Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes

*Jia Guo and Jianglin Guo*

#### **Abstract**

Malignant melanoma is one of the most invasive tumors with increasing mortality, low overall survival rates and limited effective therapeutic strategies. Ubiquitination is a post-translational protein modification, which is regulated by a series of ubiquitination-associated enzymes. Ubiquitination plays a critical role in diverse pathophysiological activities of cellular and participates in the pathogenesis of various cancers, including melanoma. This study aims to provide a conclusive of ubiquitination and deubiquitination, and their potential clinical application value in melanoma in the following aspects: melanoma pathogenesis-related components and processes in the ubuiquitin-proteasome system (UPS), ubiquitination in melanoma immunological microenvironment modulation, ubiquitination of key transcription factors in melanoma and melanoma therapeutic strategy via targeting the UPS.

**Keywords:** ubiquitination, deubiquitinating enzymes, melanoma, pathogenesis, application

#### **1. Introduction**

Malignant melanoma is one of the most invasive tumors with increasing mortality, low overall survival rates, and limited effective therapeutic strategies [1]. Although melanoma is the third most prevalent skin cancer, the two other skin malignancies, basal cell and squamous cells, are the malignant [2]. A variety of factors, including genetic mutations, sun exposure, and poor lifestyle habits, are involved in the development of melanoma [3].

There is a dynamic protein balance in cells to maintain homeostasis for cell and organism. Intracellular protein degradation by two pathways, autophagy and lysosomal degradation pathway and the ubiquitin-proteasome pathway, is primarily involved in tumor growth. Ubiquitination is one of post-translational modifications of most vital proteins. Ubiquitin, a closely conserved small protein composed of 76 amino acids, is link with the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin ligase (E3) [4]. Specifically, mono-ubiquitination was considered as only one single ubiquitin bond to the lysine, while poly-ubiquitination was considered as ubiquitin chains attached to the lysine [5]. Then the ubiquitinated proteins are transported to the 26S proteasome for degradation. Ubiquitination is involved in the development of different tumors

#### *Ubiquitin - Proteasome Pathway*

by regulation important genes or signaling pathways. However, the ubiquitination process can be reversed by the deubiquitinating enzymes (DUBs) via cleaving ubiquitin chains from substrates to prevent protein degradation, which participates in a wide range of cellular signaling pathways, such as the apoptosis, cell cycle, autophagy, DNA damage, inflammation signaling, and protein downregulation [6]. Up to date, there were reported over 600 E3 ubiquitin ligase and 100 DUBs [7].

A significant number of studies have confirmed that ubiquitination and deubiquitination play a critical role in melanoma pathogenesis, and have indicated that the key molecular goal of the mechanism may be the therapeutic strategies for the treatment of melanoma. Here, we provide a conclusive introduction about protein ubiquitin modification in relative genes, signaling pathways, and in immune system in melanoma pathogenesis, which concludes the latest DUB studies in melanoma. Besides, we summarize potential therapeutic targets of ubiquitination and de-ubiquitination in melanoma.

#### **2. Melanoma pathogenesis related components and processes of the UPS**

#### **2.1 Fbxw7**

The F-box/WD repeat-containing protein 7 (Fbxw7) belongs to the F-box protein family, which is the component of an SCF E3 ubiquitin ligase [8]. Fbxw7 is considered to be a tumor suppressor gene [9]. The degradation of Fbxw7 results in accumulation of its substrates, leading to oncogenesis. In a study, the mutation prevalence was found to be 8.1% FBXW7 in melanoma through exome sequencing in a cohort of 103 melanomas. A potential therapeutic approach for melanoma could be the loss and mutation of FBXW7 in melanoma contributing to prolonged activation of NOTCH and targeting NOTCH signaling [10]. FBXW7 deficiency can also unleash heat shock factor 1 (HSF1) and then result in melanoma invasion and metastasis [11]. Meanwhile, FBXW7 can regulate the melanoma metastasis through activating the MAPK/ERK signaling [12]. The microphthalmia-associated transcription factor (MITF) is a key regulator of melanocyte development, differentiation, and melanoma biology [13, 14]. FBXW7 is recognized as a regulator of MITF via post-transcriptional mechanisms [15].

#### **2.2 SKP2**

S-phase kinase-associated protein 2 (Skp2), also be called FBXL1 or p45, is also a member of the F-box proteins [16]. Skp2 is characterized as a cancer-related protein. In general, in primary melanoma and metastasis melanoma, SKP2 is significantly up-regulated, which is related to the prognosis, as it is reported that nuclear Skp2 expression is strongly associated with a lower survival rate during melanoma [17, 18]. In tumorigenesis, Skp2 stabilizes the MTH1 expression via K63 linked polyubiquitination, and then promotes melanoma cell survival by protecting DNA integrity upon pharmacologic oxidative stress [19]. Meanwhile, skp2 has a direct interaction with melanoma antigen-A11 (MAGE-A11), which may boost Skp2-mediated degradation of cyclin A [20].

#### **2.3 HACE1**

HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (HACE1), has been showed to act as a tumour suppressor gene in various kinds of cancers [21]. There is a significantly downregulation of HACE1 in colorectal cancer (CRC), and the decreased expression is highly associated with poor clinical features of

**57**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

patients. HACE1 inhibits YAY1 signaling and then can reverse EMT in CRC [22]. Loss of HACE1 activates RAC-family GTPases to mediate oxidative stress that increases genotoxic cellular ROS generation and then results in lung tumor formation [23]. Even though HACE1 behaves as an anti-oncogene in most reports, its function in melanoma may be cell-specific tumorigenesis. HACE1 plays a pro-oncogenic role in melanoma by

ITCH, a member of HECT-type ubiquitin E3 ligases, plays a significantly role in regulating cell growth and apoptosis [25]. In melanoma, ITCH mediates BRAF polyubiquitination through the K27-linkage result in sustained activation of BRAF/ ΜEK/ERK signaling, which leads to the survival of melanoma cells [26]. Moreover, ITCH can be regulated by microRNAs (miRNAs), such as miR-10b and miR-520f, and then be involved in the melanoma proliferation and metastasis [27, 28].

UBE2C belonging to the E2 family is operating in combination with the anaphasepromoting complex/cyclosome (APC/C) E3 ligase. It regulates the cell cycle through mitosis via destructing mitotic cyclin B1 [29]. Silence of UBE2C induces G2/M phase arrest of melanoma cells by suppressing both the level and the activity of M-phasepromoting factor (MPF), a complex consisting of CDK1 and cyclin B1 [30, 31].

Ubiquitin-conjugating enzyme E2S (UBE2S) belongs to the E2 protein family, and is involved in development of various cancers. Recently, it has been shown that UBE2S plays a vital role in regulating DNA damage-induced transcriptional silencing, by catalyzing Lys11-linkage ubiquitination [32]. Another recent research showed that UBE2S is overexpressed in melanoma, and the expression was significantly related to the cancer staging and grading, with a higher magnitude found for tumor node metastasis staging T4. Moreover, silence of UBE2S may cause melanoma cell proliferation inhibition via inducing cell cycle G1/S phase arrest, and cell apoptosis. In BALB/C nude mice, shUBE2S can suppress tumor growth and inhibit

Makorin ring finger protein 2 (MKRN2) is known as a novel ubiquitin E3 ligase, and is capable of targeting the p65 subunit of NF-κB [34]. Research indicates that there is a greater expression of MKRN2 in melanoma cell lines relative to normal skin cell lines. The silence of MKRN2 can inhibit melanoma cell growth in a P53 dependent manner. Moreover, MKRN2 can interact with ubiquitylated P53 [35]. This study suggests that MKRN2 may be a potential therapeutic target for melanoma.

There are also several ubiquitin-like proteins (UBLs) in addition to Ub, such as NEDD8 (neural precursor cell expressed, developmentally down-regulated8), SUMO (small ubiquitin-like modifiers), and ISG15 (interferon-stimulated gene 15). NEDD8 mediates the stabilization of various proteins, and plays a significant

role in the incidence and development of malignant melanoma. NEDD8 is a

epithelial-mesenchymal transition (EMT) [33].

regulating fibronectin (FN) secretion and K27 ubiquitination of FN [24].

*DOI: http://dx.doi.org/10.5772/intechopen.94512*

**2.4 ITCH**

**2.5 UBE2C**

**2.6 UBE2S**

**2.7 MKRN2**

**2.8 Ub-like proteins**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes DOI: http://dx.doi.org/10.5772/intechopen.94512*

patients. HACE1 inhibits YAY1 signaling and then can reverse EMT in CRC [22]. Loss of HACE1 activates RAC-family GTPases to mediate oxidative stress that increases genotoxic cellular ROS generation and then results in lung tumor formation [23]. Even though HACE1 behaves as an anti-oncogene in most reports, its function in melanoma may be cell-specific tumorigenesis. HACE1 plays a pro-oncogenic role in melanoma by regulating fibronectin (FN) secretion and K27 ubiquitination of FN [24].

#### **2.4 ITCH**

*Ubiquitin - Proteasome Pathway*

and de-ubiquitination in melanoma.

**2.1 Fbxw7**

**2.2 SKP2**

**2.3 HACE1**

by regulation important genes or signaling pathways. However, the ubiquitination process can be reversed by the deubiquitinating enzymes (DUBs) via cleaving ubiquitin chains from substrates to prevent protein degradation, which participates in a wide range of cellular signaling pathways, such as the apoptosis, cell cycle, autophagy, DNA damage, inflammation signaling, and protein downregulation [6]. Up to date, there were reported over 600 E3 ubiquitin ligase and 100 DUBs [7]. A significant number of studies have confirmed that ubiquitination and deubiquitination play a critical role in melanoma pathogenesis, and have indicated that the key molecular goal of the mechanism may be the therapeutic strategies for the treatment of melanoma. Here, we provide a conclusive introduction about protein ubiquitin modification in relative genes, signaling pathways, and in immune system in melanoma pathogenesis, which concludes the latest DUB studies in melanoma. Besides, we summarize potential therapeutic targets of ubiquitination

**2. Melanoma pathogenesis related components and processes of the UPS**

The F-box/WD repeat-containing protein 7 (Fbxw7) belongs to the F-box protein family, which is the component of an SCF E3 ubiquitin ligase [8]. Fbxw7 is considered to be a tumor suppressor gene [9]. The degradation of Fbxw7 results in accumulation of its substrates, leading to oncogenesis. In a study, the mutation prevalence was found to be 8.1% FBXW7 in melanoma through exome sequencing in a cohort of 103 melanomas. A potential therapeutic approach for melanoma could be the loss and mutation of FBXW7 in melanoma contributing to prolonged activation of NOTCH and targeting NOTCH signaling [10]. FBXW7 deficiency can also unleash heat shock factor 1 (HSF1) and then result in melanoma invasion and metastasis [11]. Meanwhile, FBXW7 can regulate the melanoma metastasis through activating the MAPK/ERK signaling [12]. The microphthalmia-associated transcription factor (MITF) is a key regulator of melanocyte development, differentiation, and melanoma biology [13, 14]. FBXW7 is

recognized as a regulator of MITF via post-transcriptional mechanisms [15].

Skp2-mediated degradation of cyclin A [20].

S-phase kinase-associated protein 2 (Skp2), also be called FBXL1 or p45, is also a member of the F-box proteins [16]. Skp2 is characterized as a cancer-related protein. In general, in primary melanoma and metastasis melanoma, SKP2 is significantly up-regulated, which is related to the prognosis, as it is reported that nuclear Skp2 expression is strongly associated with a lower survival rate during melanoma [17, 18]. In tumorigenesis, Skp2 stabilizes the MTH1 expression via K63 linked polyubiquitination, and then promotes melanoma cell survival by protecting DNA integrity upon pharmacologic oxidative stress [19]. Meanwhile, skp2 has a direct interaction with melanoma antigen-A11 (MAGE-A11), which may boost

HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (HACE1), has been showed to act as a tumour suppressor gene in various kinds of cancers [21]. There is a significantly downregulation of HACE1 in colorectal cancer (CRC), and the decreased expression is highly associated with poor clinical features of

**56**

ITCH, a member of HECT-type ubiquitin E3 ligases, plays a significantly role in regulating cell growth and apoptosis [25]. In melanoma, ITCH mediates BRAF polyubiquitination through the K27-linkage result in sustained activation of BRAF/ ΜEK/ERK signaling, which leads to the survival of melanoma cells [26]. Moreover, ITCH can be regulated by microRNAs (miRNAs), such as miR-10b and miR-520f, and then be involved in the melanoma proliferation and metastasis [27, 28].

#### **2.5 UBE2C**

UBE2C belonging to the E2 family is operating in combination with the anaphasepromoting complex/cyclosome (APC/C) E3 ligase. It regulates the cell cycle through mitosis via destructing mitotic cyclin B1 [29]. Silence of UBE2C induces G2/M phase arrest of melanoma cells by suppressing both the level and the activity of M-phasepromoting factor (MPF), a complex consisting of CDK1 and cyclin B1 [30, 31].

#### **2.6 UBE2S**

Ubiquitin-conjugating enzyme E2S (UBE2S) belongs to the E2 protein family, and is involved in development of various cancers. Recently, it has been shown that UBE2S plays a vital role in regulating DNA damage-induced transcriptional silencing, by catalyzing Lys11-linkage ubiquitination [32]. Another recent research showed that UBE2S is overexpressed in melanoma, and the expression was significantly related to the cancer staging and grading, with a higher magnitude found for tumor node metastasis staging T4. Moreover, silence of UBE2S may cause melanoma cell proliferation inhibition via inducing cell cycle G1/S phase arrest, and cell apoptosis. In BALB/C nude mice, shUBE2S can suppress tumor growth and inhibit epithelial-mesenchymal transition (EMT) [33].

#### **2.7 MKRN2**

Makorin ring finger protein 2 (MKRN2) is known as a novel ubiquitin E3 ligase, and is capable of targeting the p65 subunit of NF-κB [34]. Research indicates that there is a greater expression of MKRN2 in melanoma cell lines relative to normal skin cell lines. The silence of MKRN2 can inhibit melanoma cell growth in a P53 dependent manner. Moreover, MKRN2 can interact with ubiquitylated P53 [35]. This study suggests that MKRN2 may be a potential therapeutic target for melanoma.

