, patients who respond to primary treatment and relapse within six months are considered to have platinum-resistant ovarian cancer. There are two classes of drug resistance: the inherited drug resistance (intrinsic), and the acquired drug resistance, seen in patients after treatment with platinum chemotherapy (37,38).
1.1.8. Mechanisms of Drug Resistance
Over the years, the mechanism of drug resistance in OVCA has been studied in several types of resistant cell lines and appears to be multifactorial (37,38) due to (A) decreased intracellular drug accumulation by copper transporter 1 inhibition, which will result in decreasing drug uptake (39), high drug efflux by overexpression of multidrug
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Figure 1. Schematic of ovarian cancer metastasis. An illustration of the shedding of malignant cells from the ovarian surface epithelium to the surrounding cavities. The cancer cells detach from the cancerous tissues of the ovary as single cells or as MCAs which then metastasize into peritoneal cavity and organs.
resistance protein (P-glycoprotein and MDR-associated protein-1), or increased drug metabolism by thiol-containing proteins such as glutathione-S-transferase π (40); (B) increased repair of DNA damage, via increased activation of DNA repair pathways such as base excision repair or mismatch repair of cisplatin-induced intrastrand or interstrand crosslinking (38,41); (C) inhibition of apoptosis through inactivation of p53. In EOC, four tumor suppressor genes have been observed to be mutated during oncogenesis, such as p53, PTEN, BRCA1 and BRCA2 (42,43). The PTEN mutation is associated with Type I low-grade serous EOC (8). However, p53 mutation is linked with Type II high-grade EOC. Increased resistance of EOC has been linked to BRCA1 (gene on chromosome 17) and BRCA2 (gene on chromosome 13) mutations and/or overexpression as shown in several OVCA cells (44). BRCA2 repairs damaged DNA, and if there are inherited mutations in this gene, it disrupts its ability to repair damaged DNA, thereby increasing risks for cancer. Such mutations also make cancer cells more vulnerable to DNA damaging agents, e.g. platinum-based anti-cancer drugs. Initially, ovarian tumors respond well to platinum-based chemotherapy but eventually develop resistance to these drugs. When exposed to cisplatin, ovarian cancer cells develop secondary mutations on the BRCA2 gene that restores their ability to repair DNA making them resistant to chemotherapy (45).
Resistance to chemotherapy and to apoptosis has been linked to small GTPases Rac1 and Cdc42 in breast cancer (46) and OVCA (47). The role of Rac1 in the resistance to chemotherapy is that it inhibits apoptosis in breast cancer cells (46) and mediates cell spreading and migration (48) of breast cancer (46) and prostate cancer cells (49). The role of Cdc42 in cancer progression is that it enhances the motility and invasion of OVCA cancer cells (50) and metastasis in adenocarcinoma cells (51).
1.1.9. MCAs and their Resistance to Therapy
Studies showed that it is possible to generate MCAs in vitro (33,36,52,53,54,55). In vitro models of 3-D cultures of MCAs mimic in vivo MCAs in their resistance to radiotherapy and chemotherapy (56,57). When these cells were exposed to cisplatin, the apoptosis rate of MCAs was significantly less when compared to monolayer cells (35,36,56,57). Monolayer cultures treated with taxol show accumulation of cells at G2- M, sub-G1apoptotic region and drug-induced apoptosis (34). After exposure to taxol, the monolayer exhibited decreased levels of bcl-XL protein, an effect not detected in drug- exposed spheroids. These results indicate that taxol resistance in ovarian carcinoma is due to multicellular-dependent or -no associated mechanisms (33-35).
The resistance of cancer cells to chemotherapy and apoptosis is well known and studies have revealed differential expression of various genes in processes such as adhesion, cell growth, proliferation, cell death, cell cycle control and cell signaling in MCAs (46-50). Therefore, the genes belonging to the small Rho family GTPases that regulate cell motility, adhesion and tumorigenesis, will be considered next.
1.2. The Role of Small GTPases in Cancer Progression
The Rho guanosine triphosphatases (GTPases) protein family is a subset of the RAS (Rat sarcoma) super family (48,59). It is responsible for many cellular processes such as cytoskeletal dynamics, cell polarization, adhesion, invasive proliferation, cell cycle, and apoptosis (48,60). Recently, the deregulated activation of Rho GTPase has been implicated in the development of pathological conditions and cancers (49,61,62), including OVCA (63).
Studies have shown that RhoGTPases downstream signaling plays a role in tumorigenesis at multiple stages and affects tumor growth, transformation, cell invasion, and metastasis (61,62). Rho GTPases such as Rac1, Rho and Cdc42 increase tumor angiogenesis (64,65). The modulation of endothelial permeability can be increased by vasoactive agents, such as thrombin, histamine, vascular endothelial growth factor (VEGF), and others (64,65). The leaky blood vessels are a feature of tumor angiogenesis, leading to poor blood supply and increasing tumor hypoxia, which promote invasion and metastasis (64).
Several studies indicate that Rho GTPase, such as Rac1, Cdc42 and their signaling effectors, may modulate the sensitivity or resistance to radiotherapy and/or chemotherapy (66,67). The use of Rho GTPase signaling inhibitors alone or in combination with a cytotoxic drug resulted in in vivo inhibition of metastasis, tumor growth, and angiogenic responses (68-70). For example, the use of Y-27632, a selective inhibitor of the Rhoassociated protein kinase, which inhibits the induction of thrombin and histamine, resulted in decreased endothelial permeability (65). This indicates that the targeting of Rho GTPase Rac1, Cdc42, and Rho downstream signaling effectors may prove useful in designing future therapeutic approaches for cancer.
1.2.1.The Rho Small GTPase Family and their Members Rac1 and Cdc42
Several lines of evidence implicate the Rho family GTPases in cell behaviors that are central to OVCA dissemination and metastasis. They are enzymes which have roles in biological processes such as cell morphology, cell cycle, and tumor progression. Their mutation in cancers is rare, but overexpression is more common (71,72). The overexpression of these proteins cause continuous phosphorylation of the downstream effectors (63). Several studies have shown that the overexpression of Rho-family GTPases in human cancers often correlates with a poor patient prognosis and an increased tumorigenesis (61,63,64,72). Based on amino acid similarities, the Rho family consists of twenty-two members and is subdivided into six subfamilies: Rho (A/B/C), Cdc42 (cdc42/G25K, TC10, TCL, Wrch, and Chp), Rac (Rac1/2/3, RhoG), Rnd (1/2/3), Miro (1/2), and RhoBTB (1/2/3) (73), of these Rho, Rac1 and Cdc42 have been extensively studied for their role in cancer (61,63).
