The role of deubiquitinating enzymes and their inhibitors in esophageal carcinoma (Review).

Esophageal cancer (EC) ranks as the 11th most frequently diagnosed malignancy and the 7th principal cause of cancer-related mortality worldwide, with ~511,000 new cases and 445,000 fatalities recorded in 2022 ( 1 ). Furthermore, esophageal squamous cell carcinoma (ESCC) is the predominant pathological type of EC, accounting for >90% of cases ( 2 ). It has been characterized by accelerated tumor proliferation and elevated rates of metastasis and recurrence, resulting in an unfavorable prognosis for patients. The advancement of therapeutic techniques, including minimally invasive surgery, targeted therapy and immunotherapy, has improved the 5-year survival rate of patients with ESCC; however, it remains unsatisfactory ( 3 , 4 ). For certain patients with EC, the lesions' location or the disease's advanced stage may make surgery inappropriate, posing specific therapeutic challenges. Chemotherapy and targeted therapies can partially manage EC; however, certain individuals may exhibit drug resistance, resulting in diminished therapeutic effectiveness. For patients with advanced EC, current therapies demonstrate limited effectiveness, requiring the exploration of new potent therapeutic options. Therefore, comprehensive studies on EC occurrence and pathophysiology, as well as the identification of prognostic biomarkers, may yield more accurate recommendations for personalized treatment, enhancing patient prognosis, therapeutic efficacy and quality of life. Protein ubiquitination is a dynamic and reversible post-translational modification (PTM) involving the covalent attachment of one or more ubiquitin (Ub) proteins, each comprising 76 amino acids, to a substrate protein. This alteration affects various cellular proteins and is involved in various cellular processes ( 5 ). In humans, four distinct genes encode Ub, of which Ub A-52 residue ribosomal protein fusion product 1 (UBA52) and ribosomal protein S27a (UBA80) encode a solitary Ub fused at their C-termini to ribosomal proteins L40 and S27a, respectively. The Ub B and Ub C genes are polyubiquitin precursors that occur as tandem repeats ( 6 ). Ubiquitination is a cascade reaction comprising three enzymes: Ub-activating enzymes (E1s), Ub-conjugating enzymes (E2s) and Ub ligases (E3s) ( 7 ). Furthermore, Ub is activated by E1 in an ATP-dependent manner; it establishes a thioester bond between the active site cysteine of E1 and the C-terminal carboxyl group of Ub. Then, Ub is transferred to E2 through a transthiolation process, covalently bound to the amino group of a lysine residue on the substrate protein by an E3 Ub ligase ( 8 ). The following four E3 subtypes have been identified: Homologous to E6-associated protein C-terminus (HECT) type, Really Interesting New Gene (RING) type, U-box type and RING-in-between-RING (RBR) type. RING-type and U-box type E3 ligases directly promote the transfer of Ub from E2 to the substrate protein. HECT-type and RBR-type E3 ligases establish a thioester bond between the cysteine in their active site and Ub before transferring it to the substrate protein ( 9 , 10 ). Deubiquitinating enzymes (DUBs) are isopeptidases that can cleave either a single Ub or an entire Ub chain from a target protein, thus opposing protein ubiquitination, a crucial PTM that modulates protein stability, activity, subcellular localization and interactions ( 11 , 12 ). DUBs not only reverse ubiquitination but also govern various physiological pathways, such as protein trafficking, chromatin remodeling and cell cycle regulation. Therefore, they are implicated in various clinical disorders ( 13 ). Therefore, DUBs have become a research hotspot as therapeutic targets, prompting the establishment of DUB inhibitors, some of which are currently in preclinical development or clinical trials ( 14 ). To date, ~100 DUBs have been identified in and are categorized into 9 families: Ub-specific proteases (USPs), ovarian tumor proteases (OTUs), Ub C-terminal hydrolases (UCHs), Machado-Joseph disease protein domain proteases (MJDs, also referred to as Josephins), JAMM/MPN domain-associated zinc-dependent metalloproteases (JAMMs, also known as MPN+), motif interacting with Ub-containing novel DUB family, monocyte chemotactic protein-induced proteins, permuted papain fold peptidases of double-stranded RNA viruses and eukaryotes and zinc finger-containing Ub peptidase 1 ( 15 ), with a primary focus on the seven major families that include USPs ( Fig. 1 ). Several studies suggest that DUB dysfunction is markedly associated with the onset and progression of EC ( 16 - 18 ). They may function as oncogenes, enhancing critical proteins associated with proliferation, metastasis and drug resistance [e.g., β-catenin, Snail, Yes-associated protein (YAP)1, programmed death-ligand 1 (PD-L1)], or they may serve as tumor suppressor genes [e.g., BRCA1-associated protein 1 (BAP1), cylindromatosis, lysine 63 deubiquitinase (CYLD)], wherein their deletion or mutation may result in the inhibition of tumor-suppressive functions ( 16 , 17 , 19 - 62 ) ( Table I ). This review aims to systematically elucidate the complex regulatory network of DUBs in EC. This study will go beyond single-molecule descriptions to classify and analyze DUBs based on signaling pathways (TGF-β, Wnt, NF-κB, Hippo) and summarize essential biological processes [epithelial-mesenchymal transition (EMT), epigenetics, immune regulation], while assessing their viability as therapeutic targets by integrating preclinical quantitative data.