#### **2.8 Ub-like proteins**

There are also several ubiquitin-like proteins (UBLs) in addition to Ub, such as NEDD8 (neural precursor cell expressed, developmentally down-regulated8), SUMO (small ubiquitin-like modifiers), and ISG15 (interferon-stimulated gene 15).

NEDD8 mediates the stabilization of various proteins, and plays a significant role in the incidence and development of malignant melanoma. NEDD8 is a

ubiquitin-like protein composed of 81 amino acids, with around 60% of the sequence that is the same as ubiquitin [36]. The covalent binding of NEDD8 to substrates is known as neddylation. Similar to the ubiquitination, an enzyme cascade is needed for this progression. Neddylation is involved in protein ubiquitination, and is closely associated with the degradation of certain proteins in the cell cycle and apoptosisrelated factors [37]. Cullin is one of the most researched neddylation substrates [38]. Besides, studies have also investigated that NEDD8 substrates are diverse. Some proteins can be modified by NEDD8, including p53 [39], MDM2 [40], and VHL [41]. UBA3, as the subunit of NEDD8-activating enzyme, plays a critical role in the linkage of NEDD8 with cullin proteins. Previous studies have shown that in highly proliferative cell lines, NEDD8 conjugation is up-regulated and increased in melanoma cell lines [42]. After knockdown of UBA3, the proliferation of M14 melanoma cells was suppressed both in vitro and in vivo. Hence, interference of the neddylation might offer a hopeful method for melanoma therapy [43].

SUMO has been described to alter protein interactions rather than directly involving in protein degradation [44]. Sumoylation involves a 3-step pathway analogous to the ubiquitination pathway. Dysregulation of sumoylation has been implicated in multiple cancers, including melanomas. Ubc9, the single SUMO E2 conjugating enzyme, is overexpressed in advanced-stage melanomas where it protects melanoma cells from chemotherapy-induced apoptosis [45]. Moreover, SUMOylation-defective MITF germline mutation may be more susceptible to melanoma [46].

ISG15, a ubiquitin-like modifier, is implicated in both tumor oncogenic and suppressive programs [47]. It is activated by a three steps enzymatic cascade consisting of a specific E1-activating enzyme (UBE1L), E2 conjugating enzyme (typically UBCH8) and E3 ligase (commonly HERC5A), which promotes ISG15 transfer to protein substrates [48]. Previous study shows that ISG15 can be removed from its target proteins by USP18 and then the effects of ISGylation was reversed [49, 50]. A study identifies PTEN as a new substrate of the ISGylation post-translational modification pathway and USP18 can regulate PTEN stability. Inhibition of ISGylation may be a therapeutically relevant in melanoma [47].

#### **2.9 Deubiquitinating enzymes (DUBs)**

To date, several DUBs have confirmed to be consistent with melanoma tumorigenesis and metastasis. USP54 is overexpressed in intestinal stem cells, and is defined to promote cancer progression and regulate embryonic development and normal growth of adult mice. USP54 upregulates in melanoma, the loss of USP54 is dispensable for metastasis of melanoma cells [51]. An IFN stimulated to regulate type-I IFN signaling in the anti-viral immune response has been reported to be USP18 [52]. It is also reported that IFN-γ can stimulate USP18 protein expression in melanoma cells. Through IFN-γ-induced USP18 expression in melanoma cells and -regulated CTL CD8 + immune cell activity in the tumor microenvironment, endogenous IFN-γ signaling influences melanoma tumorigenesis [53]. In 2014, Harish Potu et al. reported that USP5 mediates the change in ubiquitinylated protein content and unanchors Ub chains in BRAF mutant cells treated with vemurafenib. BRAF can activate USP5, contributing by suppressing p53 and FAS induction, to inhibit cell cycle checkpoint regulation and apoptosis [54]. In 2018, USP4 upregulation in melanoma, especially in metastatic melanoma, was discovered by Weinan Guo et al. The archive of TCGA skin cutaneous melanoma (SKCM) confirms this finding. USP4 can protect melanoma cells from cisplatin-induced apoptosis in a p53-dependent manner. Moreover, USP4 up-regulation plays an important role in melanoma invasion and migration by promoting EMT [55]. The USP15 knockdown lowers the expression of MDM2 in melanoma cells, and then leads to upregulation

**59**

**modulation**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

of p53 and MDM2 target genes p21 and Puma. Moreover, Usp15−/− melanoma mice models have an increased frequency of CD8+ effector T cells tumor-infiltrating [56]. Ubiquitin specific peptidase 9, X-linked (Usp9x), a member of the USP family, is upregulated in many cancers, which has a positive and negative impact on tumorigenicity depending on the various forms of cancer [57–59]. A study shows that the growth of melanoma cells can be inhibited by Usp9x loss. The Ets-1 proteasomal, abased site-specific de-ubiquitination, is inhibited by Usp9x, which leads to Ets-1 aggregation and increases tumorigenicity of melanoma [60]. Moreover, in malignant melanoma, about 15–20% of NRAS mutations have been identified [61]. Harish Potu et al. also revealed that inhibition of BRAF and/or MEK kinase pathway can increase

Ets-1 expression. The increased Ets-1 expression upregulates NRAS levels by activating the NRAS promoter. In all, Usp9x plays a critical role in Ets-1 regulation and melanoma tumorigenicity through mediating NRAS transcription [60]. UCHL1 (ubiquitin C-terminal hydrolase 1) belongs to the ubiquitin carboxy terminal hydrolase family of DUBs. It catalyzes hydrolysis of C-terminal ubiquitin esters to regulate protein degradation [62]. Eun Young Seo et al. have investigated that UCHL1 influences melanogenesis by regulating stability of MITF in human melanocytes, which provides a framework for the further researches to evaluate potent therapeutic approaches for melanoma and other dyspigmentation disorders [63]. BAP1 (BRCA1 associated protein-1) belongs to the UCH subfamily of DUBs, and is known as a tumor suppressor gene [64, 65]. BAP1 mutations were first identified in a small number of lung and breast cancer samples, and have recently been described as leading to the pathogenesis of melanoma [66, 67]. The germline mutations in BAP1 are more prone to malignant melanoma [68]. In 2010, a study reported that 84% of inactivating somatic BAP1 mutations were identified in metastasizing uveal melanomas, including 15 premature protein termination mutations, and six affecting their ubiquitin UCH domains, which were associated with a decrease in BAP1 mRNA level [69]. However, in cutaneous melanoma, the germline mutations in BAP1 were less than 1% and its effect was unknown [70]. A recent study reported that low BAP1 mRNA predicted a better OS in older than 50 years cutaneous melanoma patients after adjusting for ulceration or Breslow depth [71]. The different function of BAP1

in cutaneous melanoma and uveal melanoma needs to be studied further.

**3. Ubiquitination in melanoma immunological microenvironment** 

lation plays a critical role in modulating immune responses and TME [73].

Tumor microenvironment (TME) is consisted of cancer cells, cancer-associated fibroblasts, immune cells, and stromal cells. TME emerges as a key mechanism that mediate tumor progression [72]. A previous study reported that protein ubiquity-

The Cbl proteins are a family of ubiquitin ligases (E3s). Cbl-b, a member of the family, functions as a negative regulator that regulates CD8 T cells costimulatory pathway and natural killer cell function [74]. In recent years, Cbl-b prone to be one of the hotspot targets of tumor immunotherapy because Cbl-b deletion can cause spontaneous or induced autoimmune call, and Cbl-b overexpression can result in the tumor immune tolerance in infiltrated lymphocytes in TME [75]. A study shows that NK cells knocking down of Cbl-b, or targeting its E3 catalytic activity, inhibit the progression of melanomas and distant melanoma metastases. Moreover, compared with WT T cells, Cbl-b−/− CD8+ and CD4+ T cell proliferation are highly suppressed by a recombinant PD-L1 Ig, and IFN-γ production is significantly less suppressed. Cbl-b deficiency in mice seems to cause a functional resistance of NK cells and T cells to PD-L1/PD-1-mediated immune suppression [76]. Adoptive cell therapy (ACT) with

*DOI: http://dx.doi.org/10.5772/intechopen.94512*

#### *Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes DOI: http://dx.doi.org/10.5772/intechopen.94512*

of p53 and MDM2 target genes p21 and Puma. Moreover, Usp15−/− melanoma mice models have an increased frequency of CD8+ effector T cells tumor-infiltrating [56].

Ubiquitin specific peptidase 9, X-linked (Usp9x), a member of the USP family, is upregulated in many cancers, which has a positive and negative impact on tumorigenicity depending on the various forms of cancer [57–59]. A study shows that the growth of melanoma cells can be inhibited by Usp9x loss. The Ets-1 proteasomal, abased site-specific de-ubiquitination, is inhibited by Usp9x, which leads to Ets-1 aggregation and increases tumorigenicity of melanoma [60]. Moreover, in malignant melanoma, about 15–20% of NRAS mutations have been identified [61]. Harish Potu et al. also revealed that inhibition of BRAF and/or MEK kinase pathway can increase Ets-1 expression. The increased Ets-1 expression upregulates NRAS levels by activating the NRAS promoter. In all, Usp9x plays a critical role in Ets-1 regulation and melanoma tumorigenicity through mediating NRAS transcription [60]. UCHL1 (ubiquitin C-terminal hydrolase 1) belongs to the ubiquitin carboxy terminal hydrolase family of DUBs. It catalyzes hydrolysis of C-terminal ubiquitin esters to regulate protein degradation [62]. Eun Young Seo et al. have investigated that UCHL1 influences melanogenesis by regulating stability of MITF in human melanocytes, which provides a framework for the further researches to evaluate potent therapeutic approaches for melanoma and other dyspigmentation disorders [63]. BAP1 (BRCA1 associated protein-1) belongs to the UCH subfamily of DUBs, and is known as a tumor suppressor gene [64, 65]. BAP1 mutations were first identified in a small number of lung and breast cancer samples, and have recently been described as leading to the pathogenesis of melanoma [66, 67]. The germline mutations in BAP1 are more prone to malignant melanoma [68]. In 2010, a study reported that 84% of inactivating somatic BAP1 mutations were identified in metastasizing uveal melanomas, including 15 premature protein termination mutations, and six affecting their ubiquitin UCH domains, which were associated with a decrease in BAP1 mRNA level [69]. However, in cutaneous melanoma, the germline mutations in BAP1 were less than 1% and its effect was unknown [70]. A recent study reported that low BAP1 mRNA predicted a better OS in older than 50 years cutaneous melanoma patients after adjusting for ulceration or Breslow depth [71]. The different function of BAP1 in cutaneous melanoma and uveal melanoma needs to be studied further.

#### **3. Ubiquitination in melanoma immunological microenvironment modulation**

Tumor microenvironment (TME) is consisted of cancer cells, cancer-associated fibroblasts, immune cells, and stromal cells. TME emerges as a key mechanism that mediate tumor progression [72]. A previous study reported that protein ubiquitylation plays a critical role in modulating immune responses and TME [73].

The Cbl proteins are a family of ubiquitin ligases (E3s). Cbl-b, a member of the family, functions as a negative regulator that regulates CD8 T cells costimulatory pathway and natural killer cell function [74]. In recent years, Cbl-b prone to be one of the hotspot targets of tumor immunotherapy because Cbl-b deletion can cause spontaneous or induced autoimmune call, and Cbl-b overexpression can result in the tumor immune tolerance in infiltrated lymphocytes in TME [75]. A study shows that NK cells knocking down of Cbl-b, or targeting its E3 catalytic activity, inhibit the progression of melanomas and distant melanoma metastases. Moreover, compared with WT T cells, Cbl-b−/− CD8+ and CD4+ T cell proliferation are highly suppressed by a recombinant PD-L1 Ig, and IFN-γ production is significantly less suppressed. Cbl-b deficiency in mice seems to cause a functional resistance of NK cells and T cells to PD-L1/PD-1-mediated immune suppression [76]. Adoptive cell therapy (ACT) with

*Ubiquitin - Proteasome Pathway*

offer a hopeful method for melanoma therapy [43].

may be a therapeutically relevant in melanoma [47].

**2.9 Deubiquitinating enzymes (DUBs)**

ubiquitin-like protein composed of 81 amino acids, with around 60% of the sequence that is the same as ubiquitin [36]. The covalent binding of NEDD8 to substrates is known as neddylation. Similar to the ubiquitination, an enzyme cascade is needed for this progression. Neddylation is involved in protein ubiquitination, and is closely associated with the degradation of certain proteins in the cell cycle and apoptosisrelated factors [37]. Cullin is one of the most researched neddylation substrates [38]. Besides, studies have also investigated that NEDD8 substrates are diverse. Some proteins can be modified by NEDD8, including p53 [39], MDM2 [40], and VHL [41]. UBA3, as the subunit of NEDD8-activating enzyme, plays a critical role in the linkage of NEDD8 with cullin proteins. Previous studies have shown that in highly proliferative cell lines, NEDD8 conjugation is up-regulated and increased in melanoma cell lines [42]. After knockdown of UBA3, the proliferation of M14 melanoma cells was suppressed both in vitro and in vivo. Hence, interference of the neddylation might

SUMO has been described to alter protein interactions rather than directly involving in protein degradation [44]. Sumoylation involves a 3-step pathway analogous to the ubiquitination pathway. Dysregulation of sumoylation has been implicated in multiple cancers, including melanomas. Ubc9, the single SUMO E2 conjugating enzyme, is overexpressed in advanced-stage melanomas where it protects melanoma cells from chemotherapy-induced apoptosis [45]. Moreover, SUMOylation-defective

ISG15, a ubiquitin-like modifier, is implicated in both tumor oncogenic and suppressive programs [47]. It is activated by a three steps enzymatic cascade consisting of a specific E1-activating enzyme (UBE1L), E2 conjugating enzyme (typically UBCH8) and E3 ligase (commonly HERC5A), which promotes ISG15 transfer to protein substrates [48]. Previous study shows that ISG15 can be removed from its target proteins by USP18 and then the effects of ISGylation was reversed [49, 50]. A study identifies PTEN as a new substrate of the ISGylation post-translational modification pathway and USP18 can regulate PTEN stability. Inhibition of ISGylation

To date, several DUBs have confirmed to be consistent with melanoma tumori-

genesis and metastasis. USP54 is overexpressed in intestinal stem cells, and is defined to promote cancer progression and regulate embryonic development and normal growth of adult mice. USP54 upregulates in melanoma, the loss of USP54 is dispensable for metastasis of melanoma cells [51]. An IFN stimulated to regulate type-I IFN signaling in the anti-viral immune response has been reported to be USP18 [52]. It is also reported that IFN-γ can stimulate USP18 protein expression in melanoma cells. Through IFN-γ-induced USP18 expression in melanoma cells and -regulated CTL CD8 + immune cell activity in the tumor microenvironment, endogenous IFN-γ signaling influences melanoma tumorigenesis [53]. In 2014, Harish Potu et al. reported that USP5 mediates the change in ubiquitinylated protein content and unanchors Ub chains in BRAF mutant cells treated with vemurafenib. BRAF can activate USP5, contributing by suppressing p53 and FAS induction, to inhibit cell cycle checkpoint regulation and apoptosis [54]. In 2018, USP4 upregulation in melanoma, especially in metastatic melanoma, was discovered by Weinan Guo et al. The archive of TCGA skin cutaneous melanoma (SKCM) confirms this finding. USP4 can protect melanoma cells from cisplatin-induced apoptosis in a p53-dependent manner. Moreover, USP4 up-regulation plays an important role in melanoma invasion and migration by promoting EMT [55]. The USP15 knockdown lowers the expression of MDM2 in melanoma cells, and then leads to upregulation

MITF germline mutation may be more susceptible to melanoma [46].

