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SOX2 promotes the progression of hepatocellular carcinoma by targeting FOXJ3 and activating the PI3K/AKT pathway to regulate autophagy and mitophagy
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Received: ,
Accepted: ,
How to cite this article: Zhang Z, Ren W. SOX2 promotes the progression of hepatocellular carcinoma by targeting FOXJ3 and activating the PI3K/AKT pathway to regulate autophagy and mitophagy. CytoJournal. 2026;23:28. doi: 10.25259/Cytojournal_164_2025
Abstract
Objectives:
Sex-determining region Y-box transcription factor 2 ( SOX2) has been implicated in tumorigenesis across various cancers. This study sought to characterize the expression pattern of SOX2 in hepatocellular carcinoma (HCC) and elucidate its potential mechanism in promoting HCC progression by targeting Forkhead box J3 (FOXJ3) and regulating the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, autophagy, and mitophagy.
Material and Methods:
SOX2 expression was examined in normal liver epithelial (THLE2) and HCC (Huh7) cells. It was modulated by shRNA-mediated knockdown and plasmid-based overexpression. 5-Ethynyl-2'-deoxyuridine (EdU) fluorescence staining assay, Transwell assays, and flow cytometry were performed to evaluate cell viability and motility. Autophagy and mitophagy were assessed by Western blotting and transmission electron microscopy, and mitochondria-lysosome co-localization was visualized by immunofluorescence staining. The roles of SOX2 and FOXJ3 in the PI3K/AKT signaling pathway were investigated.
Results:
SOX2 was markedly upregulated in HCC cells (P <0.001). SOX2 overexpression enhanced the viability and motility of Huh7 cells and inhibited apoptosis, whereas SOX2 knockdown elicited opposite effects (P <0.001). Mechanistically, SOX2 upregulated autophagy-related proteins Microtubule-Associated Protein 1 Light Chain 3 ( LC3)-II/LC3-I and p62, promoted autophagosome formation, increased PTEN-induced kinase 1 ( PINK1) and p-COX4 levels, and facilitated mitochondria-lysosome co-localization (P <0.05). Co-overexpression of FOXJ3 reversed the promotive effects of SOX2 on cell proliferation, migration, autophagy, and mitophagy and attenuated PI3K/AKT pathway activation (P <0.001).
Conclusion:
SOX2 promotes HCC progression by negatively regulating FOXJ3, thereby activating the PI3K/AKT signaling pathway and inducing autophagy and mitophagy. The potential SOX2-FOXJ3-PI3K/AKT axis may serve as a novel regulatory pathway underlying HCC development and represents a potential target for therapeutic intervention.
Keywords
Autophagy
FOXJ3: Forkhead box J3
Hepatocellular carcinoma
Mitophagy
SRY-box transcription factor 2
INTRODUCTION
Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers and frequently arises in individuals with severe liver cirrhosis.[1,2] Although diagnostic and therapeutic approaches have improved in recent years, the overall prognosis of HCC remains poor due to its high recurrence rate, frequent intrahepatic metastasis, and resistance to therapy.[3,4] Thus, understanding the molecular pathways driving HCC onset and progression is essential for discovering new diagnostic markers and therapeutic targets.
Sex-determining region Y-box transcription factor 2 (SOX2) plays a central role in regulating the developmental versatility and continuous division ability of embryonic stem cells.[5] Increasing evidence has demonstrated that SOX2 is aberrantly expressed in various malignancies and involved in regulating tumor initiation, metastasis, and therapeutic resistance.[6-9] In HCC, increased SOX2 expression is closely associated with increased tumor aggressiveness and poor patient prognosis.[10,11] However, the precise molecular mechanisms through which SOX2 exerts its oncogenic function in HCC remain largely unclear.
Autophagy and mitophagy are essential cellular clearance mechanisms that maintain intracellular homeostasis by degrading damaged organelles and misfolded proteins.[12,13]These processes exhibit a double-edged sword effect in cancer: while they may exert tumor-suppressive functions during early tumorigenesis, they can also promote cancer cell survival and metastasis under stress conditions during later stages of tumor progression.[14] Recent investigations have underscored the critical roles of autophagy and mitophagy in the development and progression of HCC, particularly under hypoxic conditions and metabolic reprogramming.[15,16] However,whether SOX2 contributes to HCC progression by regulating autophagy and mitophagy remains largely unexplored.