Despite considerable advancements in the study of DUBs in EC, bridging a gap from laboratory findings to clinical application necessitates addressing the following critical limitations: The major portion of existing data is based on immortalized EC cell lines, such as the KYSE series ( 86 ). There is insufficient validation of DUB inhibitors in patient-derived xenograft (PDX) models or patient-derived organoids. Since these models effectively maintain the heterogeneity and microenvironmental traits of the underlying tumor, the insufficient data on these models limit the predictive accuracy of the clinical efficacy of medicines ( 87 ). The data on how DUBs modulate their substrates are insufficient, and even less on how DUBs themselves are regulated by PTM (such as phosphorylation, acetylation, oxidation, ubiquitination) ( 18 , 88 , 89 ). For example, EC cells are often under oxidative stress; however, the effects of reactive oxygen species on active cysteine residues, thus altering DUBs activity, have not been comprehensively investigated ( 90 , 91 ). Certain DUBs may exhibit non-catalytic roles, such as serving as scaffold proteins in complex assembly ( 18 , 92 ). Current inhibitors predominantly focus on the catalytic active site and may fail to inhibit non-enzymatic processes, leading to ineffective therapy ( 93 ). In addition to fundamental expression level correlation analyses, prospective research on DUB mutations, splice variants or certain PTM states as predictive indicators of treatment efficacy is also limited ( 94 - 98 ).

This study followed a Narrative Review methodological framework to conduct extensive and comprehensive qualitative and quantitative analysis of existing literature and fill the gaps in the systematic understanding of DUBs in EC. The literature search was primarily based on the following key databases to ensure data comprehensiveness and rapid availability: PubMed/MEDLINE ( https://pubmed.ncbi.nlm.nih.gov/ ), Web of Science ( https://www.webofscience.com/ ), Embase ( https://www.embase.com/ ) and Google Scholar ( https://scholar.google.com/ ). Furthermore, ClinicalTrials.gov was referenced to acquire data on the ongoing clinical trials. In addition, bioinformatics analysis data were obtained from The Cancer Genome Atlas (TCGA) ( https://www.cancer.gov/ccg/ ) and Gene Expression Omnibus (GEO) ( https://www.ncbi.nlm.nih.gov/geo/ ) databases to supplement transcriptome-level evidence. i) Search timeline: Studies from 1st January 2000 to February 2025, specifically high-quality studies published in the last 5 years (2020-2025), were analyzed, reflecting the latest advances in the field. ii) Search keywords: Boolean logic combinations were employed, mainly including: iii) Disease-related: 'Esophageal cancer', 'esophageal squamous cell carcinoma' and 'esophageal adenocarcinoma'. iv) Target-related: 'Deubiquitinating enzymes', 'DUBs', 'ubiquitin-specific proteases' (USPs), 'UCH', 'OTU', 'BAP1', proteasome 26S subunit, non-ATPase 14 ('PSMD14') and specific family/member names. v) Mechanism and function: 'Signaling pathway', 'EMT', 'drug resistance', 'metastasis', 'prognosis' and 'immunotherapy'. The inclusion criteria were as follows: i) Studies with research subjects specifically identified as having EC [including ESCC and esophageal adenocarcinoma] cell lines, animal models or clinical tissue specimens; ii) original studies and reviews addressing differential expression, molecular mechanisms, prognostic significance or small-molecule inhibitors of DUBs; iii) studies indicating key quantitative data (e.g., IC 50 values, hazard ratios, P-values, tumor volume inhibition rates) were prioritized to meet the report's requirements for quantitative details; and iv) studies on DUB mechanisms in endometrial cancer were selected for lateral comparative analysis. The following exclusion criteria were applied: i) Studies only focusing on E3 Ub ligases and lacking DUB data; ii) bioinformatics prediction articles lacking biological experimental validation (unless independently validated in clinical cohorts); iii) Non-English or non-Chinese studies; and iv) conference abstracts with incomplete text or data.