**58**

autologous T cells can enforce the immune-mediated tumor cell killing, and show a promising result in various types of cancer treatments [77, 78]. However, the therapeutic efficacy of ACT is still limited because of the tumor-bearing host immune-evasion mechanisms, such as the secretion of transforming growth factor beta (TGFβ) or accumulation of Treg cells, both of which severely dampen the activation, expansion, and tumor homing of CD8+ T cells [79]. Another study reveals that silencing cbl-b reduces TGFβ sensitivity *in vitro* and enhances anti-tumor effects *in vivo*. Adoptive transfer of Cbl-b-silenced CD8+ T lymphocytes augments tumor vaccine to suppress tumor growth and prolong the survival in a B16F10 melanoma model [80].

FBXO38 belongs to the SCF family of E3 ubiquitin ligase of PD-1, and mediates Lys48-linked poly-ubiquitination and substrate proteasome degradation [81]. Previous research investigates that FBXO38 mediates PD-1 ubiquitination and maintains the anti-tumour activity of T cells in melanoma cells [82]. It offers an alternative method to block the PD-1 and highlights the clinical potential of the regulation of anti-tumour immunity through ubiquitination of FBXO38.

SIAH2, potent E3 RING finger ubiquitin ligases, mediates the cell cycle, apoptosis, and DNA repair regulation through targeting subsequent related proteins [83]. Previous study finds that hypoxia activates Siah2 E3 ligase, and then enhances the Warburg effect and pro-tumor immune response via degrading nuclear respiratory factor 1 (NRF1) through ubiquitination on lysine 230 [84]. A recent study reveals the effect and mechanism of Siah2 on the T cells and immune therapy. As is shown in this article, in the one way, Siah2-deficient mice suppress melanoma growth, increase the infiltration of T effector cells, and decrease number of FOXP3+ Treg cells. Inhibition of Siah2−/− melanoma cell proliferation is p27 dependent. Moreover, Siah2−/− BM-transplanted mice inhibit the melanoma growth, which may be a clinical potential of new adoptive cell therapy. On the other hand, loss of Siah2 exhibits synergy with anti-PD1 therapy in melanoma.

In addition to Ub, lots of Ub-related proteins display an immune regulation function in melanoma. A family of Toll-like receptors (TLRs) involves in the recognition of microbial components and regulates innate immune responses [85, 86]. TNFAIP3 (TNF-α induced protein 3), an ubiquitin-editing enzyme, can negatively regulate the TLRs via function as an ubiquitin-editing molecule [87]. E3 ligase NEDD4 mediates the function of immune regulation. Silence of NEDD4 inhibits FOXP3+ Treg cells through mediating GITR degradation, and then contributes to melanoma progression [88]. A previous study finds that USP15 was highly expressed in immune cells through analysis of the BioGPS database. In naïve CD4+ T cells, loss of USP15 stimulates the TCR + CD28 to produce cytokines, such as interleukin 2 (IL-2) and interferon-γ (IFN-γ). Moreover, USP15 inhibits the naïve CD4+ T cell activation and suppresses TH1 differentiation. MDM2, which is recognized as substrate protein of USP15, targets a T cell transcription factor, NFATc2, and negatively regulates T cell activation, which was independent of p53. Later, the author testifies the function of USP15 in B16F10 melanoma models. This study investigated that USP15−/− mice increase IFN-γ + CD4+ T cell infiltration to the tumors, and deficiency of USP15 reduces melanoma tumors size and tumor-induced lethality [89].

#### **4. Ubiquitination of key transcription factors in melanoma**

#### **4.1 Ubiquitination of p53**

The tumor suppressor protein p53 is a transcription factor that can affect cell proliferation by regulating the expression of its target protein [90]. P53 interacts with E3 ligase MDM2 in the nucleus, and is transferred from the nucleus to the cytoplasm

**61**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

following ubiquitin, resulting in proteasome degrading [91]. In 2003, Leonard Girnita et al. discovered that inhibition of p53 leads to ubiquitination and down-regulation of the IGF-1R in human malignant melanoma cells. This impact was independent of the p53 status (wild type or mutated) but can be rescued by coinhibition of MDM2. Mdm2 serves as a ligase in ubiquitination of the IGF-1R [92]. Unlike other solid tumors, malignant melanomas retain the expression of wild-type p53 and typically lack p53 mutations [93, 94]. Adil Anwar et al. reveal that the wild-type p53 is the target for the ubiquitin-proteasomal pathway (UPP) degradation. The residues serine 15 and serine 20 are also essential for the binding of MDM2, which control p53 destruction via UPP pathway. In this article, p53 stabilization mediated by UPP inhibitors is independent of phosphorylation at residues serine 15 and serine 20 of p53 in melanoma cells [95]. MKRN2 is recognized as a novel ubiquitin E3 ligase targeting the p65 subunit of NF-κB to negatively regulate inflammatory responses [96]. A recent study indicates that the MKRN2 expression increases in the human melanoma cell lines, and silence of this gene leads to the suppression of melanoma proliferation by upregulation of p53. To investigate the mechanism of this effect, authors take co-immunoprecipitation and glutathione S-transferase pulldown assays to confirm the interaction of MKRN2 with p53 and take *in vitro* ubiquitination assays to study the ubiquitination of p53 by MKRN2. The result shows that MKRN2 interacts with p53, and ubiquitylates p53, leading to the influence of melanoma cell proliferation [97].

The transcription factor c-Myc plays an important role in cell proliferation and differentiation, cell cycle, metabolism, and apoptosis [98]. C-Myc is a protein that is very unstable and vulnerable to degradation in a proteasome-dependent manner. Research has identified the E3 ligase of c-Myc in melanoma. Also, c-Myc can be

In protein degradation and melanoma pathogenesis, the UPS plays a crucial role, as shown above. The pathogenesis of malignant melanoma leads to genetic changes, irregular expression, or dysfunction [100]. Hence, targeting the UPS may be a potential therapeutic strategy for melanoma. Currently, many small molecule inhibitors targeting different components of the UPS, including the proteasome, E3 ligases, E1 enzymes, E2 enzymes, ubiquitin-like proteins, and DUBs, have been developed [101]. Bortezomib is the first proteasome inhibitor approved by FDA, which was originally used for multiple myeloma treatment [102]. However, due to the clinical safety, the study in other cancer researches, including melanoma, has been discontinued. Compared to the proteasome inhibitor bortezomib, drugs targeting a particular E3 ubiquitin ligase are expected to have better selectivity with less associated toxicity relative to the proteasome inhibitor bortezomib [103]. MDM2 is an E3 ubiquitin ligase with the ability to regulate tumor suppressor p53 and potentiate Notch signaling by degrading Numb [104, 105]. Nutlin-3a, an imidazoline compound, has been generally known as a MDM2 inhibitor. Nutlin-3a can suppress melanoma and other cancers, including retinoblastoma, leukemia, and neuroblastoma [106]. Meanwhile, WIP1 inhibitor (WIP1i), GSK2830371, can enhance p53-mediated tumor suppression by MDM2–p53 inhibitors, nutlin-3, RG7388, and HDM201 in cutaneous melanoma [107]. Therefore, more findings from the phase I clinical trials are needed to evaluate whether there exist any significant side effects.

*DOI: http://dx.doi.org/10.5772/intechopen.94512*

**4.2 Ubiquitination of c-Myc**

specifically bound by the E3 ligase SKP2 [99].

**ubiquitin-proteasome system (UPS)**

**5. Melanoma therapeutic strategy via targeting the** 

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes DOI: http://dx.doi.org/10.5772/intechopen.94512*

following ubiquitin, resulting in proteasome degrading [91]. In 2003, Leonard Girnita et al. discovered that inhibition of p53 leads to ubiquitination and down-regulation of the IGF-1R in human malignant melanoma cells. This impact was independent of the p53 status (wild type or mutated) but can be rescued by coinhibition of MDM2. Mdm2 serves as a ligase in ubiquitination of the IGF-1R [92]. Unlike other solid tumors, malignant melanomas retain the expression of wild-type p53 and typically lack p53 mutations [93, 94]. Adil Anwar et al. reveal that the wild-type p53 is the target for the ubiquitin-proteasomal pathway (UPP) degradation. The residues serine 15 and serine 20 are also essential for the binding of MDM2, which control p53 destruction via UPP pathway. In this article, p53 stabilization mediated by UPP inhibitors is independent of phosphorylation at residues serine 15 and serine 20 of p53 in melanoma cells [95]. MKRN2 is recognized as a novel ubiquitin E3 ligase targeting the p65 subunit of NF-κB to negatively regulate inflammatory responses [96]. A recent study indicates that the MKRN2 expression increases in the human melanoma cell lines, and silence of this gene leads to the suppression of melanoma proliferation by upregulation of p53. To investigate the mechanism of this effect, authors take co-immunoprecipitation and glutathione S-transferase pulldown assays to confirm the interaction of MKRN2 with p53 and take *in vitro* ubiquitination assays to study the ubiquitination of p53 by MKRN2. The result shows that MKRN2 interacts with p53, and ubiquitylates p53, leading to the influence of melanoma cell proliferation [97].

#### **4.2 Ubiquitination of c-Myc**

*Ubiquitin - Proteasome Pathway*

autologous T cells can enforce the immune-mediated tumor cell killing, and show a promising result in various types of cancer treatments [77, 78]. However, the therapeutic efficacy of ACT is still limited because of the tumor-bearing host immune-evasion mechanisms, such as the secretion of transforming growth factor beta (TGFβ) or accumulation of Treg cells, both of which severely dampen the activation, expansion, and tumor homing of CD8+ T cells [79]. Another study reveals that silencing cbl-b reduces TGFβ sensitivity *in vitro* and enhances anti-tumor effects *in vivo*. Adoptive transfer of Cbl-b-silenced CD8+ T lymphocytes augments tumor vaccine to suppress

tumor growth and prolong the survival in a B16F10 melanoma model [80].

regulation of anti-tumour immunity through ubiquitination of FBXO38.

Siah2 exhibits synergy with anti-PD1 therapy in melanoma.

reduces melanoma tumors size and tumor-induced lethality [89].

**4. Ubiquitination of key transcription factors in melanoma**

The tumor suppressor protein p53 is a transcription factor that can affect cell proliferation by regulating the expression of its target protein [90]. P53 interacts with E3 ligase MDM2 in the nucleus, and is transferred from the nucleus to the cytoplasm

FBXO38 belongs to the SCF family of E3 ubiquitin ligase of PD-1, and mediates Lys48-linked poly-ubiquitination and substrate proteasome degradation [81]. Previous research investigates that FBXO38 mediates PD-1 ubiquitination and maintains the anti-tumour activity of T cells in melanoma cells [82]. It offers an alternative method to block the PD-1 and highlights the clinical potential of the

SIAH2, potent E3 RING finger ubiquitin ligases, mediates the cell cycle, apoptosis, and DNA repair regulation through targeting subsequent related proteins [83]. Previous study finds that hypoxia activates Siah2 E3 ligase, and then enhances the Warburg effect and pro-tumor immune response via degrading nuclear respiratory factor 1 (NRF1) through ubiquitination on lysine 230 [84]. A recent study reveals the effect and mechanism of Siah2 on the T cells and immune therapy. As is shown in this article, in the one way, Siah2-deficient mice suppress melanoma growth, increase the infiltration of T effector cells, and decrease number of FOXP3+ Treg cells. Inhibition of Siah2−/− melanoma cell proliferation is p27 dependent. Moreover, Siah2−/− BM-transplanted mice inhibit the melanoma growth, which may be a clinical potential of new adoptive cell therapy. On the other hand, loss of

In addition to Ub, lots of Ub-related proteins display an immune regulation function in melanoma. A family of Toll-like receptors (TLRs) involves in the recognition of microbial components and regulates innate immune responses [85, 86]. TNFAIP3 (TNF-α induced protein 3), an ubiquitin-editing enzyme, can negatively regulate the TLRs via function as an ubiquitin-editing molecule [87]. E3 ligase NEDD4 mediates the function of immune regulation. Silence of NEDD4 inhibits FOXP3+ Treg cells through mediating GITR degradation, and then contributes to melanoma progression [88]. A previous study finds that USP15 was highly expressed in immune cells through analysis of the BioGPS database. In naïve CD4+ T cells, loss of USP15 stimulates the TCR + CD28 to produce cytokines, such as interleukin 2 (IL-2) and interferon-γ (IFN-γ). Moreover, USP15 inhibits the naïve CD4+ T cell activation and suppresses TH1 differentiation. MDM2, which is recognized as substrate protein of USP15, targets a T cell transcription factor, NFATc2, and negatively regulates T cell activation, which was independent of p53. Later, the author testifies the function of USP15 in B16F10 melanoma models. This study investigated that USP15−/− mice increase IFN-γ + CD4+ T cell infiltration to the tumors, and deficiency of USP15

**60**

**4.1 Ubiquitination of p53**

The transcription factor c-Myc plays an important role in cell proliferation and differentiation, cell cycle, metabolism, and apoptosis [98]. C-Myc is a protein that is very unstable and vulnerable to degradation in a proteasome-dependent manner. Research has identified the E3 ligase of c-Myc in melanoma. Also, c-Myc can be specifically bound by the E3 ligase SKP2 [99].