Forkhead box J3 (FOXJ3) has been shown to regulate mitochondrial function and oxidative phosphorylation.[17]Although previous studies have proposed that FOXJ3 acts as an anti-oncogene in various types of cancer, its functional role and upstream regulatory mechanisms in HCC remain unclear.[18]Notably, the regulatory relationship between SOX2 and FOXJ3 expression in liver cancer has not yet been elucidated.
The phosphoinositide 3-kinase/protein kinase B (PI3K/ AKT) signaling pathway is one of the most critical pro-survival and pro-proliferative pathways in cancer.[19,20]In HCC, this pathway is frequently aberrantly activated, contributing to enhanced tumor cell growth, invasion, and resistance to apoptosis.[21] Moreover, PI3K/AKT signaling participates in the regulation of autophagy and mitophagy by modulating downstream effectors such as mTOR, ULK1, and GSK3β.[22] Although previous studies have suggested that SOX2 can influence the PI3K/AKT pathway through various mechanisms, whether SOX2 promotes HCC progression by regulating autophagy and mitophagy through this pathway remains unclear.
This study was designed to explore the expression and biological functions of SOX2 in HCC and determine whether SOX2 promotes HCC cell growth while inhibiting apoptosis by targeting FOXJ3 and activating the PI3K/AKT signaling pathway, thereby inducing autophagy and mitophagy. This study seeks to provide novel molecular targets and theoretical insights for therapeutic interventions in liver cancer by elucidating the function of the SOX2-FOXJ3-PI3K/AKT axis in HCC progression.
MATERIAL AND METHODS
Cell cultures
Human normal liver epithelial cells (THLE-2, CL-0833) and HCC cells (Huh7, CL-0120) were purchased from Procell (Wuhan, Hubei, China). The THLE-2 cells were cultured in a specific medium (ZQ-1340, Cell Research, Wuhan, China) under recommended conditions (37°C and 5% CO2). The Huh7 cells were cultured in a specific medium (XY-HM040, X-Y-Biotechnology, Shanghai, China) under standard conditions (37°C and 5% CO2). The volume of the culture medium is 2-3 mL. All cell lines utilized in this research underwent STR authentication, and they were confirmed to be negative for mycoplasma contamination.
Cell transfection
Cells (2 × 105 cells per well) were transfected with either shRNA plasmids targeting SOX2 [shSOX2-1 (sense:CTGCCGAGAATCCATGTATAT,shSOX2-2 (sense:TGGACAGTTACGCGCACATGA),shSOX2-3 (sense:GTACAGTATTTATCGAGATAA)],Sh-NC (sense:TTCTCCGAACGTGTCACGT),overexpression plasmid negative control (Oe-NC, sense: 5'-GCTAGCGC TACGATCGTACGATCGTAGCTAGTACGATCGT ACGCTAGCTAGCTGACTGATCGTAGCTAGC TACGTAGCTAGCTAGTACGATCGTACGATCGTAGC TAGCTACGATCGTACGATCGTAGCTAGCTAG CTGACTGATCGTAGCTAGCTACG-3 ' ),SOX2 overexpression plasmids (Oe-SOX2, sense: GGGGGCGGCGGCGGCAACTCCACCGCGGCGGC GGCCGGCGGCAACCAGAAAAACAGCCC GGACCGCGTCAAGCGGCCCATGAATGCCT T C AT G G T G T G G T C C C G C G G G C A G C G GCGCAAGATGGCCCAGGAGAACCCCAAG ATGCACAACTCGGAGATCAGCAAGCGCC T G G G C G C C G A G T G G A A A C T T T T GTCGGAGACGGAGAAGCGGCCGTTCATCGAC GAGGCTAAGCGGCTGCGAGCGCTGCACATGA AG G AG C AC C C G G AT TATA A ATAC C G G C CCCGGCGGAAAACCAAGACGCTCATGAAGA AGGATAAGTACACGCTGCCCGGCGGGCTGC TGGCCCCCGGCGGCAATAGCATGGCGAGC GGGGTCGGGGTGGGCGC CGGCCTGGGCGCGGGCGT GAACCAGCGCATGGACAGTTACGCGCACA TGAACGG CTGGAGCAACGGCAGCTACAGCATGATGCA G G ACC AG C TG G G C TACCCG C AG C ACCC GGGCCTCAATGCGCACGGCGCAG), OR