1.2.2. Cellular Functions and Role of Rac1 and Cdc42 in Cancer Progression
Rac1 and Cdc42 are members of the Rho small GTPases (48,73). They are involved in the regulation of critical cellular processes such as cytoskeletal reorganization, adhesion, cell growth, apoptosis, and invasion (48,74). Their regulation involves cycling between a GTP-bound active state and GDP-bound inactive state (48,75). This cycling is highly controlled by three classes of regulators (Table IV) (48): the guanine nucleotide exchange factors (GEFs), Rho guanine nucleotide dissociation inhibitors (RhoGDIs), and GTPase-activating proteins (GAPs). The GEFs promote exchange of GDP for GTP (Figure 2). A study showed that the use of dominant negative mutant T17NRac which sequesters GEFs, resulted in blocking Rho-proteins’ activation, disruption of cytoskeletal reorganization, and causing cell cycle arrest (76). Rho GDIs work with GDP-bound Rho-GTPases to stop the exchange of GDP to GTP (77). Finally, GAPs enhance the intrinsic GTPase activity of Rho-GTPases. The active conformation where Rac1 and Cdc42 are bound to GTP results in the phosphorylation of downstream effectors (Table V) (78). Rac1 and Cdc42 are involved in actin polymerization, maintaining cell polarity, cytokinesis, actin organization, and regulation of cell to cell contacts. All these processes are involved in the progression of OVCA (42,47). The members of the Rho GTPase family that are involved in cancer include RhoA, Rac1, and Cdc42 (48,73,79). The resistance of cancer cells to chemotherapy, apoptosis, and invasiveness has been linked to Rac1, Cdc42, and their downstream effectors (46,47,50,80). For example, a study showed increased Cdc42 expression in a cisplatin-resistant OVCA cell line, IGROV-1CP compared to its parental cisplatin-senstive cell line, IGTOV-1 (47). Another study used the microarray genome analysis to identify signaling pathways involved in ovarian cancer tumorigenesis. They showed that Cdc42 is involved in cancer cells motility and invasiveness (50). In addition, inactivation of Rac1 by using a Rac1 inhibitor, NSC23766, or knocking down the expression of Tiam1, a guanine nucleotide exchange factor for Rac, using siRNA significantly reduced trastuzumab (Herceptin®) resistance in PTEN deficient and insulin-like growth factor I receptor (IGF-IR) overexpressing human breast cancer cells (80).
Overexpression of Rac1 and Cdc42 has been identified in multiple types of cancers, such as breast cancer and ovarian cancer (61,72). Their activation in cancerous cells suppresses apoptosis, and contributes to cell survival (81). After apoptosis is suppressed, abnormal tumor growth is observed through changes in cell shape, cell adhesion, cell spreading, cell migration, and cell polarity in which Rac1 and Cdc42 proteins play a role (81,82). The inhibition of apoptosis, cell polarity, and adhesion molecules makes the cancerous mass free to metastasize (81). In addition, Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and ovarian tumor cell migration (83). The activation of small GTPases leads to the construction of actin fibers in filopodia, which are slender cytoplasmic projections that extend at the leading edge of migrating cells (84). The deregulated activation of Rac1 and Cdc42 has been implicated in the development of pathological conditions and cancers (49,61,62) including OVCA (42). The Rac1 and Cdc42 downstream signaling in tumorigenesis affects tumor growth, transformation, cell invasion, and metastasis (61,62). In addition, resistance to chemotherapy and to the apoptosis pathway has been linked to Rac1 and Cdc42 in breast cancer (46) and OVCA (47). The role of Rac1 in the resistance to chemotherapy is that it inhibits apoptosis of breast cancer (46). Rac1 exerts its action by increasing the activity of transcription factor nuclear factor-kappa B (NF-κB), which regulates the transcription expression of survivin, X-linked inhibitor of apoptosis protein (XIAP), and cyclin D1 (46). In addition, the increased amount of cyclin D1 will promote the transition of G1 to S phase in breast cancer cells (46). The role of Cdc42 in cancer progression is that it enhances the motility and invasion of OVCA cancer cells (50) and metastasis in adenocarcinoma cells (51). In summary, Rac1 and Cdc42 are small GTPases that are involved in the regulation of many cellular processes through the activation of their downstream signaling (48,74,78). Overexpression of Rac1 and Cdc42 in OVCA leads to apoptosis suppression resulting in abnormal tumor growth and metastasis (50,51,72,81). Therefore, this will lead to increased resistance of OVCA cells to chemotherapy.
1.3.Types of Cell-Cell Adhesions
Cell adhesion is an essential process whereby cells interact and attach to each other. This process is formed by interactions between the molecules on the surface of the cell and it mediates intercellular communication for proper tissue or organ functioning and maintenance of epithelial structure integrity (85,86). The dynamic rearrangement of cell- cell adhesiveness is the major phenomenon for the invasive properties of cancer (87). There are four types of adhesion junctions including adherens junctions, desmosomes, gap junctions, and tight junctions. Adherens junctions are responsible for strong adhesions between adjacent epithelial cells in addition to attaching the adherens junction to actin cytoskeleton (86). Adherens junctions are composed of cadherins, β-catenin, and α-catenin. The desmosomes are another class of adhesive intercellular junctions are linked with the intermediate filament cytoskeleton in epithelial and cardiac muscle cells (88). The desmosomal cadherins, desmocollin (Dsc) and desmoglein (Dsg), are the desmosomal adhesion molecules (89). The third type is the gap junctions which are communication junctions made up of transmembrane proteins called connexons. They connect cytoplasm of two cells and allow ions to pass between the cells (91). The tight junctions are the fourth type and they are found in the apical region around the cell's circumference (91). They are made up of a network of sealing strands that hold plasma membranes together which mostly formed from a group of proteins called claudins and occludins (91). The tight junctions’ function is to prevent ion movement between cells and cell membranes. Studies showed that Rac1 and Cdc42 play a role in the regulation of desmosomal adhesions by Rho GTPases (88) and adherens junctions (48,74,81).
1.3.1.Classical Cadherins Mediated Cell-Cell Adhesions in Ovarian Cancer
The classical cadherins represent a major super family of transmembrane glycoproteins (80 kDa) that mediate calcium dependent homophilic cell-to-cell adhesion as components of the adherens junctions. They play a pivotal role in cell-cell recognition, morphogenesis, and maintenance of epithelial integrity (32,82,86,92,93). In the human genome, more than 80 members of cadherin superfamily have been identified. One type of cadherin is the classical cadherins (also known as Type I
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Figure 2. Role of Rho small GTPase family incell-cell adhesion. In the active phase: Rho small GTPases (Rac1 and Cdc42) are bound to GTP.Under these conditions, phosphorylated-IQGAP1 does not bind to β-catenin and cannot dissociate α-catenin from the cadherin-catenin complex, leading to strong adhesion. By contrast, when Rac1 and Cdc42 are bound to GDP (resting phase), IQGAP1 is freed to interact with β-catenin to dissociate α-catenin from the cadherin-catenin complex. This results in weak adhesion.
Table IV. Common upstream effectors of Rac1 and Cdc42 (48).
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Table V. Common downstream effectors for Rac1 and Cdc42 and their biological functions (78).
