ATF4 and FGFR4 cooperatively support thyroid cancer progression and CAF-associated tumor-promoting features

Abstract

Purpose

Aggressive thyroid cancer remains difficult to treat, partly because molecular programs linking malignant behavior to cancer-associated fibroblast (CAF)-related support are incompletely defined. This study investigated whether ATF4 and FGFR4 cooperate in thyroid cancer cells and whether this relationship persists in a CAF-associated context.

Methods

Stable ATF4 and/or FGFR4 knockdown was established in 8305 C thyroid cancer cells. Proliferation, migration, invasion, apoptosis, cytokine secretion, transcriptomic changes, and xenograft growth were assessed under basal and CAF-conditioned culture conditions.

Results

Silencing ATF4 or FGFR4 reduced 8305 C cell proliferation, migration, and invasion, with the strongest inhibition after combined knockdown. These effects persisted in CAF-conditioned medium and were accompanied by increased apoptosis, reduced IL-6 and TNF-α secretion, and transcriptomic changes involving cell cycle, stress-response, apoptosis, and inflammatory pathways. In vivo, ATF4 and/or FGFR4 silencing suppressed xenograft growth, increased tumor apoptosis, and reduced CAF-associated marker expression.

Conclusion

ATF4 and FGFR4 are functionally associated in thyroid cancer progression. Their cooperative program may contribute to cancer cell aggressiveness and to the maintenance of a CAF-associated tumor-promoting microenvironment.

Graphical abstract

Supplementary information

The online version contains supplementary material available at 10.1007/s12020-026-04756-8.

Introduction

Thyroid cancer is the most common endocrine malignancy, and its global incidence has increased substantially over the past decades [1, 2]. Most differentiated thyroid cancers have a favorable prognosis. However, a subset of patients develops aggressive disease: poorly differentiated thyroid cancer (PDTC), anaplastic thyroid cancer (ATC), and radioactive iodine-refractory differentiated thyroid cancer (RAIR-DTC). These tumors are characterized by dedifferentiation, weak responses to conventional treatment, and poor outcomes [3, 4]. Increasing evidence suggests that thyroid cancer dedifferentiation is accompanied by extensive remodeling of the tumor microenvironment (TME), indicating that disease progression cannot be explained solely by cancer cell-intrinsic alterations [5, 6]. Accordingly, identifying molecular determinants that connect malignant cellular programs with microenvironmental support could inform the development of effective therapeutic strategies for advanced thyroid cancer.

Among the stromal components of the TME, cancer-associated fibroblasts (CAFs) have emerged as key regulators of tumor progression [7]. Tumor cell-derived factors drive fibroblast activation; in turn, activated fibroblasts enhance thyroid cancer cell proliferation and invasion [8, 9]. Recent studies in papillary thyroid carcinoma further showed that higher fibrosis and stronger CAF infiltration at the invasive front are associated with lymph node metastasis. Specific CAF subsets can promote tumor cell proliferation, migration, and invasion while reducing apoptosis [10]. Mechanistic work has also implicated CAF-derived periostin in supporting papillary thyroid tumor growth through integrin-FAK-STAT3 signaling, further pointing to a potential tumor-promoting role for CAFs in thyroid cancer [11].

Activating transcription factor 4 (ATF4), a core effector of the integrated stress response, is induced by hypoxia, nutrient deprivation, oxidative stress, and endoplasmic reticulum stress within the tumor microenvironment [12–14]. Recent studies have shown that stromal signals can elevate ATF4 expression in cancer cells [15]. In pancreatic cancer, CAF-secreted TGF-β1 activates SMAD2/3 signaling to upregulate ATF4, thereby promoting malignant progression and chemoresistance [16]. FGFR4 may also participate in tumor–stroma interactions. In colon cancer, FGFR4-overexpressing tumor cells promoted fibroblast differentiation and CAF activation through secreted factors [17]. ER stress has also been reported to enhance ATF4-mediated transcriptional activation of FGF19, which encodes a high-affinity ligand of FGFR4 [18, 19]. These findings suggest a possible functional connection between ATF4 and FGFR4 in the regulation of CAF-associated tumor progression. However, whether such an ATF4–FGFR4 cooperative program contributes to CAF-associated progression in thyroid cancer remains unclear.

