Cell cycle regulation, uncontrolled proliferation, and inhibition of apoptosis will affect the occurrence and development of tumors. USP22, which has been described as a cancer stem cell (CSC) marker ^[23][24][68,69], is a member of the largest subfamily of DUBs ^[25][70]. The USP22 gene is located on chromosome 17 and consists of 14 exons, can indirectly affect chromatin structure through histone ubiquitination (H2A and H2B), thus regulating the transcriptional activation of many genes and widely affecting biological functions ^[21][66]. USP22 has the protein characteristics of CSCs and can promote the formation of various proteins for invasive tumor growth ^[26][71]. In addition, USP22 has been shown to regulate cellular growth and proliferation. Indeed, USP22 depletion reduces transformation of c-Myc and plays an important role in cell cycle progression and anchorage-independent growth ^[27][72]. Additionally, the downregulation of USP22 results in tumor cells remaining arrested at the G1 phase via p21 stabilization ^[28][29][73,74]. Thus, USP22 plays an important role in cell cycle regulation. The depletion of USP22 in HNSCC tissue can affect the CDK inhibitor (CDKI)/Rb signal pathway. USP22 increases the expression of cyclin-dependent kinase inhibitor 1A (CDKI) including p21 and p27, and it decreases the expression of Rb protein in HNSCC cells ^[29][30][31][74,75,76]. An immunohistochemical study showed that USP22 expression is upregulated in laryngeal carcinoma and has a close relationship with malignant behaviors including invasion, metastasis, and poor prognosis ^[32][77].

Ubiquitin-specific protease 9X (USP9X) is related to the occurrence and development of pancreatic cancer, multiple myeloma, and other tumors ^[33][78]. USP9X knockdown in HNSCC cells decreases the percentage of cells in the G0/G1 phase and increases the percentage of cells in the S and G2/M phase. The proliferation of HNSCC cells can be regulated by downstream transcription factor HES-1 (Notch pathway) through USP9X level ^[20][65]. USP9X may also regulate the proliferation of HNSCC through the mammalian target of the rapamycin (mTOR) pathway ^[32][34][77,79]. However, the loss of USP9X may be harmful to the growth of tumor cells, but its loss in primary tumor cells may accelerate the development of secondary tumors ^[35][80].

USP14 has been proven to be related to the occurrence and development of many cancers. In tumor xenograft mice, the growth of HNSCC with low USP14 was slower than that of the control group. The expression of USP14 in tongue carcinoma was higher than that in adjacent tissues. USP14 depletion can inhibit the proliferation and migration of HNSCC cells in vitro ^[36][81]. Moreover, USP14 can promote the proliferation and invasion of HNSCC both in vivo and in vitro.

Ubiquitin-specific peptidase 4 (USP4) has been identified to deubiquitinate K63-linked ubiquitin conjugates from TNF receptor-associated factor 2 (TRAF2), TNF receptor associated factor 6 (TRAF6), and TGF-beta activated kinase 1 (TAK1) and stabilizes molecules by deubiquitinating K48-linked ubiquitination ^[18][63]. USP4 is significantly upregulated in tumor tissues compared to matched non-tumor tissues in HNSCC. Co-immunoprecipitation showed that USP4 interacted with receptor-interacting protein 1 (RIPI) to remove K63-connected ubiquitin molecules to stabilize RIPI expression. Thus, it negatively regulates the activation of NF-κB mediated by deubiquitination of RIPI. These findings indicate that USP4 has tumor suppressor roles in HNSCC ^[18][63].