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cadherins). Examples of the classical cadherins are E-cadherins, VE-cadherins, and N- cadherins (94,95). The epithelial (E) cadherins are found in the cell membrane of most polarized epithelial cells. However, the neural (N) cadherins are the most prevalent in neural tissues, cardiac and skeletal muscle, mesothelium, and some mesenchymal tissues (96,97). Structurally, all cadherins are composed of an extracellular (EC) domain and a cytoplasmic domain. The EC domain (also known as cadherin repeats) is comprised of five conserved extracellular tandem repeats, which mediate homophilic interactions. Their adhesive activity is mediated by extracellular calcium which binds at junctions between the EC repeats and allows cadherin molecules to form a rigid structure that is resistant to proteolysis (98,99). Reversible modulation of cellular adhesion involving E- cadherin and N-cadherin in epithelial-mesenchymal transition likely plays a critical role in remodeling of the OSE during tumor progression, resulting in the shedding of tumor cells to the peritoneal cavity (32,100). E-cadherin interactions in OVCA are modulated by matrix metalloproteinase-9 (MMP-9). This is an enzyme that degrades structural components of the extracellular matrix, and that is overexpressed in advanced OVCA (101) and promotes invasion of OVCA 433 and OVCA 429 cell lines (102). Additionally, MMP-9 disrupts cadherin interactions by proteolytically cleaving the E- cadherin ectodomain (102). There are some indications that Rho GTPases could play a role in regulating the secretion and/or activation of secreted proteases. For example, Rac1 and Cdc42 activate 70pS6 k, a serine/threonine kinase that acts as downstream of phosphoinositide-3-kinases. The 70pS6 k is a direct transcriptional activator of MMP-9 synthesis in SKOV-3, Caov-3, and OVCAR-3 ovarian cancer cells (103,104). Another study reported that RhoA GTPases induces expression of MMP-9 (105). Different processes are involved in cancer metastasis, each of which involves different molecule, including overexpression of degrading proteases of extracellular matrix (ECM) and abnormality of cell adhesion molecules. Excessive degradation of ECM by MMP’s will result in imbalance between its expression and inhibition that is one of critical causes for cancer metastasis (101,102,105). It is important to consider what happens to cellular functions during metastasis as a result of the activation of MMP-9 expression by Rho GTPase (105).
1.4. The Relation between Rac1, Cdc42, and their Effector IQGAP1 with Cadherins
Ras GTPase-activating-like protein IQGAP1, a 189-kDa scaffolding protein, is an effector for Rac1 and Cdc42 that is accumulated at cell-cell contact sites where E- cadherin, β-catenin, and α-catenin form adherens junctions (106,107). IQGAP1 contains multiple binding sitesfor several molecules, including Rac1/Cdc42 (108,109), E-cadherin (110), actin, β-catenin, calmodulin, and components of the mitogen-activated protein kinase pathway, all of which are involved in cancer survival (107). Through interaction with these proteins, IQGAP1 plays a pivotal role in the regulation of cell-cell adhesion, migration, and signal transduction. Expression analysis indicated that IQGAP1 is overexpressed in many cancers (108-111) and in more aggressive OVCA (112). IQGAP1 is localized at the invasive front of the neoplasm, indicating a role in mobilization, progression, and spread of ovarian adenocarcinomas (112,113). Rac1 and Cdc42 phosphorylate IQGAP1 leading to its inactivation, and causing strong cell-cell adhesion (106). IQGAP1 acts as a regulator for E-cadherin-mediated cell-cell adhesion (92,93). It is localized at the sites of the E-cadherin-β-catenin-α-catenin complex (92,106,110) and, when overexpressed, it interacts with the β-catenin and dissociates α-catenin from the E- cadherin-β-catenin complex resulting in the reduction of E-cadherin-mediated cell-cell adhesion. This means that IQGAP1 acts as a negative regulator for E-cadherin-mediated cell-cell adhesion (92). Overexpressed Rac1 and Cdc42 (i.e. GTP-bound) will phosphorylate IQGAP1 and IQGAP1 will not interact with β-catenin binding site (106). This, in turn, allows α-catenin to interact with the E-cadherin-β-catenin complex and maintain intact cell-cell adhesions. Studies showed that silencing IQGAP1 by using siRNA in cancer cells resulted in strong cell-cell adhesion (108,114). This finding indicates that inactivated IQGAP1 will not interact with the β-catenin binding site and α- catenin will not dissociate from the cadherin-β-catenin complex, leading to strong adhesion (106,108,114).
1.5. Inhibitors of Rac1 and Cdc42
Inhibitors for Rac1 and Cdc42 are commercially available. For example, ML141 (CID-2950007) is demonstrated to be a potent, selective, non-reversible, and noncompetitive inhibitor of Cdc42 GTPase (IC50 ~200 nM) (115). It is suitable for in vitro assays and it has been shown to efficiently decrease GTP-Cdc42 (>95%) with low micromolar potency and selectivity against other members of the Rho family of GTPases (115). NSC23766 or 553502 (Figure 3) Rac1 inhibitor is a pyrimidine compound that is cell permeable (IC50 = 50 μM). It specifically and reversibly acts to inhibit the Rac1 GDP/GTP exchange activity. This is achieved by interfering with its interaction with the Rac1-specific guanine nucleotide exchange factors, Tiam1 and Trio. Toxin A (IC50= 300μM) is an enzyme that deactivates small GTPases, such as RhoA, Rac1, and Cdc42 by UDP-glucosylation of a threonine residue (Thr37 in RhoA, Thr35 in Rac1/Cdc42) (116). This residue is highly conserved amongst all GTPases and presumed to play a critical role for magnesium binding, which is an essential cofactor for GTP binding protein functions (116,117). The inhibition of these GTPases causes the shutdown of signal transduction cascades leading to: depolymerization of the cytoskeleton, gene transcription of certain stress-activated protein kinases, a drop in synthesis of phosphatidylinositol 4,5 bisphosphate and possibly even the loss of cell polarity (116,117). R-naproxen or R-nap (IC50 for COX-1 are 0.6-4.8 μM and 2.0-28.4 μM for COX-2) acts by inhibiting cell migration and cell to cell adhesion (118). It directly inhibits Rac1 and Cdc42 by interfering with guanine nucleotide exchange (R. Zeineldin, personal communication, January, 2012).
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Figure 3. Chemical Structures of Rac1 and Cdc42 inhibitors and a structural control (a): NSC23766; (b): ML141; (c): R-naproxen and (d): 6-methoxy-2-naphthalene acetic acid (6-MNA), a negative control with structural similarity to R-naproxen.