Here, we addressed this question by combining stable ATF4- and/or FGFR4-knockdown thyroid cancer cell models, CAF-conditioned medium, in vivo xenograft assays, and RNA sequencing to characterize transcriptomic changes following gene silencing. Through these experiments, we aimed to determine whether ATF4 and FGFR4 may represent a potential link between cancer cell aggressiveness and CAF-associated microenvironmental support in thyroid cancer.

Materials and methods

Cell culture

The human thyroid cancer cell line 8305 C and cancer-associated fibroblasts (CAFs) were purchased from Procell Life Science & Technology. Cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (ExCell Bio) and 1% penicillin-streptomycin (Beyotime) at 37 °C in a 5% CO₂ incubator. Cells were passaged at approximately 80% confluence using 0.25% trypsin and maintained under standard culture conditions. For long-term storage, cells were frozen in freezing medium and stored in liquid nitrogen.

Establishment of stable knockdown cell lines

Short hairpin RNAs (shRNAs) targeting ATF4, FGFR4, and a negative control (sh‑NC) were designed and cloned into the pLKO.1 vector (Sangon Biotech). Lentivirus packaging was performed by co-transfecting HEK293T cells with the shRNA plasmid and packaging plasmids using Lipofectamine 3000 (Thermo Fisher Scientific). Viral supernatants were collected 48 h post‑transfection, filtered through 0.45 μm filters, and stored at − 80 °C until use. 8305 C cells were infected with lentivirus and selected with puromycin (Sigma‑Aldrich) for 7 days. Knockdown efficiency was confirmed by RT-qPCR and Western blot.

RNA sequencing and bioinformatic analysis

To investigate transcriptomic alterations associated with ATF4/FGFR4 silencing, total RNA was extracted from sh-ATF4, sh-FGFR4, and sh-ATF4 + sh-FGFR4 8305 C cells, with three biological replicates per group, using the FastPure RNA Kit (Vazyme) according to the manufacturer’s instructions. RNA quality and concentration were assessed before library preparation. Strand-specific RNA-seq libraries were constructed and sequenced by LC-Bio Technologies (Hangzhou) Co., Ltd. on an Illumina NovaSeq 6000 platform using a paired-end 150-bp sequencing strategy. An average of approximately 41.47 million clean reads was obtained per sample.

Raw sequencing reads were assessed using FastQC v0.10.1 and processed with Cutadapt to remove adapter sequences and low-quality reads. Clean reads were aligned to the human reference genome using HISAT2 v2.2.1, and transcript assembly and gene-level quantification were performed using StringTie v2.1.6. Pairwise differential-expression analyses were conducted using DESeq2 v1.22.2, whereas the overall comparison among the three knockdown groups was performed using edgeR v3.22.5. For pairwise comparisons, genes with an absolute log2 fold change ≥ 1 and a nominal P value < 0.05 were considered differentially expressed. For the multiple-group comparison, genes with a nominal P value < 0.05 were considered differentially expressed, without applying a fold-change cutoff. Benjamini–Hochberg-adjusted q values were also calculated and reported in the differential-expression output tables.

Differentially expressed genes identified in the multiple-group comparison were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Gene- and transcript-expression distributions, principal component analysis, hierarchical clustering, and enrichment plots were generated to summarize and visualize the transcriptomic data. Sequencing and read-mapping quality-control metrics are provided in Supplementary Table S4.

Cell proliferation assay

Cell proliferation was measured using the EdU Apollo kit (RiboBio). Cells were seeded in 96-well plates and incubated with 50 µM EdU for 2 h. After fixation with 4% paraformaldehyde, cells were permeabilized, stained following the manufacturer’s instructions, and counterstained with DAPI. Fluorescent images were captured using an EVOS M5000 microscope (Thermo Fisher), and the percentage of EdU-positive cells was quantified with ImageJ software.

Wound-healing assay

Cells were seeded in 6-well plates and grown to confluence. Linear scratches were made using a sterile 200 µL pipette tip, and cells were washed twice with PBS to remove debris. Cells were then incubated in serum-free medium. Images were taken at 0 h and 48 h, and wound closure was quantified.

Transwell invasion assay

Transwell chambers (8 μm; Corning) were pre-coated with Matrigel. Cells (2 × 10⁵ cells/mL) in serum-free medium were added to the upper chamber, while medium containing 10% FBS was placed in the lower chamber. After 24 h, non-invading cells were removed, and invading cells on the lower surface were fixed, stained with crystal violet, and counted in three random fields under a microscope.

