SLC15A4 promotes oral cancer progression by regulating TLR9 and activating the JAK/STAT signaling pathway

Abstract

Background

Oral cancer continues to pose a major health problem worldwide, characterized by few treatment options and an unfavorable prognosis. The solute carrier family 15 member 4 (SLC15A4) has been reported to be associated with immune responses regulation and tumor progression. This study purpose was to reveal the role of SLC15A4 in regulating Toll-like receptor 9 (TLR9) activation and the subsequent JAK/STAT signaling pathway in the context of oral cancer progression.

Methods

Clinical samples from 38 oral cancer patients were analyzed for SLC15A4 and TLR9 expression using qPCR, Western blot, and immunohistochemistry (IHC). In vivo validation was performed using a xenograft mouse model. CAL27 and FaDu cell lines were used to study the effects of SLC15A4 knockdown on cell proliferation, migration, invasion, and inflammatory response via CCK8, ELISA, and transwell assays. SLC15A knockout and TLR9 overexpression were detected to explore their relationship with JAK/STAT signaling by detecting subcellular localization, JAK/STAT1 activation, cell invasion, migration, proliferation and inflammatory responses. The JAK/STAT agonist RO8191 was used to assess the impact of pathway activation on cell behavior.

Results

SLC15A4 was overexpressed in both tissue and cell line of oral cancer. Knockdown of SLC15A4 significantly reduced cell proliferation, migration, invasion, and inflammatory cytokine secretion (IL-6, IL-1β, TNF-α). Both STAT1 and STAT3 phosphorylation were suppressed, and JAK/STAT pathway activation restored these phenotypes. SLC15A4 knockdown also decreased TLR9 expression in cells. TLR9 overexpression restored the effects of SLC15A4 knockdown. TLR9 was shown to activate the JAK/STAT signaling pathway in oral cancer cells. Finally, activation of the JAK/STAT pathway reversed the effects of SLC15A4 knockdown.

Conclusion

SLC15A4 is essential in promoting oral cancer progression by regulating TLR9 activation and subsequent JAK/STAT signaling. This regulatory pathway influences cell proliferation, migration, invasion, and inflammation, highlighting SLC15A4 as a potential therapeutic target in treatment of oral cancer.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13005-026-00636-8.

Highlights

SLC15A4 is highly expressed in oral cancer tissues and cell lines.

Knockdown of SLC15A4 suppresses proliferation, migration, invasion, and inflammatory cytokine secretion (IL-6, IL-1β, TNF-α).

SLC15A4 is associated with TLR9 expression.

SLC15A4 promotes OSCC progression via TLR9-mediated activation of JAK/STAT signaling (STAT1 and STAT3).

Supplementary Information

The online version contains supplementary material available at 10.1186/s13005-026-00636-8.

Introduction

Malignancy of the oral cancer rank among the most frequently diagnosed cancers globally. There were about 377,000 new patients and 177,000 patients died in 2020, as reported [1]. The main standard of care for oral cancer consists of chemotherapy, radiotherapy, and surgery, which can be administered singly or in combination. Unfortunately, these therapeutic approaches are frequently associated with undesirable consequences, including injury to normal tissues, oropharyngeal dysfunction, and deterioration in patients’ daily living [1, 2].

The solute carrier family 15 member 4 (SLC15A4), encoded by gene SLC15A, exhibits pH-dependent oligopeptide transporter activity [3]. The molecule is known to be involved in TLR7-9 signal transmission and contributes to inflammatory responses through IFN-I cytokine secretion [4]. Pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α play critical roles in shaping the tumor microenvironment of oral cancer. These cytokines promote chronic inflammation, enhance tumor cell proliferation, and facilitate immune evasion, contributing to poor prognosis [5]. Elevated levels of IL-6 and TNF-α have been associated with increased invasiveness and metastatic potential in oral cancer patients [6, 7]. There is an unmet need to develop new therapies because current treatment options are limited, which requires understand the molecular mechanisms driving oral cancer progression.

SLC15A4 overexpression has been observed in many cancers, including colorectal [8] and lung cancers [3]. Our analysis of the TCGA database using GEPIA 2 indicated higher SLC15A4 expression in head and neck tumors (Fig. 1A). However, the regulatory mechanisms of SLC15A4 in oral cancer remain unexplored, presenting a novel area of investigation.

Fig. 1.

