**2. UPS components in ovarian cancer**

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**).

#### **2.1. E3 ligases**

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


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

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

**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.

**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.

138 Ovarian Cancer - From Pathogenesis to Treatment

RING domain for a potent E3 ligase activity and interaction with E2 UbcH5 [14, 21, 22]. BRCA1 E3 ligase substrate specificity is believed to depend on its phosphorylation-dependent binding to proteins containing phospho-SXXF motif such as CtIP, BACH1, and ABRA1 through a phospho-peptide recognition domain (BRCT) [23, 24]. The strong relation between BRCA1 tumor suppressor properties and E3 ligase activity is evident from the clustering of missense mutations that predispose to cancer in the Zn2+-binding residues of BRCA1 RING finger domain crucial for its ubiquitin ligase activity [25]. The full range of function of BRCA1/ BARD1 complex is not completely understood [18]. One of the most important functions of BRCA1 is to repair DNA double-strand breaks (DSBs). Following DNA damage, chromatinassociated histone H2AX phosphorylation by ATM and ATR at DNA damage site recruits an E3 ubiquitin ligase RFN8 and a phospho-module-binding mediator MDC1 at the damage site [17, 26–28]. RFN8 together with ubiquitin conjugase Ubc13, ubiquitinate histone H2A and H2B at chromatin lesions, which in turn translocate BRCA1 complex containing RAP80, a protein with ubiquitin-interacting motif (UIM), ABRA1, protein that interacts with BRCA1 BRCT domain and deubiquitinating enzyme, BRCC36 to Lys6- and Lys63-linked polyubiquitin chains at DSBs [17, 26, 29]. BRCA1 has also been implicated with the transcriptional activation of genes in response to DNA damage. The C-terminus of BRCA1 complexes with RNA polymerase II through RNA helicase, while N-terminus BRCA1/BRAD1 heterodimer binds to RNA polymerase II holoenzyme [30]. Identifying genes regulated by BRCA1 would shed a significant light on the transcriptional role of BRCA1. However, BRCA1 overexpression studies have shown induction in p53-responsive E3 ligase, mdm2, cell-cycle inhibitor, p21 and stress-response factor, and GADD45 in breast and small-cell lung cancer cell lines [31, 32]. Besides, BRCA1 also regulates G1/S, S-phase, and G2/M cell-cycle checkpoints through

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Over the last 10 years, significant information has been gained about the structure, function, and unique features of BRCA gene products, BRCA1 and BRCA2, which collectively contributes to the biological response to DNA damage through homologous recombination of DNA repair and regulation of cell-cycle checkpoints. BRCA1/2-deficient cancers, including ovarian cancer, are now recognized as the target for a class of drugs known as PARP (poly ADPribose polymerase) inhibitors [34]. PARP detects and initiates an immediate cellular response to metabolic or radiation-induced single-strand DNA breaks (SSB). It binds to DNA and synthesizes polymeric adenosine diphosphate ribose (poly ADP-ribose or PAR), which acts as signal to other DNA-repairing enzymes. PARP inhibition directly blocks the PARP enzymatic activity and subsequently leads to PARP accumulation on DNA, a process called PARP trapping, which converts an SSB into a double-strand DNA break through the collapse of replication fork [34]. BRCA-deficient tumor cells with impaired homologous recombination repair of double-strand DNA breaks are directed toward the error-prone repair process of non-homologous end joining which leads to genetic instability and cell death. Thus, BRCA1/2-deficient ovarian cancer cells with PARP inhibition undergo synthetically lethal cell death [34]. PARP inhibitor, Olaparib manufactured by AstraZeneca, is in phase I/II clinical trials for BRCAdeficient high-grade serous ovarian cancer [34]. Olaparib-treated ovarian cancer patients with BRCA1/2 mutation had a progression-free survival of 11.2 months compared to 4.3 months of patients receiving placebo [35]. In summary, BRCA is an ideal example of E3 ubiquitin ligase

interactions with RAD3, ATM/ATR, and Chk1/Chk2 [26, 30, 33].

playing an essential role in ovarian cancer and its intervention.

