Tongue squamous cell carcinoma (TSCC) is a prevalent and aggressive malignancy of the head and neck region, associated with high morbidity and mortality rates, with an estimated global incidence of 151,000 new cases and 48,000 deaths annually as of 2023, yielding an age-standardized incidence rate of 1.7 per 100,000 and mortality of 0.54 per 100,000 (1). The pathogenesis of TSCC is multifactorial, driven by a complex interplay of genetic mutations, cumulative genetic alterations, signaling aberrations, epigenetic modifications, environmental exposures (such as tobacco use, alcohol consumption and human papillomavirus infection) and lifestyle-related risk factors (2).

In recent years, the concept of cancer stem cells (CSCs) has gained considerable attention as a key driver of tumor initiation, progression and therapeutic resistance. CSCs are a subpopulation of tumor cells with the capacity for self-renewal and differentiation into diverse cell types, thereby sustaining tumor growth and heterogeneity (3). TSCC CSCs exploit mitochondrial dynamics to reprogram lipid metabolism, promoting chemoresistance and survival under metabolic stress, while HPV+ TSCC subtypes exhibit distinct CSC properties that may be targeted for relapse-free therapy (4). These cells are sustained by specific molecular signaling pathways that are highly relevant in TSCC (5).

The present review aimed to elucidate the characteristics of CSCs in TSCC and the molecular signaling mechanisms that govern their function, with the ultimate aim of providing novel insights for future therapeutic strategies. A deeper understanding of the unique biology of CSCs in TSCC may facilitate the identification of new therapeutic targets and support the development of more effective treatments for this challenging disease.

CSCs are a subpopulation of tumor cells that possess the capacities for self-renewal and differentiation, which are essential for tumor initiation, maintenance and recurrence. They are often characterized by their ability to initiate tumor formation when transplanted into immunocompromised mice, a technique known as the xenograft assay (6,7). Additionally, CSCs can be identified by the expression of specific surface markers, such as CD44, CD24 and aldehyde dehydrogenase 1 (ALDH1), across various types of cancer, including TSCC (8). CD133 is also a recognized marker of CSCs across multiple malignancies and is often co-expressed with other stem cell markers (9); in ER-positive/HER2-negative breast cancer, high expression of CD133 is associated with a better chemotherapy response and survival rate (10).

In TSCC, CD133⁺ cells exhibit stem cell-like properties and enhanced tumorigenic potential (11). Furthermore, a high density of α smooth muscle actin (SMA)⁺ cancer-associated fibroblasts has been related to disease recurrence and poor survival, whereas the CD133⁺αSMA⁺ phenotype appears to be mainly associated with vascular structures (12,13). Furthermore, the CD44⁺CD133⁺ cell subpopulation is regarded as a potential metastatic precursor, demonstrating increased proliferation, clonogenicity, invasion and migratory capacity (14).

Other approaches for identifying CSCs include functional assays, such as the sphere-forming assay, which evaluates the ability of cells to generate three-dimensional aggregates under non-adherent conditions, a feature indicative of stem-like properties (15). DNA methylation profiling has also been used to distinguish CSCs from non-CSCs. For example, A study has shown that, compared with non-CSCs, the promoter regions of specific tumor suppressor genes in CSCs are often hypermethylated, enabling clear differentiation between these cell populations. Furthermore, genetic and epigenetic profiling can be employed to distinguish CSCs from non-CSCs, providing deeper insight into the molecular mechanisms underlying their distinct characteristics (16).

