**3. UPS in ovarian cancer cellular signaling**

Several important factors that are implicated in the molecular pathogenesis of ovarian cancer are known to be regulated by UPS, highlighting its significance in disease progression. Some of these factors are discussed subsequently.

#### **3.1. Tumor suppressor p53 and Mdm2**

Tumor suppressor protein p53 is a multifunctional sequence-specific transcription factor that plays a key role in cellular stress response. Abrogating p53 function is a key event in human cancers, leading to the deregulation of cell cycle, genetic instability, resistance to stress signals, and resulting in cancer development [60]. Due to its growth inhibitory properties, p53 is maintained at low levels in the normal cells. The E3 ubiquitin ligase Mdm2 promotes p53 ubiquitination and subsequent proteasomal degradation [61]. In addition, E4 ubiquitin ligase p300/CBP promotes polyubiquitination of p53 to accelerate its degradation by proteasomes [61]. Although Mdm2 is the predominant E3 ligase for p53, several other E3 ligases have been identified that can promote the degradation of p53, including C-terminus of HSP70 interacting protein (CHIP), murine double minute 4 (MdmX), and p53-induced protein with a RING H2 domain (Pirh2) [60]. In addition to proteolytic ubiquitination, p53 mono-ubiquitination mediates p53 nuclear export and activity [62]. Thus, UPS plays a crucial role in maintaining and regulating p53 functions.

Several cancers, including invasive breast cancer, pediatric rhabdomyosarcoma, and soft-tissue sarcoma, exploit Mdm2-p53 pathway to maintain low p53 levels under genotoxic or oxidative-stressed environment of cancer cell. Thus, Mdm2 gene amplification and overexpression 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 clear-cell carcinomas [64].

#### **3.2. Cyclin E**

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

Several important factors that are implicated in the molecular pathogenesis of ovarian cancer are known to be regulated by UPS, highlighting its significance in disease progression. Some

Tumor suppressor protein p53 is a multifunctional sequence-specific transcription factor that plays a key role in cellular stress response. Abrogating p53 function is a key event in human cancers, leading to the deregulation of cell cycle, genetic instability, resistance to stress signals, and resulting in cancer development [60]. Due to its growth inhibitory properties, p53 is maintained at low levels in the normal cells. The E3 ubiquitin ligase Mdm2 promotes p53 ubiquitination and subsequent proteasomal degradation [61]. In addition, E4 ubiquitin ligase p300/CBP promotes polyubiquitination of p53 to accelerate its degradation by proteasomes [61]. Although Mdm2 is the predominant E3 ligase for p53, several other E3 ligases have been identified that can promote the degradation of p53, including C-terminus of HSP70 interacting protein (CHIP), murine double minute 4 (MdmX), and p53-induced protein with a RING H2 domain (Pirh2) [60]. In addition to proteolytic ubiquitination, p53 mono-ubiquitination mediates p53 nuclear export and activity [62]. Thus, UPS plays a crucial role in maintain-

Several cancers, including invasive breast cancer, pediatric rhabdomyosarcoma, and soft-tissue sarcoma, exploit Mdm2-p53 pathway to maintain low p53 levels under genotoxic or oxidative-stressed environment of cancer cell. Thus, Mdm2 gene amplification and overexpression

enhanced proteasomal activity [52].

144 Ovarian Cancer - From Pathogenesis to Treatment

**3. UPS in ovarian cancer cellular signaling**

of these factors are discussed subsequently.

**3.1. Tumor suppressor p53 and Mdm2**

ing and regulating p53 functions.

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], suggesting cyclin E inhibition as a novel therapeutic approach in ovarian cancer patients.

#### **3.3. P27, a cyclin-dependent kinase inhibitor**

Similar to cell-cycle regulatory proteins, cell-cycle inhibitors are frequently altered in cancer [72, 73]. p27Kip1 inhibits cell-cycle G1 phase by interacting with CDK2/cyclin A or CDK2/ cyclin E complexes [73, 74]. Low levels of p27Kip1 protein are associated with tumor progression and growth resulting in poor prognosis of ovarian and breast cancer patients [74–76]. The evaluation of subcellular localization of p27Kip1 in tissue microarray of late-stage ovarian cancer patients revealed that patients with nuclear-only expression of p27Kip1 had a better overall survival than those with negative expression or cytoplasmic localization of the marker (p-value = 0.0002; n = 355) [77]. p27Kip1 level is an important prognostic marker of malignant transformation. Genetically altered mice with p27Kip1 haploinsufficiency are predisposed to cancer [78]. p27Kip1 protein levels are regulated by SCF E3 ligase-associated protein Skp2. Skp2 binds to p27Kip1 and mediates its ubiquitination and subsequent proteasomal degradation [79, 80]. Skp2 levels in different cancers correlate with tumor grade and inversely correlate with p27Kip1 levels and cancer prognosis. Skp2 levels were upregulated in ovarian cancer patients and were associated with advanced FIGO stage III and IV and high grade of the tumor [81]. Skp2 levels were also associated with downregulation of both p27 and p21 in these patients, suggesting an important role of Skp2- p27Kip1 pathway in ovarian cancer pathogenesis. A strong negative correlation between Skp2 levels and FOXO3a (r = −0.743; p < 0.05) in immunohistochemical analysis of ovarian cancer patients indicates that it is another potential target of Skp2 in ovarian cancer [82]. These findings and Skp2 overexpression or amplification in serous ovarian cancer characterize it as an oncogene and its inhibition a plausible approach in ovarian cancer management.

