Abstract

Background

   Oral squamous cell carcinoma (OSCC) is the most lethal oral malignant
   tumor, however, clinical outcomes remain unsatisfactory. The
   Hedgehog/Gli2 pathway plays a pivotal role in tumor progression, yet
   the regulatory mechanism governing its involvement in the malignant
   evolution process of OSCC remains elusive.

Methods

   OSCC animal tissue samples were used to detect the activation of the
   Hedgehog/Gli2 pathway in OSCC. Based on the clinical information of
   oral cancer patients in TCGA database, the role of this pathway in
   patients was analyzed, and the activation status of this pathway was
   verified in human OSCC cells. After activating or inhibiting the
   Hedgehog pathway, the effects of this pathway on the biological
   function of OSCC cells and its regulatory mechanism were examined.
   Interfering the expression of Gli2, a key transcription factor in this
   pathway, revealed the role of Hedgehog/Gli2 pathway in the malignant
   evolution of OSCC cells.

Results

   The Hedgehog pathway exhibits abnormal activation in animal models of
   OSCC. Clinical data from TCGA demonstrate a significant enrichment of
   the Hedgehog pathway in patients with OSCC, and Gli2, a key downstream
   factor of this pathway, is closely associated with the occurrence and
   progression of OSCC. Cellular studies have revealed aberrant activation
   of this pathway in human OSCC cells, which exerts its function by
   modulating the activation of epithelial-mesenchymal transition (EMT)
   and Wnt/β-catenin pathways. Subsequent investigations further confirm
   the pivotal involvement of Gli2 in the Hedgehog pathway activation,
   underscoring its potential as a therapeutic target for inhibiting
   malignant proliferation and metastasis of OSCC cells through modulation
   of EMT and Wnt/β-catenin pathways.

Conclusion

   The Hedgehog/Gli2 pathway induces EMT and activates Wnt/β-catenin
   pathway to trigger the malignant proliferation and metastasis of OSCC
   cells, and Gli2 plays a key role in this process, which suggests that
   targeting Gli2 may be a promising therapeutic strategy for inhibiting
   the proliferation and metastasis of OSCC.

   Keywords: OSCC, Hedgehog/Gli2 pathway, Cell proliferation, Cell
   metastasis, EMT, Wnt/β-catenin

1. Introduction

   The effective control of the increasing morbidity and mortality rates
   of cancer remains a significant global challenge in the 21st century.
   Oral cancer ranks as the sixth most prevalent form of cancer worldwide
   [[37]1,[38]2]. As an underestimated public health concern, oral cancer
   poses a serious threat to people's lives and well-being globally.
   Existing studies indicate that there will be an estimated 31,733 new
   cases and 15,745 deaths from oral cancer in China in 2022 [[39]3].
   Globally, over 350,000 new cases of oral cancer are diagnosed annually,
   resulting in approximately 145,000 deaths [[40]4]. Among various types
   of oral cancer, oral squamous cell carcinoma (OSCC) is particularly
   detrimental and accounts for more than 90 % of all cases [[41]5]. OSCC
   exhibits high malignancy potential along with susceptibility to
   metastasis and recurrence, leading to poor prognosis and adverse
   effects on patients' daily interpersonal communication [[42]6].
   Currently available clinical treatment methods for OSCC include
   surgery, chemotherapy, radiotherapy, and immunotherapy. These
   approaches have significantly advanced our understanding of OSCC
   pathogenesis and treatment options [[43]7]. However their efficacy
   remains limited for patients with OSCC accompanied by lymph node
   metastasis. Therefore it is crucial to explore novel therapeutic
   strategies. Targeted therapy has demonstrated positive therapeutic
   outcomes in clinical tumor treatments; however effective targeted
   therapies specifically designed for OSCC are currently scarce. Further
   investigation into targeted therapies for OSCC is warranted.

