**1. Introduction**

Ovarian cancer is the most lethal gynecologic malignancy with a high case-to-fatality ratio [1]. According to American Cancer Society, approximately 22,440 new cases of ovarian cancer will be diagnosed in the year 2017 and about 14,080 women in the United States will die from this deadly disease [2]. About 90% of ovarian carcinomas are heterogeneous epithelial neoplasms with distinctive biology and clinicopathologic features at cellular and molecular

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

levels [1, 3]. The clinical management of ovarian cancer has addressed this heterogeneity and classified ovarian cancer into high-grade and low-grade serous, endometrioid, clear cell, and mucinous subtypes based on the histology, tissue of origin, prognosis, and genetic alterations that deregulate specific signaling pathways in these tumor cells [4, 5] (**Figure 1**). Of these, high-grade serous ovarian cancer (HGSOC) is the most prevalent and lethal subtype of ovarian cancer. It accounts for 70–80% of ovarian cancer deaths [1]. The low *five-year survival rate* of HGSOC patients is attributed to the late detection of extensively metastasized disease, especially to omentum, which is the primary site of ovarian cancer metastasis. Moreover, about 80–90% of HGSOC patients eventually develop chemo-resistant tumors, after an initial positive response to cytoreductive surgery and chemotherapy, which are important prognosticators of the survival of HGSOC patients [1, 3]. The initiation and development of HGSOC is known to proceed through the early acquisition of genetic alterations in the tumor suppressor gene *TP53* [3, 6]. About 96% of HGSOC patients carry gain-of-function (GOF) mutations in *TP53* gene [3]. It is believed that *TP53* mutations lead to the precursor lesions in fallopian tube fimbria, which develop into serous tubal intraepithelial carcinoma (STIC) and ultimately to HGSOC [7, 8]. The reduced risk of ovarian cancer in BRCA1 mutation carriers after salpingooophorectomy supports the theory of HGSOC origin from STIC [9]. Mutant p53 orchestrates a distinct pro-tumorigenic signaling network and confer chemo-resistance through transcription-dependent and independent mechanisms in cancer cells. A recent study in triple-negative breast cancer cells revealed the role of mutant p53-proteasome axis in regulating global effects on cancer cell's protein homeostasis, inhibiting tumor suppressive pathways or turning on the oncogenic signaling in cancer cells [10]. A growing number of evidences suggest the role of ubiquitin signaling in tumor progression and growth. This chapter discusses the role of ubiquitin-mediated signaling in ovarian cancer pathogenesis. The different components of ubiquitin proteasome system, which are involved in this regulation, will be highlighted.

**1.1. Conceptual overview of ubiquitin modifications**

SUMO, phosphorylation, and acetylation [13].

Protein ubiquitination is a dynamic multifaceted posttranslational modification (PTM), which is involved in nearly all biological functions in a eukaryotic cell. Similar to phosphorylation, it functions as a signaling device and can be activated by extracellular stimuli, DNA damage, phosphorylation, ligand-dependent receptor activation, and signal transduction. Ubiquitin is a highly conserved 76-amino acid protein, which is expressed in all cell types. It has seven lysine (Lys or K) residues, K6, K11, K27, K29, K33, K48, and K63. Each lysine residue can result in a linkage-specific ubiquitin chain of certain topology [11, 12], which when bound to the target protein (substrate) dictates the fate of the protein (**Figure 2**). For example, the most predominant K48-linked polyubiquitin chains, which have a compact conformation, lead to the proteasomal degradation of the bound substrate. By contrast, the second most abundant K63-linked chains, which have an open conformation, are involved in non-proteolytic regulatory functions [13]. The K11-linked ubiquitin chains act as an additional proteasomal degradation signal, particularly in cell-cycle regulation [13]. The functions of the other lysine-specific ubiquitin chains remain less well characterized. K6-linked chains are shown to be upregulated with UV genotoxic stress and are known to be associated with BRCA1/BARD1 complex [14]. Similarly, K27 chains act to serve as scaffolds for protein recruitment such as p53-binding protein 1 in the DNA damage response. In addition, ubiquitin chain of mixed topology with different linkage at succeeding positions is also seen as in NF-κB signaling or in protein trafficking (**Figure 2F**) [13]. Moreover, branched ubiquitin chains of unknown function are generated when a single ubiquitin is modified with multiple molecules [12, 13]. These ubiquitin chains creating a multitude of signals with distinct cellular outcomes are referred to as "ubiquitin code" [13]. New layers of the ubiquitin code are emerging, based on findings that revealed the modification of ubiquitin chains with small ubiquitin-like (Ubl) modifier such as