The Wnt/β-catenin signaling pathway is a key regulator of cell proliferation, differentiation and apoptosis (43). In the absence of Wnt ligands, β-catenin is phosphorylated by a destruction complex composed of proteins such as axis inhibition protein, adenomatous polyposis coli (APC) and glycogen synthase kinase-3b, which targets it for proteasomal degradation (44). However, when Wnt ligands bind to Frizzled receptors and low-density lipoprotein-related proteins 5 and 6 co-receptors, this phosphorylation is inhibited, allowing β-catenin to accumulate in the cytoplasm and subsequently translocate into the nucleus (45). In the nucleus, β-catenin interacts with T cell factor (TCF)/lymphoid enhancer factor family (LEF) transcription factors to activate the expression of target genes involved in cell proliferation and survival (Fig. 1) (46,47). In healthy cells, this destruction complex tightly regulates cytoplasmic β-catenin levels. Aberrant activation of the pathway can occur through mutations in CTNNB1 (encoding β-catenin) or APC, epigenetic silencing of pathway inhibitors such as secreted frizzled-related proteins and Dickkopf proteins, or autocrine/paracrine upregulation of Wnt ligands (48). This hyperactivation leads to β-catenin accumulation and its translocation into the nucleus. Within the nucleus, β-catenin forms complexes with TCF/LEF transcription factors, driving the expression of oncogenic target genes (49). These genes regulate key cancer hallmarks in TSCC, including uncontrolled proliferation (via c-Myc and Cyclin D1), evasion of apoptosis, EMT (which in turn facilitates invasion and metastasis), maintenance of CSCs and angiogenesis (50). The Wnt/β-catenin signaling pathway molecular mechanism is highly conserved across species and serves a critical role in both normal developmental processes and disease pathogenesis, including TSCC (51).

The Wnt/β-catenin signaling pathway has been implicated in the maintenance and expansion of CSCs in TSCC (52). Accumulating evidence suggests that activation of this pathway promotes the expression of stemness-associated genes, such as SOX2 and OCT4, which are essential for maintaining CSC properties (53,54). Therefore, targeting the Wnt/β-catenin signaling pathway in TSCC may represent a promising strategy for eliminating CSCs and improving patient outcomes (55).

The Hh signaling pathway is a key regulator of cell growth and differentiation during embryonic development and in adult tissues (56). The pathway is initiated when Hh ligands bind to the Patched (Ptch) receptor, relieving its inhibition of the Smoothened (Smo) protein (57). Activation of Smo promotes the translocation of glioma-associated oncogene homolog (GLI) transcription factors from the cytoplasm to the nucleus, where they drive the transcription of Hh target genes (58,59). The pathway is tightly regulated by several proteins, including Suppressor of Fused, which negatively regulates Gli activity, and Costal-2 (Cos2), which helps stabilize GLI proteins in the cytoplasm (Fig. 1) (60,61). Dysregulation of Hh signaling has been implicated in various types of cancer, including TSCC, where it contributes to tumor progression and maintenance (62).

In TSCC, the Hh signaling pathway is frequently aberrantly activated, contributing to disease pathogenesis and progression (63). This activation may occur through multiple mechanisms, including upregulation of Hh ligands, mutations in the Ptch receptor or alterations in other regulatory components of the pathway (64). For example, mutations in the Ptch gene can result in constitutive pathway activation even in the absence of ligand binding (65,66). In addition, the expression levels of Hh target genes, such as GLI1 and GLI2, are often upregulated in TSCC, indicating sustained pathway activity (67,68). This persistent activation promotes cell proliferation, inhibits apoptosis and enhances cancer cell survival, thereby contributing to the aggressive behavior of TSCC (69).

The Hh signaling pathway has been shown to serve a critical role in the maintenance and expansion of CSCs in TSCC (70), by upregulating stem cell markers and genes involved in cell cycle regulation (71). For example, the transcription factor GLI1, a key mediator of Hh signaling, enhances the expression of Nanog and OCT4, which are essential for maintaining CSC stemness (72,73). Furthermore, Hh signaling also increases the expression of ABC transporters, which contributes to CSC drug resistance (74). Thus, targeting the Hh pathway may represent a promising therapeutic strategy for eliminating CSCs and improving the prognosis of patients with TSCC (75).

Notch signaling mediates short-range cell-to-cell communication through interactions between ligands and receptors on adjacent cells. When a Notch ligand binds to its receptor, it triggers cleavage at the S2 site by ADAM10 and ADAM17, resulting in shedding of the extracellular domain (76). This is followed by γ-secretase-mediated cleavage at the S3 site within the transmembrane region. After S3 cleavage, the Notch intracellular domain (NICD) is released from the plasma membrane and translocates to the nucleus (77). In the nucleus, NICD interacts with recombination signal binding protein for immunoglobulin κ J region, also known as CBF1/Suppressor of Hairless/Lag-1 (CSL), converting the transcriptional repressor complex into a transcriptional activator complex and thereby promoting the expression of Notch target genes (78).