   Accumulating evidence has demonstrated the increasing significance of
   the Hedgehog signaling pathway in tumorigenesis. The aberrant
   activation of the Hedgehog pathway is closely associated with tumor
   proliferation, differentiation, apoptosis, angiogenesis, invasion,
   metastasis, and other cellular functions [[44]8,[45]9]. Guo et al.
   confirmed that Smad4 regulates TGF-Beta1-mediated Hedgehog activation
   by inhibiting Gli1 activity, thereby promoting epithelial-mesenchymal
   transition (EMT) of pancreatic cancer cells [[46]10]. Yang et al.
   verified that oncogene MYBL2 promotes a malignant phenotype and
   suppresses apoptosis through the Hedgehog signaling pathway in clear
   cell renal cell carcinoma [[47]11]. Wu et al. demonstrated that PARD3
   drives tumorigenesis by activating the Hedgehog signaling pathway in
   liver cancer initiating cells [[48]12]. Zhu et al. established that
   HERC4 regulates ovarian cancer cell proliferation by modulating
   Smo-triggered Hedgehog signaling [[49]13]. Wu et al. validated USP5 as
   a promoter of tumorigenesis through activation of the Hedgehog
   signaling pathway in osteosarcoma [[50]14]. Importantly, Gong et al.
   substantiated allotrypine's effect on OSCC proliferation and EMT via
   M6A-mediated modulation of the Hedgehog pathway [[51]15]. Niu et al.
   confirmed EHMT2's promotion of OSCC's malignant phenotype and stemness
   properties by suppressing ARRB1 transcription and activating the
   Hedgehog signaling [[52]16]. In summary, while it is evident that the
   Hedgehog pathway plays a crucial role in various cancers including
   OSCC; its regulatory mechanisms within OSCC lesions remain elusive and
   warrant further investigation.

   It is important to emphasize that the Hedgehog pathway activation can
   be categorized into two distinct pathways: canonical and non-canonical
   Hedgehog pathway regulatory networks, depending on the activation of
   the Hedgehog pathway and its dependence on Gli protein for biological
   effects [[53]17]. The canonical activation pathway refers to the signal
   transduction pathway mediated by Hedgehog's regulation of Gli family
   transcription factors. In the absence of Shh ligand protein, Ptch
   releases a protein that inhibits Smo activity, thereby blocking Smo
   activation and suppressing the Hedgehog pathway. Upon binding of
   Hedgehog ligand protein to Ptch, Ptch ceases secretion, leading to
   uninhibited Smo activity and subsequent activation of the Hedgehog
   signaling system. This ultimately results in the activation of Gli
   transcription factor members and their translocation into the nucleus,
   influencing downstream target gene expression within the Hedgehog
   pathway and promoting tumor proliferation, invasion, metastasis, and
   angiogenesis [[54][18], [55][19], [56][20]]. The non-canonical
   activation pathway refers to signal responses involving one or more
   components of the Hedgehog signaling cascade. It can be primarily
   classified into three categories based on its regulatory mechanism:
   Ptch-mediated signal transduction, Smo-mediated signal transduction,
   and Gli-mediated Hh signaling [[57]21]. Ptch-mediated signal
   transduction encompasses Ptch's role in inducing apoptosis by
   recruiting pro-apoptotic factors, as well as its involvement in cell
   cycle regulation through interaction with CyclinB1 [[58]22].
   Smo-mediated signal transduction refers to the Smo-dependent pathway
   that operates independently of Gli, wherein it binds to heterotrimeric
   G proteins of the Gi family and activates several crucial protein
   kinases, second messengers, and Ca2^+ [[59]21,[60]22]. Gli-mediated
   signal transduction is not reliant on either Smo or upstream signals of
   Gli; instead, it engages multiple tumor-related signaling pathways such
   as TGF-β, MAPK, PI3K-AKT, and TNF-α to activate Gli activity [[61]23].
   Thus, the Hedgehog pathway represents a complex regulatory network
   rather than a linear cascade that synergistically interacts with
   various signaling pathways to regulate tumor progression.