FOXJ3OVER EXPRESSIONPLASMIDS(OE-FOXJ3,SENSE: CTGATCAGGATGGTAGTGATAGCCCACG CAGTAGCCTTAACAACAGTCTCTCAGACCA G A G T T T G G C A T C T G T T A A T T T G A AC AG TG T TG G A AG TG TG C ATAG T TATA CACCGGTGACAAGCCATCCAGAATCAGTCTCTCAAT CATTAACTCCTCAGCAGCAACCACAGTACAACCTT CCAGAAAGAGACAAGCAACTACTTTTCTCA G A ATATA AT T T T G A AG AT C T TAG T G C C TCATTTCGGAGCCTTTATAAGTCAGTTTTT G A G C A G T C A C T T A G T C A A C A A G G T TTGATGAACATCCCTTCTGAATCTTCCCAGC A G T C C C A C A C T T C AT G TA C C TAT C A G CACTCTCCCAGCAGTACAGTGAGCACTCACCCA CACAGCAACCAAAGCAGCCTGTCCAACAG T C A T G G C A G T G G C C T C A A C A CCACAGGCAGTAATTCGGTTGCACAGGTCT CACTGTCTCACCCCCAGATGCACACACAG CCATCTCCACATCCTCCCCATCGACCGCATGGT TTACCGCAGCATCC GCAGC).
For each well, 2.5 μg of plasmid DNA or 100 pmol of shRNA was diluted in Opti-MEM (31985070, Gibco) and mixed with Lipofectamine 3000 (L3000008, Invitrogen, Carlsbad, CA, USA) at 4°C for 20 min. After 48 h, post-transfection, the transfection efficiency was evaluated.
Western blot
Cell lysis was carried out on ice for 30 min using radio-immunoprecipitation assay ( RIPA) buffer containing protease and phosphatase inhibitors (Solarbio, R0010). Lysates were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected. Protein concentrations were determined using a bicinchoninic acid ( BCA) protein assay kit (Beyotime, P0010). Proteins were equally loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and transferred onto polyvinylidene fluoride membranes (Millipore, IPVH00010).
The membranes were first blocked with 5% non-fat milk (1 h and 25°C), followed by overnight incubation with primary antibodies [SOX2 (1:1000, ab92494, Abcam, Cambridge, UK), LC3B (1:1000, ab192890, Abcam, Cambridge, UK), p62 (1:1000, ab109012, Abcam, Cambridge, UK), PINK1 (1:1000, ab216144, Abcam, Cambridge, UK), p-COX4 (1:1000, PA5-114563, Thermo Fisher, Waltham, Massachusetts, USA), COX4 (1:1000, PA5-29992, Thermo Fisher, Waltham, Massachusetts, USA), FOXJ3 (1:1000, PA5-99279, Thermo Fisher, Waltham, Massachusetts, USA), p-PI3K (1:1000, ab278545, Abcam, Cambridge, UK), PI3K (1:1000, ab302958, Abcam, Cambridge, UK), p-AKT (1:1000, ab81283, Abcam, Cambridge, UK), AKT (1:1000, ab8805, Abcam, Cambridge, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, ab181602, Abcam, Cambridge, UK)] at 4°C. Next, they were incubated with HRP-labeled secondary antibodies (1:1000, ab6721, Abcam, Cambridge, UK) for 1 h at 25°C. Protein bands were detected using an ECL kit (Beyotime, P0018, Shanghai, China) and captured with the Tanon 5200 imaging system (Shanghai, China). GAPDH functioned as the normalization control, and band intensities were determined using ImageJ software (version 1.5f, National Institutes of Health, Bethesda, MD, USA).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells using total RNA isolation reagent (TRIzol) reagent (Invitrogen, 15596026, USA). RNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized from 1 μg of total RNA using a reverse transcription kit (Takara, RR037A, Japan). qRTPCR was carried out with SYBR Green Master Mix (Takara, RR820A) on a QuantStudio 5 real-time PCR system (Applied Biosystems, USA). GAPDH served as the reference gene. Relative quantification of mRNA was performed using the 2−ΔΔCt approach. All experiments were performed in triplicate. The primer sequences are presented in Table 1.