**Figure 4.** Key players of each UPS component involved in regulating ovarian cancer signaling. (A and B) DUBs and E3 ligases as candidate genes in ovarian cancer, (C) proteasomal activity and inhibitors in ovarian cancer, and (D) regulation of ovarian cancer oncogenic signaling by UPS.

terminus (HECT) domain transfer ubiquitin to the substrate in a two-step process—ubiquitin is first transferred to a catalytic cysteine on E3 and then to the substrate. Based on their N terminus extensions, HECTs are further classified into three subfamilies: Nedd4 family, HERC family, and other HECT that contain various domains. The RBR E3s are characterized by the presence of three RING domains, RING1 and RING2, separated by an in-between-RING (IBR) domain. RING1 recruits the ubiquitin-charged E2, RING2 possess catalytic cysteine. The IBR is called benign-catalytic domain as it lacks catalytic cysteine residue [15]. Given their cellular specificity and complexity, E3s are implicated in a number of pathophysiological conditions, which makes them an attractive therapeutic target in human diseases, including cancer [16].

#### *2.1.1. BRCA1*

The breast and ovarian cancer susceptibility gene, BRCA1, is a tumor suppressor gene [17]. Heterozygous mutations in BRCA1 gene predispose women to both familial and sporadic breast and ovarian cancers [18, 19]. Nonetheless, BRCA1 mutations are also associated with other cancers like stomach, pancreas, prostate, and colon [20]. BRCA1 acts as a hub protein, which participates in several different protein complexes to coordinate a diverse range of cellular functions including DNA repair, cell-cycle regulation, apoptosis, transcriptional regulation, and centrosome duplication to maintain genomic stability [17]. The structural analysis of BRCA1 protein suggested that it has a RING finger domain that harbors E3 ubiquitin ligase activity [14]. In addition, BRCA1 forms a heterodimer complex with BARD1, a protein with a RING finger domain [14]. BARD1 interaction stabilizes the proper conformation of BRCA1 RING domain for a potent E3 ligase activity and interaction with E2 UbcH5 [14, 21, 22]. BRCA1 E3 ligase substrate specificity is believed to depend on its phosphorylation-dependent binding to proteins containing phospho-SXXF motif such as CtIP, BACH1, and ABRA1 through a phospho-peptide recognition domain (BRCT) [23, 24]. The strong relation between BRCA1 tumor suppressor properties and E3 ligase activity is evident from the clustering of missense mutations that predispose to cancer in the Zn2+-binding residues of BRCA1 RING finger domain crucial for its ubiquitin ligase activity [25]. The full range of function of BRCA1/ BARD1 complex is not completely understood [18]. One of the most important functions of BRCA1 is to repair DNA double-strand breaks (DSBs). Following DNA damage, chromatinassociated histone H2AX phosphorylation by ATM and ATR at DNA damage site recruits an E3 ubiquitin ligase RFN8 and a phospho-module-binding mediator MDC1 at the damage site [17, 26–28]. RFN8 together with ubiquitin conjugase Ubc13, ubiquitinate histone H2A and H2B at chromatin lesions, which in turn translocate BRCA1 complex containing RAP80, a protein with ubiquitin-interacting motif (UIM), ABRA1, protein that interacts with BRCA1 BRCT domain and deubiquitinating enzyme, BRCC36 to Lys6- and Lys63-linked polyubiquitin chains at DSBs [17, 26, 29]. BRCA1 has also been implicated with the transcriptional activation of genes in response to DNA damage. The C-terminus of BRCA1 complexes with RNA polymerase II through RNA helicase, while N-terminus BRCA1/BRAD1 heterodimer binds to RNA polymerase II holoenzyme [30]. Identifying genes regulated by BRCA1 would shed a significant light on the transcriptional role of BRCA1. However, BRCA1 overexpression studies have shown induction in p53-responsive E3 ligase, mdm2, cell-cycle inhibitor, p21 and stress-response factor, and GADD45 in breast and small-cell lung cancer cell lines [31, 32]. Besides, BRCA1 also regulates G1/S, S-phase, and G2/M cell-cycle checkpoints through interactions with RAD3, ATM/ATR, and Chk1/Chk2 [26, 30, 33].