   However, in the two activation pathways of Hedgehog pathway, Gli family
   transcription factors play a key role as the convergence point of
   activation at all levels of Hedgehog pathway, which can transmit
   extracellular signals into the cell, regulate the expression of related
   genes at the transcriptional level by directly acting on downstream
   targets, and thus affect cell biological functions [[62]24,[63]25].
   Gli2 plays a crucial role in embryonic cell differentiation, tissue
   development, organ formation and disease regulation (including
   tumorigenesis) [[64]26]. Gli2 functions as a highly active
   transcription factor within the Hedgehog pathway [[65]27]. It possesses
   five conserved zinc finger DNA binding domains in tandem repeats,
   predominantly localizes to the nucleus, and exhibits high affinity for
   its DNA binding sites [[66]28]. Acting as a downstream transcription
   factor of this pathway, Gli2 serves as a convergence point for
   activation at all levels of the Hedgehog signaling cascade. Following
   activation, Gli2 undergoes post-translational modifications (such as
   phosphorylation and ubiquitination) to stabilize its nuclear presence
   [[67]29,[68]30]. This enables it to transmit extracellular signals to
   the nucleus and directly modulate downstream targets by regulating
   target gene expression at the transcriptional level, thereby affecting
   various biological processes including cell proliferation,
   differentiation and survival [[69]31,[70]32]. It is crucial to
   emphasize that Alexaki et al. have confirmed the direct involvement of
   Gli2 in melanoma invasion and metastasis [[71]33]. Cannonier et al.
   have demonstrated a correlation between Gli2 levels in clinical OSCC
   samples and bone infiltration [[72]34]. Yan et al. have revealed a
   close association between Gli2 expression and poor prognosis in
   patients with oral cancer [[73]35]. These findings suggest that Gli2,
   as a pivotal downstream factor of the Hedgehog pathway, plays a
   fundamental role in the pathogenesis of this disease. However, the
   potential therapeutic targeting of Gli2 for OSCC treatment remains
   uncertain, necessitating further investigation into its precise role in
   OSCC development.

   To elucidate the underlying mechanism of the Hedgehog/Gli2 pathway
   regulation in OSCC development, we initially employed bioinformatics
   methods to predict Hedgehog/Gli2 pathway potential regulatory role in
   OSCC patients. Subsequently, we investigated the activation status of
   the Hedgehog pathway at both cellular and animal levels. Furthermore,
   we examined the impact of modulating Hedgehog pathway activation on the
   biological functions of OSCC cells. To ascertain whether Gli2, a key
   downstream transcription factor of this pathway, drives its activation
   in OSCC cells, we interfered with Gli2 expression and evaluated its
   influence on co-functional aspects of OSCC cells. Our findings confirm
   that aberrant Hedgehog pathway activation promotes malignant
   proliferation and metastasis in OSCC cells by inducing EMT and
   Wnt/β-catenin pathways. Importantly, Gli2 emerges as a pivotal driver
   mediating Hedgehog's regulatory role in tumorigenesis. These results
   suggest that targeting Gli2 could be a promising therapeutic strategy
   for inhibiting proliferation and metastasis in OSCC.

2. Materials and methods

2.1. Animal tissue samples

   Female Chinese hamsters (8 weeks old, weighing 20–25g, n = 48) were
   procured and housed in a controlled environment at the Laboratory
   Animal Center of Taiyuan Medical University, Shanxi Province. The
   animals were randomly divided into two groups: control group (n = 24)
   and experimental group (n = 24). The control group received regular
   feeding, while the experimental group was subjected to thrice-weekly
   application of DMBA with a concentration of 0.005 g/L on the bilateral
   buccal pouch mucosa. To investigate OSCC pathogenesis comprehensively,
   eight Chinese hamsters from each group were randomly selected at the
   9th, 15th, and 21st week of continuous DMBA application for collection
   of cheek pouch tissues and cancerous tissues. Some tissue samples
   underwent pathological examination while the remaining samples were
   stored at −80 °C. All animal procedures adhered to ethical guidelines
   approved by the Institutional Animal Care and Use Committee of Shanxi
   Medical University (IACUC 2021012), ensuring compliance with animal
   welfare principles based on the 3R principles without compromising
   experimental requirements or outcomes.

2.2. Data Source

   Clinical data of patients with OSCC were downloaded from the Cancer
   Genome Atlas (TCGA) database
   ([74]https://www.cancer.gov/ccg/research/genome-sequencing/tcga),
   excluding those without clinical information. Among the clinical data,
   samples from various oral cancer sites including alveolar ridge, tongue
   base, buccal mucosa, floor of mouth, hard palate, mouth, and tongue
   were retained while non-oral cancer site samples such as hypopharynx,
   larynx lip oropharynx tonsil were excluded. Pathway enrichment analysis
   for differential genes in patients with oral cancer was conducted using
   clusterProfiler package. Paired sample t-test was employed to analyze
   the expression level of functional genes in clinical samples.

2.3. Cell lines

   The human oral cancer cell lines CAL27 and SCC9 were obtained from the
   American Type Culture Collection (Manassa, VA, USA). Both CAL27 and
   SCC9 cells were cultured using DMEM (Boster, China) supplemented with
   10 % FBS (Gibco, USA), along with 1 % penicillin/streptomycin (Boster,
   China). Human normal oral epithelial cells (HOK) were generously
   provided by the Key Laboratory of the School of Stomatology at Shanxi
   Medical University. HOK cells were cultured using RPMI1640 (Boster,
   China) supplemented with 10 % FBS and 1 % penicillin/streptomycin. All
   cell lines were incubated at 37 °C under a CO[2] concentration of 5 %.