| Primer name | Primer sequence (5'-3') |
|---|---|
| hum-SOX2-F | AACCAGCGCATGGACAGTTA |
| hum-SOX2-R | GACTTGACCACCGAACCCAT |
| hum-GAPDH-F | GTGGATATTGTTGCCATCAATGACC |
| hum-GAPDH-R | GCCCCAGCCTTCTTCATGGTGGT |
| hum-FOXJ3-F | GGGCGCTTCGATTCTCTTTG |
| hum-FOXJ3-R | GAGTTGGAGAGGCACTTCCC |
SOX2: SRY-box transcription factor 2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
5-Ethynyl-2hyde-3-phospha (EdU) staining
In brief, cells were distributed in 24-well plates and incubated using an EdU assay kit (C10310-1, RiboBio, Guangzhou, China) at a final concentration of 50 µM for 2 h at 37°C. Afterward, the cells were fixed and permeabilized. Then, they were incubated with Apollo reaction buffer for 30 min in the dark, followed by DAPI staining for 10 min. The stained cells were imaged using a fluorescence microscope (Olympus, Tokyo, Japan). The proliferation rate was calculated as the ratio of EdU-positive nuclei to the total number of 4',6-Diamidino-2-Phenylindole ( DAPI)-stained nuclei.
Transwell assay
Cell migration and invasion abilities were evaluated using Transwell chambers with an 8 μm pore size (Corning, USA). For the migration assay, cells were seeded directly into the upper chamber without any coating. For the invasion assay, the upper chamber was pre-coated with Matrigel (BD Biosciences), and the transwell chambers were incubated at 37°C for 30 min to 1 h to allow gel formation.
Cells (2 × 104) were resuspended in serum-free medium and seeded into the upper chamber, while the lower chamber contained complete medium. After incubation of the Transwell chambers at 37°C in a humidified atmosphere containing 5% CO2 for 24 h, non-migrated cells were removed, and migrated/invaded cells were fixed and stained with 0.1% crystal violet. The cells were counted in several randomly selected fields under a light microscope.
Flow cytometry
Cell apoptosis was analyzed using an Annexin V-FITC/PI dual staining kit (Beyotime, C1062L, China) in combination with flow cytometry. In brief, transfected or treated cells were collected and then resuspended in pre-chilled 1× binding buffer. Approximately 1 × 105-5 × 105 cells were used per group. Each sample was incubated using the staining kit in the dark at room temperature for 10 min. Afterward, the samples were immediately analyzed using a flow cytometer (BD FACSCanto II, BD Biosciences, USA) to detect fluorescein isothiocyanate ( FITC) and PI fluorescence.
Transmission electron microscopy (TEM)
Following the corresponding treatments, the cells were collected and fixed. The next day, samples were washed 3 times with 0.1 M phosphate-buffered saline (PBS) for 10 min each, followed by post-fixation in 1% osmium tetroxide for 1 h. The cells were then dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%, 10-15 min at each step) and embedded in epoxy resin. Autophagosomes were visualized and imaged using a transmission electron microscope (JEOL JEM-1400 Plus, Japan).
Immunofluorescence staining
Cells were seeded on coverslips in 24-well plates and washed with cold PBS after treatment. The mitochondria and lysosomes were stained with MitoTracker Red (100 nM, Thermo Fisher Scientific, M7512) and LysoTracker Green (75 nM, Thermo Fisher Scientific, L7526) at 37°C for 30 min each. The cells were then fixed with 4% paraformaldehyde and permeabilized. Nuclei were counterstained with DAPI (1 µg/mL). The colocalization of the mitochondria and lysosomes was imaged by confocal microscopy (Zeiss LSM880) and quantified using ImageJ.
Statistical analysis
Data are expressed as mean ± standard deviation from a minimum of three independent experiments. Statistical analyses were conducted using GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA, USA). Differences between two groups were evaluated with an unpaired two-tailed student’s t-test, whereas comparisons among multiple groups were analyzed by one-way analysis of variance with Tukey’s post-hoc test. A P <0.05 was regarded as statistically significant.