SLC15A4 overexpression in oral cancer tissues and cell lines. A TCGA analysis of SLC15A4 expression in oral cancer. The expression levels in oral cancer tissues (n=519) were compared to adjacent normal tissues (n=44). B qPCR detection of SLC15A4 expression in clinical samples (n=38, t=15.030). C Western blot and D immunohistochemistry validation of SLC15A4 expression in OSCC tissues versus adjacent normal tissues (t=5.325). E qPCR detection of SLC15A4 expression in oral cancer cell lines (n=3, F=54.600). F Western blot analysis of SLC15A4 protein expression in oral cancer cell lines (n=3, F=11.68). Statistical analysis: Unpaired t-test for clinical sample comparisons; one-way ANOVA with Tukey’s post-hoc test for multiple cell line comparisons. *, P<0.05; **, P<0.01; ***, P<0.001

SLC15A4 is implicated in the activation of Toll-like receptors (TLRs) within lysosomes, particularly TLR9 [9]. TLRs are essential for recognizing pathogens and triggering immune responses [9]. The protein SLC15A4 is a critical component of the lysosomes TLR machinery involved in immune regulation. The absence of SLC15A4 specifically disrupts the TLR pathway, establishing it as a key adaptor for TLR7, TLR8, and TLR9 in mediating immune responses [4]. The interaction between SLC15A4 and TLR9 were extremely important for inflammatory response in tumor microenvironments. Multiple studies have demonstrated that SLC15A4 regulates cellular inflammatory responses through TLR9 [9]. Elevated TLR9 levels are linked to a poor prognosis in oral cancer patients and may promote the progression of oral cancer. A study involving 30 oral squamous cell carcinoma (OSCC) patients found that those with high TLR9 levels had a decreased overall survival rate. The analysis also revealed that high TLR9 expression may contribute to a pathogenic loop influencing disease progression through altered immune responses and tumor promotion [10]. In OSCC, TLR9 is highly expressed and its suppression significantly inhibits invasion, proliferation and migration. Furthermore, TLR9 promotes tumor progression and immune escape by regulating the PARP1/STAT3 pathway and the expression of PD-L1, contributing to immunosuppression and tumorigenesis [11]. The activation of TLR9 is known to influence cancer progression, particularly through its role in the inflammatory response and metastasis in a lot of types of cancer[ 12, 13].

Cell fate decisions involving proliferation, differentiation, and survival are tightly controlled through Janus kinase/signal transducer and activator of transcription (JAK-STAT) mediated signaling events [14, 15]. Signal transducer and activator of transcription 3 and 1(STAT3 or STAT1) serve as a pivotal regulator of immune function, mediating anti-tumor immunity while demonstrating constitutive activation across malignant and stromal cell populations in the tumor microenvironment. In the context of oral cancer, it was reported that STAT3 hyperactivation is usually observed with higher tumor aggression and those patients would harbor worse prognosis. STAT3 inhibits immune activation and promotes immunosuppression, making it a promising target for cancer therapy [16]. JAK/STAT pathway activation is widely observed in different types of cancers, for instance, prostate, non-small cell lung, and oral cancers [15, 17]. Previous publications and data suggest that sustained activation of STATs could promote tumorigenesis by regulating the expression of numerous genes that were involved in the regulation of for tumor metastasis, invasion, proliferation and survival. For instance, flavopereirine has shown promise as an anti-cancer agent in oral cancer by significantly inhibiting tumor progression. The mechanism involves suppression of JAK/STAT signaling through inhibition of STAT3 phosphorylation, a critical mediator of both tumor progression and immune modulation [17]. Research indicates TLR9 can activate the JAK/STAT cascade via phosphorylation [18]. Therefore, it is pertinent to investigate whether SLC15A4 was associated with regulation of the phosphorylation of JAK/STAT pathway mediated by TLR9 in oral cancer progression. However, the interplay between SLC15A4-mediated immune regulation and inflammatory cytokines in OSCC remains poorly understood.

In summary, we hypothesize that in oral tumors, high SLC15A4 expression promotes tumor progression and inflammatory cytokine production (IL-6, IL-1β, TNF-α) by upregulating TLR9 and activating the JAK/STAT signaling pathway, including STAT1 and STAT3. Our study targeted to find out mechanisms of this regulatory pathway, potentially identifying novel therapeutic targets for oral cancer treatment. Understanding this pathway could not only shed light on the mechanisms of oral cancer progression but also provide information supporting novel therapy development.

Materials and methods

Clinical sample

We collected the clinical samples from 38 oral cancer patients at the hospital. During surgical resection, paired samples of tumor and normal adjacent tissue were acquired, promptly frozen in liquid nitrogen, and stored at -80 °C. Ethics Committee of the hospital approved the study. All participants signed informed consents. According to the median expression of SLC15A4, patients were divided into SLC15A4 high expression patients and SLC15A4 low expression patients. Detailed clinical parameters are provided in Table 1.

Table 1.

Correlation between SLC15A4 expression and clinicopathological parameters of OSCC patients

Clinical parameters	SLC15A4 expression level		P value
	Low (19)	High (19)
Sex
Male	10	11	0.4698
Femal	9	8
Age (year)
≤ 57	9	6	0.0752
> 57	10	13
Site
Tongue	7	7	0.965
Gingiva	8	7
Other	4	5
Differentiation
Well	9	9	0.7226
Moderate	5	6
Poor	5	4
TNM stage
I	9	2	0.0108*
II	4	5
III	5	4
IV	1	8

*=significant differences

In vivo xenograft assay

4–6-week-old female BALB/c nude mice (approximately 20 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. Randomly divide the mice into groups (sh-NC, sh-SLC15A4, sh-SLC15A4 + RO8191), with 5 mice in each group, and inject corresponding CAL27 (5 × 10⁶) cells subcutaneously. RO8191(2 mg/kg/day, beyotime, Y061799) was administered intraperitoneally every day after subcutaneous tumor injection. The assessment of tumor volume commenced on day 8 post-injection. Checking tumor growth every four days. After the measurement on the 36th day, the nude mice were euthanized using carbon dioxide anesthesia, and the tumors were removed and photographed. Then, the tumor tissue was embedded and sliced in paraffin, and the expression levels of SLC15A4 and Ki67 were determined by immunohistochemistry. The Ethics Committee approved all animal experiments.