2. Chapter Two: Rationale and Goals
Ovarian cancer is the leading cause of gynecological cancer mortality, resulting in an estimated 15,500 deaths in the USA in 2012 (1). A significantly high mortality rate and a five-year survival rate of less than 40% are due to recurrence and poor response to chemotherapy (1-3). Low-grade serous OVCA starts from the OSE whereas high-grade serous OVCA originates in the fimbria of the fallopian tubes (8-10). Diagnosis is rarely made in the early stages, with approximately 75% of patients presenting with OVCA in the advanced stages (6). OVCA patients with advanced disease frequently develop malignant ascitis that contains detached cancer cells in the ascitic fluid as SCs and/or MCAs (31,32,58). Studies showed that the disruption of chemoresistant MCAs could improve their response to chemotherapy (119,120,121). For example, blocking of E- cadherin-mediated adhesion by using SHE78-7 monoclonal (murine IgG2a) antibody against E-cadherin, disrupted the human colon adenocarcinoma MCAs cells and sensitized them to chemotherapy (119). The use of nitric oxide-mimetic agent disrupted breast cancer MCAs. Crystal violet staining of these cells showed a significant decrease in spheroids survival following exposure to doxorubicin indicating that nitric oxide- mimetic agent attenuated breast cancer cells resistance to doxorubicin through decreasing breast cancer cells’ hypoxia (121). A recent study examining the early response of MCAs to chemotherapy revealed a differential expression of genes that play a role in adhesion, cell growth, proliferation, cell death, cell cycle control, and cell signaling (122). Among these genes were genes that encode small Rho family GTPases (122) that normally regulate cell motility and adhesion (48,123) and play a role in tumorigenesis (61,63,64). Not only was the early response related to small GTPases but also resistance to chemotherapy. In addition, resistance to chemotherapy and to apoptosis has been linked to small GTPases Rac1 and Cdc42 in breast cancer (46) and OVCA (47). The resistance to chemotherapy and to apoptosis is due to their role in the regulation of various cytoskeleton-dependent processes such as changes in cell shape, cell adhesion, cell spreading, cell migration and cell polarity (81). Both Rac1 and Cdc42 are overexpressed in OVCA (47,50,63) and their inhibition significantly decreases cell proliferation, migration (123), and improves the sensitivity to chemotherapy in in vitro breast cancer cells (46,124). We propose that inhibiting Rac1 and Cdc42 causes their effector IQGAP1 to act as a negative regulator for E-cadherin mediated cell-cell adhesions. IQGAP1 interacts with the β-catenin binding domain which causes dissociation of the α-catenin from the β-catenin/cadherin complex resulting in weak adherens junctions (Figure 2). Such interactions are expected to cause a disruption of MCAs’ aggregation.
Hypothesis: The inhibition of Rac1 and Cdc42 in OVCA will lead to the disruption of MCAs/spheroids.
Aim 1: To investigate if small inhibitors of Rac1 and/or Cdc42 disrupt in vitro formation of MCAs/spheroids.
Aim 2: To confirm decreased activity of Rac1 and Cdc42 as well as their effector IQGAP1 after inhibiting Rac1 and Cdc42.
Significance and Innovation: Inhibiting cell-cell adhesion or proteins that control adhesion disperses MCAs/spheroids (Figure 4) and sensitizes them to chemotherapy (52,58,67,119,120,124,125). Inhibition of Rac1 and Cdc42 in OVCA might lead to disruption of MCAs, making them more sensitive to chemotherapy. This could increase the effectiveness of IP chemotherapy and improve patient outcome and survival. R-Naproxen is an FDA approved off-patent NSAID drug which inhibits Rac1 and Cdc42, prevents phosphorylation of IQGAP1 and acts as a negative regulator for E-cadherin mediated cell-cell adhesions resulting in disruption of MCAs’ and sensitizing the OVCA cells to chemotherapy.
Innovation: Firstly, small GTPases are overexpressed in cancers so they may serve as key biomarkers for screening for OVCA. Secondly, despite their critical role in cellular functions, GTPases have not yet been extensively examined as therapeutic targets specifically in OVCA. The characterization of Rac1 and Cdc42 as targets and biomarkers offers new opportunities for disease detection, therapeutic monitoring, and targeting.
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Figure 4. Disruption of MCAs by small inhibitors. The small inhibitors, such as NSC 23766, R-naproxen, ML 141, and toxin A, are expected to disrupt the MCAs which may sensitize them to chemotherapy.
3. Research Design/Methodology
3.1. Specific Aim 1
To investigate if small inhibitors of Rac1 and/or Cdc42 disrupt in vitro formation of MCAs/spheroids.
3.1.1. Rationale for Specific Aim 1
Studies indicated that inhibitors of adhesion disperse MCAs and sensitize them to chemotherapy (119,120,121,125). The blocking of E-cadherin-mediated adhesion by using SHE78-7 monoclonal (murine IgG2a) antibody against E-cadherin, disrupted the human colon adenocarcinoma MCAs cells and sensitized them to chemotherapy (119). Supression of β1 integrin by laminin-1 derived scrambled peptide AG73T results in disruption of TAC3 OVCA cell aggregates. Laminins are trimeric proteins that promote the formation of spheroid by enhancing the expression of β1 integrin (125). This suppression of β1 integrin is associated with a two-fold increased sensitivity to cisplatin in comparison to aggregated spheroids (125). In addition, the disruption of aggregates in breast cancer cells by nitric oxide-mimetic agent showed a significant decrease in spheroids survival following exposure to doxorubicin as detected by crystal violet staining through decreasing breast cancer cells’ hypoxia (121). These studies provide an evidence for sensitizing MCAs to chemotherapy by disrupting them, using inhibitors of adhesion. Furthermore, bortezomib, which degrades several cytosolic proteins, such as the proteasome, caused a disruption of MCAs aggregation and led to decreased proliferation along with induced cell cycle arrest in OVCA 429 and SKOV-3 cells (120). A recent study examining early response of MCAs to chemotherapy revealed a differential expression of genes that play a role in adhesion, cell growth, proliferation, cell death, cell cycle control, and cell signaling (122). Among these genes were genes that encode small Rho family GTPases (122) that normally regulate cell motility and adhesion (48,74) and play a role in tumorigenesis (61,63,64). We predict that inhibiting Rac1 and Cdc42 will disrupt the MCAs. We will test the effect of small inhibitors of Rac1 and/or Cdc42 such as, NSC 23766 (as a Rac1-specific inhibitor), ML141 (as a Cdc42-specific inhibitor), R-napoxen, and toxin A (inhibitor of both) on disruption of MCAs formation in vitro (Figure 5).
3.1.2. Research Design for Specific Aim 1
3.1.2 a. Selecting OVCA Cell Lines that can Form MCAs and are Resistant to Paclitaxel or Cisplatin
The aim is to investigate if treatment with Rac1 and/or Cdc42 inhibitors (NSC 23766, R-nap, Toxin A, and ML 141) reduces MCAs formation in vitro (Figure 6). Cell lines that are resistant to cisplatin or paclitaxel such as OVCA 429 and SKOV-3ip will be used so as to generate resistant MCAs (33-35). SKOV-3ip produces fast deteriorating peritoneal disease and malignant ascites (35). Studies reported that cells that express E- cadherin (epithelial), such as OVCA 429 or N-cadherin (mesenchymal), e.g. SKOV-3ip, generate MCAs that are resistant to both paclitaxel and cisplatin (33,34,126). Moreover, another study demonstrated that OVCA 429 and SKOV-3 (the cell line from which SKOV-3ip is derived) are capable of forming MCAs/spheroids and have an invasive capacity due to the expression of MMP proteolytic activity (53,102). Therefore, we will
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Figure 5. A sketch of proposed Aim1 & Aim 2. R-nap = R-naproxen (inhibits Rac1 & Cdc42); Toxin A (inhibits Rac1 & Cdc42); NSC 23766: inhibits Rac1; ML141: inhibits Cdc42.