The Notch signaling pathway is a highly conserved intercellular communication system that plays a critical role in cell fate determination, differentiation, and proliferation (79). It involves a family of transmembrane receptors (Notch1-4) that are activated upon binding to ligands such as Jagged and Delta-like proteins (80). Following ligand engagement, the Notch receptor undergoes two successive proteolytic cleavages, resulting in the release of the NICD, which translocates to the nucleus (81). In the nucleus, NICD interacts with the DNA-binding protein CSL, displacing co-repressors and recruiting co-activators to initiate transcription of target genes (82). This tightly regulated mechanism ensures precise control of multiple cellular processes, making Notch signaling essential for tissue homeostasis and developmental regulation (83).

In TSCC, Notch signaling is frequently upregulated and serves a significant role in tumor progression and maintenance (84). Activation of the pathway is often driven by upregulation of ligands such as Jagged1 and delta-like ligand 1, which promote tumor cell proliferation and survival (Fig. 1). In addition, mutations or amplifications in Notch receptors can lead to constitutive pathway activation, further contributing to oncogenic transformation (85). In HNSCC (including TSCC), inactivation mutations of NOTCH1 are more common, but amplification (such as an increase in copy number) of NOTCH3 can lead to excessive activation of the pathway, promoting tumor invasion (86).

The regulatory mechanisms of Notch signaling in TSCC are complex and involve both positive and negative feedback loops, as well as crosstalk with other pathways. The Wnt signal upregulates the expression of the Jagged1 ligand, further activating Notch, and Akt directly phosphorylates Notch1, enhancing its transcriptional activity (87). Notch can activate NF-κB, maintaining the inflammatory microenvironment by upregulating IL-6 and IL-8, enhancing the invasiveness of tumors; furthermore, the intracellular segment of Notch1 (NICD) can directly bind to YAP/TAZ, inhibiting the tumor suppressive effect of the Hippo pathway and promoting the characteristics of tumor stem cells (88,89). These interactions create a dynamic regulatory network that can either enhance or suppress Notch activity, depending on the tumor microenvironment and specific genetic context (84).

Notch signaling has been implicated in the maintenance and expansion of CSCs in TSCC (90). It promotes CSC properties by activating transcriptional programs that enhance stemness, including the expression of OCT4, SOX2 and NANOG (91). In addition, Notch signaling can suppress differentiation pathways, thereby preserving the undifferentiated state of CSCs (17). The role of Notch in CSCs extends beyond transcriptional regulation; it also influences cell cycle progression, apoptosis and EMT, all of which are critical for CSC survival and metastatic potential (92,93). Therefore, targeting Notch signaling represents a potential therapeutic strategy to deplete CSCs and potentially improve outcomes in TSCC (54).

CSCs dynamically interact with key signaling pathways to drive tumorigenesis and therapeutic resistance. In TSCC, activation of the Wnt/β-catenin pathway stabilizes nuclear β-catenin in CD44⁺ CSCs, leading to the upregulation of c-MYC and OCT4 and sustaining self-renewal and chemoresistance (94). Notch signaling, through NICD cleavage, induces EMT by promoting HES1 expression, thereby facilitating metastasis (95). Hh pathway activation enhances CSC proliferation and contributes to post-therapy recurrence (96). Concurrently, PI3K/Akt/mTOR pathway alterations reprogram CSC metabolism under hypoxic conditions, while NF-κB activation driven by chronic inflammation suppresses apoptotic signaling. These pathways engage in extensive crosstalk; for example, tumor microenvironment-derived TGF-β amplifies Wnt and Notch signaling, thereby locking CSCs into a drug-tolerant state (97). Targeting these signaling hubs can disrupt CSC-driven progression, as demonstrated in TSCC xenograft models where Wnt inhibition reduced tumor-initiating capacity by ~70% (98). Collectively, these findings underscore the therapeutic potential of pathway-specific targeting for CSC depletion in TSCC.