Over the last 10 years, significant information has been gained about the structure, function, and unique features of BRCA gene products, BRCA1 and BRCA2, which collectively contributes to the biological response to DNA damage through homologous recombination of DNA repair and regulation of cell-cycle checkpoints. BRCA1/2-deficient cancers, including ovarian cancer, are now recognized as the target for a class of drugs known as PARP (poly ADPribose polymerase) inhibitors [34]. PARP detects and initiates an immediate cellular response to metabolic or radiation-induced single-strand DNA breaks (SSB). It binds to DNA and synthesizes polymeric adenosine diphosphate ribose (poly ADP-ribose or PAR), which acts as signal to other DNA-repairing enzymes. PARP inhibition directly blocks the PARP enzymatic activity and subsequently leads to PARP accumulation on DNA, a process called PARP trapping, which converts an SSB into a double-strand DNA break through the collapse of replication fork [34]. BRCA-deficient tumor cells with impaired homologous recombination repair of double-strand DNA breaks are directed toward the error-prone repair process of non-homologous end joining which leads to genetic instability and cell death. Thus, BRCA1/2-deficient ovarian cancer cells with PARP inhibition undergo synthetically lethal cell death [34]. PARP inhibitor, Olaparib manufactured by AstraZeneca, is in phase I/II clinical trials for BRCAdeficient high-grade serous ovarian cancer [34]. Olaparib-treated ovarian cancer patients with BRCA1/2 mutation had a progression-free survival of 11.2 months compared to 4.3 months of patients receiving placebo [35]. In summary, BRCA is an ideal example of E3 ubiquitin ligase playing an essential role in ovarian cancer and its intervention.

terminus (HECT) domain transfer ubiquitin to the substrate in a two-step process—ubiquitin is first transferred to a catalytic cysteine on E3 and then to the substrate. Based on their N terminus extensions, HECTs are further classified into three subfamilies: Nedd4 family, HERC family, and other HECT that contain various domains. The RBR E3s are characterized by the presence of three RING domains, RING1 and RING2, separated by an in-between-RING (IBR) domain. RING1 recruits the ubiquitin-charged E2, RING2 possess catalytic cysteine. The IBR is called benign-catalytic domain as it lacks catalytic cysteine residue [15]. Given their cellular specificity and complexity, E3s are implicated in a number of pathophysiological conditions, which makes them an attractive therapeutic target in human diseases, including cancer [16].

**Figure 4.** Key players of each UPS component involved in regulating ovarian cancer signaling. (A and B) DUBs and E3 ligases as candidate genes in ovarian cancer, (C) proteasomal activity and inhibitors in ovarian cancer, and (D) regulation

The breast and ovarian cancer susceptibility gene, BRCA1, is a tumor suppressor gene [17]. Heterozygous mutations in BRCA1 gene predispose women to both familial and sporadic breast and ovarian cancers [18, 19]. Nonetheless, BRCA1 mutations are also associated with other cancers like stomach, pancreas, prostate, and colon [20]. BRCA1 acts as a hub protein, which participates in several different protein complexes to coordinate a diverse range of cellular functions including DNA repair, cell-cycle regulation, apoptosis, transcriptional regulation, and centrosome duplication to maintain genomic stability [17]. The structural analysis of BRCA1 protein suggested that it has a RING finger domain that harbors E3 ubiquitin ligase activity [14]. In addition, BRCA1 forms a heterodimer complex with BARD1, a protein with a RING finger domain [14]. BARD1 interaction stabilizes the proper conformation of BRCA1

*2.1.1. BRCA1*

of ovarian cancer oncogenic signaling by UPS.

140 Ovarian Cancer - From Pathogenesis to Treatment

#### *2.1.2. Cullin-RING ligases: cullin 4*

Cullin-RING ubiquitin ligases (CRLs), composed of CUL1, 2, 3, 4A, 4B, 5, and 7, are the largest family of E3s that ubiquitinate a wide array of substrates involved in cell-cycle, DNAdamage response, chromatin remodeling, and gene expression. Cullin (CUL) neddylation, a process of adding ubiquitin-like protein—NEDD8 to the cullin [36], is crucial for their activation. Neddylation is catalyzed by NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8-E3 ligase. The genome-wide analysis of human cancers revealed *CUL4A* amplification in 20% of the basal-like breast cancer subtype, characterized as "triple negative," and CUL4A levels were associated with aggressive growth and poor prognosis. Dysregulation of CUL4A in multiple tumor types leads to the hypothesis that CUL4A plays a role in promoting oncogenesis [36]. High CUL4A expression and activity in ovarian cancer is implicated with cancer cell proliferation and survival. NEDD8-activating enzyme inhibitor, MLN4924, which blocks cullin neddylation activation, is reported to induce cellcycle arrest, apoptosis, and tumor cell growth in epithelial ovarian cancer cells. In addition, MLN4924 sensitized ovarian cancer cells to chemotherapeutic drug treatments [37].

