Itch E3 ubiquitin-protein ligase (Itch) has been reported to be highly expressed in several tumors, including lung cancer, breast cancer, pancreatic cancer, skin cancer, and neuroblastoma, and its inhibition leads to the induction of apoptosis and the inhibition of cell proliferation [38,59,62,63,64]. Itch selectively binds and ubiquitinates p73α via the PY motif (Met 452–Ala 489) but not p53, thereby leading to a proteasome-dependent p73 degradation [43]. The use of the proteasome inhibitor MG132 or the depletion of Itch using siRNA causes the inhibition of the Itch-mediated p73 degradation and increases p73 expression levels [43]. Similarly, treating cancer cells with DNA-damaging agents, such as doxorubicin, cisplatin, and etoposide, decreases the expression of endogenous Itch in a time- and dose-dependent manner, and this effect is associated with an increase in endogenous p73 levels and enhanced apoptosis [43].

Itch and p73 expressions are controlled through several mechanisms. For example, the Itch–p73 interaction was proposed to be under the control of the transcriptional co-activator Yes-associated protein 1 (Yap1), another partner of p73 [40]. Indeed, Yap1 competes with Itch for binding to p73, thereby preventing the Itch-mediated ubiquitination and degradation of p73. Cisplatin increases p73 expression and apoptosis, while the depletion of Yap1 using siRNA blocks these effects [40]. Yap1 interacts with another transcription activator, Runx, to target the promoter of Itch and enhance its transcription, with subsequent support of the degradation of p73 [41].Nedd4-binding partner-1 (N4BP1) has been also found to compete with Itch for binding to p73α, thus reducing the ability of Itch to interact with and ubiquitylate p73α [65]. The Itch/p73 axis is also regulated in chronic lymphocytic leukemia (CLL) cells through the direct regulation of Itch by microRNA 106b (miR106b) [42]. Indeed, the induction of miR106b in CLL cells is associated with a decrease in Itch expression and an increase in the expression of p73 and PUMA, as well as induction of CLL apoptosis [42].

MDM2 is a negative regulator of p53; its interaction with the TAD of p53 leads to a ubiquitination-mediated proteasomal degradation of p53 [13,66]. MDM2 also interacts with p73α and inhibits its function, but it does so without promoting p73 degradation [55,56]. MDM2 also forms a specific complex in vitro and in vivo with p73β through at least two residues (F15 and W19) [57]. MDM2 overexpression reduces the ability of p73β to activate transcription but without promoting its degradation, indicating that MDM2 can directly induce the degradation of the p53 protein but not of its homolog p73 [57]. However, MDM2 has been shown to induce p73 degradation in HeLa cells through a mechanism involving the interaction of MDM2 with Itch [45].

Adriamycin can increase p73 expression and HeLa cell apoptosis, most likely by decreasing the expression of both MDM2 and Itch. Similarly, the MDM2-mediated p73 degradation is inhibited in cells treated with the inhibitor proteasome MG132 [45]. In the same context, MDM2 was reported to use its RING domain to bind to p73α and p73β at multiple Lys residues of ubiquitin, thereby promoting the ubiquitination of both isoforms but without inducing their degradation [44]. MDM2 overexpression induces Itch-mediated p73 degradation in MDM2-deficient mouse embryo fibroblasts, whereas its depletion using siRNA induces p73-dependent transactivation and apoptosis [44].

A recent study showed that the MDM2/p73 interaction is established by specific binding between the N-terminus TA domain of MDM2 and the SAM domain of p73 [58]. MDMX, the structural homolog of MDM2, was also shown to negatively regulate p73 [46,59,67]. MDMX physically interacts with p73α, and its overexpression specifically induces alterations in the subcellular localization of p73α but not of the p73 homologs p53 and p63 [59]. Both MDM2 and MDMX are capable of binding to the TA domain of p73 [46].

Pirh2 is a RING finger E3 ligase that induces p53 degradation by promoting its ubiquitination [68,69]. Several studies have reported that Pirh2 acts as an oncogene, and its overexpression in cancer is associated with decreased p53 expression levels [48,49,70]. Pirh2 has also been shown to interact with the DNA-binding domain of both p73α and p73β [68,71,72], and its E3 ligase activity promotes p73 ubiquitination both in vivo and in vitro [47,73]. Pirh2 ubiquitinates p73 in vitro via K63-linked chains, while it promotes p73 ubiquitination in vivo by targeting K11-, K29-, K48-, and K63-linked chains [47]. The downregulation of Pirh2 significantly decreases p73 ubiquitination, whereas its overexpression is correlated with an inhibition of p73-dependent translational activity [47]. Pirh2 knockdown in the RKO colon carcinoma cell line inhibits cell growth and increases the levels of p73 and the cell cycle inhibitor p21^WAF1 [73]. Conversely, the overexpression of Pirh2 in colon and breast cells induces the degradation of p73 via the proteasomal pathway [73]. The exposure of RKO cells to either doxorubicin or camptothecin decreases Pirh2-mediated p73 polyubiquitination [73].

Tripartite Motif Containing 32 (TRIM32) a RING protein, is overexpressed in several tumors, including skin tumors. It has E3 ubiquitin ligase proprieties and plays a role in tumor growth, metastasis, and resistance to anti-cancer drugs [74]. In this context, TRIM32 is involved in the ubiquitination of the tumor-suppressor Abl-interactor 2 (Abi2), resulting in an enhancement of cell proliferation and metastases [75]. TRIM32 also regulates the expression of p73 through the ubiquitination pathway, thereby promoting cell survival and tumor growth [50,76]. A regulatory feedback loop has been reported between TP73 and TRIM32 in human embryonic kidney 293 cells [50]. In HEK293 cells, p73 can act as a transcriptional regulator of TRIM32, and the depletion of p73 significantly decreases TRIM32 expression. TRIM32, in turn, binds to p73α and promotes its ubiquitination and degradation, thus inhibiting p73 transcriptional activity [50]. The overexpression of TRIM32 inhibits the p73 transcriptional activation of the p21^WAF1 promoter, thereby enhancing cell cycle progression and cell survival [50].

