Autophagy is an essential catabolic process that orchestrates cellular homeostasis and plays dual roles in tumor promotion and suppression. However, the mechanism by which autophagy affects the self-renewal of cancer stem cells (CSCs) remains unclear. In this study, we investigated whether autophagy activation contributes to CSC properties of head and neck squamous cell carcinoma (HNSCC). The results showed that the autophagy level and CSC properties of HNSCC cells were elevated in response to several adverse conditions, including treatment with cisplatin, starvation, and hypoxia. Pretreatment with autophagy inhibitors, such as 3-MA and chloroquine, diminished the CSC properties acquired under adverse conditions. In addition, the isolated CSCs were endowed with stronger autophagic activity than non-CSCs, and the CSC properties were dampened when autophagy was inhibited either by 3-MA, chloroquine, or Beclin1 knockdown. Notably, the tumor-initiating activity of CSCs was decreased upon knocking down Beclin1. Further study revealed that FOXO3, a substrate for autophagy, was enriched in the nucleus of cells with lower autophagy levels. Nuclear FOXO3 directly bound to the promoter region of SOX2 and negatively regulated its transcriptional activity. Overexpression of FOXO3 decreased the expression of SOX2 and thereby impaired the CSC phenotype both in vitro and in vivo. Taken together, our findings suggest that the activation of autophagy is essential for the acquisition of CSC properties in adverse conditions and the self-renewal of CSCs. We clarify the role of autophagy in regulating the CSC phenotype and demonstrate that the noncanonical FOXO3/SOX2 axis is the intrinsic regulatory mechanism.

Background Epithelial-mesenchymal transition (EMT) and cell stemness are implicated in the initiation and progression of oral squamous cell carcinoma (OSCC). Revealing the intrinsic regulatory mechanism may provide effective therapeutic targets for OSCC. Results In this study, we found that Forkhead box D1 (FOXD1) was upregulated in OSCC compared with normal samples. Patients with a higher FOXD1 expression had a poorer overall survival and disease-free survival. Immunohistochemical staining results showed that FOXD1 expression was related to the clinical stage and relapse status of OSCC patients. When FOXD1 expression was knocked down in CAL27 and SCC25 cells, the migration, invasion, colony formation, sphere formation, and proliferation abilities decreased. Moreover, EMT and stemness-related markers changed remarkably, which indicated that the EMT process and cell stemness were inhibited. Conversely, overexpression of FOXD1 promoted EMT and cell stemness. Further study demonstrated that FOXD1 could bind to the promoter region and activate the transcription of SNAI2. In turn, the elevated SNAI2 affected EMT and cell stemness. An in vivo study showed that FOXD1-overexpressing CAL27 cells possessed a stronger tumorigenic ability. Conclusions Our findings revealed a novel mechanism in regulating EMT and cell stemness and proposed FOXD1 as a potential marker for the diagnosis and treatment of OSCC.

Hypoxia is a main feature of most solid tumors, but how melanoma cells under hypoxic conditions exploit tumor microenvironment (TME) to facilitate tumor progression remains poorly understood. In this study, we found that hypoxic melanoma-derived small extracellular vesicles (sEVs) could improve the proangiogenic capability of cancer-associated fibroblasts (CAFs). This improvement was due to the activation of the IKK/IκB/NF-κB signaling pathway and upregulation of CXCL1 expression and secretion in CAFs. By proteomic analysis, we verified that hypoxia could promote enrichment of chaperone HSP90 and client protein phosphorylated IKKα/β (p-IKKα/β) in melanoma-derived sEVs. Delivery of the HSP90/p-IKKα/β complex by sEVs could activate the IKK/IκB/NF-κB/CXCL1 axis in CAFs and promote angiogenesis in vitro and in vivo. Taken together, these findings deepen the understanding of hypoxic response in melanoma progression and provide potential targets for melanoma treatment.

Tumor volume increases continuously in the advanced stage, and aside from the self-renewal of tumor cells, whether the oncogenic transformation of surrounding normal cells is involved in this process is currently unclear. Here, we show that oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) promote the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of normal epithelial cells but delay their apoptosis. In addition, nuclear-cytoplasmic invaginations and multiple nucleoli are observed in sEV-treated normal cells, both of which are typical characteristics of premalignant lesions of OSCC. Mechanistically, miR-let-7c in OSCC-derived sEVs is transferred to normal epithelial cells, leading to the transcriptional inhibition of p53 and inactivation of the p53/PTEN pathway. In summary, we demonstrate that OSCC-derived sEVs promote the precancerous transformation of normal epithelial cells, in which the miR-let-7c/p53/PTEN pathway plays an important role. Our findings reveal that cancer cells can corrupt normal epithelial cells through sEVs, which provides new insight into the progression of OSCC.

BackgroundEpithelial-mesenchymal transition (EMT) and cell stemness are implicated in the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Revealing the intrinsic regulatory mechanism may provide effective therapeutic targets for HNSCC.ResultsIn this study, we found Forkhead box D1 (FOXD1) was upregulated in HNSCC when compared with normal samples. Patients with higher FOXD1 expression had poorer overall survival and disease-free survival. Immunohistochemistry results showed that FOXD1 expression was related to the clinical stage and relapse status of HNSCC patients. When knockdown the expression of FOXD1 in CAL27 and SCC25 cells, the migration, invasion, colony formation, sphere formation, and proliferation abilities decreased. Moreover, the EMT and stemness-related markers changed remarkably, which indicated the EMT process and cell stemness were inhibited. Conversely, overexpression of FOXD1 promoted EMT and cell stemness. Further study demonstrated that FOXD1 could bind to the promoter region and activate the transcription of SNAI2. The elevated SNAI2, in turn, affected the EMT and cell stemness. The in vivo study showed FOXD1 overexpressed CAL27 cells possessed stronger tumorigenic ability.ConclusionsOur findings revealed a novel mechanism in regulating EMT and cell stemness, and proposed FOXD1 as a potential marker for diagnosis and treatment of HNSCC.