CAF-conditioned medium treatment

CAFs were cultured to 80% confluence, and the supernatant was collected, centrifuged, and filtered. 8305 C cells from the sh-NC, sh-ATF4, sh-FGFR4, or sh-ATF4 + sh-FGFR4 groups were then cultured in this CAF-conditioned medium for 48 h before subsequent assays, including proliferation, migration, invasion, apoptosis, molecular expression analysis, immunofluorescence, and ELISA.

Apoptosis assay

Apoptosis was detected using the Annexin V-FITC/PI kit (Beyotime). Treated cells were collected, washed, and stained with Annexin V-FITC and PI for 15 min in the dark. Samples were analyzed by flow cytometry (Attune NxT, Thermo Fisher).

RNA extraction and RT-qPCR

Total RNA was extracted using the FastPure RNA Kit (Vazyme) and reverse-transcribed into cDNA using HiScript III (Vazyme). RT-qPCR was performed on a CFX96 Touch (Bio-Rad) using SYBR Green mix (Vazyme). Primers were as follows: ATF4: Forward 5’-GGGATCGGGAAAGCGTAGTC-3’, Reverse 5’-TCTTGGTTCCTGCCACGTTT-3’. FGFR4: Forward 5’-GAGGAGGACCCCACATGGA-3’, Reverse 5’-AGGGATGAGCTTGACTTGCC-3’. GAPDH: Forward 5’-AATGGGCAGCCGTTAGGAAA-3’, Reverse 5’-GCGCCCAATACGACCAAATC-3’. Relative expression was calculated using the 2⁻ΔΔCt method.

Western blotting

Total protein was extracted from cells using RIPA lysis buffer (Beyotime) containing PMSF (Beyotime), and protein concentrations were determined using a BCA Protein Assay Kit (NCM Biotech). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Sigma-Aldrich). After blocking with 5% skim milk, membranes were incubated overnight at 4 °C with primary antibodies against ATF4 (Proteintech, 81798-1-RR, 1:2,000) and FGFR4 (Proteintech, 81069-1-RR, 1:8,000), followed by HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (Bioss, bs-0296G-HRP, 1:20,000). Protein bands were visualized using an ECL substrate (NCM Biotech, P2100) and quantified with ImageJ.

Immunofluorescence staining of cultured cells

Treated 8305 C cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with goat serum. Cells were incubated overnight at 4 °C with anti-ATF4 (Proteintech, 60035-1-Ig, 1:200) and anti-FGFR4 (Proteintech, 11098-1-AP, 1:200), followed by CoraLite Plus 555-conjugated goat anti-mouse IgG (Proteintech, RGAM003, 1:800) and CoraLite 488-conjugated goat anti-rabbit IgG (Proteintech, SA00013-2, 1:800). Nuclei were counterstained with DAPI, and images were captured under identical settings and quantified using ImageJ.

Enzyme-linked immunosorbent assay

Culture supernatants were collected after CAF-conditioned medium treatment and centrifuged to remove debris. IL-6 and TNF-α levels were measured using commercial ELISA kits (Hnybio) according to the manufacturers’ instructions. Absorbance was read at 450 nm, and cytokine concentrations were calculated from standard curves.

In vivo xenograft model

Four-week-old male BALB/c nude mice were randomly divided into four groups. After pre-culture in CAF-conditioned medium, 5 × 10⁶ 8305 C cells from each group (sh-NC, sh-ATF4, sh-FGFR4, or sh-ATF4 + sh-FGFR4) were injected subcutaneously into the flank of each mouse. After four weeks, mice were euthanized, tumors were excised, weighed, and processed for histological analysis. All animal experiments were approved by the Animal Ethical and Welfare Committee (AEWC) of Guangzhou Miles Biosciences (Approval No. MIS2026006) and were performed in accordance with institutional guidelines for the care and use of laboratory animals.