Ubiquitin Signaling in Ovarian Cancer: From Potential to Challenges

http://dx.doi.org/10.5772/intechopen.75485

137

Ubiquitination is an orchestrated enzymatic reaction of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and ubiquitin E3 ligase (E3). It is the most coordinated and conserved multistep process of covalently tagging a protein with mono- or polyubiquitin chain. The process begins with the ATP-dependent activation of ubiquitin by E1 ubiquitin-activating enzyme (E1s), which then transfers it to the active site cysteine of E2 ubiquitin-conjugating enzymes (E2s) forming a thioester linkage between ubiquitin and cysteine. Ubiquitin E3 ligases (E3s) have a central role in this process, as they recognize the specific protein substrates and facilitate the transfer of ubiquitin from the E2 onto the target protein [11, 12]. Deubiquitinating

**Box 1.** The discovery of ubiquitin-mediated protein degradation in the late 1970s by Drs. Avram Hershko, Aaron Ciechanover, and Irwin Rose was awarded 2004 Nobel Prize in Chemistry. Their study highlighted the role of protein ubiquitination in selective protein breakdown, regulating the cellular functions by modulating the levels of key enzymes, regulatory proteins and removal of abnormal proteins that arise by biosynthetic errors or post synthetic damages. Ubiquitin was first isolated from bovine thymus in 1975 by Goldstein et al. (PNAS, 1975;72:11-15) [88] and found to be covalently attached to histone 2A (Goldknopf and Busch, PNAS, 1977;74:864-868) [89]. Subsequently, Drs. Hershko, Ciechanover, and Rose in a series of biochemical studies discovered and characterized the ATP-dependent, ubiquitin-mediated protein degradation using the reticulocyte lysate system (PNAS, 1979;76:3107-3110) [90].

**Figure 1.** A schematic representation of molecular drivers of low- and high-grade ovarian cancer initiation and progression. Low-grade tumors are low malignant potential (LMP) tumors associated with KRAS or BRAF mutation and loss of PTEN. High-grade serous tumors frequently have mutated *TP53* gene as well as activated members of PI3K/ Akt pathway. Highly invasive tumors originate from the fallopian tube precursor lesion, STIC, and spread to the ovary and other peritoneal surfaces. Genotoxic stresses in BRCA1/2 carriers predispose them to ovarian cancer.

#### **1.1. Conceptual overview of ubiquitin modifications**

levels [1, 3]. The clinical management of ovarian cancer has addressed this heterogeneity and classified ovarian cancer into high-grade and low-grade serous, endometrioid, clear cell, and mucinous subtypes based on the histology, tissue of origin, prognosis, and genetic alterations that deregulate specific signaling pathways in these tumor cells [4, 5] (**Figure 1**). Of these, high-grade serous ovarian cancer (HGSOC) is the most prevalent and lethal subtype of ovarian cancer. It accounts for 70–80% of ovarian cancer deaths [1]. The low *five-year survival rate* of HGSOC patients is attributed to the late detection of extensively metastasized disease, especially to omentum, which is the primary site of ovarian cancer metastasis. Moreover, about 80–90% of HGSOC patients eventually develop chemo-resistant tumors, after an initial positive response to cytoreductive surgery and chemotherapy, which are important prognosticators of the survival of HGSOC patients [1, 3]. The initiation and development of HGSOC is known to proceed through the early acquisition of genetic alterations in the tumor suppressor gene *TP53* [3, 6]. About 96% of HGSOC patients carry gain-of-function (GOF) mutations in *TP53* gene [3]. It is believed that *TP53* mutations lead to the precursor lesions in fallopian tube fimbria, which develop into serous tubal intraepithelial carcinoma (STIC) and ultimately to HGSOC [7, 8]. The reduced risk of ovarian cancer in BRCA1 mutation carriers after salpingooophorectomy supports the theory of HGSOC origin from STIC [9]. Mutant p53 orchestrates a distinct pro-tumorigenic signaling network and confer chemo-resistance through transcription-dependent and independent mechanisms in cancer cells. A recent study in triple-negative breast cancer cells revealed the role of mutant p53-proteasome axis in regulating global effects on cancer cell's protein homeostasis, inhibiting tumor suppressive pathways or turning on the oncogenic signaling in cancer cells [10]. A growing number of evidences suggest the role of ubiquitin signaling in tumor progression and growth. This chapter discusses the role of ubiquitin-mediated signaling in ovarian cancer pathogenesis. The different components of ubiquitin proteasome system, which are involved in this regulation, will be highlighted.