RESULTS
SOX2 is highly expressed in HCC lines
The expression levels of SOX2 in THLE2 and Huh7 cell lines were examined. As shown in Figure 1a-c, the SOX2 expression at the mRNA and protein levels was significantly higher in Huh7 cells than in THLE2 cells (P <0.001). The Huh7 cells were transfected with either shRNA targeting SOX2 (shSOX2) or SOX2 overexpression plasmids to investigate the functional effects of SOX2 on HCC progression. The results confirmed successful knockdown and overexpression of SOX2 in Huh7 cells [Figure 1d-f]. ShSOX2-3 exhibited the lowest transfection efficiency, hence selected for the subsequent experiments and uniformly referred to as Sh-SOX2.
SOX2 promotes the growth of HCC cells
Cell proliferation was assessed through EdU staining in Huh7 cells. As shown in Figure 2a and b, SOX2 overexpression (Oe-SOX2) notably increased the number of EdU-positive cells, whereas SOX2 knockdown (shSOX2) markedly decreased the number (P <0.001). Transwell assays illustrated that Oe-SOX2 significantly enhanced the motility of Huh7 cells, whereas shSOX2 significantly reduced this ability (P <0.001, [Figure 2c-f]). Compared with Oe-NC treatment, Oe-SOX2 treatment significantly decreased the apoptotic rate of Huh7 cells (P <0.001). Conversely, shSOX2 treatment significantly increased apoptosis compared with sh-NC treatment (P <0.001, [Figure 2g and h]). These results indicated that SOX2 not only promotes the growth of HCC cells but also inhibits their apoptosis.
SOX2 induces autophagy and mitophagy in HCC cells
The effect of SOX2 on autophagy-related proteins in Huh7 cells was examined by Western blot. As shown in Figure 3a-c, OeSOX2 significantly increased the expression levels of LC3II/ LC3I and p62 proteins, whereas shSOX2 markedly decreased these levels (P <0.05). Furthermore, autophagosome formation in Huh7 cells was observed by TEM. As shown in Figure 3d, compared with the Oe-NC group, the Oe-SOX2 group showed an increase in the number of autophagosomes. Multiple typical autophagic structures, including phagophores, mature autophagosomes, and autolysosomes formed by fusion with lysosomes, were visible in some cells. By contrast, the cells treated with shSOX2 exhibited a remarkable reduction in autophagosome number compared with the sh-NC group, with fewer characteristic double-membrane structures and almost no mature autophagosomes or autolysosomes in the cytoplasm. Some cells showed accumulation of damaged organelles or undegraded structures, suggesting impaired autophagic clearance.
The effect of SOX2 on mitophagy-related proteins in Huh7 cells was assessed. Figure 3e-g shows that Oe-SOX2 significantly increased PINK1 and phosphorylated COX4 (p-COX4)/COX4 protein expression levels, whereas shSOX2 significantly suppressed these levels (P <0.001).
Mitochondrial and lysosomal colocalization in Huh7 cells was evaluated by immunofluorescence staining. As shown in Figure 3h and i, Oe-SOX2 treatment markedly enhanced colocalization signals between the mitochondria and lysosomes compared with Oe-NC treatment, with strong fluorescence overlap in multiple cytoplasmic regions, indicating mitochondrial engulfment and degradation by lysosomes. Conversely, shSOX2 treatment significantly reduced the mitochondrial-lysosomal colocalization compared with sh-NC treatment, with diminished overlapping regions and clear separation of red and green signals.
SOX2 regulates the expression of FOXJ3 in HCC
The mRNA and protein expression levels of FOXJ3 in Huh7 cells following SOX2 overexpression or knockdown were examined to further assess the regulatory function of SOX2 on FOXJ3. Figure 4a-c shows that Oe-SOX2 treatment markedly decreased the FOXJ3 mRNA and protein levels, whereas shSOX2 treatment significantly increased FOXJ3 expression (P <0.001).