Histological, immunohistochemical, and immunofluorescence analyses

Tumor tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 4 μm. H&E staining was performed for morphological evaluation. For immunohistochemistry, sections were incubated with antibodies against ATF4 (Abcam, ab184909, 1:200) and FGFR4 (Abcam, ab41948, 1:200), followed by a ready-to-use secondary antibody reagent (ZSGB-BIO) and DAB visualization kit (ZSGB-BIO). For immunofluorescence, sections were incubated with antibodies against α-SMA (Abcam, ab7817, 1:200), vimentin (Abcam, ab92547, 1:200), and FAP (R&D Systems, MAB3715, 1:200), followed by Alexa Fluor 555 goat anti-mouse IgG (Thermo Fisher, A-21422, 1:500), Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, A-11008, 1:500), and Alexa Fluor 594 donkey anti-rat IgG (Thermo Fisher, A-21209, 1:500). Nuclei were counterstained with DAPI and images were analyzed using ImageJ.

TUNEL assay

Apoptotic cells in tumor sections were detected using a TUNEL Apoptosis Detection Kit (Beyotime) according to the manufacturer’s instructions. Briefly, paraffin sections were deparaffinized, rehydrated, incubated with TUNEL reaction mixture at 37 °C for 60 min in the dark, and observed under a fluorescence microscope. TUNEL-positive cells were quantified in three random fields per section.

Statistical analysis

All data were expressed as mean ± SD from at least three independent experiments. Comparisons between two groups were performed using Student’s t-test, and multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. A P value < 0.05 was considered statistically significant.

Results

Knockdown of ATF4 and FGFR4 suppressed proliferation, migration, and invasion of thyroid cancer cells in vitro

To determine whether ATF4 and FGFR4 cooperatively drive thyroid cancer cell aggressiveness, we generated 8305 C cells with stable silencing of ATF4, FGFR4, or both. Stable silencing of ATF4 and FGFR4 was confirmed at both the mRNA and protein levels. Notably, ATF4 silencing reduced FGFR4 expression, whereas FGFR4 silencing likewise diminished ATF4, and the dual-knockdown cells displayed the lowest expression of both factors (Fig. 1a, b), suggesting coordinated expression between ATF4 and FGFR4. Functionally, loss of ATF4 or FGFR4 was sufficient to restrain 8305 C cell proliferation, migration, and invasion, as shown by EdU, wound-healing, and Transwell assays, with the greatest impairment observed after dual silencing (Fig. 1c–e). Together, these data identified ATF4 and FGFR4 as functionally linked determinants of the malignant phenotype of thyroid cancer cells.

Fig. 1.

Silencing ATF4 and/or FGFR4 restrains the malignant phenotype of 8305 C thyroid cancer cells in vitro. a Western blot analysis of ATF4 and FGFR4 protein expression in 8305 C cells infected with sh-NC, sh-ATF4, sh-FGFR4, or sh-ATF4 + sh-FGFR4. b RT-qPCR analysis of ATF4 and FGFR4 mRNA expression in the indicated groups. c EdU incorporation assay showing cell proliferation in the indicated groups. Scale bars, 50 μm. d Wound-healing assay performed at 0 and 48 h to assess cell migration. Scale bars, 70 μm. e Transwell invasion assay of 8305 C cells in the indicated groups. Data are presented as mean ± SD from three independent experiments (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sh-NC

Knockdown of ATF4 and FGFR4 attenuated the pro-tumorigenic effects of CAF-conditioned medium on thyroid cancer cells

Having shown that ATF4 and FGFR4 sustained the malignant phenotype of thyroid cancer cells under basal conditions, we next asked whether this dependency was preserved in a CAF-conditioned context. Under CAF-conditioned medium, depletion of ATF4 or FGFR4 markedly attenuated EdU incorporation and reduced the migratory and invasive capacities of 8305 C cells, with the most pronounced effects observed in the dual-knockdown group (Fig. 2a–c). Consistent with these changes, flow cytometric analysis demonstrated a significant increase in apoptosis following ATF4 and/or FGFR4 silencing (Fig. 2d). Importantly, western blot and RT-qPCR confirmed that the knockdown remained effective under CAF-conditioned culture, and the reciprocal decrease in ATF4 and FGFR4 expression was retained in the corresponding groups (Fig. 2e, f). Together, these data indicated that persistent suppression of ATF4 and/or FGFR4 continued to limit the malignant phenotype of thyroid cancer cells despite exposure to CAF-derived cues, suggesting that ATF4 and/or FGFR4 remained required in a CAF-associated setting.

Fig. 2.