results were confirmed by knocking down BRCA1 in ovarian cancer cells [86]. However, inhibitors targeting this pathway have little effect on cancer cells as a single agent due to the presence of alternative pathways affecting the cancer phenotype, particularly the activation of the PI3K/Akt/mTOR and mitogen-activated protein kinases (MAPKs) pathway [83], suggest-

Ubiquitin Signaling in Ovarian Cancer: From Potential to Challenges

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

147

It is now well known that UPS not only mediates protein degradation but is also involved in the extensive regulation of cellular functions and signaling. A large number of studies in various cancers have uncovered the diverse and intricate role of ubiquitin in oncogenic signaling. The alterations in the genes involved in UPS support its role in cancer development and progression. However, the lack of information on DUBs specificity and multiple targets of E3s raise a question on the use of DUBs or E3s inhibitors in cancer treatment. One possible way forward is to characterize the cancer-specific and tissue-specific expression of DUBs as certain DUBs are predominantly expressed in certain tissues and cancer, suggesting the cancer-specific use of a DUB inhibitor. Moreover, most DUBs studied thus far appear to regulate a small number of targets. It is also possible that only a fraction of ubiquitinated proteins are regulated by a specific DUB family. Similarly, the E3s can be manipulated in cancer if their role is characterized in cancer-specific aberrant molecular signaling. Moreover, further characterization of mutations in DUBs or E3s in cancer patients can be used for cancer screening. In addition, proteasomes carry a great potential in cancer treatment. Although Bortezomib did not show promising results against solid tumors, the advent of next-generation proteasome inhibitors opens new possibilities. Currently, five different types of next-generation proteasome inhibitors are in phase I or phase IIb clinical trials. Moreover, understanding the regulation of proteasomal activity by altered proteasome composition may open novel ways

Compared to breast cancer, ovarian cancer is a rare but far more lethal cancer. It is estimated that 69% of all patients with ovarian carcinoma will succumb to their disease as compared with 19% of those with breast cancer [1]. Ovarian cancer heterogeneity is represented by several genetic (BRCA1/2), epigenetic, and signaling (p53, CDK/p27, CCNE1) alterations, and various UPS components are implicated in these ovarian cancer-specific alterations. Several studies have established a link between UPS and ovarian cancer. However, further studies are needed to identify potential inhibitors for proteasome-based or E3s/DUBs-based therapies in

The author acknowledges the support of the Indiana University School of Medicine, Biomedical Research Grant, Showalter Research Grant, and Ovarian Cancer Research Fund

ing a combined use of EGFR and PI3K inhibitors in ovarian cancer [87].

**4. Concluding remarks**

to target proteasomes in cancer.

**Acknowledgements**

Alliance.

ovarian cancer, which can be taken to clinical trials.

#### **3.4. The epidermal growth factor receptor (also known as HER or ERBB) family**

The EGFR family of receptor tyrosine kinases plays an important role in the pathogenesis of several cancers [83]. The four members: EGFR, HER2, HER3, and HER4 (or ERBB1–4), of EGFR family structurally consist of an extracellular ligand-binding domain, a single transmembrane-spanning region, and an intracellular tyrosine kinase domain. More than 30 ligands have been identified that bind to the EGFR family receptors, including EGF- and EGF-like ligands, transforming growth factor (TGF)-α, and heregulins (HRGs) [83]. The activated EGFR receptors undergo C-terminal phosphorylation of cytoplasmic tyrosine residues after receptor dimerization to mediate cell regulatory signaling. E3 ubiquitin ligase CBL binds to EGFR receptor at specific phosphotyrosine residues and mediates its ubiquitination subsequent internalization in clatherin-coated endosomes, which then lead to lysosomemediated degradation of EGFR [84].

Amplifications and overexpression of various EGFR family members, including EGFR, Her2, and ErbB3, have been reported in epithelial ovarian cancer. Attenuated ubiquitination and HER2 gene amplification favor the formation of EGFR/HER2 heterodimers that recruit CBL to a lesser degree, thus stabilizing and recycling the receptor to cell surface [85]. BRCA1 mutations are known to be associated with an increased EGFR expression in serous ovarian cancer patients. EGFR expression was not only increased in BRCA1 mutated cancer tissues but was also high in BRCA1-mutated normal tissues compared to respective control tissues. These results were confirmed by knocking down BRCA1 in ovarian cancer cells [86]. However, inhibitors targeting this pathway have little effect on cancer cells as a single agent due to the presence of alternative pathways affecting the cancer phenotype, particularly the activation of the PI3K/Akt/mTOR and mitogen-activated protein kinases (MAPKs) pathway [83], suggesting a combined use of EGFR and PI3K inhibitors in ovarian cancer [87].