#### **5. Melanoma therapeutic strategy via targeting the ubiquitin-proteasome system (UPS)**

In protein degradation and melanoma pathogenesis, the UPS plays a crucial role, as shown above. The pathogenesis of malignant melanoma leads to genetic changes, irregular expression, or dysfunction [100]. Hence, targeting the UPS may be a potential therapeutic strategy for melanoma. Currently, many small molecule inhibitors targeting different components of the UPS, including the proteasome, E3 ligases, E1 enzymes, E2 enzymes, ubiquitin-like proteins, and DUBs, have been developed [101].

Bortezomib is the first proteasome inhibitor approved by FDA, which was originally used for multiple myeloma treatment [102]. However, due to the clinical safety, the study in other cancer researches, including melanoma, has been discontinued. Compared to the proteasome inhibitor bortezomib, drugs targeting a particular E3 ubiquitin ligase are expected to have better selectivity with less associated toxicity relative to the proteasome inhibitor bortezomib [103]. MDM2 is an E3 ubiquitin ligase with the ability to regulate tumor suppressor p53 and potentiate Notch signaling by degrading Numb [104, 105]. Nutlin-3a, an imidazoline compound, has been generally known as a MDM2 inhibitor. Nutlin-3a can suppress melanoma and other cancers, including retinoblastoma, leukemia, and neuroblastoma [106]. Meanwhile, WIP1 inhibitor (WIP1i), GSK2830371, can enhance p53-mediated tumor suppression by MDM2–p53 inhibitors, nutlin-3, RG7388, and HDM201 in cutaneous melanoma [107]. Therefore, more findings from the phase I clinical trials are needed to evaluate whether there exist any significant side effects.

Besides, Siah2 is known as a RING finger E3 ubiquitin ligase. Inhibition of Siah2 activity using a peptide is reported to be able to weaken its effect on hypoxia, effectively leading to melanoma metastasis inhibition, while suppression of Siah2 activities prevents the tumorigenicity of melanoma by disrupting Ras/MAPK signaling pathways [108]. Menadione (MEN), also known as vitamin K3, is a quinone used for cancer chemotherapeutic agents. A recent research identifies MEN as a novel Siah2 inhibitor, which attenuates hypoxia and MAPK signaling, and blocks melanoma tumorigenesis [109]. This study revealed that targeting Siah2 by MEN may be a new therapeutic strategy in melanoma treatment.

Cullin-RING ligases (CRLs) are a subgroup of the E3 ligases, and play an important role in the degradation of oncology relative proteins. It is activated by the neddylation pathway, such as NEDD8 conjugation [110]. The NEDD8-activating enzyme (NAE) is a critical regulator of the neddylation pathway [111]. Pevonedistat (TAK-924/MLN4924) is reported as a first-in-class, small-molecule inhibitor of NAE. Previous preclinical studies reveal that Pevonedistat is associated with tumor growth inhibition of a range of cell lines and primary human cancer cells derived from solid tumors, including malignant melanoma [112, 113]. A phase I study of Pevonedistat about patients with advanced solid tumors was undertaken. The results find that, in nine melanoma patients, one achieved a partial response (PR) while another 8 patients achieved stable disease (SD) lasting 6 months [114]. In addition, another phase I study (NCT01011530) was conducted to assess the safety, pharmacokinetics (PK), pharmacodynamic (PD), and antitumor activity of Pevonedistat in metastatic melanoma patients. The maximum tolerated dose (MTD) is reported as 209 mg/m2 . Most patients have a well toleration to Pevonedistat, only 16% patients experience drug-related serious adverse event (SAE), such as drug-related grade 4 acute renal failure, grade 3 myocarditis, and grade 3 small intestinal obstruction. At the end of the research, the research results show that one patient achieves a partial response, and stable disease is reported in 15 patients with lasting for 6.5 months or more in 4 patients [115].

As mentioned, ubiquitination removes the process of Ub and plays an important role in genomic instability regulation and tumorigenesis processes. Thus, several DUB inhibitors have been developed and identified as potential anticancer agents [116]. G9 is described as small molecule Usp9x inhibitor suppressing Usp9x activity [117]. G9 can inhibit NRAS mutant melanoma growth by decreasing Ets-1 protein content and NRAS expression. G9 also has a synergistic effect with PD0325901, a MEK inhibitor [60]. For specific Usp9x inhibitors such as G9 targeting two other DUBs, namely Usp24 and Usp5, more drug testing is needed [117, 118]. In addition, Spautin-1 is recognized a potent USP10/13 deubiquitinating activity antagonist. A recent study revealed that Spautin-1 plays an anti-tumor role in melanoma suppression via DNA damage by increasing ROS levels and has a synergistic effect with Cisplatin [119]. Targeting USP10/13 by Spautin-1 may be a new therapeutic strategy in melanoma patient treatment.

#### **6. Conclusions**

Melanoma has a low 5-year survival rate due to being susceptible to invasion and metastasis. Recently, growing evidence identified the critical role of ubiquitination and de-ubiquitination in malignant melanoma progression, which may be the novel targets for cancer therapy. In this article, we make a brief conclusion that the misregulated expressions of the E2 ubiquitin conjugating-enzymes, E3 ubiquitin ligases, and DUBs lead to aberrant oncogenic signaling in malignant melanoma (**Figure 1**). The ubiquitination plays a vital role in melanoma not only through ubiquitination of key transcription factors or key cell signaling but also immunological

**63**

**Figure 1.**

**Reference UPS** 

**component**

[10] Fbxw7 Targeting

[22, 24] HACE1 Targeting

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

microenvironment modulation. We also make a conclusion of the target UPS components, the corresponding therapeutic drugs or potential therapeutic targets, and the molecular mechanism (**Table 1**). Understanding of ubiquitination and

*Important UPS components and therapeutic targets toward melanoma pathogenesis.*

**Potential therapeutic targets or drugs**

NOTCH signaling

HACE1

[26] ITCH None Cells Mediating BRAF

[30, 31] UBE2C None Cells Suppressing both the

[19] SKP2 None Human and

**Experimental model**

Human and cells

cells

Human and cells

**Molecular mechanism**

Inhibiting NOTCH activation, unleashing HSF1, and activating the MAPK/ERK signaling

Stabilizing the MTH1 expression via K63-linked polyubiquitination, and mediating degradation of cyclin A

Inhibiting YAY1 signaling, and activating RAC-Family GTPases, and regulating K27 ubiquitination of FN

polyubiquitination

level and the activity

of MPF

**Clinical trial**

None

None

None

None

None

*DOI: http://dx.doi.org/10.5772/intechopen.94512*

microenvironment modulation. We also make a conclusion of the target UPS components, the corresponding therapeutic drugs or potential therapeutic targets, and the molecular mechanism (**Table 1**). Understanding of ubiquitination and

#### **Figure 1.**

*Ubiquitin - Proteasome Pathway*

therapeutic strategy in melanoma treatment.

dose (MTD) is reported as 209 mg/m2

Besides, Siah2 is known as a RING finger E3 ubiquitin ligase. Inhibition of Siah2 activity using a peptide is reported to be able to weaken its effect on hypoxia, effectively leading to melanoma metastasis inhibition, while suppression of Siah2 activities prevents the tumorigenicity of melanoma by disrupting Ras/MAPK signaling pathways [108]. Menadione (MEN), also known as vitamin K3, is a quinone used for cancer chemotherapeutic agents. A recent research identifies MEN as a novel Siah2 inhibitor, which attenuates hypoxia and MAPK signaling, and blocks melanoma tumorigenesis [109]. This study revealed that targeting Siah2 by MEN may be a new

Cullin-RING ligases (CRLs) are a subgroup of the E3 ligases, and play an important role in the degradation of oncology relative proteins. It is activated by the neddylation pathway, such as NEDD8 conjugation [110]. The NEDD8-activating enzyme (NAE) is a critical regulator of the neddylation pathway [111]. Pevonedistat (TAK-924/MLN4924) is reported as a first-in-class, small-molecule inhibitor of NAE. Previous preclinical studies reveal that Pevonedistat is associated with tumor growth inhibition of a range of cell lines and primary human cancer cells derived from solid tumors, including malignant melanoma [112, 113]. A phase I study of Pevonedistat about patients with advanced solid tumors was undertaken. The results find that, in nine melanoma patients, one achieved a partial response (PR) while another 8 patients achieved stable disease (SD) lasting 6 months [114]. In addition, another phase I study (NCT01011530) was conducted to assess the safety, pharmacokinetics (PK), pharmacodynamic (PD), and antitumor activity of Pevonedistat in metastatic melanoma patients. The maximum tolerated

Pevonedistat, only 16% patients experience drug-related serious adverse event (SAE), such as drug-related grade 4 acute renal failure, grade 3 myocarditis, and grade 3 small intestinal obstruction. At the end of the research, the research results show that one patient achieves a partial response, and stable disease is reported in

As mentioned, ubiquitination removes the process of Ub and plays an important role in genomic instability regulation and tumorigenesis processes. Thus, several DUB inhibitors have been developed and identified as potential anticancer agents [116]. G9 is described as small molecule Usp9x inhibitor suppressing Usp9x activity [117]. G9 can inhibit NRAS mutant melanoma growth by decreasing Ets-1 protein content and NRAS expression. G9 also has a synergistic effect with PD0325901, a MEK inhibitor [60]. For specific Usp9x inhibitors such as G9 targeting two other DUBs, namely Usp24 and Usp5, more drug testing is needed [117, 118]. In addition, Spautin-1 is recognized a potent USP10/13 deubiquitinating activity antagonist. A recent study revealed that Spautin-1 plays an anti-tumor role in melanoma suppression via DNA damage by increasing ROS levels and has a synergistic effect with Cisplatin [119]. Targeting USP10/13 by Spautin-1

Melanoma has a low 5-year survival rate due to being susceptible to invasion and

metastasis. Recently, growing evidence identified the critical role of ubiquitination and de-ubiquitination in malignant melanoma progression, which may be the novel targets for cancer therapy. In this article, we make a brief conclusion that the misregulated expressions of the E2 ubiquitin conjugating-enzymes, E3 ubiquitin ligases, and DUBs lead to aberrant oncogenic signaling in malignant melanoma (**Figure 1**). The ubiquitination plays a vital role in melanoma not only through ubiquitination of key transcription factors or key cell signaling but also immunological

15 patients with lasting for 6.5 months or more in 4 patients [115].

may be a new therapeutic strategy in melanoma patient treatment.

. Most patients have a well toleration to

**62**

**6. Conclusions**

*Important UPS components and therapeutic targets toward melanoma pathogenesis.*



**65**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

de-ubiquitination mechanisms and their regulation in melanoma will help us to better understand the pathogenesis of this cancer, and develop effective therapeutic approaches, which lets us see a promising future for the application of these advancements owing to the prosperity and success of drugs targeting ubiquitination

This work was supported by the National Natural Science Foundation of China

JLZ developed the ideas and revised the manuscript. JG wrote the main

HACE1 HECT domain and ankyrin repeat-containing E3 ubiquitin protein

NEDD8 Neural precursor cell expressed, developmentally down-regulated8

*DOI: http://dx.doi.org/10.5772/intechopen.94512*

and de-ubiquitination in melanoma.

The authors declare no conflict of interest.

**Acknowledgements**

(Grants No. 81772917).

**Conflict of interest**

**Authors' contributions**

**Acronyms and abbreviations**

CRC Colorectal cancer

E3 The ubiquitin ligase

ligase 1 HSF1 Heat-shock factor 1

ISG15 Interferon-stimulated gene 15 MAGE-A11 Melanoma antigen-A11

MKRN2 Makorin ring finger protein 2 MPF M-phase-promoting factor

NRF1 Nuclear Respiratory Factor 1 Skp2 S-phase kinase-associated protein 2 SUMO Small ubiquitin-like modifiers

TME Tumor microenvironment

UBLs Ubiquitin-like proteins

UBE2S Ubiquitin-conjugating enzyme E2S

TLRs Toll-like receptors

MITF Microphthalmia-associated transcription factor

FN Fibronectin

MEN Menadione miRNAs MicroRNAs

ACT Adoptive cell therapy BAP1 BRCA1-associated protein-1

DUB Deubiquitinating enzymes E1 The ubiquitin-activating enzyme E2 The ubiquitin-conjugating enzyme

EMT Epithelial-mesenchymal transition Fbxw7 F-box/WD repeat-containing protein 7

manuscript.

#### **Table 1.** *Summarization of the target UPS components.*

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes DOI: http://dx.doi.org/10.5772/intechopen.94512*

de-ubiquitination mechanisms and their regulation in melanoma will help us to better understand the pathogenesis of this cancer, and develop effective therapeutic approaches, which lets us see a promising future for the application of these advancements owing to the prosperity and success of drugs targeting ubiquitination and de-ubiquitination in melanoma.

#### **Acknowledgements**

*Ubiquitin - Proteasome Pathway*

**component**

[32] UBE2S Targeting

[35] MKRN2 Targeting

[50] USP54 Targeting

[52] USP18 Targeting

[53] USP5 Targeting

[54] USP4 Targeting

[55] USP15 Targeting

[62] UCHL1 Targeting

[67, 68] BAP1 Targeting

**Potential therapeutic targets or drugs**

UBE2S

MKRN2

USP54

USP18

USP5

USP4

USP15

UCHL1

BAP1

[105, 106] MDM2 Nutlin-3a Human, animal

[111–113] NEDD8 Pevonedistat Human, animal

[118] USP10/13 Spautin-1 Animal and

*Summarization of the target UPS components.*

**Experimental model**

Human, animal and cells

Animal and cells

Animal and cells

Animal and cells

Human, animal and cells

and cells

and cells

cells

[107, 108] Siah2 Menadione Cells Attenuating hypoxia

[59, 116] Usp9x G9 Cells Decreasing Ets-1

**Molecular mechanism**

Catalyzing Lys11-linkage ubiquitination, and inhibiting EMT

ubiquitylated P53

Bing stimulated by IFN-γ, and regulating CTL CD8+ immunecell function

FAS induction, and then suppressing cell cycle checkpoint and

apoptosis

Cells Promoting EMT None

Downregulating MDM2 expression, and increasing frequency of CD8+ effector T cell tumor-infiltrating

of MITF in human melanocytes

Inhibiting MDM2 and cyclin B1/CDK1 phosphorylated nuclear iASPP

and MAPK signaling

protein content and NRAS expression, and having a synergistic effect with PD0325901

Inhibiting the activity of cullin E3 ligases and then stabilizing cullin substrates

Inducing DNA damage by increasing ROS levels, and having synergistic effect with Cisplatin

Unknown None

Unknown None

Cells Interacting with

Cells Blocking p53 and

Cells Regulating stability

**Clinical trial**

None

None

None

None

None

None

None

None

None

None

NCT01011530

**Reference UPS** 

**64**

**Table 1.**

This work was supported by the National Natural Science Foundation of China (Grants No. 81772917).