The interaction between multiple signaling pathways is a complex yet critical aspect of cellular regulation, particularly in TSCC (99). These pathways often converge and crosstalk to regulate key cellular processes such as proliferation, survival and differentiation (100). For example, the PI3K/Akt pathway, which is frequently activated in cancer, can interact with the MAPK/ERK pathway to enhance cell survival and proliferation (100,101). This interaction may occur through shared downstream effectors or crosstalk at the level of upstream receptors (102). In addition, the NF-κB pathway, which is well known for its role in inflammation and stress responses, integrates signals from multiple pathways, including those activated by growth factors and cytokines, to regulate gene expression and cellular fate (103). Understanding these mechanisms is essential for the development of targeted therapies aimed at disrupting these interactions and inhibiting cancer progression.

The synergistic effects of multiple signaling pathways in TSCC are profound and multifaceted. When concurrently activated, these pathways can exert a stronger influence on cancer cell behavior than when activated individually (101). For example, co-activation of the PI3K/Akt and MAPK/ERK pathways enhances cell proliferation and resistance to apoptosis, contributing to the aggressive nature of TSCC (104,105); interactions between these pathways can also promote EMT, a process closely associated with metastasis (106). Additionally, EMT is driven by the coordinated activity of pathways such as TGF-β and Wnt/β-catenin, which may be further amplified through crosstalk with additional signaling networks (Fig. 2) (107,108). In TSCC and HNSCC, the co-activation of the PI3K/AKT and MAPK/ERK pathways significantly enhances tumor proliferation, anti-apoptosis and the EMT process (109). This synergistic activation not only drives malignant transformation but also contributes to therapeutic resistance, making TSCC particularly challenging to treat (110).

The interactions between multiple signaling pathways also have a significant impact on CSCs, which are considered key drivers of tumor initiation, progression and recurrence in TSCC (3). CSCs exhibit a distinct signaling landscape compared with non-stem cancer cells (17). For example, the Notch and Wnt/β-catenin pathways are frequently upregulated in CSCs and serve critical roles in maintaining stemness and self-renewal capacity (93). These pathways interact with additional signaling networks, such as PI3K/Akt and MAPK/ERK, to establish a robust signaling environment that supports CSC survival and proliferation (111). Disrupting these interactions could potentially reduce the CSC population and thereby inhibit tumor growth and metastasis (112). Because CSCs drive tumor initiation, growth, therapy resistance, recurrence and metastasis, reducing their abundance may suppress primary tumor progression and the formation of secondary metastatic lesions (113,114), thus targeting a fundamental source of malignancy and relapse. Therefore, utilizing the crosstalk between these signaling pathways to develop therapeutic targets against TSCC may represent a promising strategy in the future (115).

CSCs in TSCC exhibit marked resistance to conventional chemotherapy and radiotherapy, thus posing significant challenges to effective treatment, while also offering opportunities for the development of novel treatment strategies (116). A study has shown that TSCC-CSCs express specific biomarkers, including ALDH, CD44, NANOG, OCT4 and BMI1. These markers not only facilitate the identification of CSCs, but may also serve as potential therapeutic targets (117).

The present review of CSC characteristics in TSCC and key signaling pathways such as Wnt/β-catenin, Notch and Hh aimed to advance the understanding of TSCC pathogenesis (130). These findings highlight the critical role of CSCs in TSCC initiation and progression, providing a theoretical basis for the development of targeted therapies against CSCs and their regulatory networks (8). A balanced interpretation of existing research is essential; while a study emphasized the role of Wnt/β-catenin in CSC maintenance, others highlight the synergistic effects of Notch and Hh signaling (108). CSCs resist therapy by leveraging Wnt/β-catenin for self-renewal, while Notch and Hh signaling synergistically enhance survival and immune evasion (131). In pancreatic cancer models where dual inhibition of Notch and Hh reduced CSC frequency by 70% compared to single-pathway targeting (132).

It could be considered that simultaneous targeting of multiple pathways may be more effective in eliminating CSCs and preventing recurrence (133). Integrating molecular insights with clinical data is crucial for translating these findings into practical therapies (134). Future research should further elucidate pathway mechanisms and interactions to support the development of personalized medicine approaches (135), potentially leading to more effective, less toxic treatments for TSCC and improved patient outcomes (75). Overall, the exploration of CSCs in TSCC holds promise for transforming current cancer treatment strategies (136). Bridging laboratory findings with clinical application will be essential for the development of targeted therapies that improve prognosis and quality of life for patients (137).