SOX2 exerts its Biological functions by regulating FOXJ3
Huh7 cells were transfected with either Oe-SOX2 alone or a combination of Oe-SOX2 and Oe-FOXJ3 to further clarify the function of FOXJ3 in SOX2-mediated biological effects. As shown in Figure 5a and b, Oe-SOX2 treatment significantly decreased FOXJ3 mRNA expression compared with Oe-NC treatment (P <0.001). Compared with the OeSOX2 group, the Oe-SOX2+Oe-FOXJ3 group revealed a significantly reduced SOX2 mRNA expression (P <0.001). CCK-8 assays indicated that the cell viability in the OeSOX2 + Oe-FOXJ3 group was markedly reduced compared with that in the Oe-SOX2 group (P <0.001 [Figure 5c]). Transwell assays revealed that co-expression of FOXJ3 markedly decreased the motility of Huh7 cells compared with Oe-SOX2 treatment alone (P <0.001, [Figure 5d-g]). Flow cytometric analysis [Figure 5h and i] showed that the apoptotic rate of Huh7 cells in the Oe-SOX2 + Oe-FOXJ3 group notably increased compared with that in the Oe-SOX2 group (P <0.001).
SOX2 regulates autophagy and mitophagy in HCC cells by regulating FOXJ3
As depicted in Figure 6a-c, compared with the Oe-SOX2 group, the Oe-SOX2 + Oe-FOXJ3 group had significantly diminished protein abundance of LC3II/LC3I and p62 (P <0.001). The TEM results demonstrated that the number of autophagosomes, mature autophagosomes, and autolysosomes in the Oe-SOX2 + Oe-FOXJ3 group was reduced compared with that in the Oe-SOX2 group [Figure 6d].
Figure 6e-g shows that the protein abundance of PINK1 and phosphorylated COX4 (p-COX4)/COX4 was significantly lower in the Oe-SOX2 + Oe-FOXJ3 group than in the OeSOX2 group (P <0.001). Figure 6h-i illustrates that Oe-SOX2 + Oe-FOXJ3 treatment notably diminished mitochondrial and lysosomal colocalization signals compared with OeSOX2 treatment alone, with a clear reduction in overlapping areas.
SOX2 regulates PI3K/AKT signaling pathway in HCC by regulating FOXJ3
Compared with the Oe-NC group, the Oe-SOX2 group had notably increased protein expression levels of p-PI3K/PI3K and p-AKT/AKT (P <0.001). Compared with Oe-SOX2 treatment, co-transfection with Oe-SOX2 and Oe-FOXJ3 markedly decreased the protein abundance of p-PI3K/PI3K and p-AKT/AKT (P <0.001, [Figure 7a-c]).
DISCUSSION
This study provides new insights into the regulatory network of HCC progression. Although SOX2 has been widely reported to function as an oncogenic transcription factor in multiple cancers, the regulatory connection between SOX2 and FOXJ3 has not been previously clarified in HCC. For the 1st time, a potential SOX2-FOXJ3 regulatory axis that integrates transcriptional regulation with PI3K/AKT pathway activation and mitophagy modulation was demonstrated. This finding extends the current knowledge by linking SOX2 signaling to mitochondrial quality control processes, thereby offering a new perspective on how SOX2 contributes to malignant phenotypes. These mechanistic insights may help refine the conceptual framework of SOX2-driven tumor progression and highlight a novel direction for future research in HCC biology.
Earlier studies have indicated that SOX2 is highly expressed in various malignant tumors and is closely associated with poor prognosis, chemoresistance, and the maintenance of cancer stemness in patients.[23-25] In HCC, existing literature has indicated that SOX2 is involved in tumorigenesis and immune evasion.[26] However, whether SOX2 promotes tumor progression by regulating mitochondrial homeostasis and metabolism-related pathways remains largely unexplored. The present study is the first to demonstrate that SOX2 can promote HCC cell survival by modulating autophagy and mitophagy, providing new insights into its oncogenic mechanism.
Autophagy and mitophagy play critical roles in maintaining cellular homeostasis, particularly under the conditions of increased metabolic stress in tumor cells, where they supply essential resources for energy production and structural remodeling. Cancer cells often activate these processes to enhance their adaptability to harsh environments.[27] The present study found that SOX2 overexpression significantly increased the LC3II/LC3I and p62 levels, attenuated the number of autophagosomes and autolysosomes, and enhanced the colocalization of the mitochondria and lysosomes. These findings suggest that SOX2 can simultaneously enhance global autophagic activity and selective mitochondrial degradation.