3. Relationship between Deubiquitinating EnzymeUBs and Prognosis of Head and Neck Squamous Cell NSCCarcinoma

The prognosis of HNSCC is related to its stage of recurrence and lymph node metastasis. USP7 participates in intracellular tumor regulation, DNA repair, immune response, and epigenetic effects ^[37][82]. An immunohistochemical study showed that USP7 overexpression is correlated with histone-lysine N-methyltransferase (EZH2) in HNSCC ^[16][61]. EZH2 can promote the epithelial-mesenchymal transformation (EMT) in vivo and inhibits cellular senescence and differentiation ^[38][83]. EZH2 is widely expressed in various malignant tumors, including nasopharyngeal carcinoma ^[39][84]. USP7 expression in poorly differentiated HNSCC is higher than that in well differentiated HNSCC, and the high expression of USP7 was significantly related to a high degree of lymphatic invasion and high TNM stage. Survival analysis showed that USP7 overexpression is correlated with poor prognosis in HNSCC patients ^[16][61]. USP2a is one of the two splicing variants of USP2, which regulates the stability and function of many important cell growth and differentiation regulators and signal transduction factors in vivo ^[40][85]. In HNSCC, increased USP2a is associated with poor prognosis. The expression of ErbB2-interacting protein (Erbin) and USP2a increased in HNSCC, indicating that the expression of Erbin and USP2a was positively correlated with lymph node metastasis. Moreover, USP2a expression is related to epidermal growth factor (EGF) in HNSCC ^[41][86].

4. The Role of Deubiquitinating EnzymeUBs in Radiosensitivity of Head and Neck Squamous Cell NSCCarcinoma 

For the patients with HNSCC in the advanced stage, surgery combined with radiotherapy is needed to improve the survival rate of the patients. The radiation target and dose should be determined according to the location of the primary tumor, clinical-stage, postoperative pathological diagnosis, and postoperative imaging evaluation. The tolerance of HNSCC to radiotherapy is the main factor affecting the prognosis. BRCA1-associated protein-1 (BAP1), also known as UCHL2 can regulate various cellular processes including cell cycle, cell differentiation, transcription, DNA damage response, and resistance to tumor radiotherapy. BAP1 mutation can increase the sensitivity of HNSCC to radiotherapy and lead to tumor susceptibility syndrome, which is not related to the status of high-risk human papillomavirus (HPV) and p53 tumor suppressor protein. It was found that the survival fraction of BAP1 knockout HNSCC was significantly lower than that of HNSCC cells after radiotherapy, the sensitivity to radiotherapy was increased, and this sensitivity could be reversed by forced re-expression ^[22][67]. BAP1 can enhance the stability of histone H2A by deubiquitination and increase its expression level, thus interfering with the modification of chromatin and histone at the double strand break, reducing the rate of cell proliferation and increasing the sensitivity of radiotherapy ^[42][87]. HNSCC that is driven by HPV is more sensitive to DNA-damaging therapy than HPV-negative HNSCC. A recent study revealed that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via the p16–HUWE1–USP7–TRIP12 pathway ^[43][88].