136 Ovarian Cancer - From Pathogenesis to Treatment

**Figure 1.** A schematic representation of molecular drivers of low- and high-grade ovarian cancer initiation and progression. Low-grade tumors are low malignant potential (LMP) tumors associated with KRAS or BRAF mutation and loss of PTEN. High-grade serous tumors frequently have mutated *TP53* gene as well as activated members of PI3K/ Akt pathway. Highly invasive tumors originate from the fallopian tube precursor lesion, STIC, and spread to the ovary

and other peritoneal surfaces. Genotoxic stresses in BRCA1/2 carriers predispose them to ovarian cancer.

Protein ubiquitination is a dynamic multifaceted posttranslational modification (PTM), which is involved in nearly all biological functions in a eukaryotic cell. Similar to phosphorylation, it functions as a signaling device and can be activated by extracellular stimuli, DNA damage, phosphorylation, ligand-dependent receptor activation, and signal transduction. Ubiquitin is a highly conserved 76-amino acid protein, which is expressed in all cell types. It has seven lysine (Lys or K) residues, K6, K11, K27, K29, K33, K48, and K63. Each lysine residue can result in a linkage-specific ubiquitin chain of certain topology [11, 12], which when bound to the target protein (substrate) dictates the fate of the protein (**Figure 2**). For example, the most predominant K48-linked polyubiquitin chains, which have a compact conformation, lead to the proteasomal degradation of the bound substrate. By contrast, the second most abundant K63-linked chains, which have an open conformation, are involved in non-proteolytic regulatory functions [13]. The K11-linked ubiquitin chains act as an additional proteasomal degradation signal, particularly in cell-cycle regulation [13]. The functions of the other lysine-specific ubiquitin chains remain less well characterized. K6-linked chains are shown to be upregulated with UV genotoxic stress and are known to be associated with BRCA1/BARD1 complex [14]. Similarly, K27 chains act to serve as scaffolds for protein recruitment such as p53-binding protein 1 in the DNA damage response. In addition, ubiquitin chain of mixed topology with different linkage at succeeding positions is also seen as in NF-κB signaling or in protein trafficking (**Figure 2F**) [13]. Moreover, branched ubiquitin chains of unknown function are generated when a single ubiquitin is modified with multiple molecules [12, 13]. These ubiquitin chains creating a multitude of signals with distinct cellular outcomes are referred to as "ubiquitin code" [13]. New layers of the ubiquitin code are emerging, based on findings that revealed the modification of ubiquitin chains with small ubiquitin-like (Ubl) modifier such as SUMO, phosphorylation, and acetylation [13].

**Box 1.** The discovery of ubiquitin-mediated protein degradation in the late 1970s by Drs. Avram Hershko, Aaron Ciechanover, and Irwin Rose was awarded 2004 Nobel Prize in Chemistry. Their study highlighted the role of protein ubiquitination in selective protein breakdown, regulating the cellular functions by modulating the levels of key enzymes, regulatory proteins and removal of abnormal proteins that arise by biosynthetic errors or post synthetic damages. Ubiquitin was first isolated from bovine thymus in 1975 by Goldstein et al. (PNAS, 1975;72:11-15) [88] and found to be covalently attached to histone 2A (Goldknopf and Busch, PNAS, 1977;74:864-868) [89]. Subsequently, Drs. Hershko, Ciechanover, and Rose in a series of biochemical studies discovered and characterized the ATP-dependent, ubiquitin-mediated protein degradation using the reticulocyte lysate system (PNAS, 1979;76:3107-3110) [90].