Knockdown of ATF4 and/or FGFR4 attenuates the pro-tumorigenic effects of CAF-conditioned medium in 8305 C thyroid cancer cells. a EdU incorporation assay showing the proliferative capacity of 8305 C cells cultured with CAF-conditioned medium. Scale bars, 50 μm. b Wound-healing assay performed at 0 and 48 h under CAF-conditioned culture. Scale bars, 70 μm. c Transwell invasion assay of 8305 C cells cultured with CAF-conditioned medium. Scale bars, 100 μm. d Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in the indicated groups. e Western blot analysis of ATF4 and FGFR4 protein expression in 8305 C cells cultured with CAF-conditioned medium. f RT-qPCR analysis of ATF4 and FGFR4 mRNA expression in the indicated groups under CAF-conditioned culture. Data are presented as mean ± SD from three independent experiments (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sh-NC

Transcriptomic profiling and cytokine analyses suggested altered inflammatory and stress-related programs associated with ATF4/FGFR4 silencing

To gain mechanistic insight into the phenotypic changes induced by ATF4/FGFR4 depletion, we next performed transcriptomic profiling in the knockdown groups. GO enrichment analysis showed that the differentially expressed genes were mainly involved in cell cycle regulation, DNA damage response, innate immune and inflammatory signaling, protein folding, and endoplasmic reticulum-associated transport (Fig. 3a). KEGG analysis further highlighted pathways related to PI3K-Akt signaling, cell cycle progression, TNF/NF-κB signaling, protein processing in the endoplasmic reticulum, and apoptosis (Fig. 3b). These findings indicated that transcriptomic differences among the three knockdown conditions involved coordinated changes in proliferative, stress-adaptive, and survival-related programs. Immunofluorescence analysis further confirmed reduced ATF4 and FGFR4 expression in the corresponding knockdown groups (Fig. 3c). Consistent with these transcriptomic and protein-level changes, ELISA showed that IL-6 and TNF-α levels in the culture supernatant were significantly reduced after ATF4 or FGFR4 silencing, with the lowest levels observed in the dual-knockdown group (Fig. 3d). These data suggested that silencing of ATF4 and/or FGFR4 was associated with reduced inflammatory output and disruption of stress-related programs that may contribute to thyroid cancer progression. Additional gene- and transcript-expression distribution plots, principal component analysis, a differentially expressed gene heatmap, GO and KEGG enrichment plots, differential-expression and enrichment-analysis results, and RNA-sequencing and read-mapping quality-control metrics are provided in the Electronic Supplementary Material.

Fig. 3.

Silencing ATF4 and/or FGFR4 is associated with transcriptomic reprogramming and reduced inflammatory cytokine secretion in 8305 C cells. a GO enrichment analysis of differentially expressed genes among the sh-ATF4, sh-FGFR4, and sh-ATF4 + sh-FGFR4 groups. b KEGG pathway enrichment analysis of differentially expressed genes among the three knockdown groups. c Immunofluorescence staining of ATF4 (red) and FGFR4 (green) in 8305 C cells. Nuclei were counterstained with DAPI (blue). Scale bars, 150 μm. d ELISA quantification of IL-6 and TNF-α levels in culture supernatants from the indicated groups. Data are presented as mean ± SD from three independent experiments (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sh-NC

Silencing ATF4 and FGFR4 inhibited tumor growth in a xenograft mouse model

To validate the tumor-suppressive effects of ATF4 and FGFR4 knockdown in vivo, we established a xenograft mouse model by subcutaneously injecting sh-NC, sh-ATF4, sh-FGFR4, or sh-ATF4 + sh-FGFR4 8305 C cells pre-cultured with CAF-conditioned medium into nude mice (Fig. 4a). Tumors derived from ATF4- or FGFR4-silenced cells were consistently smaller than those formed by control cells, with the most pronounced inhibition observed in the dual-knockdown group (Fig. 4b). This reduction was further supported by significant decreases in maximal tumor diameter, tumor weight, and tumor weight/body weight ratio across the knockdown groups (Fig. 4c–e). Histological examination by H&E staining showed that tumors from the knockdown groups displayed looser architecture and reduced cellular density compared with the densely packed tumors in the control group (Fig. 4f). These in vivo findings supported a critical role for ATF4 and FGFR4 in sustaining thyroid tumor growth.