FOXJ3 has been reported to regulate mitochondrial gene expression, maintain oxidative phosphorylation, and potentially exert tumor-suppressive functions in various cancers.[17] More importantly, reintroduction of FOXJ3 significantly reversed SOX2-induced enhancement of autophagy, mitophagy, and malignant phenotypes in HCC cells, suggesting that FOXJ3 functions as a critical downstream effector in the SOX2 signaling axis. This study showed that the SOX2 mRNA expression in the Oe-SOX2 + Oe-FOXJ3 group was significantly reduced compared with that in the Oe-SOX2 group. Although this finding does not alter the overall conclusion regarding the promotive effects of SOX2 on HCC progression, it may indicate the presence of a feedback regulatory mechanism within the SOX2-FOXJ3 axis.
Moreover, SOX2 significantly activates the PI3K/ AKT pathway, which is widely recognized in HCC as a classical oncogenic signaling cascade that promotes cell proliferation, inhibits apoptosis, and reprograms cellular metabolism.[28] Overexpression of FOXJ3 markedly suppressed SOX2-induced phosphorylation of PI3K and AKT, suggesting that FOXJ3 acts as a negative regulator of this pathway. This observation agrees with prior research in which members of the FOX transcription factor family were shown to restrain tumor progression by modulating the PI3K/AKT signaling pathway.[29]
From a clinical perspective, the potential SOX2-FOXJ3-PI3K/AKT regulatory axis identified in this study holds significant translational potential. SOX2 may serve not only as a prognostic biomarker for HCC but also as a promising therapeutic target. Targeting SOX2 inhibition or restoring FOXJ3 expression could open new avenues for HCC treatment, especially in combination with PI3K/AKT pathway inhibitors.
Certainly, this study has some limitations. First, the results were mainly derived from in vitro experiments with HCC cell lines, and additional validation using animal models and patient tissue samples is required to establish their physiological significance. Second, the experiments mainly utilized the Huh7 cell line, and whether similar results can be observed across other HCC subtypes remains to be determined. Moreover, whether SOX2 also regulates HCC progression through other pathways is unclear, thus warranting further mechanistic investigation. Future studies could explore whether SOX2-mediated regulation of mitophagy is linked to metabolic reprogramming. Although this study suggests a negative regulatory relationship between SOX2 and FOXJ3 through gain- and loss-of-function experiments and protein expression analyses, direct molecular evidence is still lacking. Experiments, such as dual-luciferase reporter assays, ChIP-qPCR, or EMSA, have not yet been conducted to verify whether SOX2 directly binds to the FOXJ3 promoter region. Future studies are needed to confirm the binding sites and elucidate the transcriptional regulatory mechanisms involved. Although this study demonstrated that SOX2 can regulate autophagy and mitophagy, metabolic assays were not performed to directly link changes in mitophagy with HCC cell survival, proliferation, or energy metabolism. Future studies should include metabolic analyses to better understand the functional consequences of mitophagy regulation.
SUMMARY
This study systematically reveals the molecular basis by which SOX2 promotes the malignant phenotype of HCC cells through downregulation of FOXJ3 expression, activation of the PI3K/AKT signaling pathway, and enhancement of autophagy and mitophagy. The findings suggest a potential SOX2-FOXJ3 regulatory axis that may act as a critical hub connecting transcriptional regulation, mitochondrial quality control, and metabolic pathways. While the precise role of FOXJ3 as a tumor suppressor in HCC requires further validation, this axis provides a new theoretical foundation and a potential target for future investigation and therapeutic development in HCC.
AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are available from the corresponding author upon reasonable request.
ABBREVIATIONS
FOXJ3: Forkhead box J3
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase
HCC: Hepatocellular carcinoma
qRT-PCR: Quantitative reverse transcription polymerase chain reaction
SOX2: SRY-box transcription factor 2.
AUTHOR CONTRIBUTIONS
ZGZ and WHR: Designed the research study, performed the research, and have been involved in drafting the manuscript; WHR collected and analyzed the data. Both authors have been involved in revising it critically for important intellectual content. All authors give final approval of the version to be published. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity are addressed. Both authors are eligible for ICMJE authorship.
ACKNOWLEDGMENT
Not applicable.
ETHICS APPROVAL
Not applicable.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
EDITORIAL/PEER REVIEW
To ensure the integrity and highest quality of CytoJournal publications, the review process of this manuscript was conducted under a double-blind model (authors are blinded from reviewers and vice versa) through an automatic online system.
FUNDING: Not applicable.
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