Several studies have reported the overexpression of FBXO45 in human cancer and an essential role for FBXO45 in tumorigenesis and progression [77,78,79]. FBXO45 induces the ubiquitination–proteasomal degradation of several tumor-suppressor proteins, such as Par4 (prostate apoptosis response protein 4) [80], F-box/WD repeat-containing protein 7 (FBXW7) [81], and p73 [51]. Through its spla and the ryanodine receptor (SPRY) domain, FBXO45 specifically binds to the SAM domain of p73α, but not to its homolog p53, thus leading to the proteasome-dependent degradation of p73 both in vitro and in vivo [51]. The silencing of FBXO45 in the BT-20 cells, a p53-mutated cell line, induces the expression of p73 at the protein level [51]. The exposure of FBO45-depleted BT-20 cells to doxorubicin induces apoptosis, indicating that FBXO45 specifically binds to p73 and induces apoptosis via a p73-dependent mechanism [51]. Similar to p73, FBXO45 is also able to interact with ΔNp73 to play a crucial role in its stability in apoptosis and the DNA damage response. However, mutant FBXO45 has shown an impaired function against p73 and does not cause ubiquitination-associated p73 degradation [51].

Ubiquitin factor 2A (UFD2a), also known as Ubiquitin factor E4B (UBE4B), is an E3/E4 ligase belonging to the U box-type ubiquitin protein ligase family that can catalyze the elongation of the polyubiquitin chain. In response to apoptosis, UFD2a is cleaved by caspase 6, resulting in impaired enzymatic activity and dysfunction, as well as confirming the role of UFD2a in apoptotic signaling [82]. UFD2a physically interacts with p53 in brain tumors to promote p53 polyubiquitination and degradation, thus decreasing p53-mediated apoptotic activity [83]. Similar to its effects on p53, UFD2a regulates the stability of p73 via ubiquitination-independent degradation [60]. UFD2a also physically interacts with the SAM domain of p73α [60]. The treatment of SH-SY5Y neuroblastoma cells with cisplatin results in a decrease in the expression of UFD2a, while p73 protein expression and apoptosis are increased [60]. The expression of the p73 protein is also increased in UFD2a-depleted cells [60]. Alternatively, the overexpression of UFD2a decreases both p73α expression and p73-mediated pro-apoptotic activity [60]. Similar findings have been reported in squamous cell carcinoma (SCC) cells [84]. UFD2a knockdown in SCC cells was found to result in p73 accumulation and sensitized the SCC cells to the inhibitory effects of 1,25D3 (the most active metabolite of vitamin D) on cell growth, suggesting that the UFD2a/p73 pathway contributes to the antiproliferative effects of anticancer drugs [84,85].

Hades, also referred to as mitochondrial ubiquitin ligase activator of NFKB1 (MULAN), is an E3 ubiquitin ligase. Hades is anchored to the outer membrane of mitochondria through its transmembrane domains, with a conserved C-terminal RING-finger domain with an E3 ligase activity facing the cytosol [61]. Hades interacts with p53 in the mitochondria to reduce p53 stability [86]. Hades polyubiquitinates p53 through an MDM2-independent mechanism, thereby inhibiting the p53-dependent mitochondrial cell death pathway by blocking the interaction between p53 and Bcl-2 [86]. Through its RING-finger domain, Hades also interacts with p73 in etoposide-treated H1299 cells [61]. In response to etoposide treatment, p73 is translocated to the mitochondria to colocalize with Hades [61]. Hades induces the ubiquitination of p73 and its degradation, whereas its depletion using siRNA significantly inhibits p73 degradation, indicating that Hades is involved in the ubiquitination-dependent degradation of p73 [61].

The CDL4A complex targets various proteins that are involved in several signaling pathways, including the DNA damage response and the cell cycle [87,88,89], and it contributes to the degradation of several proteins through the ubiquitin-proteasome pathway. In this context, Cul4A has been reported to interact with p53 to promote its proteasomal degradation [90]. Through damage specific DNA binding protein 1(DDB1), the CDL4A complex can directly bind to the N-terminal region of p73, which encompasses residues 1–320 and includes the DNA-binding domain, thereby inducing p73 mono-ubiquitination and inhibiting p73-dependent transcriptional activity without affecting p73 stability at the protein level [52]. The depletion of DDB1 induces the expression of several known target genes of p73, such as p21^WAF1 and PUMA [52]. Clinically, high expression levels of Cul4A in human breast carcinomas are associated with the repression of target genes of p73, indicating that Cul4A is a potent inhibitor of p73 transcriptional activity in cancer [52].

WWP2, an E3 ubiquitin ligase, has an important role in different cellular functions and ubiquitinates, and it decreases the expression of the tumor-suppressor genes PTEN [91] and p73 [53] through the proteasomal pathway. Through its WW3 domain, WWP2 specifically interacts with the oligomerization domain of both p73α and p73β but not with p53 [51]. This results in p73 ubiquitination via a K48 linkage, with the subsequent induction of p73 degradation in the proteasome [53]. Interestingly, WWP2 depletion dramatically decreases p73 ubiquitination and increases its expression at the protein level [53].

3. Conclusions