the master regulator of ovarian cancer metabolism [41]. They reported the co-amplification of USP13 gene with PIK3CA (phosphatidylinositol-3-kinase catalytic subunit, α-isoform) in 29.3% of high-grade serous ovarian cancer patients and its association with poor clinical outcome. USP13 stabilized the protein levels of two key metabolic enzymes, ATP citrate lyase and oxoglutarate dehydrogenase, which in turn regulate the mitochondrial respiration, glutaminolysis, and fatty acid synthesis in ovarian cancer cells. USP13 inhibition suppressed ovarian tumor progression and sensitized the tumor cells to PI3K/AKT inhibitor [41]. Similarly, USP7 (also known as HAUSP, herpes virus-associated ubiquitin protease) plays a crucial role in ovarian cancer [42]. USP7 is a DUB for MDM2, which prevents MDM2 autoubiquitination, leading to its stabilization and consequent induction of p53 degradation. Treating an ovarian cancer xenograft model with a novel inhibitor of USP7, CDDO-Me suppressed tumor growth. CDDO-Me directly binds to USP7, which leads to a decrease in its substrate Mdm2, Mdmx protein levels [42]. USP4 overexpression is reported in invasive breast carcinoma, enhancing TGFβ signaling by stabilizing SMAD2/SMAD4 complex but not much is known about its role in ovarian cancer [11]. USP36 expression is increased in ovarian cancer cells compared to normal ovarian surface epithelium; however, further studies are needed to understand its role in ovarian cancer [43]. DUB UCHL1 (ubiquitin-carboxyl terminal hydrolase 1) plays a contradicting role in different cancers [11]; it is reported as a methylated tumor suppressor gene in ovarian cancer [44, 45], while it is overexpressed in lymphoma, esophageal squamous cell carcinoma, renal, lung cancers, and acts as an oncogene [46–48]. Under hypoxic conditions, UCHL1 is shown to deubiquitinate and stabilize HIF-1α and promote tumor metastasis [49, 50]. We for the first time identified the oncogenic overexpression of UCHL1 in high-grade serous ovarian cancer and association with poor clinical outcome (unpublished data). These studies suggest the emerging role of DUBs in ovarian cancer and the potential of DUB inhibi-

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The efficient and selective degradation of cellular proteins is essential for protein quality control and maintenance of cellular homeostasis [51]. Impaired protein quality control and degradation is associated with many human diseases such as cancer, cardiovascular diseases, and aging-related pathophysiological conditions such as Alzheimer's and Parkinson's. UPS mediates targeted protein degradation under both normal and malignant conditions [52]. However, cancer cells are more dependent on UPS-mediated degradation to promote the degradation of tumor suppressors and various cell-cycle checkpoint proteins as well as to reduce proteotoxic stress accumulated due to genomic aberrations [53]. The 26S proteasome is a multi-subunit complex that contains one barrel-shaped 20S catalytic core particle (CP) and 19S regulatory particle (RP) that binds to one or both the ends of barrel-shaped CP. The active degradation of proteins is regulated by 20S CP harboring proteolytic active sites while 19S RP regulates substrate binding and target protein entry

The amazing efficacy and clinical use of proteasome inhibitor Bortezomib (PS-341, Velcade) for the treatment of multiple myeloma and mantle cell lymphoma has encouraged researchers to explore the possibility of targeting other components of the UPS for cancer treatment [54]. However, Bortezomib has not demonstrated a significant activity against other solid

tors in neo-adjuvant therapies for ovarian cancer.

**2.3. Proteasomes**

into 20S [52].

The role of Skp2 and FBXW7 in ovarian cancer signaling is discussed in the next section.

#### **2.2. Deubiquitinating enzymes**

Reversibility is an important aspect of ubiquitin system, which is mediated by deubiquitinating enzymes or deubiquitinases (DUBs). DUBs are essential components of UPS that possess ubiquitin-isopeptidase activity and catalyze the removal of ubiquitin from the target proteins. Thus, DUBs play a crucial role in the regulation of ubiquitin-mediated regulatory and proteolytic signaling [11, 38]. DUBs activity affect the activation, recycling, localization, and turnover of multiple proteins, which in turn regulate cellular homeostasis, protein stability, and a wide range of signaling pathways [39]. DUBs also maintain ubiquitin homeostasis in the cell by generating free ubiquitin monomers, which is essential for ubiquitin-mediated regulation of cell function [38]. Consistent with this, an altered DUB expression or activity has been implicated with several diseases including cancer. Numerous DUBs have been characterized as oncogenes mediating cancer initiation and progression [11, 40]. Therefore, pharmacological interventions targeting DUB activity using small molecule inhibitors are being used as a rationale to search for novel anticancer drugs [11].