Cell sources, transfection, and treatment

The human oral cell lines used in this study: normal epithelial cells (HOEC, BFN6072012669, BLUEFBIO) and cancer lines: CAL27 (CRL-2095, ATCC, RRID: CVCL_1107), HSC3 (SNL-624, Sunncell, RRID: CVCL_1288), FaDu (HTB-43, ATCC, RRID: CVCL_1218), UM-SCC-1 (CL-0907, Procell, RRID: CVCL_7707), CA9-22 (ZY-H351, Zeye Biotechnology, RRID: CVCL_1102). All cell lines were grown in DMEM containing 10% FBS and 1% antibiotics, incubated at 37 °C with 5% CO₂ in a humidified chamber.

For transfection, cells were cultured to 70–80% confluence. Lipofectamine 2000 (Invitrogen) was used for SLC15A4 knockdown (sh-SLC15A4), TLR9 knockdown (sh-TLR9) or TLR9 overexpression (oe-TLR9) plasmids transfection according to the manufacturer’s protocol. Plasmids were obtained from GenePharma (Shanghai). After 48 h, transfected cells were treated with RO8191 (beyotime, Y061799), a JAK/STAT agonist, at a concentration of 10 µM for 24 h.

Western blot

Cell lysates were prepared using RIPA buffer supplemented with protease inhibitors, with lysis performed on ice for 30 min. Following centrifugation (12,000 × g, 15 min, 4 °C), supernatants were collected and protein concentrations determined via BCA assay (Thermo Fisher). Equal amounts (20 µg) of protein were resolved by 10% SDS-PAGE and electrotransferred to PVDF membranes. After blocking with 5% non-fat milk/TBST (1 h, RT), membranes were probed overnight at 4 °C with primary antibodies: anti-SLC15A4 (0.2 µg/mL, Invitrogen PA5-42513), anti-TLR9 (1:1000, Cell Signaling Technology, CST13674), anti-STAT1 (1:1000, Cell Signaling Technology, CST14994), anti-p-STAT1 (1:1000, Cell Signaling Technology, CST9167), anti-STAT3 (1:1000, Cell Signaling Technology, CST9139), anti-p-STAT3 (1:1000, Cell Signaling Technology, CST9145), anti-JAK (1:1000, Cell Signaling Technology, CST3331), anti-p-JAK (1:1000, Cell Signaling Technology, CST3332), and anti-β-actin (1:5000, Sigma-Aldrich). Following TBST washes, membranes were incubated with HRP-conjugated secondary antibodies (1:5000, Jackson ImmunoResearch) for 1 h at RT. Protein bands were visualized using ECL substrate (Bio-Rad) after final washes.

Co‑immunoprecipitation (Co‑IP)

Co‑immunoprecipitation assays were performed to examine potential protein–protein interactions as indicated. Briefly, cells were lysed in IP lysis buffer supplemented with protease inhibitors, and the lysates were clarified by centrifugation at 12,000 × g for 15 min at 4 °C. Equal amounts of total protein were incubated with anti-SLC15A4 or normal IgG (as a negative control) overnight at 4 °C with gentle rotation. Subsequently, Protein A/G agarose beads were added and incubated for an additional 2–4 h at 4 °C. The immunocomplexes were then washed extensively with lysis buffer, eluted by boiling in SDS loading buffer, and subjected to SDS–PAGE followed by Western blot analysis using the indicated antibodies.

Quantitative Real-Time PCR (qPCR)

Cellular RNA was isolated using TRIzol reagent (Invitrogen) following the recommended protocol. The quality and integrity were assessed using a NanoDrop spectrophotometer. Reverse transcription was conducted using PrimeScript RT reagents (TaKaRa). SYBR Green-based quantitative PCR (Applied Biosystems) was performed under these conditions: 40 cycles (95°C 15 sec, 60°C 1 min) after initial 95°C activation for 10 min. Expression levels were normalized to GAPDH. Primer sequence (5’-3’): SLC15A4-F-TTGTGGTCTTCCTCTGTGGC, SLC15A4-R-TCCACTTCGCTTCTGGGAAC; TLR7-F-CACAGCCGTCCCTACTGTTT, TLR7-R-TTTTTACACGGCGCACAAGG; TLR8-F-CACATGTGCCACCCAAACTG, TLR8-R-TGAAGCACCTCGGACAGTTC; TLR9-F-GCAGACTGGGTGTACAACGA, TLR9-R-CAAAGAGGGTTTTGCCAGGC; GAPDH-F-GATTTGGTCGTATTGGGCGC, GAPDH-R-TTCCCGTTCTCAGCCTTGAC.