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Authors' contributions**

JLZ developed the ideas and revised the manuscript. JG wrote the main manuscript.

#### **Acronyms and abbreviations**


#### *Ubiquitin - Proteasome Pathway*


### **Author details**

Jia Guo and Jianglin Guo\* Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan, China

\*Address all correspondence to: zhangjianglin123@hotmail.com

© 2020 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.

**67**

*Ubiquitination and Deubiquitination in Melanoma Research and Clinically Relevant Outcomes*

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[11] Kourtis N, Moubarak RS, Aranda-Orgilles B, Lui K, Aydin IT, Trimarchi T, et al. FBXW7 modulates cellular stress response and metastatic potential through HSF1 post-translational modification. Nature Cell Biology. 2015;**17**:322-332. DOI: 10.1038/ncb3121

[12] Cheng Y, Chen G, Martinka M, Ho V, Li G. Prognostic significance of Fbw7 in human melanoma and its role in cell migration. The Journal of Investigative Dermatology. 2013;**133**:1794-1802. DOI:

[13] King R, Weilbaecher KN, McGill G,

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*Ubiquitin - Proteasome Pathway*

WIP1i WIP1 inhibitor

UCHL1 Ubiquitin C-terminal hydrolase 1 UPP Ubiquitin proteasomal pathway UPS Ubuiquitin-proteasome system

Usp9x Ubiquitin specific peptidase 9, X-linked

**66**

**Author details**

Jia Guo and Jianglin Guo\*

Changsha, Hunan, China

provided the original work is properly cited.

Department of Dermatology, Xiangya Hospital, Central South University,

© 2020 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,

\*Address all correspondence to: zhangjianglin123@hotmail.com

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10.1042/BJ20121249

10.1016/j.ccr.2013.03.013

10.1016/j.ccr.2013.03.013.

overexpression in therapeutic resistance of malignant tumors. Cancer Cell International. 2019;**19**:216. DOI: 10.1186/s12935-019-0937-4

[111] Soucy TA, Dick LR, Smith PG, Milhollen MA, Brownell JE. The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy. Genes & Cancer. 2010;**1**:708-716. DOI: 10.1177/1947601910382898

Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;**458**:732-736. DOI: 10.1038/

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[114] Sarantopoulos J, Shapiro GI, Cohen RB, Clark JW, Kauh JS, Weiss GJ, et al. Phase I Study of the Investigational NEDD8-Activating Enzyme Inhibitor Pevonedistat (TAK-924/MLN4924) in Patients with Advanced Solid Tumors. Clinical Cancer Research. 2016;**22**:847-

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**77**

**Chapter 5**

**Abstract**

ubiquitin, ubiquitylation

**1. Introduction**

New Discoveries on the Roles

*Emma I. Kane and Donald E. Spratt*

of "Other" HECT E3 Ubiquitin

Ligases in Disease Development

HECT E3 ubiquitin ligases selectively recognize, bind, and ubiquitylate their substrate proteins to target them for 26S proteasomal degradation. There is increasing evidence that HECT E3 ubiquitin ligase dysfunction due to misfolding and/or the gene encoding the protein being mutated is responsible for the development of different diseases. Apart from the more prominent and well-characterized E6AP and members of the NEDD4 family, new studies have begun to reveal how other members of the HECT E3 ubiquitin ligase family function as well as their links to disease and developmental disorders. This chapter provides a comprehensive discussion on the more mysterious members of the HECT E3 ubiquitin ligase family and how they control intracellular processes. Specifically, AREL1, HACE1, HECTD1, HECTD4, G2E3, and TRIP12 will be examined as these enzymes have

recently been identified as contributors to disease development.

**1.1 HECT E3 ubiquitin ligase-dependent ubiquitylation**

**Keywords:** apoptosis, AREL1, cancer, HECT E3 ubiquitin ligase, G2E3, HACE1, HECT, HECTD1, HECTD4, neurodevelopment, proteasomal degradation, TRIP12,

Ubiquitylation is an essential post-translational modification that regulates numerous intracellular processes including protein localization and trafficking, DNA damage response, immune system and viral response, apoptosis and proteolysis [1, 2]. E3 ubiquitin ligases play an important role in recognizing, binding, and covalently attaching ubiquitin to their various substrates to elicit a specific cellular response [3]. The homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases are a unique subfamily that use a multistep pathway to selectively target substrate proteins for ubiquitylation [4]. HECT-dependent ubiquitylation requires the recruitment of an E2 ubiquitin conjugating enzyme charged with ubiquitin to the N-terminal lobe of the HECT domain on the E3 ligase [5, 6]. The ubiquitin cargo is then transferred from the E2 enzyme to the conserved catalytic cysteine within the C-terminal lobe of the HECT domain *via* a transthiolation reaction to form a thioester bond. The HECT E3~ubiquitin complex will then bind to a substrate and

#### **Chapter 5**

## New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development

*Emma I. Kane and Donald E. Spratt*

### **Abstract**

HECT E3 ubiquitin ligases selectively recognize, bind, and ubiquitylate their substrate proteins to target them for 26S proteasomal degradation. There is increasing evidence that HECT E3 ubiquitin ligase dysfunction due to misfolding and/or the gene encoding the protein being mutated is responsible for the development of different diseases. Apart from the more prominent and well-characterized E6AP and members of the NEDD4 family, new studies have begun to reveal how other members of the HECT E3 ubiquitin ligase family function as well as their links to disease and developmental disorders. This chapter provides a comprehensive discussion on the more mysterious members of the HECT E3 ubiquitin ligase family and how they control intracellular processes. Specifically, AREL1, HACE1, HECTD1, HECTD4, G2E3, and TRIP12 will be examined as these enzymes have recently been identified as contributors to disease development.

**Keywords:** apoptosis, AREL1, cancer, HECT E3 ubiquitin ligase, G2E3, HACE1, HECT, HECTD1, HECTD4, neurodevelopment, proteasomal degradation, TRIP12, ubiquitin, ubiquitylation

#### **1. Introduction**

#### **1.1 HECT E3 ubiquitin ligase-dependent ubiquitylation**

Ubiquitylation is an essential post-translational modification that regulates numerous intracellular processes including protein localization and trafficking, DNA damage response, immune system and viral response, apoptosis and proteolysis [1, 2]. E3 ubiquitin ligases play an important role in recognizing, binding, and covalently attaching ubiquitin to their various substrates to elicit a specific cellular response [3]. The homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases are a unique subfamily that use a multistep pathway to selectively target substrate proteins for ubiquitylation [4]. HECT-dependent ubiquitylation requires the recruitment of an E2 ubiquitin conjugating enzyme charged with ubiquitin to the N-terminal lobe of the HECT domain on the E3 ligase [5, 6]. The ubiquitin cargo is then transferred from the E2 enzyme to the conserved catalytic cysteine within the C-terminal lobe of the HECT domain *via* a transthiolation reaction to form a thioester bond. The HECT E3~ubiquitin complex will then bind to a substrate and

#### *Ubiquitin - Proteasome Pathway*

covalently attach ubiquitin on to a lysine residue of the substrate protein forming a stable isopeptide bond between the C-terminus of ubiquitin and the ε-amine of the substrate lysine [3, 5, 6]. This process can be repeated numerous times to form different polyubiquitin chain linkages with the specific HECT E3 ubiquitin ligase dictating the type(s) of ubiquitin linkages that are built [2, 7].


#### **Table 1.**

*Ubiquitin conjugation determines the intracellular fate of a substrate protein.*

**79**

embryogenesis.

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development*

**1.2 Ubiquitin attachment site(s) and chain type linkages determine the fate** 

The destiny of a ubiquitin-tagged protein is dependent on (i) the site(s) of ubiquitin attachment on the substrate, (ii) the number of ubiquitin moieties attached to the substrate (i.e. mono-, multi-mono-, or polyubiquitin), and (iii) the specific type(s) of linkages between the different ubiquitin molecules in a polyubiquitin chain (i.e. K48, K63, branched, etc.) [1, 2, 7]. Potential fates of a ubiquitin-tagged substrate include changes in cellular localization/trafficking, enhanced/inhibited protein activity, changes in protein–protein affinity/interactions, and proteolysis [1, 2, 6–8].

Differences in ubiquitin lysine linkage specificity determine the destination and/

or fate of the targeted protein in the cell (**Table 1**). For example, the well-established K48-polyubiquitylation chain, heterotypic K11/K48-polyubiquitin, K29/ K48-polyubiquitin monoubiquitin tagged peptides and multiple monoubiquitin tagged proteins have also been found to signal for 26S proteasomal degradation [7, 8]. K63-polyubiquitin chains signal for protein degradation through the initiation of K48/K63 polyubiquitin branch formation [5] but cannot be recognized by the 26S proteosome [26]. To date, many different varieties of ubiquitin chain types have

**2. The "other" HECT E3 ubiquitin ligases: important players in disease,** 

The HECT E3 ubiquitin ligases can be categorized into three subfamilies – NEDD4, HERC, and "other" – based on their sequence/structure similarity and domain architecture [4, 5]. Of the 28 HECT E3 ubiquitin ligases identified in humans, there are 12 "other" HECT E3 ubiquitin ligases that do not fall under the well-studied NEDD4 or HERC subfamilies. Each member of the "other" HECTs have variable N-terminal domains that are thought to be involved in protein–protein interactions and/or intracellular localization [4, 5]. Having prominent responsibilities in cellular homeostasis would leave the impression there is ample research on the HECT E3 ubiquitin ligase family as a whole, however, there remain many unanswered questions about the biological functions and mechanisms of this important E3 ligase family, particularly for members of the more mysterious "other" subfamily. With new research and discoveries becoming available, there is mounting evidence that the lesser known HECT E3 ubiquitin ligases play critical roles in regulating intracellular processes and their dysfunction have been suggested to contribute to the onset of many diseases and disorders [4, 29–32]. Here we discuss the latest discoveries on these lesser known members of the HECT E3 ubiquitin ligases and on their emerging roles in developmental and neurological abnormalities, cancers, and

**2.1 AREL1, a key regulator of apoptosis and potential oncogenic drug target**

Apoptosis resistant E3 ubiquitin protein ligase 1(AREL1; 823 residues) is a cytosolic enzyme responsible for regulating apoptosis through the inhibition of

been identified, but their distinct biological functions remain unclear. Monoubiquitylation can occur at one site or at multiple sites (multimonoubiquitylation) on a substrate. Polyubiquitylation can build off of a monoubiquitin attachment site with a specific lysine linkage (homotypic) or have multiple chains with different lysine linkages (branch) at the end of a growing ubiquitin chain (heterotypic). These modifications can also influence signaling pathways, whether it is through enhancing or inhibiting participating proteins and processes.

*DOI: http://dx.doi.org/10.5772/intechopen.91770*

**of a substrate protein**

**yet poorly understood**

#### **1.2 Ubiquitin attachment site(s) and chain type linkages determine the fate of a substrate protein**

The destiny of a ubiquitin-tagged protein is dependent on (i) the site(s) of ubiquitin attachment on the substrate, (ii) the number of ubiquitin moieties attached to the substrate (i.e. mono-, multi-mono-, or polyubiquitin), and (iii) the specific type(s) of linkages between the different ubiquitin molecules in a polyubiquitin chain (i.e. K48, K63, branched, etc.) [1, 2, 7]. Potential fates of a ubiquitin-tagged substrate include changes in cellular localization/trafficking, enhanced/inhibited protein activity, changes in protein–protein affinity/interactions, and proteolysis [1, 2, 6–8].

Differences in ubiquitin lysine linkage specificity determine the destination and/ or fate of the targeted protein in the cell (**Table 1**). For example, the well-established K48-polyubiquitylation chain, heterotypic K11/K48-polyubiquitin, K29/ K48-polyubiquitin monoubiquitin tagged peptides and multiple monoubiquitin tagged proteins have also been found to signal for 26S proteasomal degradation [7, 8]. K63-polyubiquitin chains signal for protein degradation through the initiation of K48/K63 polyubiquitin branch formation [5] but cannot be recognized by the 26S proteosome [26]. To date, many different varieties of ubiquitin chain types have been identified, but their distinct biological functions remain unclear.

Monoubiquitylation can occur at one site or at multiple sites (multimonoubiquitylation) on a substrate. Polyubiquitylation can build off of a monoubiquitin attachment site with a specific lysine linkage (homotypic) or have multiple chains with different lysine linkages (branch) at the end of a growing ubiquitin chain (heterotypic). These modifications can also influence signaling pathways, whether it is through enhancing or inhibiting participating proteins and processes.

#### **2. The "other" HECT E3 ubiquitin ligases: important players in disease, yet poorly understood**

The HECT E3 ubiquitin ligases can be categorized into three subfamilies – NEDD4, HERC, and "other" – based on their sequence/structure similarity and domain architecture [4, 5]. Of the 28 HECT E3 ubiquitin ligases identified in humans, there are 12 "other" HECT E3 ubiquitin ligases that do not fall under the well-studied NEDD4 or HERC subfamilies. Each member of the "other" HECTs have variable N-terminal domains that are thought to be involved in protein–protein interactions and/or intracellular localization [4, 5]. Having prominent responsibilities in cellular homeostasis would leave the impression there is ample research on the HECT E3 ubiquitin ligase family as a whole, however, there remain many unanswered questions about the biological functions and mechanisms of this important E3 ligase family, particularly for members of the more mysterious "other" subfamily. With new research and discoveries becoming available, there is mounting evidence that the lesser known HECT E3 ubiquitin ligases play critical roles in regulating intracellular processes and their dysfunction have been suggested to contribute to the onset of many diseases and disorders [4, 29–32]. Here we discuss the latest discoveries on these lesser known members of the HECT E3 ubiquitin ligases and on their emerging roles in developmental and neurological abnormalities, cancers, and embryogenesis.