**Box 2.** About 98 DUBs are reported in human genome, which are mainly divided into five families based on their sequence and structural homology: Ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado Joseph disease proteases (MJD), and JAB1/MPN/Mov34 (JAMM) metallopeptidases. Most DUBs are cysteine proteases except JAMMs, which belong to catalytic class of metalloproteases. The recent discovery of DUBs with the selectivity of cleaving extended Lys-48-linked polyubiquitin chains belongs to new family of DUBs named Mindy. The DUB-substrate specificity somewhat depends on ubiquitin chain linkage and topology; however, by large, given the complexity of ubiquitin system, it remains unknown [38].

DUBs role is evident in several cancers including Fanconi anemia (USP1), prostate cancer (USP2), adenocarcinoma (USP4), non-small-cell lung carcinoma (USP7), glioblastoma (USP15), myeloma, and leukemia (USP9x) [11, 39]. Han et al. identified the role of USP13 as the master regulator of ovarian cancer metabolism [41]. They reported the co-amplification of USP13 gene with PIK3CA (phosphatidylinositol-3-kinase catalytic subunit, α-isoform) in 29.3% of high-grade serous ovarian cancer patients and its association with poor clinical outcome. USP13 stabilized the protein levels of two key metabolic enzymes, ATP citrate lyase and oxoglutarate dehydrogenase, which in turn regulate the mitochondrial respiration, glutaminolysis, and fatty acid synthesis in ovarian cancer cells. USP13 inhibition suppressed ovarian tumor progression and sensitized the tumor cells to PI3K/AKT inhibitor [41]. Similarly, USP7 (also known as HAUSP, herpes virus-associated ubiquitin protease) plays a crucial role in ovarian cancer [42]. USP7 is a DUB for MDM2, which prevents MDM2 autoubiquitination, leading to its stabilization and consequent induction of p53 degradation. Treating an ovarian cancer xenograft model with a novel inhibitor of USP7, CDDO-Me suppressed tumor growth. CDDO-Me directly binds to USP7, which leads to a decrease in its substrate Mdm2, Mdmx protein levels [42]. USP4 overexpression is reported in invasive breast carcinoma, enhancing TGFβ signaling by stabilizing SMAD2/SMAD4 complex but not much is known about its role in ovarian cancer [11]. USP36 expression is increased in ovarian cancer cells compared to normal ovarian surface epithelium; however, further studies are needed to understand its role in ovarian cancer [43]. DUB UCHL1 (ubiquitin-carboxyl terminal hydrolase 1) plays a contradicting role in different cancers [11]; it is reported as a methylated tumor suppressor gene in ovarian cancer [44, 45], while it is overexpressed in lymphoma, esophageal squamous cell carcinoma, renal, lung cancers, and acts as an oncogene [46–48]. Under hypoxic conditions, UCHL1 is shown to deubiquitinate and stabilize HIF-1α and promote tumor metastasis [49, 50]. We for the first time identified the oncogenic overexpression of UCHL1 in high-grade serous ovarian cancer and association with poor clinical outcome (unpublished data). These studies suggest the emerging role of DUBs in ovarian cancer and the potential of DUB inhibitors in neo-adjuvant therapies for ovarian cancer.

#### **2.3. Proteasomes**

*2.1.2. Cullin-RING ligases: cullin 4*

142 Ovarian Cancer - From Pathogenesis to Treatment

**2.2. Deubiquitinating enzymes**

rationale to search for novel anticancer drugs [11].

Cullin-RING ubiquitin ligases (CRLs), composed of CUL1, 2, 3, 4A, 4B, 5, and 7, are the largest family of E3s that ubiquitinate a wide array of substrates involved in cell-cycle, DNAdamage response, chromatin remodeling, and gene expression. Cullin (CUL) neddylation, a process of adding ubiquitin-like protein—NEDD8 to the cullin [36], is crucial for their activation. Neddylation is catalyzed by NEDD8-activating enzyme E1 (NAE), NEDD8-conjugating enzyme E2 (UBC12), and NEDD8-E3 ligase. The genome-wide analysis of human cancers revealed *CUL4A* amplification in 20% of the basal-like breast cancer subtype, characterized as "triple negative," and CUL4A levels were associated with aggressive growth and poor prognosis. Dysregulation of CUL4A in multiple tumor types leads to the hypothesis that CUL4A plays a role in promoting oncogenesis [36]. High CUL4A expression and activity in ovarian cancer is implicated with cancer cell proliferation and survival. NEDD8-activating enzyme inhibitor, MLN4924, which blocks cullin neddylation activation, is reported to induce cellcycle arrest, apoptosis, and tumor cell growth in epithelial ovarian cancer cells. In addition,

MLN4924 sensitized ovarian cancer cells to chemotherapeutic drug treatments [37].