2.4. Cell treatment

   Cells in the exponential growth phase were inoculated into a 6-well
   plate (50,000 cells/well) and cultured at 37 °C with 5 % CO[2]. The
   Hedgehog pathway inhibitor, GANT61 (MCE, USA), and agonist, SAG
   (Beyotime, China), were employed to modulate the activity of this
   signaling cascade. Furthermore, we conducted a drug concentration
   gradient experiment to screen the optimal treatment concentration. The
   cells were treated with either GANT61 (20 μM) or SAG (5 μM). After 48h
   of drug treatment, other assays were performed to assess the impact of
   the Hedgehog pathway on various biological functions of oral cancer
   cells. Detailed information regarding these small-molecule drugs can be
   found in [75]Supplementary Table 1.

2.5. Cell transfection

   Cells in the exponential growth phase were inoculated into a 6-well
   plate (50,000 cells/well) and cultured at 37 °C with 5 % CO[2].
   Specific siRNAs targeting Gli2 and a negative control (si-NC) were
   designed and synthesized by GenePharma. The optimal concentration for
   siRNA transfection was determined through a drug concentration gradient
   experiment. Transfection of siRNA (100 nM) and si-NC (100 nM) was
   performed the following day using Lipofectamine™ 3000 (Invitrogen,
   USA). The medium was replaced with fresh medium after 6h of
   transfection. At 48h post-siRNA transfection, the efficiency of
   cellular transfection was assessed using quantitative reverse
   transcription polymerase chain reaction (qRT-PCR). [76]Supplementary
   Table 2 provides the sequences of the siRNAs used to knockdown Gli2.

2.6. qRT-PCR

   Total RNA was extracted by using a TRIzol kit (Takara, Japan), and cDNA
   was synthesized using a reverse transcription kit (Takara, Japan).
   qRT-PCR was performed on an Applied Biosystems 7500 (ABI 7500) system
   (Thermofisher, USA) using a SYBR Green PCR Kit (Takara, Japan). Using
   GAPDH as internal reference, the gene expression was normalized. The
   2^−ΔΔCT method was used to quantify gene expression. The primer
   sequences were obtained from Shanghai GenePharma Co., Ltd., China, and
   listed in [77]Supplementary Table 3.

2.7. Western blot analysis

   Protein collection were performed using RIPA Lysis Buffer (Boster,
   China). Total protein content was detected using a BCA Protein
   Quantification Kit (Boster, China). Total protein was subjected to
   sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
   and transferred to a nitrocellulose filter membrane (Boster, China).
   After the nitrocellulose filter membranes were blocked using 5 % skim
   milk at room temperature for 60min, the primary antibody was added and
   incubated overnight in a shaker at 4 °C. The next day, the
   nitrocellulose filter membranes were washed with washing buffer (TBST)
   (Boster, China) and incubated with the secondary antibodies at 37 °C
   for 60min. An Enhanced Chemiluminescent Substrate Kit (Boster, China)
   and an automatic gel imaging system were used to detect the luminescent
   signals of the protein bands. β-actin was used as a standardization
   control. Details on the antibody is provided in [78]Supplementary Table
   4.

2.8. Cell counting Kit-8 (CCK-8) assay

   Cell toxicity was detected using the CCK-8 assay: the cell suspension
   (5000 cells/well) was seeded into 96-well plates, and incubated at
   37 °C and 5 % CO[2] for 24h. Each group had at least 3 replicate wells,
   and five concentration gradients were set for each siRNA and pathway
   agonist and inhibitor. In addition, CCK-8 assay was used to detect cell
   proliferation. Cell suspensions (5000 cells/well) were seeded onto
   96-well plates and incubated at 37 °C with 5 % CO[2,] with a minimum of
   three replicate wells per group. At predetermined time points (0h, 24h,
   48h, and 72h), each well containing 90 μl of culture medium was
   supplemented with 10 μl CCK-8 solution (MCE, USA) and further incubated
   at 37 °C with 5 % CO[2] for a duration of 2 h. The absorbance at the
   wavelength of 450 nm was measured using a microplate reader (BioTek,
   USA).