#### **2.1 AREL1, a key regulator of apoptosis and potential oncogenic drug target**

Apoptosis resistant E3 ubiquitin protein ligase 1(AREL1; 823 residues) is a cytosolic enzyme responsible for regulating apoptosis through the inhibition of

*Ubiquitin - Proteasome Pathway*

Monoubiquitylation Monoubiquitylation/ multi-monoubiquitylation

Polyubiquitylation

covalently attach ubiquitin on to a lysine residue of the substrate protein forming a stable isopeptide bond between the C-terminus of ubiquitin and the ε-amine of the substrate lysine [3, 5, 6]. This process can be repeated numerous times to form different polyubiquitin chain linkages with the specific HECT E3 ubiquitin ligase

Endocytosis [9]

DNA damage repair [10–15] Histone regulation [10–15] Mitophagy [10–15] Protein localization [10–15] Protein interactions [10–15] Protein transportation [10–15] Transcription activation [10–15]

Linear chain formation [9] NF-κB activation [9, 16] Signaling cascades [9, 16]

NF-κB regulation [14] Mitophagy [14]

Kinase activation [19] Protein degradation [20] Protein scaffolding [21] Protein trafficking [22]

Kinase activation [19] Protein degradation [9]

Kinase activation [23] Post-golgi trafficking [24] T-cell signaling [23]

NF-κB activation [9, 16] Protein trafficking [9]

Mitophagy [17] NF-κB activation [16] Protein degradation [17]

DNA damage response [18]

K6 DNA damage response [14]

K11 Cell cycle regulation [17]

K27 DNA damage response [18]

K29 DNA damage response [18]

K33 DNA damage response [10–15, 18]

K48 Protein degradation [1, 2, 25] K63 DNA damage response [9, 18]

K11/K48 Protein degradation [26, 27] K29/K48 Protein degradation [26] K48/K63 Protein degradation [26] K11/K63 Endocytosis [28]

dictating the type(s) of ubiquitin linkages that are built [2, 7].

**Chain types Linker Proposed function**

Chain (homotypic) M1 Innate immunity [2, 9, 16]

Chain (heterotypic; branched) M1/K63 NF-κB activation [16]

*Ubiquitin conjugation determines the intracellular fate of a substrate protein.*

**78**

**Table 1.**

proapoptotic proteins *via* ubiquitylation [33]. AREL1 contains two immunoglobin-like folds (IGF) near its N-terminus that potentially mediate substrate binding and recognition (**Figure 1**) [35, 36]. IGF domains assemble into a sandwich-like form consisting of antiparallel β-strands that allow for protein–protein interactions [36, 37].

Apoptosis (aka programmed cell death) is an important and highly regulated biological process that occurs during early embryonic development and the immune response [38, 39]. Once apoptosis is initiated the cell is committed to die, which is mediated by a caspase cascade [40]. Intrinsic apoptosis is turned on by the release of intermembrane mitochondrial proteins when cells are under oxidative stress [38, 39, 41]. In contrast, the extrinsic apoptotic pathway is activated by extracellular signaling at the cell membrane leading to the formation of the death-inducing signaling complex (DISC) [42–44].

AREL1 was first identified in 2013 and was immediately recognized as an oncogenic target due to its inhibitory role in apoptosis [33]. Identified substrates for AREL1 include second mitochondrial activator of caspase (SMAC), HtrA serine peptidase 2 (HtrA2) and septin 4 (ARTS), which are known antagonists of inhibitor of apoptosis proteins (IAPs) [45]. Studies have shown that AREL1 can build K48 and K63 polyubiquitin chains to target substrates for proteolysis, as well as atypical K33 polyubiquitin chains whose biological function is still being clarified [35, 46]. Various IAPs, including SMAC, HtrA1, and ARTS, are released from the mitochondrial intermembrane into the cytosol when the cell is triggered or stressed. AREL1 inhibits apoptosis by ubiquitylating these IAP antagonists with K48-linked polyubiquitin chains targeting the IAPs for proteasomal degradation [33].

The induction of apoptosis is thought to require the release of numerous IAPs in the cytosol to allow different signaling pathways to initiate apoptosis depending on the cell's specific stress. For example, the release of SMAC into the cytosol allows it to bind cellular inhibitor of apoptosis protein 1/2 (cIAP1/2), which then targets cIAP1/2 for proteasomal degradation to initiate apoptosis. However, when AREL1 is present, SMAC is ubiquitylated by AREL1 and degraded, thus blocking SMACcIAP1/2 complex formation enabling cell survival [33]. Many cancer therapies are interested in specifically turning on apoptosis through IAPs in cancer cells [47–49],

#### **Figure 1.**

*AREL1 domain architecture. AREL1 contains a putative immunoglobulin fold domain (IGF, residues 52-158) and the canonical HECT domain (436-823) as annotated on UniProt and InterPro. Representative crystal structures of an IGF fold (human IGF FAB in yellow/orange; PDB 7FAB [34]), which is suggested to mediate AREL1 substrate binding and recognition, while the AREL1 HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue, catalytic cysteine C790 in red; PDB 6JX5 [35]) is required for ubiquitylation activity. Structures were visualized using PyMol.*

**81**

**Figure 2.**

*using PyMol.*

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development*

thus AREL1 could prove to be a novel enzyme in drug development. Continued

First identified in 2004, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1; 909 residues) has been shown to take part in various cellular processes. For example, HACE1 is best known as a tumor suppressor as altered HACE1 expression levels have been observed in various cancers including colorectal, breast, liver, kidney, osteosarcoma, lymphoma and gastric cancer [50–54]. HACE1 contains six ankyrin repeats near its N-terminus that likely take part in HACE1-substrate recognition and protein–protein interactions (**Figure 2**). While it is not yet fully understood how the ankyrin repeats support HACE1 function, ankyrin repeats in other proteins have been shown to instigate the development of a wide array of diseases including cancer [56]. HACE1 also supports Golgi membrane biogenesis during cell division by ubiquitylating members of the

Studies have shown that HACE1 expression levels are altered when comparing normal and cancerous tissue. Specifically, in the Wilms' tumor cell line, HACE1 expression was essentially nonexistent, whereas in other cancer cell lines expression levels were lower than average [50]. This study concluded that HACE1 was essential in repressing cancer development as the lowered expression levels of HACE1 were not mutation dependent. Low expression levels of HACE1 have also been observed in other cancer cell lines. For instance, it was found that the methylation of the HACE1 promoter resulted in decreased HACE1 expression in liver cancer cells, which in turn decreased HACE1's ability to ubiquitylate its identified substrates optineurin (OPTN) and microtubule-associated proteins 1A/1B light chain 3B protein [53]. Many different substrates of HACE1 have been identified to date (summarized in [4]), including β2-adrenergic receptor (ß2AR) [58], OPTN [59], retinoic acid receptor beta (RAR-β) [57], tumor necrosis factor receptor-2 (TNFR2) [60], and various Ras-related

*Predicted domain architecture of HACE1. HACE1 contains six putative ankyrin-repeats (ANK; residues 64-258) and a HECT domain (574-909) as annotated on UniProt and InterPro. Representative crystal structures of an ANK repeat fold (in shades of pink, PDB 4O60 [55]), which is likely involved in HACE1 substrate binding and recognition, and a HECT domain (HECTN-lobe in green, HECTC-lobe in blue; PDB 6JX5 [35]) found at HACE1's C-terminus that is required of ubiquitylation activity. Structures were visualized* 

studies on AREL1's mechanisms in controlling cell death are warranted.

**2.2 HACE1, a prominent tumor suppressor with dual function**

*DOI: http://dx.doi.org/10.5772/intechopen.91770*

Ras-related superfamily of small G proteins [57].

thus AREL1 could prove to be a novel enzyme in drug development. Continued studies on AREL1's mechanisms in controlling cell death are warranted.

#### **2.2 HACE1, a prominent tumor suppressor with dual function**

First identified in 2004, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1; 909 residues) has been shown to take part in various cellular processes. For example, HACE1 is best known as a tumor suppressor as altered HACE1 expression levels have been observed in various cancers including colorectal, breast, liver, kidney, osteosarcoma, lymphoma and gastric cancer [50–54]. HACE1 contains six ankyrin repeats near its N-terminus that likely take part in HACE1-substrate recognition and protein–protein interactions (**Figure 2**). While it is not yet fully understood how the ankyrin repeats support HACE1 function, ankyrin repeats in other proteins have been shown to instigate the development of a wide array of diseases including cancer [56]. HACE1 also supports Golgi membrane biogenesis during cell division by ubiquitylating members of the Ras-related superfamily of small G proteins [57].

Studies have shown that HACE1 expression levels are altered when comparing normal and cancerous tissue. Specifically, in the Wilms' tumor cell line, HACE1 expression was essentially nonexistent, whereas in other cancer cell lines expression levels were lower than average [50]. This study concluded that HACE1 was essential in repressing cancer development as the lowered expression levels of HACE1 were not mutation dependent. Low expression levels of HACE1 have also been observed in other cancer cell lines. For instance, it was found that the methylation of the HACE1 promoter resulted in decreased HACE1 expression in liver cancer cells, which in turn decreased HACE1's ability to ubiquitylate its identified substrates optineurin (OPTN) and microtubule-associated proteins 1A/1B light chain 3B protein [53]. Many different substrates of HACE1 have been identified to date (summarized in [4]), including β2-adrenergic receptor (ß2AR) [58], OPTN [59], retinoic acid receptor beta (RAR-β) [57], tumor necrosis factor receptor-2 (TNFR2) [60], and various Ras-related

#### **Figure 2.**

*Ubiquitin - Proteasome Pathway*

signaling complex (DISC) [42–44].

proapoptotic proteins *via* ubiquitylation [33]. AREL1 contains two immunoglobin-like folds (IGF) near its N-terminus that potentially mediate substrate binding and recognition (**Figure 1**) [35, 36]. IGF domains assemble into a sandwich-like form consisting

Apoptosis (aka programmed cell death) is an important and highly regulated biological process that occurs during early embryonic development and the immune response [38, 39]. Once apoptosis is initiated the cell is committed to die, which is mediated by a caspase cascade [40]. Intrinsic apoptosis is turned on by the release of intermembrane mitochondrial proteins when cells are under oxidative stress [38, 39, 41]. In contrast, the extrinsic apoptotic pathway is activated by extracellular signaling at the cell membrane leading to the formation of the death-inducing

AREL1 was first identified in 2013 and was immediately recognized as an oncogenic target due to its inhibitory role in apoptosis [33]. Identified substrates for AREL1 include second mitochondrial activator of caspase (SMAC), HtrA serine peptidase 2 (HtrA2) and septin 4 (ARTS), which are known antagonists of inhibitor of apoptosis proteins (IAPs) [45]. Studies have shown that AREL1 can build K48 and K63 polyubiquitin chains to target substrates for proteolysis, as well as atypical K33 polyubiquitin chains whose biological function is still being clarified [35, 46]. Various IAPs, including SMAC, HtrA1, and ARTS, are released from the mitochondrial intermembrane into the cytosol when the cell is triggered or stressed. AREL1 inhibits apoptosis by ubiquitylating these IAP antagonists with K48-linked

polyubiquitin chains targeting the IAPs for proteasomal degradation [33].

The induction of apoptosis is thought to require the release of numerous IAPs in the cytosol to allow different signaling pathways to initiate apoptosis depending on the cell's specific stress. For example, the release of SMAC into the cytosol allows it to bind cellular inhibitor of apoptosis protein 1/2 (cIAP1/2), which then targets cIAP1/2 for proteasomal degradation to initiate apoptosis. However, when AREL1 is present, SMAC is ubiquitylated by AREL1 and degraded, thus blocking SMACcIAP1/2 complex formation enabling cell survival [33]. Many cancer therapies are interested in specifically turning on apoptosis through IAPs in cancer cells [47–49],

*AREL1 domain architecture. AREL1 contains a putative immunoglobulin fold domain (IGF, residues 52-158) and the canonical HECT domain (436-823) as annotated on UniProt and InterPro. Representative crystal structures of an IGF fold (human IGF FAB in yellow/orange; PDB 7FAB [34]), which is suggested to mediate AREL1 substrate binding and recognition, while the AREL1 HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue, catalytic cysteine C790 in red; PDB 6JX5 [35]) is required for ubiquitylation activity. Structures were* 

of antiparallel β-strands that allow for protein–protein interactions [36, 37].

**80**

**Figure 1.**

*visualized using PyMol.*

*Predicted domain architecture of HACE1. HACE1 contains six putative ankyrin-repeats (ANK; residues 64-258) and a HECT domain (574-909) as annotated on UniProt and InterPro. Representative crystal structures of an ANK repeat fold (in shades of pink, PDB 4O60 [55]), which is likely involved in HACE1 substrate binding and recognition, and a HECT domain (HECTN-lobe in green, HECTC-lobe in blue; PDB 6JX5 [35]) found at HACE1's C-terminus that is required of ubiquitylation activity. Structures were visualized using PyMol.*

#### *Ubiquitin - Proteasome Pathway*

proteins [57, 58, 61–65]. Expanded studies on how HACE1 binds and recognizes its substrates are needed to further clarify the role of HACE1 in cancer development.

HACE1 also plays an essential role in neurodevelopment as it was recently shown to be involved in the spastic paraplegia and psychomotor retardation with or without seizures (SPPRS) phenotype [66]. HACE1 also has cardiac protection function during hemodynamic stress when it was shown in mice with *HACE1* deficiency, their susceptibility to accelerated heart failure greatly increased [67]. This suggests that HACE1 has a critical role in protecting the heart from various stresses, thus making it a potential cardiac drug target.