Ubiquitination is an orchestrated enzymatic reaction of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and ubiquitin E3 ligase (E3). It is the most coordinated and conserved multistep process of covalently tagging a protein with mono- or polyubiquitin chain. The process begins with the ATP-dependent activation of ubiquitin by E1 ubiquitin-activating enzyme (E1s), which then transfers it to the active site cysteine of E2 ubiquitin-conjugating enzymes (E2s) forming a thioester linkage between ubiquitin and cysteine. Ubiquitin E3 ligases (E3s) have a central role in this process, as they recognize the specific protein substrates and facilitate the transfer of ubiquitin from the E2 onto the target protein [11, 12]. Deubiquitinating

ubiquitin proteasome system (UPS). UPS plays an indispensable role in regulating ubiquitinmediated proteolytic and non-proteolytic regulatory signaling to control cellular homeostasis,

Ovarian cancer is characterized by multiple genetic and epigenetic abnormalities and several major (about seven) activated signaling pathways, which are directly or indirectly implicated with UPS. Moreover, several UPS components, E1s, E2s, E3s, DUBs and proteasomes are known to be deregulated or mutated in cancer (**Table 1**), suggesting their role in cancer signaling and cancer progression. This section discusses each UPS component implicated in ovarian cancer and the role of key players of each component in regulating ovarian cancer signaling (**Figure 4**).

E3 ligases (E3s) are the most heterogeneous class of enzymes in UPS as they facilitate ubiquitination with exquisite spatial, temporal, and substrate specificity. There are more than 600 E3s in a human genome, indicating the precise substrate specificity of E3s [15]. E3s can be classified into three main types, RING E3s, HECT E3s, and RBR E3s depending on the presence of type-specific domains and on the mechanism of ubiquitin transfer to the substrate protein. RING E3s are the most abundant type of ubiquitin ligases. They are characterized by the presence of zinc-binding domain called Really Interesting New Gene (RING) and U-box domain. RING E3s mediate a direct transfer of ubiquitin to substrate, functioning as a scaffold to orient the ubiquitin-charged E2, whereas E3s with homologous to the E6AP carboxyl

Ovarian and breast cancers [19, 20]

Ubiquitin Signaling in Ovarian Cancer: From Potential to Challenges

http://dx.doi.org/10.5772/intechopen.75485

139

Ovarian cancer and various malignancies [63, 64]

Ovarian, breast, and prostate cancers [76–81]

Ovarian, breast, gastric, lymphoma, lung, Esophageal squamous cell carcinoma [44–48]

Clear-cell carcinoma, lung cancer [49]

Ovarian and endometrial cancer, leukemia [71]

**Gene. Role Effect Cancer [references]**

USP13 DUB Amplification, oncogene Ovarian cancer [41]

USP7 DUB Overexpression, oncogene Ovarian cancer [42]

suppressor function of p27

BRCA1 E3 ligase Mutation, loss of tumor suppressor function

Mdm2 E3 ligase Overexpression, loss of p53 tumor

Skp2 E3 ligase Overexpression, loss of tumor

UCHL1 DUB Overexpression or methylation, role varies with cancer

FBW7 E3 ligase Mutation, loss of tumor suppressor function

VHL E3 ligase Mutation, loss of tumor suppressor function

**Table 1.** Cancer-associated alterations in UPS.

suppressor function

protein stability, and a wide range of signaling pathways.

**2. UPS components in ovarian cancer**

**2.1. E3 ligases**

**Figure 2.** Linkage-specific ubiquitin chains of different topologies. Each circle represents one ubiquitin moiety. (A) Monoubiquitination, (B) multi-monoubiquitination, (C) K48-linked chain, (D) K63-linked chain, (E) branched chain, and (F) mixed chain.

**Figure 3.** Enzymatic cascade of ubiquitin proteasome system. Ubiquitin is activated and conjugated to target protein by a conserved action of E1-ubiquitin-activating enzyme, E2-ubiquitin-conjugating enzyme, and E3 ubiquitin ligase.

enzymes (DUBs) are another class of enzymes, which removes or edits the ubiquitin chains attached to a protein, making this a highly reversible process and thus highlighting the dynamic regulation of ubiquitin signaling in the cell (**Figure 3**). These enzymes together with proteasomes, a cellular machinery involved in ubiquitin-mediated protein degradation, comprise the ubiquitin proteasome system (UPS). UPS plays an indispensable role in regulating ubiquitinmediated proteolytic and non-proteolytic regulatory signaling to control cellular homeostasis, protein stability, and a wide range of signaling pathways.