2.9. Cell migration assay

   Cell suspensions (20,000 cells/pores) were added to the upper chamber
   of the transwell chamber and cultured with 1 % FBS. Then, 600 μl medium
   containing 10 % FBS was added to the lower cavity. The cells were
   cultured at 37 °C and 5 % CO[2] with at least 3 repeat wells per group.
   After incubation for 24h, the cell medium and non-migrated cells in the
   upper chamber were removed and fixed with 4 % paraformaldehyde (Boster,
   China) for 30min, and 0.1 % crystal purple solution (Beyotime, China)
   was added to the migrated cells for 15min for staining. Images of
   migrating cells were captured under a microscope (Olympus, Japan) and
   counted using the ImageJ software.

2.10. Cell invasion assay

   A pre-cooled 100 μL solution of matrigel (Corning, USA) was added to
   the upper chamber of a transwell system and incubated at 37 °C for
   30min. Subsequently, a cell suspension containing 20,000 cells/well was
   introduced into the upper chamber and cultured with 1 % FBS.
   Additionally, 600 μl medium supplemented with 10 % FBS was added to the
   lower compartment. The cells were then incubated in a controlled
   environment at 37 °C with 5 % CO[2] concentration following identical
   steps as those employed for cell migration assays.

2.11. Colony formation assay

   Cell suspensions (1000 cells/well) were added to 6-well plates and
   cultured at 37 °C and 5 % CO[2]. Cells were grown in a medium
   containing 20 % FBS, and the medium was changed every five days. Two
   weeks later, cells were fixed with 4 % paraformaldehyde for 30min. The
   colonies were then stained with a 0.1 % crystal violet aqueous solution
   at room temperature for 15min. Images were acquired under a microscope,
   and the number of colonies (clusters of >50 cells) was counted.

2.12. Statistical analysis

   The statistical analyses were conducted using SPSS Statistics 20.0, and
   the data were presented as mean ± deviation. The t-test was employed to
   assess differences between two groups, while a one-way analysis of
   variance followed by Tukey's post hoc test was used to determine
   differences among multiple groups. Statistical significance was defined
   as *P < 0.05, **P < 0.01. All experiments were performed at least three
   times.

3. Results

3.1. Hedgehog is abnormally activated in OSCC

   Our preliminary histopathological analysis showed that after 9 weeks of
   continuous DMBA induction, the Chinese hamster OSCC model group
   exhibited simple epithelial hyperplasia. After 15 weeks of continuous
   DMEM induction, the Chinese hamster OSCC model group entered the
   abnormal proliferation stage. After 21 weeks of continuous DMBA
   induction, the Chinese hamster OSCC model group displayed the
   characteristics of squamous cell carcinoma and invasive carcinoma, and
   the OSCC animal model was successfully established. To further validate
   the involvement of the Hedgehog pathway in OSCC development, we built
   on our previous research and used qRT-PCR and western blotting to
   detect the mRNA and protein expression of key Hedgehog pathway factors
   in the simple hyperplasia, abnormal proliferation, and OSCC groups
   [[79]36]. qRT-PCR results demonstrated increased expression of Shh,
   Ptch1, Gli1, and Gli2 in dysplasia and OSCC groups ([80]Fig. 1A).
   Additionally, western blotting analysis indicated elevated protein
   expression of Gli2 in simple hyperplasia group; increased protein
   expression of Shh, Gli1, and Gli2 in dysplasia group; as well as
   enhanced protein expression of Shh, Ptch1, Gli1, and Gli2 in OSCC group
   ([81]Fig. 1B).

Fig. 1.

   [82]Fig. 1
   [83]Open in a new tab

   Hedgehog/Gli2 is abnormally activated in OSCC (A) mRNA expression of
   key components of the Hedgehog pathway in OSCC animal samples. (B)
   Protein expression of key components of the Hedgehog pathway in OSCC
   animal samples (western blotting is related to [84]Fig. S2). (C) GSEA
   analysis of oral cancer samples. (D) Key gene networks enriched by GSEA
   (the higher the degree, the bluer the color, and the lower the degree,
   the yellower the color). (E) Gli2 expression in oral cancer samples
   from TCGA database (the lines represent pairs of samples). (F) mRNA
   expression of key components of the Hedgehog pathway in OSCC cells. (G)
   Protein expression level of key components of the Hedgehog pathway in
   OSCC cell lines (western blotting is related to [85]Fig. S3). (Data are
   represented as mean ± standard deviation, n = 3; *P < 0.05, **P < 0.01,
   compared with the control group). (For interpretation of the references