Cell counting kit-8 (CCK8) assay

To perform CCK8 assay (Beyotime) and determine cell proliferation, we seeded cells in 96-well plates and incubated overnight. The density was 5 × 10³ cells per well. At 24, 48, and 72 h, we added 10 µL of CCK8 solution to each well, and the plates were then incubated for another two hours. Absorbance at 450 nm was detected. The background absorbance of wells containing only medium and CCK8 solution was subtracted from the sample readings.

Transwell assay

For migration assays, cells were serum-starved for 24 h and then harvested. 50,000 cells suspended in 200 µL serum-free medium were loaded into Corning Transwell inserts (8 μm pores). The chemoattractant gradient was established using 600 µL of 10% FBS-supplemented medium in the lower chamber. Following a 24 h incubation period, cells that did not migrated and remained on the upper membrane side were gently wiped away using a cotton swab. whereas transmigrated cells were fixed (4% PFA, 15 min), stained (0.1% crystal violet, 20 min), and manually counted in five random microscopic fields per well.

For invasion analysis, transwell inserts were pre-coated with 50 µL of Matrigel (BD Biosciences, 1:8 dilution in serum-free medium) and incubated at 37 °C for 2 h to form a barrier. Cells were then seeded in the upper chamber. The chemoattractant gradient was established using 600 µL of 10% FBS-supplemented medium in the lower chamber. Following a 24 h incubation period, cells that did not migrated and remained on the upper membrane side were gently wiped away using a cotton swab. whereas transmigrated cells were fixed (4% PFA, 15 min), stained (0.1% crystal violet, 20 min), and manually counted in five random microscopic fields per well.

Immunofluorescence and immunohistochemistry

For immunofluorescence analysis, cells were grown overnight on coverslips in 24-well plates. Subsequent processing included: fixation (4% PFA, 15 min), permeabilization (0.1% Triton X-100, 10 min), and blocking (5% BSA, 1 h). Cells were then incubated with primary antibodies against TLR9 (1:1000, Cell Signaling Technology #13674) overnight at 4 °C. Primary antibody detection was performed using Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Fisher R37117, 1 h), followed by DAPI nuclear staining (5 min). Mounted specimens were examined with a Zeiss LSM 710 confocal imaging system.

For immunohistochemical detection, clinical or mouse tissue sections are boiled in citric acid solution after dewaxing and rehydration to recover antigens. Then incubate with peroxidase inhibitor at room temperature for 10 min. Apply primary antibody and incubate overnight at 4 °C, then incubate with enzyme labeled secondary antibody at 37 °C for 20 min. Finally, DAB and hematoxylin staining were performed and observed under a microscope for recording. Primary antibody: anti-SLC15A4 (5 µg/mL, Invitrogen, PA5-42513), anti-Ki67 (1:20, Cell Signaling Technology, CST34330).

Enzyme-Linked Immunosorbent Assay (ELISA)

The assessment of pro-inflammatory cytokines, including TNF-alpha and IL-1beta, IL-6 was performed by ELISA (R&D Systems kits) on collected culture supernatants, with readings obtained at 450 nm as specified in the manufacturer’s guidelines. The concentrations of cytokines were determined using a standard curve.

Statistical analysis

Triplicate measurements were performed for all experiments, with results shown as mean ± standard deviation. GraphPad Prism was employed for all statistical computations. Intergroup differences were evaluated using either unpaired t-tests (two groups) or one-way ANOVA with Tukey’s multiple comparisons test (≥ 3 groups). Full reporting includes F-values, degrees of freedom, exact p-values. Data were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

SLC15A4 overexpression in oral cancer tissues and cell lines

Initial investigation of SLC15A4 expression patterns in oral cancer was performed using TCGA dataset analysis. As shown in Fig. 1A, a significant upregulation of SLC15A4 mRNA and protein levels, validated by qPCR, western blot, and immunohistochemistry was observed in tumor tissues relative to matched normal controls. Consistent with the TCGA data, a significant increase in SLC15A4 mRNA expression in oral cancer tissues was also observed on clinical samples from 38 oral cancer patients and their corresponding adjacent normal tissues (Fig. 1B, C, D; Table 1).

Next, we compared SLC15A4 mRNA levels in normal oral epithelial cells (HOEC) with those in various oral cancer cell lines (CAL27, HSC3, FaDu, UM-SCC-1, CA9-22). Our results indicated that SLC15A4 was overexpressed in all tested oral cancer cell lines compared to HOEC, with the highest expression observed in CAL27 and FaDu cell lines (Fig. 1E). Consistent with the mRNA data, Western blot results demonstrated elevated SLC15A4 protein levels in oral cancer cell lines, particularly in CAL27 and FaDu cells, compared to HOEC (Fig. 1F). These results collectively indicate that SLC15A4 is overexpressed in both oral cancer tissues and cell lines, suggesting a potential role for SLC15A4 in the pathogenesis of oral cancer.