#### **2.3 HECTD1, an important regulator in neurodevelopment**

HECT domain containing E3 ubiquitin protein ligase 1 (HECTD1; 2610 residues) was discovered in 2007 as a novel and important regulator of neurodevelopment [68]. HECTD1 has similar domain architecture to HACE1 with four ankyrin repeats near its N-terminus and a C-terminal HECT domain (**Figure 3**). HECTD1 plays an important role in pulmonary fibrosis during endothelial-mesenchymal transition (EndMT) with reports of increased circular RNA HECTD1 (circHECTD1) transcription, which causes decreased HECTD1 protein expression in lung tissue [72, 73]. Elevated circHECTD1 gene expression has also been found in patients with acute ischemic stroke (AIS) [74]. HECTD1 also contains a Smad4 activation SAD1/ UNC (SUN) domain and a mind bomb (MIB) domain, with each having unique roles in intracellular signaling due to Smad-DNA complex formation and cellular interactions through the Notch pathway, respectively [75, 76].

HECTD1 supports fetal growth and proper placenta development. Specifically, HECTD1 aids in the development of the labyrinthine and junctional zones of the placenta, regions where the fetus acquires nutrients and disposes of waste, as well as a bilayer between the labyrinthine and decidual cells, respectively [77, 78]. HECTD1 ensures the proper size development of the labyrinthine, yet the mechanisms to ensure this are still not fully understood. Mutations within HECTD1 lead to the onset of irregular labyrinthine development, which in turn depletes nutrients for the fetus. Fetal fatality can occur without proper maintenance of these placental regions, suggesting

#### **Figure 3.**

*HECTD1 domain architecture. HECTD1 contains putative protein-protein interaction domains including two armadillo-repeat containing domains (ARM1, residues 8-254; ARM2 residues 892-925 in purple; PDB 4DB8 [69]), four ankyrin-repeats (residues 395-612 in shades of pink; PDB 4O60 [56]), a SAD1/UNC domain (SUN, residues 1115-1244 in yellow; PDB 3UNP [70]), a mind bomb domain (MIB, residues 1266–1338 in red; PDB 2DK3), a helix-bundle domain (H, residues 1896–1968 in magenta; PDB 2KZS [71]) and a HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]). Domain boundaries are denoted according to UniProt and InterPro. Structures were visualized using PyMol.*

**83**

**features**

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development*

that HECTD1 expression is essential for the proper development and survival of fetuses *in utero*. HECTD1 also plays a role in proper neural tube closure. Anencephaly occurs when the neural tube does not close properly, which has been linked to

**2.4 HECTD4, a genetically linked precursor to cancer and cardiovascular** 

that need to be identified and functionally examined.

still needed to verify this relationship.

sion and DNA damage response [85, 87, 88].

HECTD1 control of heat shock protein 90 (Hsp90) levels [79]. When Hsp90 secretion levels are not properly regulated by HECTD1 ubiquitylation, abnormal neural tube development can occur. The continued examination of this important enzyme will hopefully clarify the molecular basis for HECTD1's role in neurodevelopment.

HECT domain containing E3 ubiquitin protein ligase 4 (HECTD4; 3996 residues) was recently discovered in 2014. A pleiotropic gene screen showed that there were links between metabolic syndromes and inflammation, specifically with single nucleotide polymorphisms (SNPs) in the *HECTD4* gene [80]. Since 2014, HECTD4 has also been found to be associated with diabetes, hypertension and cardiovascular disease, lung adenocarcinoma, urothelial carcinoma and ovarian endometriosis [81–84]. Being one of the larger enzymes within the "other" HECT E3 ubiquitin ligase subfamily, it is intriguing that there have been no putative domains annotated for HECTD4 except the C-terminal HECT domain (residues 3627–3996). There are likely different domains located in the N-terminal region of the HECTD4 protein

Genetic screening has identified various mutations in *HECTD4* in cancer cells. For example, *HECTD4* was recently identified as one of nine genes that correlated with the onset of lung adenocarcinoma [83]. Tumor genetic screens have revealed in patients with urothelial carcinoma in the bladder (UCB) that mutations in *HECTD4*, Fibrillin-3 Precursor (*FBN3*) and Citron Rho-Interacting Kinase (*CIT*) were correlated to UCB disease progression [81]. *HECTD4* may also be linked to the development of ovarian endometriosis (OEM) [82]. However, future studies are

Already having various genetic links to cancers and cardiovascular disease, HECTD4 deserves more attention by the research community to further clarify its biological and functional roles in the cell. Currently very little is known about HECTD4, therefore it will be imperative to first identify potential similarities in protein sequence and/or domain architecture. To better clarify HECTD4's role in ubiquitin biology, it will also be important to discover HECTD4 substrates and annotate the sites of HECTD4-dependent ubiquitylation to answer how this myste-

**2.5 G2E3, a unique multifunctional HECT E3 ubiquitin ligase with RING-like** 

G2E3 contains three plant homeodomain (PHD)-type zinc finger repeats, a domain typically known to bind to modified histones and act as epigenetic readers [89], making it the only known HECT E3 ubiquitin ligase to possess "RING"-like

G2/M-phase specific E3 ubiquitin protein ligase (G2E3; 706 residues) was first identified in 2006 and named for its role during the G2/M phase of cell division and for having a conserved C-terminal HECT domain [85]. Knockout studies of G2E3 in mice demonstrated that this enzyme is essential in preventing apoptotic cell death during early embryonic development [86]. Expression levels in G2E3 were also observed to increase during early embryogenesis, specifically during central nervous system development. This enzyme is also implicated in cell cycle progres-

rious HECT E3 ubiquitin ligase contributes to disease development.

*DOI: http://dx.doi.org/10.5772/intechopen.91770*

**disease**

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development DOI: http://dx.doi.org/10.5772/intechopen.91770*

that HECTD1 expression is essential for the proper development and survival of fetuses *in utero*. HECTD1 also plays a role in proper neural tube closure. Anencephaly occurs when the neural tube does not close properly, which has been linked to HECTD1 control of heat shock protein 90 (Hsp90) levels [79]. When Hsp90 secretion levels are not properly regulated by HECTD1 ubiquitylation, abnormal neural tube development can occur. The continued examination of this important enzyme will hopefully clarify the molecular basis for HECTD1's role in neurodevelopment.

#### **2.4 HECTD4, a genetically linked precursor to cancer and cardiovascular disease**

HECT domain containing E3 ubiquitin protein ligase 4 (HECTD4; 3996 residues) was recently discovered in 2014. A pleiotropic gene screen showed that there were links between metabolic syndromes and inflammation, specifically with single nucleotide polymorphisms (SNPs) in the *HECTD4* gene [80]. Since 2014, HECTD4 has also been found to be associated with diabetes, hypertension and cardiovascular disease, lung adenocarcinoma, urothelial carcinoma and ovarian endometriosis [81–84].

Being one of the larger enzymes within the "other" HECT E3 ubiquitin ligase subfamily, it is intriguing that there have been no putative domains annotated for HECTD4 except the C-terminal HECT domain (residues 3627–3996). There are likely different domains located in the N-terminal region of the HECTD4 protein that need to be identified and functionally examined.

Genetic screening has identified various mutations in *HECTD4* in cancer cells. For example, *HECTD4* was recently identified as one of nine genes that correlated with the onset of lung adenocarcinoma [83]. Tumor genetic screens have revealed in patients with urothelial carcinoma in the bladder (UCB) that mutations in *HECTD4*, Fibrillin-3 Precursor (*FBN3*) and Citron Rho-Interacting Kinase (*CIT*) were correlated to UCB disease progression [81]. *HECTD4* may also be linked to the development of ovarian endometriosis (OEM) [82]. However, future studies are still needed to verify this relationship.

Already having various genetic links to cancers and cardiovascular disease, HECTD4 deserves more attention by the research community to further clarify its biological and functional roles in the cell. Currently very little is known about HECTD4, therefore it will be imperative to first identify potential similarities in protein sequence and/or domain architecture. To better clarify HECTD4's role in ubiquitin biology, it will also be important to discover HECTD4 substrates and annotate the sites of HECTD4-dependent ubiquitylation to answer how this mysterious HECT E3 ubiquitin ligase contributes to disease development.

#### **2.5 G2E3, a unique multifunctional HECT E3 ubiquitin ligase with RING-like features**

G2/M-phase specific E3 ubiquitin protein ligase (G2E3; 706 residues) was first identified in 2006 and named for its role during the G2/M phase of cell division and for having a conserved C-terminal HECT domain [85]. Knockout studies of G2E3 in mice demonstrated that this enzyme is essential in preventing apoptotic cell death during early embryonic development [86]. Expression levels in G2E3 were also observed to increase during early embryogenesis, specifically during central nervous system development. This enzyme is also implicated in cell cycle progression and DNA damage response [85, 87, 88].

G2E3 contains three plant homeodomain (PHD)-type zinc finger repeats, a domain typically known to bind to modified histones and act as epigenetic readers [89], making it the only known HECT E3 ubiquitin ligase to possess "RING"-like

*Ubiquitin - Proteasome Pathway*

making it a potential cardiac drug target.

**2.3 HECTD1, an important regulator in neurodevelopment**

interactions through the Notch pathway, respectively [75, 76].

proteins [57, 58, 61–65]. Expanded studies on how HACE1 binds and recognizes its substrates are needed to further clarify the role of HACE1 in cancer development. HACE1 also plays an essential role in neurodevelopment as it was recently shown to be involved in the spastic paraplegia and psychomotor retardation with or without seizures (SPPRS) phenotype [66]. HACE1 also has cardiac protection function during hemodynamic stress when it was shown in mice with *HACE1* deficiency, their susceptibility to accelerated heart failure greatly increased [67]. This suggests that HACE1 has a critical role in protecting the heart from various stresses, thus

HECT domain containing E3 ubiquitin protein ligase 1 (HECTD1; 2610 residues) was discovered in 2007 as a novel and important regulator of neurodevelopment [68]. HECTD1 has similar domain architecture to HACE1 with four ankyrin repeats near its N-terminus and a C-terminal HECT domain (**Figure 3**). HECTD1 plays an important role in pulmonary fibrosis during endothelial-mesenchymal transition (EndMT) with reports of increased circular RNA HECTD1 (circHECTD1) transcription, which causes decreased HECTD1 protein expression in lung tissue [72, 73]. Elevated circHECTD1 gene expression has also been found in patients with acute ischemic stroke (AIS) [74]. HECTD1 also contains a Smad4 activation SAD1/ UNC (SUN) domain and a mind bomb (MIB) domain, with each having unique roles in intracellular signaling due to Smad-DNA complex formation and cellular

HECTD1 supports fetal growth and proper placenta development. Specifically, HECTD1 aids in the development of the labyrinthine and junctional zones of the placenta, regions where the fetus acquires nutrients and disposes of waste, as well as a bilayer between the labyrinthine and decidual cells, respectively [77, 78]. HECTD1 ensures the proper size development of the labyrinthine, yet the mechanisms to ensure this are still not fully understood. Mutations within HECTD1 lead to the onset of irregular labyrinthine development, which in turn depletes nutrients for the fetus. Fetal fatality can occur without proper maintenance of these placental regions, suggesting

*HECTD1 domain architecture. HECTD1 contains putative protein-protein interaction domains including two armadillo-repeat containing domains (ARM1, residues 8-254; ARM2 residues 892-925 in purple; PDB 4DB8 [69]), four ankyrin-repeats (residues 395-612 in shades of pink; PDB 4O60 [56]), a SAD1/UNC domain (SUN, residues 1115-1244 in yellow; PDB 3UNP [70]), a mind bomb domain (MIB, residues 1266–1338 in red; PDB 2DK3), a helix-bundle domain (H, residues 1896–1968 in magenta; PDB 2KZS [71]) and a HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]). Domain boundaries are denoted according* 

*to UniProt and InterPro. Structures were visualized using PyMol.*

**82**

**Figure 3.**

#### **Figure 4.**

*Domain architecture of G2E3. G2E3 contains three putative plant homeodomain (PHD)-type domains (residues 78-128) and a unique HECT-like domain (371-678) as annotated on UniProt and InterPro. Representative crystal structures of a PHD domain (human PHD finger protein 13 in cyan with Zn-coordinating residues in yellow; PDB 3O70 [90]), which is suggested to interact with DNA and/or proteins in the nucleus, while the G2E3 HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]) may have lost its ability to catalyze the transfer of ubiquitin. Structures were visualized using PyMol.*

characteristics (**Figure 4**). G2E3 is primarily found within the nucleus due to its N-terminal nucleolar localization signal sequence, while the PHD domains have been suggested to cause the translocation of G2E3 to the cytoplasm [85, 86]. Previous biochemical studies showed that the ubiquitin ligase activity of G2E3 was exclusively found in two of the putative N-terminal PHD domains while the C-terminal HECT domain of G2E3 had apparently lost its ubiquitylation activity [86]. Since the PHD domains of G2E3 appear to be capable of recruiting E2 enzymes to build K48-linked polyubiquitin chains [86], this suggests that the HECT domain of G2E3 may have become vestigial through evolutionary pressure. Intriguingly, G2E3 has aspects of the RING and HECT E3 ubiquitin ligase families, analogous to members of the RING-between-RING (aka RING-BRcat-Rcat) E3 ubiquitin ligases that includes parkin and HOIL-interacting protein (HOIP) of the LUBAC complex [91, 92].

G2E3 was recently identified as a potential drug target to increase the efficacy of chemotherapy drugs, specifically with Cisplatin [88]. Since Cisplatin is the most common chemotherapy drug, much research has been dedicated to increasing Cisplatin's ability to specifically trigger the DNA damage response in cancer cells to initiate apoptosis while limiting its exposure time and prescribed duration for patients [93]. Clearly, G2E3 is an important nuclear protein whose mechanism is currently unresolved. Further studies are needed to clarify how this divergent HECT-domain containing E3 ubiquitin ligase works in the cell.