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Figure 6. A sketch of Aim 1 research design.
use OVCA 429 and SKOV-3ip, as examples of an epithelial and a mesenchymal cell lines that form MCAs that are resistant to treatment with chemotherapeutic agents paclitaxel or cisplatin.
3.1.2.b. Generation of MCAs for Testing their Disruption
MCAs/spheroids will be first generated from resistant cell lines and we will confirm that they are resistant to chemotherapeutic agents such as paclitaxel and cisplatin. For those reasons, we will generate MCAs through a hang-drop method, where MCAs are generated by being suspended within a drop that is hanging from the cover of cell culture plate (54) and a liquid overlay assay, where MCAs are generated on an agarose layer. In both methods, cells will appear as clustered cellular spheroids (33,52). We will generate MCAs using the epithelial cell line OVCA 429, and the mesenchymal cell line SKOV- 3ip. After that, we will investigate if treatment with the small inhibitors (NSC 23766, ML 141, R-napoxen (R-nap) and toxin A) will reduce MCAs/spheroids’ formation. The negative controls will be the vehicle and 6-methoxy-2-naphthalene acetic acid (6MNA) which is a NSAID structural analog. We will examine the MCAs’ disruption with or without inhibitors under the light microscope. All techniques will be repeated three times.
3.1.2.c. Examination of Cytotoxicity of the Inhibitors
A cytotoxicity detection kit will be used to confirm that the inhibitors (NSC 23766, R-nap, ML 141, and toxin A) are not toxic to cells. The assay uses a colorimetric assay based on the measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells during cell death or cell lysis. Monolayer cells will be seeded in 96-well culture plates and after overnight incubation at 37oC, 5% CO2, allowing the cells to adhere tightly. After that, cells will be treated with different concentration of inhibitors. The positive control for inducing cell death will be treatment with camptothecin. The negative control will be 6MNA as a NSAID sturacural analog. The percentage of cytotoxicity will be determined by calculating the average absorbance values of the triplicate samples and controls.
3.1.3. Methods for Specific Aim 1
3.1.3.a. OVCA Cell Lines
Two ovarian carcinoma cell lines OVCA 429 and SKOV-3ip will be obtained from MD Anderson Cancer Center (Houston, TX) and grown as described previously (35,53). Briefly, SKOV-3ip cells will be grown in RPMI-1640 media (Sigma, St. Louis, MO) and OVCA 429 cells will be grown in Medium Essential Medium Eagle (MEME) media (Sigma, St. Louis, MO). Both media will be supplemented with 10% FBS (Invitrogen, Camarillo, CA), 2 mM L-glutamine (Sigma, St. Louis, MO), 1 mM sodium pyruvate (Sigma, St. Louis, MO), and penicillin G [50 U/mL]/streptomycin [50 μg/mL] (Sigma, St. Louis, MO). SKOV-3ip and OVCA 429 will be incubated at 37°C in a humidified 5% CO2 atmosphere to 80% confluence and cell counting protocols will be followed.
3.1.3.b. Cell Treatments
For small inhibitors (NSC 23766, R-nap, ML 141, toxin A), the concentrations that will be tested are 100 μM NSC23766 (Tocris, Bristol, UK) (R. Zeineldin, personal communication, January, 2012), 0.4 Μm ML141 (Tocris, Bristol, UK), 600 μM toxin A (Tocris, Bristol, UK), and 0, 0.1, 1, 3, 10, 100, 300, and 1000 μM of R-nap or 6-MNA (Cayman, Ann Arbor, Michigan).
3.1.3.c. Formation of Multicellular Aggregates
We will generate MCAs for the two ovarian carcinoma cell lines OVCA 429 and SKOV-3ip by using two assays, hanging drop suspension cultures and liquid overlay system.
3.1.3.c.1. Formation of MCAs using Hang Drop Assay
The method that will be used to form a MCAs (Figure 7) will follow that described previously (53,54). SKOV-3ip and OVCA 429 cells will be cultured to ~90% confluency. Then, cells will be counted and diluted to a concentration of 25x105 cells/mL (R. Zeineldin, personal communication, January, 2012) with or without treatments (NSC 23766, or R-nap, or ML 141, or toxin A). After that, 25 μL will be taken of each cell suspension to be dropped onto a 10 cm cell culture plate lid that will be marked with the treatment. A volume of 8 mL sterile phosphate buffer saline (PBS) will be placed in the bottom of the plate. The cover of the plate will be inverted on the plate and the drops will hang down towards the PBS where cells will from MCAs within the drops. Cells will be allowed to aggregate for 48 hours at 37°C, and will be examined under the light microscope to determine the effect of the treatment on aggregation after 24 and 48 hours.
3.1.3.c.2. Formation of MCAs using Liquid Overlay Assay
The method that will be used to form a MCAs on agarose (Figure 8) will follow that described previously (33,36,52,55). A twenty-four well cell culture plates will be coated with 500 μL of 0.5 % (w/v) agarose in serum-deprived medium (contains all the additives specific for each cell line but replacing the serum with 0.1% (w/v) bovine serum albumin), and the agarose will be allowed to solidify for 30 min at room temperature. A volume of 300 μL cells will be suspended in complete media, and transferred to the agarose-coated wells at 50,000 cells/well (R. Zeineldin, personal communication, January, 2012). Cells will be allowed to aggregate for 48 hours at 37°C, and will be examined under the light microscope to determine the effect of the treatment on aggregation after 24 and 48 hours.
3.1.3.d. Use of LDH Assay to Examine Cytotoxicity of Rac1 and Cdc42 Inhibitors
The LDH assay will be used according to the manufacturer’s protocol (Roche, Indianapolis, IN) to confirm that the inhibitors (NSC 23766, R-nap, ML 141, toxin A) are not toxic to cells (Figure 9). Briefly, A volume of 100 μL SKOV-3ip (1 x 104 cells per well) or OVCA 429 cells (with or without treatments) will be pipetted in triplicates onto a 96-well plate. Cells will be incubated at 37°C in a humidified 5% CO2 atmosphere for 4 hours to allow cells’ adherence to the wells; then the treatments NSC 23766 or R-nap or ML 141 or toxin A will be added and cells will be incubated for 20 hours. The LDH assay kit will be used to evaluate cytotoxicity and absorbance readings will be then taken at 490 nm using a plate reader. The percentage of cytotoxicity will be determined by
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Figure 7. Steps of the hang drop assay. Cells will be cultured to ~90% confluency, then will be counted and diluted to 25 x 105 cells/mL. Cells will be treated with inhibitors (NSC 23766, R-naproxen, ML141, and Toxin A). Then, 25 μL of each cell suspension will be dropped onto a 10 cm cell culture plate lid. The bottom of the cell culture plate will be filled with 8 mL sterile PBS. Cells will be incubated for 24 and 48 hours and then examined under a light microscope.