SLC15A4 knockdown reduces proliferation, migration, invasion, and inflammation in oral cancer cells

To explore the functional impact of SLC15A4 in oral cancer cells, we knocked down SLC15A4 in CAL27 and FaDu cells. A significant reduction in SLC15A4 expression in cells transfected with sh-SLC15A4 compared to the control and sh-NC groups were observed (Fig. 2A). Subsequently, SLC15A4 knockdown resulted in a significant decrease in cell viability in both CAL27 and FaDu cells (Fig. 2B). To evaluate the inflammatory response, we measured the levels of inflammatory cytokines (IL-6, IL-1β, TNF-α) in the cell culture supernatant. Our results showed that SLC15A4 knockdown led to a reduction in the inflammatory response (Fig. 2C). Furthermore, SLC15A4 knockdown significantly reduced both migration and invasion in CAL27 and FaDu cells. These findings suggest that SLC15A4 plays a crucial role in promoting proliferation, migration, invasion, and inflammation in oral cancer cells.

Fig. 2.

SLC15A4 knockdown reduces proliferation, migration, invasion, and inflammation in oral cancer cells. A Western blot and qPCR analysis of SLC15A4 in CAL27 (F=66.450) and FaDu (F=32.400) cells. B CCK-8 analysis of cell viability in CAL27 (F=8.484) and FaDu (F=12.920) cells. C ELISA detection of inflammatory cytokines in supernatant of CAL27 (F=19.240, F=29.940, F=30.500) and FaDu (F=154.900, F=19.770, F=30.600) cells. D Transwell analysis of cell migration and invasion in CAL27 (F=93.330, F=147.400) and FaDu (F=262.900, F=85.240) cells. **, P<0.01; ***, P<0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for comparisons among control, sh-NC, and sh-SLC15A4 groups. Data were presented as mean ± SD. F-values and p-values were reported. n=3 for cell-based experiments

SLC15A4 knockdown decreases TLR9 expression in oral cancer cells and tissues

To investigate the relationship between SLC15A4 and TLR9, we performed SLC15A4 knockdown on CAL27 and FaDu cells, which significantly reduced TLR9 mRNA and protein expression, while the expression of TLR7 and TLR8 remained unaffected (Fig. 3A and B). To validate these findings in clinical samples, we analyzed TLR9 expression in oral cancer tissues with high and low SLC15A4 expression (Fig. 3C and D). TLR9 expression was significantly higher in tissues with high SLC15A4 expression compared to those with low SLC15A4 expression and adjacent normal tissues. These results demonstrate that SLC15A4 is associated with TLR9 expression in oral cancer cells and tissues. Additionally, Co-IP assays revealed no direct physical interaction between SLC15A4 and TLR9 or NOD1 (Figure S1A, S1B). Overexpression of SLC15A4 upregulated TLR9 expression, which was reversed by NOD1 knockdown, indicating that SLC15A4 may regulate TLR9 via a NOD1-dependent pathway (Figure S1C).

Fig. 3.

SLC15A4 knockdown decreases TLR9 expression in oral cancer cells and tissues. A qPCR analysis of TLR7, TLR8, and TLR9 in CAL27 (F=3.418, F=1.564, F=30.810) and FaDu (F=3.255, F=0.766, F=34.110) cells. B Western blot analysis of TLR9 in CAL27 (F=69.070) and FaDu (F=15.040) cells. C qPCR analysis of TLR9 in adjacent normal tissues, SLC15A4 high expression tissues, and SLC15A4 low expression tissues (n=38, F=140.800). D Western blot analysis of TLR9 in adjacent normal tissues, SLC15A4 high expression tissues, and SLC15A4 low expression tissues (F=26.530). *, P<0.05; **, P<0.01; ***, P<0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for three-group comparisons. Data were presented as mean ± SD. F-values or t-values and p-values were reported. n=3 for cell-based experiments; n=38 for clinical sample

SLC15A4 regulates proliferation, migration, invasion, and inflammation via TLR9 in oral cancer cells

To further elucidate the mechanism by which SLC15A4 influences oral cancer cell behavior, we conducted experiments to assess the role of SLC15A4/TLR9 axis. SLC15A4 knockdown reduced both SLC15A4 and TLR9 expression, while TLR9 overexpression did not affect SLC15A4 expression (Fig. 4A and B). In addition, SLC15A4 knockdown decreased cell viability, reduced the inflammatory response, decreased cell migration and invasion, and these effects were all reversed by TLR9 overexpression (Fig. 4C, D, E). These findings indicate that SLC15A4 regulates proliferation, migration, invasion, and inflammation in oral cancer cells through its effect on TLR9.

Fig. 4.