The role of Skp2 and FBXW7 in ovarian cancer signaling is discussed in the next section.

Reversibility is an important aspect of ubiquitin system, which is mediated by deubiquitinating enzymes or deubiquitinases (DUBs). DUBs are essential components of UPS that possess ubiquitin-isopeptidase activity and catalyze the removal of ubiquitin from the target proteins. Thus, DUBs play a crucial role in the regulation of ubiquitin-mediated regulatory and proteolytic signaling [11, 38]. DUBs activity affect the activation, recycling, localization, and turnover of multiple proteins, which in turn regulate cellular homeostasis, protein stability, and a wide range of signaling pathways [39]. DUBs also maintain ubiquitin homeostasis in the cell by generating free ubiquitin monomers, which is essential for ubiquitin-mediated regulation of cell function [38]. Consistent with this, an altered DUB expression or activity has been implicated with several diseases including cancer. Numerous DUBs have been characterized as oncogenes mediating cancer initiation and progression [11, 40]. Therefore, pharmacological interventions targeting DUB activity using small molecule inhibitors are being used as a

DUBs role is evident in several cancers including Fanconi anemia (USP1), prostate cancer (USP2), adenocarcinoma (USP4), non-small-cell lung carcinoma (USP7), glioblastoma (USP15), myeloma, and leukemia (USP9x) [11, 39]. Han et al. identified the role of USP13 as

**Box 2.** About 98 DUBs are reported in human genome, which are mainly divided into five families based on their sequence and structural homology: Ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado Joseph disease proteases (MJD), and JAB1/MPN/Mov34 (JAMM) metallopeptidases. Most DUBs are cysteine proteases except JAMMs, which belong to catalytic class of metalloproteases. The recent discovery of DUBs with the selectivity of cleaving extended Lys-48-linked polyubiquitin chains belongs to new family of DUBs named Mindy. The DUB-substrate specificity somewhat depends on ubiquitin chain linkage and topology; however, by large, given the complexity of ubiquitin system, it remains unknown [38].

The efficient and selective degradation of cellular proteins is essential for protein quality control and maintenance of cellular homeostasis [51]. Impaired protein quality control and degradation is associated with many human diseases such as cancer, cardiovascular diseases, and aging-related pathophysiological conditions such as Alzheimer's and Parkinson's. UPS mediates targeted protein degradation under both normal and malignant conditions [52]. However, cancer cells are more dependent on UPS-mediated degradation to promote the degradation of tumor suppressors and various cell-cycle checkpoint proteins as well as to reduce proteotoxic stress accumulated due to genomic aberrations [53]. The 26S proteasome is a multi-subunit complex that contains one barrel-shaped 20S catalytic core particle (CP) and 19S regulatory particle (RP) that binds to one or both the ends of barrel-shaped CP. The active degradation of proteins is regulated by 20S CP harboring proteolytic active sites while 19S RP regulates substrate binding and target protein entry into 20S [52].