In summary, DUBs are the key regulators of the onset, malignant progression, treatment resistance and immune evasion of EC. DUBs intricately regulate signaling pathways such as TGF-β, Wnt and NF-κB, as well as the stability of crucial oncoproteins including Snail, YAP1 and PD-L1, forming a complex oncogenic network. USP7, USP14 and PSMD14 demonstrate significant potential as therapeutic targets due to their critical roles in numerous carcinogenic pathways and the availability of previous inhibitor data. Comparative analyses with endometrial cancer further validated the role of certain DUBs (such as USP14) as general prognostic markers for poorer prognosis, while also highlighting their functional specificity in distinct tissue environments. Current DUB inhibitors (e.g., P5091, Thiolutin and Degrasyn) have promising anti-tumor efficacy and sensitizing effects to chemotherapy and irradiation in preclinical models, specifically Thiolutin, which achieved an in vivo tumor inhibition rate exceeding 50%, and Degrasyn's ability to reverse radiation resistance ( 17 , 22 , 60 , 78 , 83 ). However, translating these insights into clinical advantages requires addressing the selectivity challenges of inhibitors and performing thorough validation in PDX or organoid models that more accurately replicate clinical conditions ( 94 ). Future research must concentrate on developing highly selective allosteric inhibitors, utilizing innovative technologies such as proteolysis targeting chimera/deubiquitinase-targeting chimera to degrade or target DUBs ( 99 , 100 ), and investigating targeted immune combination therapy strategies informed by DUB expression profiles, to overcome the persistent challenge posed by EC ( 19 ). Although organoids offer benefits in primary in vitro efficacy assessment, PDX models are essential for investigating the effects of DUBs on tumor angiogenesis, distant metastasis and systemic pharmacokinetics ( 101 ). By directly implanting patients' tumor tissue into immunodeficient mice (such as NOD-Prkdcem26Il2rgem26/Gpt or NOD scid gamma), PDX provides the same stromal architecture and intercellular connections ( 102 ). The oncogenic roles of Josephin domain containing 2 (JOSD)2 and PSMD14 have been thoroughly confirmed in PDX models within ESCC research. For instance, JOSD2 knockdown can significantly limit volumetric expansion in PDX tumors and enhance tumor susceptibility to chemotherapeutic agents ( 61 ). Furthermore, PDX models serve as the gold standard for evaluating in vivo target engagement of allosteric inhibitors that target the non-catalytic functions of DUBs. Utilizing activity-based protein profiling in conjunction with mass spectrometry, researchers can quantitatively assess the extent to which an inhibitor restricts the specific in vivo activity of a DUB ( 103 ). The development of humanized PDX models has emerged as a leading approach to more accurately replicate the immune microenvironment. Researchers can ascertain whether DUB inhibitors, such as USP14 inhibitors, alter tumor development dynamics by altering CAFs or influencing macrophage polarization by transplanting human hematopoietic stem cells or peripheral blood mononuclear cells to reconstruct the murine immune system ( 95 , 98 ). Furthermore, to ensure accurate clinical use of DUB inhibitors, it is essential to develop a standardized protocol comprising antibody validation, scoring systems, clinical threshold determination and multi-indicator joint detection ( 104 ). Companion diagnostics can identify the patient populations most likely to benefit from specific DUB inhibitors. For instance, the Food and Drug Administration's Combined Positive Score standard for PD-L1 expression has become the criterion for the administration of pembrolizumab in ESCC ( 105 , 106 ). Future research for developing DUB inhibitors should establish a 'DUBome panel' that comprehensively categorizes patients by including the mRNA or protein expression profiles of 10-20 essential DUBs ( 107 , 108 ). Accurate prognostic prediction models can be developed by integrating IHC data with gene microarray or next-generation sequencing results, and employing Artificial Intelligence methods for dimensionality reduction ( 109 - 111 ). This multimodal integrated analysis can reveal patients with moderate single-target expression but significantly elevated pathway activity, thus broadening the pool that may benefit ( 112 - 114 ).