SLC15A4 regulates proliferation, migration, invasion, and inflammation via TLR9 in oral cancer cells. A qPCR analysis of SLC15A4 and TLR9 in CAL27 (F=20.230, F=14.260) and FaDu (F=39.680, F=22.790) cells. B Western blot analysis of SLC15A4 and TLR9 in CAL27 (F=42.760, F=11.870) and FaDu (F=26.120, F=16.090) cells. C CCK-8 analysis of cell viability in CAL27 (F=10.800) and FaDu (F=10.850) cells. D ELISA detection of inflammatory cytokines in supernatant of CAL27 (F=388.400, F=132.300, F=110.500) and FaDu (F=39.940, F=225.900, F=66.920) cells. E Transwell analysis of cell migration and invasion in CAL27 (F=57.000, F=139.000) and FaDu (F=415.500, F=56.330) cells. *, P<0.05; **, P<0.01; ***, P<0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for comparisons among control, sh-SLC15A4, and sh-SLC15A4 + TLR9 overexpression groups. Data were presented as mean ± SD. F-values and p-values were reported. n=3 for cell-based experiments

TLR9 activates the JAK/STAT signaling pathway in oral cancer cells

TLR9 is primarily expressed in the cytoplasm in CAL27 cells (Fig. 5A). Knockdown of TLR9 inhibited the phosphorylation of JAK/STAT1 and STAT3, while overexpression of TLR9 promoted their activation (Fig. 5B). These results demonstrate that TLR9 plays a critical role in activating the JAK/STAT1 and STAT3 signaling pathway in oral cancer cells.

Fig. 5.

TLR9 activates the JAK/STAT signaling pathway in oral cancer cells. A Immunofluorescence detection of TLR9 in CAL27 cells. B Western blot analysis of TLR9 and the phosphorylation status of JAK/STAT1 and STAT3 in CAL27 cells (F=16.670, F=40.430, F=18.950, F=29.330). *, P<0.05; **, P<0.01; ***, P<0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for comparisons among sh-NC, sh-TLR9, oe-NC, and oe-TLR9 groups. Data were presented as mean ± SD. F-values and p-values were reported. n=3 for cell-based experiments

SLC15A4 influences oral cancer cell proliferation, migration, invasion, and inflammation through TLR9 and JAK/STAT pathway activation

To comprehensively understand how SLC15A4 affects oral cancer cell behavior, we treated CAL27 and FaDu cells with the JAK/STAT agonist (RO8191). SLC15A4 knockdown reduced SLC15A4 and TLR9 expression and inhibited JAK/STAT1 activation. Treatment with RO8191 did not affect SLC15A4 or TLR9 expression but activated the JAK/STAT1 and STAT3 pathway (Fig. 6A and B). SLC15A4 knockdown decreased cell viability, inflammatory response, migration and invasion which were all restored upon activation of the JAK/STAT1 and STAT3 pathway by RO8191 (Fig. 6C, D E). These findings suggest that SLC15A4 influences oral cancer cell proliferation, migration, invasion, and inflammation through TLR9-mediated activation of the JAK/STAT1 and STAT3 signaling pathway.

Fig. 6.

SLC15A4 influences oral cancer cell proliferation, migration, invasion, and inflammation through TLR9 and JAK/STAT pathway activation. A qPCR analysis of SLC15A4 and TLR9 in CAL27 (F=12.160, F=15.680) and FaDu (F=20.540, F=24.550) cells. B Western blot analysis of SLC15A4, TLR9, and the phosphorylation status of JAK/STAT1 and STAT3 in CAL27 (F=16.750, F=11.810, F=9.185, F=16.610, F=12.870) and FaDu (F=12.920, F=26.230, F=12.240, F=18.810, F=19.830) cells. C CCK-8 analysis of cell viability in CAL27 (F=3.637) and FaDu (F=4.066) cells. D ELISA detection of inflammatory cytokines in supernatant of CAL27 (F=24.080, F=23.230, F=136.700) and FaDu (F=55.050, F=15.270, F=23.930) cells. E Transwell analysis of cell migration and invasion in CAL27 (F=17.960, F=111.900) and FaDu (F=148.200, F=73.940) cells. *, P<0.05; **, P<0.01; ***, P<0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for comparisons among control, sh-SLC15A4, and sh-SLC15A4 + RO8191 groups. Data were presented as mean ± SD. F-values and p-values were reported. n=3 for cell-based experiments

SLC15A4 activates the JAK/STAT signaling pathway through TLR9 to promote the growth of oral cancer xenografts in mice

To further validate the role of SLC15A4 in oral cancer progression, we performed in vivo experiments using a xenograft mouse model. As shown in Fig. 7A and B, knockdown of SLC15A4 markedly suppressed tumor growth, resulting in significantly smaller tumor volumes and weights compared to the control group (p < 0.001). In contrast, activation of the JAK/STAT pathway by RO8191 partially restored tumor growth, with a statistically significant difference observed between the sh-SLC15A4 and sh-SLC15A4 + RO8191 groups (p = 0.0247), indicating that JAK/STAT signaling mediates the oncogenic effects of SLC15A4. Western blot analysis of tumor tissues revealed that SLC15A4 knockdown reduced the expression of SLC15A4 and TLR9, as well as the phosphorylation of JAK, STAT1, and STAT3. Treatment with RO8191 did not alter SLC15A4 or TLR9 expression but significantly increased phosphorylation of JAK, STAT1, and STAT3, confirming pathway activation (Fig. 7C). Immunohistochemistry further demonstrated decreased SLC15A4 and Ki67 expression in tumors derived from sh-SLC15A4 cells, whereas RO8191 treatment restored Ki67 levels, consistent with enhanced proliferative activity (Fig. 7D). Collectively, these in vivo findings corroborate our in vitro results, supporting the conclusion that SLC15A4 promotes oral cancer progression through TLR9-mediated activation of the JAK/STAT signaling pathway.