The amazing efficacy and clinical use of proteasome inhibitor Bortezomib (PS-341, Velcade) for the treatment of multiple myeloma and mantle cell lymphoma has encouraged researchers to explore the possibility of targeting other components of the UPS for cancer treatment [54]. However, Bortezomib has not demonstrated a significant activity against other solid tumors [55]. This conundrum has spurred the development of next-generation proteasome inhibitors, including MLN9708 (Millennium Pharmaceuticals), Carfilzomib and ONX0912 (Onyx Pharmaceuticals, South San Francisco, CA), and CEP18770 (Cephalon, Frazer, PA) [56]. Although these compounds target the same 20S CP, they differ in targeted active site and enzyme kinetics, resulting in activity differences based on tumor type and tumor location. Bazzaro et al. reported elevated levels of ubiquitinated proteins and 19S and 20S proteasome subunits in both low-grade and high-grade ovarian carcinoma tissues and cell lines compared to benign ovarian tumors and immortalized normal ovarian surface epithelium controls. They reported an increased sensitivity to apoptosis in proteasome inhibitor, PS-341 treated cells, and a reduced growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice [57]. In a similar study, proteasome inhibitor, MG132—a peptide aldehyde—showed an enhanced sensitivity of ovarian cancer cells, SKOV3 to cisplatin both *in vitro* and *in vivo* [58]. The effect of Bortezomib on ovarian cancer cells is also supported by the increased sensitivity of Bortezomib-treated chemoresistant ovarian cancer cells to TRAIL-induced apoptosis [59]. Together, these results indicate the essential role of proteasomes in mediating prosurvival signaling in cancer, which may also be due to altered proteasome composition resulting in an enhanced proteasomal activity [52].

have been reported in many cancers [63]. In addition, the expression and activity of Usp7, a deubiquitinating enzyme for Mdm2, is increased in several cancers including breast and ovarian cancer, which prevents Mdm2 ubiquitination and promotes its stability. Reduced tumor growth was seen in an ovarian cancer xenograft model treated with Usp7 inhibitor [42]. On the other hand, when p53 acquires gain-of-function (GOF) mutations as in the case of nearly half of the cancers, it gains oncogenic functions and loses its wild-type tumor suppressor properties. Thus, in these cancer cells, several mechanisms stabilize mutant p53 through its activation or by inhibition of its degradation by disrupting Mdm2 and mutant p53 binding. Several splice variants of Mdm2 are reported in cancer, which lack a p53-binding domain and thus stabilizes mutant p53 expression [63]. In addition, GOF mutation-induced conformational changes in mutant p53 allow the binding of Hsp90 (heat shock protein 90) to mutant p53, which prevents Mdm2 binding and Mdm2-mediated degradation of mutant p53 [60]. It is now well established that elevated mutant p53 levels correlate with more aggressive tumors and poor prognosis. About 96% of high-grade serous ovarian cancer patients have GOF p53 mutations, which orchestrate a distinct pro-tumorigenic transcription and oncogenic programs. Knowledge of a UPS component responsible for mutant p53 stabilization, which could be chemically manipulated, will be useful in HGSOC. Nonetheless, Mdm2 is a great therapeutic target and prognostic factor for ovarian cancer with wild-type p53, such as

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Genomic alterations in cell-cycle regulatory genes have been reported in almost every human carcinoma. Cyclins are the crucial regulators of cell-cycle progression [65]. A periodic increase in cyclin levels and their timed interplay with cyclin-dependent kinases (CDKs) is essential for the proper progression of cell cycle [65]. Their levels are regulated by a combination of transcription and ubiquitin-mediated degradation [18, 66]. About 30% of high-grade serous ovarian cancer patients have amplification of the CCNE1 gene, which encodes for G1/Sspecific cyclin E. Cyclin E-CDK2 interactions commit the cell to S-phase genome duplication [3]. Aberrant accumulation and overabundance of cyclin E leads to premature entry of the cell into S-phase, resulting in chromosome instability and tumor formation [67]. Cyclin E amplification is likely to be an early event in the development of high-grade serous ovarian cancer [3]. This subclass of patients has no apparent defect in homologous recombination as seen in patients with BRCA1 and BRCA2 mutations with defect in DNA repair pathways [3]. The overexpression of cyclin E is an indicator of poor overall survival of ovarian cancer patients. Cyclin E protein levels are maintained by a multi-subunit SCF ubiquitin ligase, which mediates its ubiquitination and degradation [68]. Cyclin E auto-phosphorylation after its association with CDK2 is recognized by the SCF-associated F-box protein 7 (FBXW7), which binds to cyclin E and facilitates its ubiquitination and degradation [68, 69]. More than 30% of human cancers have a deleted FBXW7 gene located on chromosome 4q32. FBXW7 also regulates mTOR, Myc, and Notch1 degradation, depending upon the type of tumor [70, 71]. FBXW7 is known to be mutated in breast and ovarian cancer cell lines with high cyclin E levels [3]. The loss of cyclin E or CDK2 results in cell-cycle arrest or apoptosis in HGSOC cell lines [3], sug-

gesting cyclin E inhibition as a novel therapeutic approach in ovarian cancer patients.

clear-cell carcinomas [64].

**3.2. Cyclin E**