5. The Role of Deubiquitinating EnzymeUBs in Immune Mechanisms of Head and Neck Squamous Cell NSCCarcinoma 

Cancer is considered to be an immunosuppressive disease that is characterized by dysfunction of immune cells and disturbance of cytokine excretion ^[44][89]. EBV is closely related to the occurrence of HNSCC ^[45][90]. BPLF1 is the largest protein in EBV. There is deubiquitinating activity in the first 205 amino acids of EBV proteins. Its deubiquitinating activity can cleave K63 and K48 polyubiquitin chains. These two regulatory functions play a role in preventing virus protein degradation ^[46][91]. When the BPLF1 of EBV was knocked out, When the BPLF1 of EBV was knocked out, the replication quantity and infectivity of its genome decreased. The BPLF1 protein of EBV and its conserved deubiquitinating activity regulate cellular DNA repair enzyme polymerase and recruit it to potential virus destruction and replication sites, thus enhancing the ability to produce infectious viruses. BPLF1 can increase the fault tolerance rate of EBV virus after infecting human DNA by deubiquitinating and stabilizing EBV virus polymerase Pol, so that the DNA of the virus can continue to replicate in the injured site, thus increasing the infectivity of the virus ^[47][92]. It has been found that BPLF1 inhibits NF-κB signal transduction during lysozyme infection via deubiquitinating TNF receptor-related factor 6 (TRAF6) ^[48][93]. As HNSCC is closely related to EBV infection, the existence of BPLF1 may promote the HNSCC development. USP9X upregulation can lead to programmed death receptor-ligand 1 (PD-L1) deubiquitination and PD-L1 stable expression in HNSCC tissues. PD-L1 inhibits T-cell activation and proliferation and allows cancer cells to escape T-cell immune surveillance. Preventing USP9X from locating PD-L1 may be an effective strategy for the treatment of HNSCC, especially metastatic tumors ^[19][64]. The conserved cylindromatosis (CYLD) belongs to the family of USPs, and its deletion can activate NF-κB and MAPK signaling pathways in HNSCC. NF-κB transcription factors can promote cell survival and induce innate and adaptive immunity to pathogens, such as viruses or bacterial pathogens, inflammatory cytokines, which are abnormally activated during the occurrence, and the development of cancer. The main target of CYLD is the classical NF-κB signal transduction that is mediated by NF-κB essential regulatory protein (NEMO) and cytokine reactive IκB kinase (IKK) complex subunit IKK-γ. CYLD deubiquitinates NEMO and prevents the phosphorylation of IκB and NF-κB activation ^[49][94]. The loss of CYLD in HNSCC can also increase the invasiveness of HNSCC by promoting transforming growth factor beta receptor 1 (ALK5) to stabilize NF-κB.

6. The Therapeutic Effect of Deubiquitinating EnzymeUBs on Head and Neck Squamous Cell NSCCarcinoma

As DUBs have become a clinical target for tumor treatment, many people are paying more and more attention to the application of DUBs targeting drugs in cancer therapy. As most ubiquitin enzymes are cysteinases, it is easier to develop into a candidate drug. The effects of DUB inhibitors are as follows: (1) to increase the aggregation of polyubiquitin molecules, (2) to reduce the mono-ubiquitin part, and (3) to change some molecular activities, especially the persistence of carcinogenic proteins that are mediated by DUBs ^[50][16]. The inhibition of DUBs can lead to functional impairment of the proteasome and the accumulation of misfolded proteins, resulting in tumor cytotoxicity and death ^[51][95]. Some DUBs are associated with cancer and can be used as targets for new inhibitors ^[52][53][96,97]. At present, by screening the DUB inhibitor library, it was found that compound 2X-0324 had a potential radio-sensitizing effect on laryngeal cancer (RER > 1–1.86). Vif1 and Vif2 are protease inhibitors of USP7, which can regulate the anticancer effect that is mediated by p53 and have the potential to become molecular targeted drugs for the treatment of laryngeal carcinoma ^[54][98]. Azepan-4-ones and b-AP15 are protease inhibitors of USP14 and UCHL5. They can induce the accumulation of high molecular weight polyubiquitin proteins, resulting in the enhancement of the unfolded protein response (UPR). Both can inhibit the development of HNSCC ^[55][99]. In tongue cancer, b-AP15 can significantly inhibit the growth of tumor cells and increase apoptosis in a dose-dependent manner, thereby overcoming the drug resistance of bortezomib in vitro ^[56][100]. Squamous cell carcinoma of the head and neck is the most common malignant tumor of the head and neck, and its occurrence and development are related to a variety of DUBdeubiquitination enzymes ^{[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]}[49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67]. Some DUBs are not only functional in head and neck squamous cell carcinomas but are also important in other squamous cell carcinomas. For example, CYLD is associated with HPV infection and inhibits NF-κB signaling in HNSCC, further promoting the growth and metastasis of head and neck cancer. The same functions of CYLD and HPV occur in cervical cancer cells ^[57][101]. In addition, USP46 is also associated with HPV infection in esophageal squamous cell carcinoma, but it has not been reported in HNSCC ^[58][102]. The Hippo/YAP signaling pathway plays an important role in HNSCC ^[59][103]. USP36 promotes proliferation and invasion of esophageal squamous cell carcinoma by deubiquitination of YAP ^[60][104]. USP36 may also play an important role in head and neck cancer.