Fig. 7.

In vivo validation of SLC15A4 function in oral cancer progression. A Images and weight of xenograft tumors (F = 25.350). B Tumor growth curves in xenograft mouse model (sh-NC, sh-SLC15A4, sh-SLC15A4 + RO8191 groups) (F = 58.820). C Western blot analysis of SLC15A4, TLR9, and JAK/STAT pathway components in tumor tissues (F = 12.070, F = 27.440, F = 21.600, F = 17.120, F = 25.060). D Immunohistochemistry detection of SLC15A4 and Ki67 expression in xenograft tumors. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analysis: One-way ANOVA with Tukey’s post-hoc test for comparisons among three experimental groups. Data were presented as mean ± SD. F-values and p-values were reported. n = 5 for animal experiments

Discussion

The role of SLC15A4 in oral cancer has been increasingly recognized due to its involvement in immune responses and tumor progression [19, 20]. Our study aimed to elucidate the specific mechanisms through which SLC15A4 influences oral cancer development. Our main findings include the overexpression of SLC15A4 in oral cancer tissues and cell lines, its impact on cell proliferation, migration, invasion, and inflammation, and its regulation of TLR9 and the JAK/STAT signaling pathway. Our findings indicate that SLC15A4 knockdown significantly reduced IL-6, IL-1β, and TNF-α secretion, suggesting that the SLC15A4–TLR9–JAK/STAT axis may regulate inflammatory signaling in OSCC. These cytokines have been reported to activate downstream signaling pathways associated with tumor growth and immune suppression, including STAT3-mediated mechanisms [5–7].

Our results demonstrated that SLC15A4 is overexpressed in both oral cancer tissues and cell lines (Fig. 1), suggesting its potential as a biomarker for oral cancer. Previous studies have also reported the overexpression of SLC15A4 in various cancers, including colorectal and lung cancers [3, 8], which is consistent with our findings in oral cancer. However, the role of SLC15A4 in oral cancer has not been extensively explored, highlighting the novelty of our research. The potential mechanisms by which SLC15A4 influences inflammation in oral cancer could involve other pathways or molecular interactions, which warrant further investigation. Furthermore, the knockdown of SLC15A4 significantly reduced cell proliferation, migration, invasion, and inflammation in CAL27 and FaDu cells (Fig. 2). These findings underscore the critical role of SLC15A4 in promoting the aggressive behavior of oral cancer cells, highlighting its potential as a therapeutic target. While our results align with previous research showing that SLC15A4 influences prognosis in other cancer types [3, 21, 22], our study uniquely demonstrates its impact on inflammation in oral cancer, suggesting a broader role for SLC15A4 in cancer progression.

Our investigation extended to understanding how SLC15A4 affects oral cancer through the regulation of TLR9. We found that SLC15A4 knockdown in oral cancer cells led to a significant decrease in TLR9 expression (Fig. 3), indicating a direct regulatory relationship between these two proteins. This finding is consistent with previous reports that SLC15A4 is involved in the regulation of TLR9-mediated signaling 4, reinforcing the notion that SLC15A4 plays a pivotal role in modulating immune responses in cancer [23]. This relationship was further validated in clinical samples, where high SLC15A4 expression correlated with increased TLR9 levels. SLC15A4 may regulate TLR9 through lysosomal trafficking, as reported in previous studies [9]. The solute carrier family 15A4 regulates TLR9 and NOD1 functions in the innate immune system and promotes colitis in mice [23]. We demonstrated that overexpression of SLC15A4 significantly increased both the mRNA and protein levels of TLR9. Importantly, this upregulation was effectively abolished by NOD1 knockdown, indicating that NOD1 is required for SLC15A4-mediated TLR9 induction. Mechanistically, SLC15A4, an endolysosomal transporter, is known to export microbial- or tumor-derived metabolites into the cytosol, thereby activating NOD1. Our rescue experiments strongly support a model in which SLC15A4-driven functional activation of NOD1 in OSCC cells initiates a downstream pro‑inflammatory transcriptional program, likely mediated through NF‑κB, which subsequently enhances transcription of the TLR9 gene. Thus, we speculate that SLC15A4 does not exert its effects through direct physical interaction with TLR9, but rather functions upstream to establish a NOD1-dependent feed‑forward transcriptional loop. This crosstalk between the cytosolic NOD1 axis and the endosomal TLR9 pathway ensures sustained and amplified tumor‑promoting inflammatory signaling, which is critical for OSCC progression and survival.