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Figure 8. Steps of the liquid overlay assay. Twenty-four-well cell culture plates will be coated with 500 μL of 0.5% agarose in serum-free media and will be allowed to solidify for 30 minute at room temperature. Cells will be suspended in complete media, transferred to the agarose-coated wells at 50,000 cells/well and will be incubated for 48 hours at 37oC.
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Figure 9. The Lactate dehydrogenase (LDH) assay protocol. A volume of 100 μL of cells (with or without treatments) will be pipetted in triplicates onto a 96-well plate. Cells will be incubated at 37°C in a humidified 5% CO2 atmosphere for 4 hours to allow for cellular adherence to the well, then treatments with NSC 23766, R-nap, ML 141, and toxin A will be added and cells will be incubated for 20 hours. The reaction mixture (dye solution and lysis solution) will be added and the reading will be taken at 490 nm.
calculating the average absorbance values of the triplicate samples and controls (see below). After that, the background absorbance value (contains media only) will be subtracted from the absorbance reading of the sample. Then substitute the resulting values in the following equation:
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The low control: Determines the LDH activity released from the untreated normal cells (spontaneous LDH release). The high control: Determines the maximum releasable LDH activity in the cells via lysis (maximum LDH activity).
3.1.3.e. Statistical Tests and Data Analysis
Data will be collected in triplicates for all experiments. Results for cells with small inhibitors will be compared to control cells without treatments using a nonparametric Welch’s two sample t-test. Any p-value below 0.05 will be considered statistically significant.
3.1.3.f. Anticipated Results, Potential Problems, and Alternative Approaches for Aim 1
We expect that the OVCA 429 and SKOV-3ip ovarian cancer cell lines will generate clustered cellular spheroids or MCAs as reported in the literature. We expect that small inhibitors NSC 23766, ML141, R-naproxen, and toxin A will disrupt the MCAs of both cell lines. We do not anticipate problems forming MCAs through the hang drop assay or the liquid overlay assay. For the LDH assay, we do not anticipate problems examining cytotoxicity of Rac1 and Cdc42 inhibitors, e.g. NSC 23766, or R- nap, or ML 141, or toxin A. We expect that the LDH assay will show that the small inhibitors are not toxic for the cells. If we face any problem with LDH assay the alternative will be an alamar blue assay. The alamar blue assay (#DAL1025, Invitrogen. Camarillo, CA) is a fluorometric/colorimetric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation- reduction (REDOX) indicator that both fluoresces and changes color in response to a chemical reduction of growth medium resulting from cell growth.
3.2. Specific Aim 2
To confirm decreased activity of Rac1 and Cdc42 as well as their effector IQGAP1 after inhibiting Rac1 and Cdc42.
3.2.1. Rationale for Aim 2
Overexpression of Rho-family GTPases in human cancers often correlates with a poor patient prognosis (72). They are proposed therapeutic targets for cancer due to their regulatory roles in adhesion, migration, and rearrangement of actin cytoskeleton (48,74). Studies showed that interference with Rac1 and Cdc42 activity resulted in significant inhibition in cell viability, migration, survival, and apoptosis (46,123,124). Studies demonstrated that silencing IQGAP1, an effector of Rac1 and Cdc42 using siRNA in cancer cells, resulted in strong cell-cell adhesions (108,114). Overexpressed Rac1 and Cdc42 (GTP-bound) phosphorylates IQGAP1, leading to inactivation of IQGAP1 and inhibiting its interaction with β-catenin and leading to strong cell-cell adhesion (106). Collectively, we predict that inhibiting Rac1 and Cdc42 resulting in increased nphosphorylated IQGAP1 will decrease E-cadherin-mediated adherens junctions leading to weak cell-cell adhesions and resulting in MCAs’ disruption. The goal is to confirm decreased Rac1 and Cdc42 activity by measuring the levels of GTP-bound Rac1 and Cdc42 by a G-LISA activation assay (Figure 5). We will also evaluate if their downstream effector IQGAP1 is involved by examining if the phosphorylated IQGAP1 is decreased by immunoblot analysis. We will examine the cellular localization of IQGAP1 and of adherens junctions’ molecules such as E-cadherins, β-catenin, and α-catenin by fluorescence microscopy.
3.2.2. Research Design for Specific Aim2
3.2.2.a. Evaluating Activation of Rac1 and Cdc42
The same cell lines will be used to confirm that Rac1 and Cdc42 activity levels are low in the presence of small inhibitors (NSC 23766, R-nap, ML141, and toxin A). The activation of Rac1 and Cdc42 will be measured by G-LISA assay (Figure 10). It will be used for detecting GTP-bound (i.e. active) Rac1 and/or Cdc42 in the presence of small inhibitors. This is because G-LISA is highly sensitive as it can detect very low concentrations, ranging form 0.5 ng to 10 ng for Cdc42 and 10 pg to 2 ng for Rac1. The kit contains a specific binding protein for either Rac1-GTP or Cdc42-GTP linked to the wells of a 96-well plate. Active GTP bound Rho GTPase in cell lysates will bind to the wells, while inactive GDP bound Rho GTPase will be removed during washing steps. The attached and active GTPase, Rac1 or Cdc42, will be identified by a Rac1- or Cdc42- specific antibody. We will use a purified protein or a peptide that contains the immunogen sequence for the antibody as positive control. A sample known not to express the protein will be used as negative control. The degree of Rac1 or Cdc42 activation will be determined by comparing readings from cell lysates with or without inhibitors. This experiment will be repeated three times.
3.2.2.b. Measuring the Expression of Phosphorylated and Total IQGAP1
We will measure the expression of phosphorylated and total IQGAP1 by Western blot (Figure 11) analysis using amouse anti-IQGAP1 antibody (#33-8900, Invitrogen. Camarillo, CA) to confirm the decreased activity of Rac1 and Cdc42 (in Aim1 section), as IQGAP1 is one of their downstream effectors. This technique will be repeated three times.
