Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 800,000 new cases and 450,000 fatalities in 2020 (Sung et al., 2021). The prognosis of patients with advanced HNSCC is poor with 5-year survival rate of barely 30% and little improvement in recent years (Monnerat et al., 2002). High local recurrence and distal metastasis associated with tumor progression lead to unexpected failure of conventional treatments for HNSCC including surgery, radiotherapy, and chemotherapy. Therefore, explorations of the key regulators of HNSCC progression are imperative for improving the survival of HNSCC patients.

The up-regulation of ALG-2 interacting protein X (ALIX) has been reported to be associated with the progression of various cancers. The expression of ALIX in prostate tumor tissues was found involved in the regulation of tumor growth, thus negatively correlating with prognosis (Duijvesz et al., 2019). The level of ALIX was also elevated in advanced glioma and upper gastrointestinal cancer, thus potentially applicable for survival or prognostic forecast (Husi et al., 2015, Zhao et al., 2016). Apart from ALIX detected in tumor tissues, ALIX carried on extracellular vesicles (EVs) also exhibited great values in diagnosis of pancreatic cancer at an early stage, implying the close correlation between ALIX and the onset of tumors (Yang et al., 2021). Recent studies also pointed out that EV-carried ALIX from serum or saliva could be a potential diagnostic biomarker for oral cancer and a candidate predictor of therapeutic response (Nakamichi et al., 2021). However, the mechanisms underlying the tumor progression mediated by ALIX, especially in HNSCC, has not been fully elucidated.

ALIX is first recognized as an adaptor of the calcium-binding protein apoptosis-linked gene-2 (ALG-2), which mediates programmed cell death. It is also reported that ALIX participates in virus egress, protein sorting, cytokinesis, and membrane repair (Jimenez et al., 2014, Katoh et al., 2003, Morita et al., 2007, Strack et al., 2003). All these biological progresses are initiated with the interaction between ALIX and protein members of the endosomal sorting complex required for transport (ESCRT) family. Proteins selectively sorted by ESCRT undergo either lysosomal degradation for intracellular signal transduction termination or extracellular secretion for intercellular communication (Bissig & Gruenberg, 2014). Previously studies reported that aberrant expression of ESCRT could regulate receptor trafficking of growth factors like EGF and TGF-β, which could be important for tumorigenesis (Baldys and Raymond, 2009, Miller et al., 2018). Additionally, degradation of adhesion molecules mediated by ESCRT could also lead to a change in cell motility or migration, which contributes to tumor metastasis (Lobert et al., 2010, Tu et al., 2010). Moreover, ESCRT-depended secretion of EVs is widely confirmed in tumor progression (Bissig and Gruenberg, 2014, Wang et al., 2021). Thus, we speculated that ALIX may participate in tumor progression by regulating protein sorting and trafficking.

In the present study, we compared the expression levels of ALIX in normal mucosal, HNSCC tissues, and metastatic lymph nodes to identify the effects of ALIX in tumor progression. A series of functional proteins regulating biological process were also investigated to explore the potential mechanisms of ALIX regulating the progression of HNSCC.