#### **2.6 TRIP12, the multifunctioning E3 ubiquitin ligase essential for embryogenesis and DNA damage repair**

Thyroid hormone receptor interactor 12 (TRIP12; 2040 residues), was first identified in 2001 for containing a unique tryptophan-tryptophan-glutamate (WWE) domain that is predicted to be involved in ubiquitylation and ADP-ribosylation [94] (**Figure 5**). It also contains two N-terminal ARM domains, similar to HECTD1, and

**85**

**3. Conclusion**

**Figure 5.**

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development*

a conserved HECT domain at its C-terminus. TRIP12 is a novel HECT E3 ubiquitin ligase that has been shown to take part in various cellular pathways and processes including embryogenesis, DNA damage response and the neddylation pathway [95–97]. It has been reported that TRIP12 preferentially builds mono- as well as K48 and K63 polyubiquitin chains to tag its substrates for degradation and for DNA

*TRIP12 domain architecture. TRIP12 contains two putative armadillo-repeat containing domains (ARM1, residues 437-713; ARM2, residues 826-938), a tryptophan-tryptophan-glutamate (WWE)-domain (residues 749-836) and a conserved HECT domain (1885-1992) as annotated on UniProt and InterPro. Representative crystal structures of an ARM domain (in purple; PDB 4DB8 [69]) and WWE domain (in orange; PDB 6MIW), both of which are suggested to be involved in protein-protein and substrate interactions, as well as a HECT domain (HECTN<sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]) required for ubiquitylation activity.* 

TRIP12 has been shown to be directly and/or indirectly involved in cancer progression. For example, TRIP12 may serve as an oncogenic drug target for patients with acute myeloid leukemia (AML) by blocking a TRIP12 alternative splicing event, specifically excising exon3 from the mature TRIP12 mRNA [98]. TRIP12 also targets pancreas transcription factor 1a (PTF1a) for proteasomal degradation, a protein essential for pancreatic cancer development [99]. TRIP12 forms a ternary complex with deubiquitylase ubiquitin-specific protease 7 (USP7) that aids in hepatocellular carcinoma (HCC) proliferation; when USP7 expression levels are heightened, TRIP12 cannot tag ARF tumor suppressor (p14ARF) for ubiquitylation [100]. Furthermore, TRIP12 is associated with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) due to its mediation of

Members of the HECT E3 ubiquitin ligase family play important roles in neurodevelopment and their malfunction may be causative in different neurological diseases and disorders (reviewed in [4]). Recent genetic screens have been looking to identify genetic markers for autism spectrum disorder (ASD) and intellectual disability (ID). Interestingly, a *de novo* mutation in *TRIP12* was found in patients with or without ASD and displaying characteristics of ID [102]. Further studies to clarify the specific mechanism(s) for how mutations in the *TRIP12* gene contribute

Although much research has and continues to be performed for E6AP and members of the NEDD4 family, greater attention on the mysterious "other"

*DOI: http://dx.doi.org/10.5772/intechopen.91770*

damage site recruitment, respectively [96].

*Structures were visualized using PyMol.*

p16-related radiation efficacy [101].

to ASD and ID phenotypes are needed.

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development DOI: http://dx.doi.org/10.5772/intechopen.91770*

#### **Figure 5.**

*Ubiquitin - Proteasome Pathway*

characteristics (**Figure 4**). G2E3 is primarily found within the nucleus due to its N-terminal nucleolar localization signal sequence, while the PHD domains have been suggested to cause the translocation of G2E3 to the cytoplasm [85, 86]. Previous biochemical studies showed that the ubiquitin ligase activity of G2E3 was exclusively found in two of the putative N-terminal PHD domains while the C-terminal HECT domain of G2E3 had apparently lost its ubiquitylation activity [86]. Since the PHD domains of G2E3 appear to be capable of recruiting E2 enzymes to build K48-linked polyubiquitin chains [86], this suggests that the HECT domain of G2E3 may have become vestigial through evolutionary pressure. Intriguingly, G2E3 has aspects of the RING and HECT E3 ubiquitin ligase families, analogous to members of the RING-between-RING (aka RING-BRcat-Rcat) E3 ubiquitin ligases that includes parkin and HOIL-interacting protein (HOIP) of the LUBAC complex [91, 92].

*have lost its ability to catalyze the transfer of ubiquitin. Structures were visualized using PyMol.*

*Domain architecture of G2E3. G2E3 contains three putative plant homeodomain (PHD)-type domains (residues 78-128) and a unique HECT-like domain (371-678) as annotated on UniProt and InterPro. Representative crystal structures of a PHD domain (human PHD finger protein 13 in cyan with* 

*Zn-coordinating residues in yellow; PDB 3O70 [90]), which is suggested to interact with DNA and/or proteins in the nucleus, while the G2E3 HECT domain (HECT<sup>N</sup><sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]) may* 

G2E3 was recently identified as a potential drug target to increase the efficacy of chemotherapy drugs, specifically with Cisplatin [88]. Since Cisplatin is the most common chemotherapy drug, much research has been dedicated to increasing Cisplatin's ability to specifically trigger the DNA damage response in cancer cells to initiate apoptosis while limiting its exposure time and prescribed duration for patients [93]. Clearly, G2E3 is an important nuclear protein whose mechanism is currently unresolved. Further studies are needed to clarify how this divergent

Thyroid hormone receptor interactor 12 (TRIP12; 2040 residues), was first identified in 2001 for containing a unique tryptophan-tryptophan-glutamate (WWE) domain that is predicted to be involved in ubiquitylation and ADP-ribosylation [94] (**Figure 5**). It also contains two N-terminal ARM domains, similar to HECTD1, and

HECT-domain containing E3 ubiquitin ligase works in the cell.

**embryogenesis and DNA damage repair**

**2.6 TRIP12, the multifunctioning E3 ubiquitin ligase essential for** 

**84**

**Figure 4.**

*TRIP12 domain architecture. TRIP12 contains two putative armadillo-repeat containing domains (ARM1, residues 437-713; ARM2, residues 826-938), a tryptophan-tryptophan-glutamate (WWE)-domain (residues 749-836) and a conserved HECT domain (1885-1992) as annotated on UniProt and InterPro. Representative crystal structures of an ARM domain (in purple; PDB 4DB8 [69]) and WWE domain (in orange; PDB 6MIW), both of which are suggested to be involved in protein-protein and substrate interactions, as well as a HECT domain (HECTN<sup>−</sup>lobe in green, HECTC<sup>−</sup>lobe in blue; PDB 6JX5 [35]) required for ubiquitylation activity. Structures were visualized using PyMol.*

a conserved HECT domain at its C-terminus. TRIP12 is a novel HECT E3 ubiquitin ligase that has been shown to take part in various cellular pathways and processes including embryogenesis, DNA damage response and the neddylation pathway [95–97]. It has been reported that TRIP12 preferentially builds mono- as well as K48 and K63 polyubiquitin chains to tag its substrates for degradation and for DNA damage site recruitment, respectively [96].

TRIP12 has been shown to be directly and/or indirectly involved in cancer progression. For example, TRIP12 may serve as an oncogenic drug target for patients with acute myeloid leukemia (AML) by blocking a TRIP12 alternative splicing event, specifically excising exon3 from the mature TRIP12 mRNA [98]. TRIP12 also targets pancreas transcription factor 1a (PTF1a) for proteasomal degradation, a protein essential for pancreatic cancer development [99]. TRIP12 forms a ternary complex with deubiquitylase ubiquitin-specific protease 7 (USP7) that aids in hepatocellular carcinoma (HCC) proliferation; when USP7 expression levels are heightened, TRIP12 cannot tag ARF tumor suppressor (p14ARF) for ubiquitylation [100]. Furthermore, TRIP12 is associated with human papillomavirus (HPV) positive head and neck squamous cell carcinoma (HNSCC) due to its mediation of p16-related radiation efficacy [101].

Members of the HECT E3 ubiquitin ligase family play important roles in neurodevelopment and their malfunction may be causative in different neurological diseases and disorders (reviewed in [4]). Recent genetic screens have been looking to identify genetic markers for autism spectrum disorder (ASD) and intellectual disability (ID). Interestingly, a *de novo* mutation in *TRIP12* was found in patients with or without ASD and displaying characteristics of ID [102]. Further studies to clarify the specific mechanism(s) for how mutations in the *TRIP12* gene contribute to ASD and ID phenotypes are needed.

#### **3. Conclusion**

Although much research has and continues to be performed for E6AP and members of the NEDD4 family, greater attention on the mysterious "other"

HECT E3 ubiquitin ligases is warranted due to their emerging involvement in various diseases and neurological disorders. Combining genetic, cell biology, biochemical, and biophysical approaches to study these unique HECT E3 ubiquitin ligases will help to decipher their specific roles and/or functions in the cell as well as potentially aid in novel therapy development to treat rare conditions caused by the dysfunction of these lesser known members of the HECT E3 ubiquitin ligase family.

### **Acknowledgements**

This work was supported by the National Institutes of Health (R15GM126432 to D.E.S.) and start-up funds from Clark University (to D.E.S.).

### **Conflict of interest**

The authors declare that there is no conflict of interest regarding the publication of this chapter.

### **Abbreviations**


**87**

**Author details**

Emma I. Kane and Donald E. Spratt\*

provided the original work is properly cited.

Clark University, Worcester, Massachusetts, United States

© 2020 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,

\*Address all correspondence to: dspratt@clarku.edu

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development*

SPPRS spastic paraplegia and psychomotor retardation with or without

NEDD4 neuronal precursor cell-expressed developmentally down-

*DOI: http://dx.doi.org/10.5772/intechopen.91770*

OEM ovarian endometriosis

p14ARF ARF tumor suppressor

seizures

OPTN optineurin

regulated 4

SMAC second mitochondrial activator of caspase

PHD plant homeodomain-type zinc finger PTF1a pancreas transcription factor 1a RAR-β retinoic acid receptor beta RING really interesting new gene TNFR2 tumor necrosis factor receptor-2 SNPs single nucleotide polymorphisms

SUN Smad4 activation SAD1/UNC domain TRIP12 thyroid hormone receptor interactor 12 UCB urothelial carcinoma in the bladder

USP7 deubiquitylase ubiquitin-specific protease 7 WWE tryptophan-tryptophan-glutamate domain

*New Discoveries on the Roles of "Other" HECT E3 Ubiquitin Ligases in Disease Development DOI: http://dx.doi.org/10.5772/intechopen.91770*


### **Author details**

*Ubiquitin - Proteasome Pathway*

**Acknowledgements**

**Conflict of interest**

of this chapter.

**Abbreviations**

AIS acute ischemic stroke AML acute myeloid leukemia

ARM armadillo-repeat domain

ASD autism spectrum disorder β2AR adrenergic receptor β2AR

circHECTD1 circular RNA HECTD1 CIT citron rho-interacting kinase EndMT endothelial-mesenchymal transition

FBN3 fibrillin-3 precursor

H helix-bundle domain

ligase 1 HCC hepatocellular carcinoma HECT hmologous to E6AP C-terminus

HOIP HOIL-interacting protein HPV human papillomavirus Hsp90 heat shock protein 90 HtrA2 HtrA serine peptidase 2 ID intellectual disability IGF immunoglobulin-like fold MIB mind bomb domain

HERC HECT and RLD domain-containing HNSCC head and neck squamous cell carcinoma

ANK ankyrin repeat

ARTS septin 4

family.

HECT E3 ubiquitin ligases is warranted due to their emerging involvement in various diseases and neurological disorders. Combining genetic, cell biology, biochemical, and biophysical approaches to study these unique HECT E3 ubiquitin ligases will help to decipher their specific roles and/or functions in the cell as well as potentially aid in novel therapy development to treat rare conditions caused by the dysfunction of these lesser known members of the HECT E3 ubiquitin ligase

This work was supported by the National Institutes of Health (R15GM126432 to

The authors declare that there is no conflict of interest regarding the publication

D.E.S.) and start-up funds from Clark University (to D.E.S.).

AREL1 apoptosis resistant E3 ubiquitin protein ligase 1

G2E3 G2/M-phase specific E3 ubiquitin protein ligase

HECTD1 HECT domain containing E3 ubiquitin protein ligase 1 HECTD4 HECT domain containing E3 ubiquitin protein ligase 4

HACE1 HECT domain and ankyrin repeat containing E3 ubiquitin protein

cIAP1/2 cellular inhibitor of apoptosis protein 1/2

**86**

Emma I. Kane and Donald E. Spratt\* Clark University, Worcester, Massachusetts, United States

\*Address all correspondence to: dspratt@clarku.edu

© 2020 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.

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**95**

**Chapter 6**

**Abstract**

drugs targeting UPS.

**1. Introduction**

(UPS)

Abnormal Ubiquitination of

*Xianquan Zhan and Miaolong Lu*

Ubiquitin-Proteasome System in

Lung Squamous Cell Carcinomas

Ubiquitination is an important post-translational modification. Abnormal ubiquitination is extensively associated with cancers. Lung squamous cell carcinoma (LUSC) is the most common pathological type of lung cancer, with unclear molecular mechanism and the poor overall prognosis of LUSC patient. To uncover the existence and potential roles of ubiquitination in LUSC, label-free quantitative ubiquitomics was performed in human LUSC vs. control tissues. In total, 627 ubiquitinated proteins (UPs) with 1209 ubiquitination sites were identified, including 1133 (93.7%) sites with quantitative information and 76 (6.3%) sites with qualitative information. KEGG pathway enrichment analysis found that UPs were significantly enriched in ubiquitin-mediated proteolysis pathway (hsa04120) and proteasome complex (hsa03050). Further analysis of 400 differentially ubiquitinated proteins (DUPs) revealed that 11 subunits of the proteasome complex were differentially ubiquitinated. These findings clearly demonstrated that ubiquitination was widely present in the ubiquitin-proteasome pathway in LUSCs. At the same time, abnormal ubiquitination might affect the function of the proteasome to promote tumorigenesis and development. This book chapter discussed the status of protein ubiquitination in the ubiquitin-proteasome system (UPS) in human LUSC tissues, which offered the scientific data to elucidate the specific molecular mechanisms of abnormal ubiquitination during canceration and the development of anti-tumor

**Keywords:** lung squamous cell carcinoma, ubiquitination, ubiquitinated protein (UP), differentially ubiquitinated protein (DUP), ubiquitin-proteasome system

Ubiquitination is one of the important protein post-translational modifications (PTMs) in human body, in which ubiquitin, a 76-amino-acid protein with a molecular weight of 8.5 KDa, is covalently attached its C-terminus to the ε-amino group of the substrate protein lysine residue through a multi-step enzymatic reaction cascade catalyzed by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) [1]. As substrate proteins commonly contain multiple lysine residues, there are a variety of ubiquitination forms such as monoubiquitination (only one ubiquitin attached to a protein), multiubiquitination

#### **Chapter 6**