3.2.2.c. Examination of the Adhesion Molecules by Fluorescence Microscopy
Finally, the cellular localization of adherens junctions’ molecules such as E- cadherins, β-catenin, and α-catenin by fluorescence microscopy will be examined to confirm the dissociation of α-catenin from E-cadherin/β-catenin complex through the interaction of IQGAP1 with β-catenin in presence of Rac1 and Cdc42 inhibitors (Figure 12). We will use immunofluorescence microscopy to examine localization of α-catenin and IQGAP1 by goat anti-IQGAP1 antibody (#361598, Everest Biotech, Ramona, CA) and mouse anti-α-catenin antibody (#13-9700, Invitrogen. Camarillo, CA). Localization of the E-cadherin and α-catenin will be also examined by goat anti-E-cadherin antibody (#102137, EMD Milipore, Billerica, MA) and mouse anti-α-catenin antibody (#13- 9700, Invitrogen. Camarillo, CA). The localization of β-catenin and IQGAP1 will be examined by using mouse anti-IQGAP1 antibody (#33-8900, Invitrogen. Camarillo, CA) and rabbit anti-β-catenin antibody (#19022, EMD Milipore, Billerica, MA). In addition, the localization of E-cadherins and IQGAP1 will be examined by mouse anti- IQGAP1 antibody (#33-8900, Invitrogen. Camarillo, CA) and goat anti-E-cadherin antibody (#102137, EMD Milipore, Billerica, MA). Secondary antibodies against the primary antibodies conjugated to fluorescein (green) or alexa fluor (red) will be used to detect fluorescence using a confocal microscopy. Images will be generated using a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging, Inc.; Thornwood, NY), with excitation at 488 nm, using an argon laser, and at 543 nm, using helium neon 1, and LSM Image Browser software (Carl Zeiss MicroImaging, Inc.). Images will be compiled using Adobe Photoshop software (Adobe; San Jose, CA). The analysis of colocalization will be examined by determining Pearson’s correlation coefficient using LSM imaging software (Carl Zeiss MicroImaging, Inc.). Pearson’s correlation coefficient value ranges from -1 to +1, where +1 indicates perfect colocalization, while - 1 indicates total lack of colocalization, and a value of 0 indicates no correlation. In case IQGAP1, β-catenin, and E-cadherins colocalize in the presence of the small inhibitors, a value of +1 or close to +1 will be observed and that indicates significant colocalization. If α-catenin did not show any colocalize, it will be observed by a value of -1 or close to -1. Any values either +ve or -ve close to 0 will not show significant colocalization. If α- catenin, β-catenin, and E-cadherins colocalize in absence of small inhibitors and since IQGAPI will not be colocalize, it will be observed under fluorescence microscope to get the pearsons values.
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Figure 10. Steps of the G-LISA assay. Cells will be serum-deprived for 24 hours in media that contains the treatment (R-nap, NSC 23766, ML141, and toxin A) and will be incubated at 37oC. Then the lysis buffer will be added and transfer the lysate collection to Rac1 or Cdc42 GTP-binding 96-well plate. After that, the primary antibody (anti-Rac1 or Cdc42 antibody) will be added. The secondary antibody conjugated to the HRP will be added. The reaction will be detected by luminescence.
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Figure 11. Evaluating the expression of phosphorylated and total IQGAP1 by Western blot. Cells will be serum deprived for 24 hours in media that contains the treatment (Rnap, NSC 23766, ML141, and toxin A) and will be incubated at 37oC. Then the lysis buffer will be added followed by running the proteinson gel electrophoresis followed by transferring onto a membrane. Then the membrane will be incubated with the primary antibody (anti-IQGAP1 or p-IQGAP1) followed by the secondary antibody conjugated to HRP. The reaction will be detected by luminescence.
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Figure 12. Examining the cellular localization of adherens junctions’ molecules. Adhesion molecules, such as E-cadherins, β-catenin, and α-catenin, will be examined using primary antibodies against each, which in turn will be detected by secondary antibodies conjugated either to fluorescein (green appearance) or alexa fluor (red appearance) will attach to primary antibody. The fluorescence microscopy will be used to confirm that IQGAP1 is interacting with β-catenin and the α-catenin is dissociated from the E-cadherin-β-catenin complex. Cells will be fixed with 3.7% (w/v) paraformaldehyde, blocked with bovine serum albumin in PBS, and stained for 1 hour with primary antibodies to Rac1 or Cdc42 diluted in the block solution. Then secondary fluorescently labeled antibodies will be diluted in the block solution and added to slides colocalize, then it will be shown by a - value. If α-catenin, β-catenin, and E-cadherins colocalize in absense of small inhibitors and IQGAP1 will not colocalize in the absence of small inhibitors, it will be observed under fluorescence microscope to get the Pearsons values. for 1 hour and then slides will be mounted with confocal microscopy and images will be obtained.
3.2.3. Methods for Specific Aim 2
3.2.3. a. G-LISA Assay for Quantitating Activation of Rac1 and Cdc42
Before the G-LISA assay protocol, cell lysates will be collected from cells with or without inhibitors. The culture vessel will be placed on ice, media will be aspirated, and cells will be washed with ice-cold PBS. Next, the cells will be lysed in an appropriate volume of cell lysis buffer. After that, lysates will be collected with aid of a cell scraper and transferred into pre-labeled sample tubes on ice and will be centrifuged at 14,000 Xg, 4 oC for 2 minutes. The protein conentraction will be determined using the protein assay reagent kit. The calculation will be used to modify the volumes of the cell extracts with ice-cold lysis buffer to give identical protein concentrations. The volume of ice- cold cell lysis buffer to be added to the more concentrated samples can be calculated as follows:
illustration not visible in this excerpt
Where A is the higher concentration lysates (mg/mL) and B is the concentration of the most dilute sample (mg/mL).
The G-LISA assay will be used according to the manufacturer’s protocol (Cytoskeleton, Denver, CO) to detect GTP-bound Rac1 and/or Cdc42. A buffer blank control will be prepared with 60 μL of lysis buffer and 60 μL of cold binding buffer, which will be kept on ice. The positive control will be prepared by aliquoting 24 μL of Rac1 control protein into a labeled microcentrifuge tube and will be diluted with 96 μL of cold binding buffer, and will be kept on ice. First, the powder (Rac1 or Cdc42 binds GTP) in the wells will be dissolved with 100 μL ice-cold water. The snap frozen cell lysates will be thawed in room temperature water bath then will be placed on ice after they have thawed. Based on calculations for obtaining identical protein concentrations, calculated volume of ice-cold lysis buffer will be added to the respective tubes to make all lysate protein concentrations identical. Sufficient lysates will be aliquoted for duplicate (60 μL) or triplicate (90 μL) assays into new ice-cold microcentrifuge tubes. An equal volume of ice-cold binding buffer will be added to each tube and mixed well with a pipette and then kept on ice. The water will be completely removed from the microplate wells by flicking and patting the inverted plate, and then the plate will be put back on ice. Immediately, 50 μL of lysates will be added to the respective wells. After that, 50 μL of buffer blank control will be pipetted to duplicate wells. A 50 μL of Rac1 positive control will be pipetted into duplicate wells. Immediately, the plate will be placed on a cold orbital microplate shaker at 400 rpm, 4 oC for 30 minute. During the incubation, the anti-Rac1 primary antibody will be diluted to 1/250 in antibody dilution buffer by adding 2 μL of antibody to every 500 μL antibody dilution buffer. After 30 minute, the solution will be flicked out from the wells and wash twice with 200 μL wash buffer at room temperature. After each wash, the plate will be inverted, flicked and patted vigorously to remove the wash buffer and the plate will be placed on the bench. A 200 μL of room temperature antigen presenting buffer will be pipetted into each well and will be incubated at room temperature for 2 minute. The plate will be inverted, flicked, and patted to remove the antigen presenting buffer. After that, the wells will be washed three times with 200 μL wash buffer each at room temperature. Then, 50 μL of diluted anti- Rac1 primary antibody will be added to each well and the plate will be left on the orbital microplate shaker (200-400 rpm) at room temperature for 45 minute. During the primary antibody incubation, the secondary horseradish peroxidase (HRP) labeled antibody will be diluted to 1/200 in antibody dilution buffer by adding 2.5 μL of antibody to every 500 μL antibody dilution buffer. After that, the plate will be inverted and patted to remove the anti-Rac1 primary antibody. The plate will be washed three times with 200 μL room temperature wash buffer and will be removed after each wash. A 50 μL of diluted secondary antibodies conjugated to HRP obtained from Promega (Madison, WI) will be added to each well and the plate will be placed on a microplate shaker (200-400 rpm) at room temperature for 45 minute, and then the inverted plate will be flicked and patted to remove the secondary antibody. The wells will be washed three times with 200 μL room temperature wash buffer, and will be removed after each wash. Finally, the detection of luminescence signal will be done using a microplate luminescence reader.