Moreover, our experiments demonstrated that the effects of SLC15A4 knockdown on cell proliferation, migration, invasion, and inflammation could be reversed by TLR9 overexpression (Fig. 4). These results suggest that SLC15A4 influences oral cancer progression by modulating TLR9, which in turn affects the inflammatory microenvironment and metastatic potential of cancer cells. While the role of TLR9 in cancer has been well-documented, particularly in promoting inflammation and metastasis [10, 11], our study provides new evidence that SLC15A4 directly regulates TLR9 in oral cancer, offering a novel mechanism for its oncogenic effects. Potential therapeutic strategies targeting the SLC15A4-TLR9 interaction, such as small molecule inhibitors or gene therapy approaches, could be explored to disrupt this pathway. The broader implications of the SLC15A4-TLR9 axis in other cancers should be considered, as similar interactions have been reported in different tumor types.

Having established the link between SLC15A4 and TLR9, we further investigated the downstream signaling pathways involved, focusing on the JAK/STAT pathway. Our findings revealed that TLR9 is primarily localized in the cytoplasm of oral cancer cells and plays a crucial role in activating the JAK/STAT pathway (Fig. 5). This is in line with previous research showing that TLR9 can activate JAK/STAT signaling in various cellular contexts [18], supporting the idea that this pathway is a common downstream effector of TLR9 activation. This activation is essential for the cellular responses mediated by SLC15A4 and TLR9. Previous studies have demonstrated that pro-inflammatory cytokines can activate JAK/STAT signaling, which is critical for tumor progression and immune evasion [24].

Importantly, when we treated cells with the JAK/STAT agonist RO8191, we observed a restoration of cell proliferation, migration, invasion, and inflammation that had been suppressed by SLC15A4 knockdown (Fig. 6). These results align with previous studies that have shown the involvement of the JAK/STAT pathway in various cancers, including oral cancer, and suggest that SLC15A4-mediated regulation of TLR9 influences oral cancer progression through the activation of JAK/STAT signaling. Although the JAK/STAT pathway has been implicated in oral cancer progression [17], our study uniquely demonstrates that SLC15A4 and TLR9 are key regulators of this pathway, providing a new perspective on the molecular mechanisms driving oral cancer. Other signaling pathways, such as PI3K/AKT or MAPK, might also be influenced by SLC15A4 and TLR9, and their roles in oral cancer progression should be explored.

In summary, our study provides evidence that SLC15A4 promotes oral cancer progression by regulating TLR9 and activating the JAK/STAT signaling pathway. Given the involvement of SLC15A4 in regulating TLR9 and JAK/STAT signaling, therapeutic strategies targeting this axis may hold promise. Small-molecule inhibitors of SLC15A4 or JAK/STAT pathway modulators could potentially suppress tumor progression and inflammation.

The overexpression of SLC15A4 in oral cancer tissues and cell lines, coupled with its role in enhancing cell proliferation, migration, invasion, and inflammation, positions it as a potential target for therapeutic intervention. Our findings not only corroborate the existing literature on the role of SLC15A4 and TLR9 in cancer but also extend the understanding of their interactions in the context of oral cancer, suggesting a more targeted approach to cancer therapy. The identification of the SLC15A4-TLR9-JAK/STAT axis offers new insights into the molecular mechanisms underlying oral cancer progression and suggests novel strategies for targeted therapies. Future research should focus on investigating the therapeutic potential of targeting SLC15A4, assessing the efficacy of inhibitors specific to the SLC15A4-TLR9-JAK/STAT axis, and exploring the role of this pathway in different subtypes of oral cancer. Future studies should explore whether combining anti-inflammatory agents with JAK/STAT inhibitors enhances treatment efficacy in OSCC.

Authors’ contributions

Meiling Feng: Conceptualization, Data Curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing - Original Draft and Writing - Review & Editing. Siguleng Bayi: Data Curation, Formal analysis, Methodology, Resources, Software and Visualization. Dema Deji: Data Curation, Investigation, Methodology and Resources. Tana Han: Data Curation, Formal analysis, Methodology and Resources. Yulan Zhang: Project administration and Supervision. All authors have read and approved the final version of this manuscript to be published.

Funding

This study was supported by the Standardization Project of Mongolian Medicine in Inner Mongolia Autonomous Region (No. 2023-[MB015]).

Data availability

All data generated or analysed during this study are included in this article.

Declarations

Ethics approval and consent to particiapte

This study was approved by the Ethics Committee of Affiliated Hospital of Inner Mongolia Minzu University (No. NM-LL-2025-06-05-01). All procedures adhered to the principles in the Declaration of Helsinki, and written informed consent were obtained from all participants. All animal experiments received approval from the Ethics Committee of Affiliated Hospital of Inner Mongolia Minzu University.

Competing interests

The authors declare no competing interests.