3.2.3.b. Western Blot Analysis to Quantify the IQGAP1
Western blot will be used to measure the expression of phosphorylated and total IQGAP1. Cells with and without inhibitors (NSC 23766, R-nap, toxin A, and ML141) will be washed with PBS, then cell lysates will be collected using a lysis buffer (10 mM Tris-HCl, pH 7.4, 1% sodium dodecyl sulfate (SDS), 5 mM ethylene diamine tetraacetic acid (EDTA), 0.1 mM dithiothreitol, 1mM phenylmethanesulfonylfluoride (PMSF), and 5 mM sodium orthovanadate). An amount of 10 μg of total cell lysate will be resolved by electrophoresis through 10% SDS-polyacrylamide gel, transferred to polyvinylidene difluouride (PVDF) membranes (Millipore Corp, Bedford, MA), and probed with the indicated antibodies. These include mouse antibody for intracellular and phosphorylated IQGAP1 (Invitrogen, Camarillo, CA) at 1-2 μg/mL dilution. Secondary antibodies conjugated to horseradish peroxidase will be obtained from Promega (Madison, WI). Detection will be performed by direct luminescence detection with Image Quant LAS 400 (GE Healthcare Biosciences, Pittsburgh, PA). Quantitation on band intensities will be performed on samples obtained from three independent experiments.
3.2.3.c. Cellular Colocalization of IQGAP1 with Į-catenin and E- cadherin-ȕ-catenin Complex
The OVCA 429 and SKOV-3ip cells lines will be seeded in a Lab Tek II chamber slide system (Nalge Nunc Int., Naperville, IL). The cells will be treated with small inhibitors NSC 23766, ML 141, R-naproxen, and toxin A will be fixed with 3.7% (w/v) formaldehyde in PBS for 10 minute and then will be washed with PBS. Blocking will be done by using 2 mg/mL bovine serum albumin for 1 hour. The fixed cells will be stained with the indicated antibodies used in immunofluorescence, including goat anti-E- cadherin antibody (#102137, EMD Milipore, Billerica, MA), rabbit anti-β-catenin antibody (#19022, EMD Milipore, Billerica, MA), mouse anti-IQGAP1 antibody (#33- 8900, Invitrogen. Camarillo, CA). All primary antibodies will be prepared in the blocking solution at 1:500 dilution and will be incubated for 1 hour at room temperature. Secondary antibodies conjugated to alexa fluor or fluorescein (Cell Signaling Technology, Danvers, MA) will be used at a 1:500 dilution in the blocking solution for 1 hour at room temperature in the dark to detect primary antibodies. The confocal images will be generated using a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY), with excitation at 488 nm, using an argon laser, and at 543 nm, using helium neon 1, and LSM Image Browser software (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Images will be compiled using Adobe Photoshop software (Adobe; San Jose, CA). Analysis of colocalization will be examined by determining Pearson’s correlation coefficient using LSM imaging software (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Quantitation will be performed on samples obtained from three independent experiments and values will be represented the mean ± standard deviation.
3.2.3.d. Statistical Tests and Data Analysis
Data will be collected in triplicates for all experiments. Results for cells with treatments will be compared to control cells without treatments using a non-parametric Welch’s two sample t- test. Any p-value below 0.05 will be considered statistically significant.
3.2.3.e. Anticipated Results, Potential Problems, and Alternative Approaches for Aim 2
We expect that treating OVCA 429 and SKOV-3ip cells with the small inhibitors NSC 23766, ML 141, R-naproxen, and toxin A will cause a reduction of the activity level of Rac1 and Cdc42 in G-LISA assay reflecting successful inhibition of these GTPase in the cell. We expect that Western blot analysis will show the presence of total IQGAP1 and absence or reduction in phosphorylated IQGAP1. Also, we expect that upon inhibition of Rac1 and Cdc42, IQGAP1 will localize with β-catenin and E-cadherin which will be detected by immunofluorescence and also α-catenin will not localize with β-catenin in presence of small inhibitors, which indicates weak cell-cell adhesion. However, in the absence of inhibitors, α-catenin will localize with E-cadherin and β-catenin, but IQGAP1 will not localize β-catenin which confirms that IQGAP1 is not interacting with the β-catenin binding site in absence of small inhibitors. If we face problems with immunofluorescence, the alternative is immunocolorimetric detection where enzymes such as alkaline phosphatase or HRP are conjugated to secondary antibodies and substrates are added to generated a colored product that is detected by light microscopy. An alternative to G-LISA is to use GTP-bound specific antibodies specific for GTP-bound Rac1 and Cdc42 (Life Span BioSciences, Inc, Seattle, WA).
4. Summary and Future Directions
There is a great need to identify new drugs and drug targets for treatment and management of residual and recurrent metastatic OVCA. Of particular importance are drugs that inhibit the formation of MCAs cell population, as this could increase the effectiveness of chemotherapy. We expect that the inhibition of Rac1 and Cdc42 will disperse MCAs. Our future goal is to examine if dispersing MCAs/spheroids by inhibiting Rac1 and Cdc42 will (Figure 4) sensitize the MCAs to chemotherapy (52,58,67,119,120,124,125). This will improve the efficacy of paclitaxel or cisplatin treatment, two therapeutic agents to which the MCA subpopulation of ascitic carcinoma cells are resistant.
In the future, we will silence Rac1 or Cdc42 or through knocking down their RNA to check if the effect is equivalent to treatment with small inhibitors. Also, depending on the outcome of Aim 2, we may explore effects on expression or interactions of adhesion molecules, such as β-catenin, E-cadherin, and integrins or metalloproteases, and examine signaling pathways involved in their regulations. This will help us understand the mechanism of action of MCAs dispersion and how it sensitizes them to chemotherapy. On the other hand, other adhesion molecules such as desmosomal adhesions can be considered as another target molecule as they are regulated by Rho GTPases (88).
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- Citation du texte
- Ahmed Ashour (Auteur), 2012, The Role Of Rac1 And Cdc42 In the Aggregation of Ovarian Cancer Spheroids, Munich, GRIN Verlag, https://www.grin.com/document/282089
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