Fig. 4.

ATF4–FGFR4 silencing inhibits thyroid tumor growth in vivo. a Schematic illustration of the xenograft experimental design. b Representative images of excised tumors from each group at the experimental endpoint (n = 4 mice per group). c–e Maximum tumor diameter, tumor weight, and tumor weight/body weight ratio in the indicated groups. Data are presented as mean ± SD (n = 4 mice per group). f Representative H&E staining of xenograft tumor sections (n = 3 tumors per group). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sh-NC+CAFs

Knockdown of ATF4 and FGFR4 promoted tumor apoptosis and suppressed CAF-associated microenvironment activation in vivo

To further define the tissue-level changes accompanying tumor growth inhibition, we examined apoptosis and CAF-associated features in xenograft tissues. Immunohistochemistry confirmed that ATF4 and FGFR4 expression remained markedly reduced in the corresponding knockdown tumors (Fig. 5a). TUNEL staining showed a clear increase in apoptotic cells in the sh-ATF4, sh-FGFR4, and dual-knockdown groups, with the highest apoptotic index observed in tumors subjected to combined silencing (Fig. 5b). In parallel, immunofluorescence staining revealed decreased expression of the CAF-associated markers α-SMA, Vimentin, and FAP in tumors derived from ATF4- and/or FGFR4-silenced cells (Fig. 5c), consistent with reduced CAF-related microenvironmental activation. These data indicated that suppression of ATF4 and/or FGFR4 in vivo coincided with increased tumor apoptosis and reduced CAF-associated stromal activation.

Fig. 5.

Silencing ATF4 and/or FGFR4 enhances tumor apoptosis and reduces CAF-associated marker expression in vivo. a Immunohistochemical staining of ATF4 and FGFR4 in xenograft tumor sections. Scale bars, 20 μm. b TUNEL staining of xenograft tumors showing apoptotic cells (green); nuclei were counterstained with DAPI (blue). Scale bars, 20 μm. c Immunofluorescence staining of the CAF-associated markers α-SMA, Vimentin, and FAP in xenograft tumor sections. Nuclei were stained with DAPI. Scale bars, 20 μm. Data are presented as mean ± SD (n = 3 mice per group). * P < 0.05, ** P < 0.01, *** P < 0.001 versus sh-NC+CAFs

Discussion

Our study identifies ATF4 and FGFR4 as functionally coupled regulators of thyroid cancer progression and suggests that their contribution extends beyond tumor cell-intrinsic aggressiveness to a CAF-associated tumor-promoting context. This interpretation is supported by the observation that silencing ATF4 and/or FGFR4 suppressed malignant phenotypes not only under basal conditions but also in the presence of CAF-conditioned medium. These findings are consistent with the broader view that thyroid cancer progression, particularly dedifferentiation and aggressive disease evolution, is accompanied by extensive remodeling of the tumor microenvironment [5, 6]. CAFs have also been recognized as important stromal regulators of thyroid cancer progression, and previous studies have shown that fibroblast activation or CAF infiltration can promote thyroid cancer cell proliferation, invasion, and metastatic behavior [8–11]. In this context, our findings suggest that ATF4 and FGFR4 may represent tumor-cell-associated regulators that help maintain malignant behavior in a CAF-associated microenvironment.

A key observation in the present study is that the effects of ATF4 and FGFR4 were evident not only under basal culture conditions but also in the presence of CAF-conditioned medium. CAF-derived factors have been reported to enhance thyroid cancer cell proliferation and invasion and to contribute to a tumor-supportive stromal context [8–11]. Our data extend these observations by showing that depletion of ATF4 or FGFR4 reduced proliferation, migration, and invasion while increasing apoptosis despite exposure to CAF-derived cues. The reciprocal reduction of ATF4 and FGFR4 after silencing either factor is also noteworthy, as it suggests that the two molecules may participate in a coordinated regulatory program rather than functioning as entirely independent regulators of tumor progression. However, the present data support a functional link between ATF4 and FGFR4 without establishing their direct molecular relationship.

The reduction in IL-6 and TNF-α secretion is meaningful in this setting. Inflammatory signaling is an important component of tumor–stroma communication, and CAF-associated tumor-promoting effects are often accompanied by cytokine-mediated remodeling of the microenvironment. In the context of the phenotypic changes observed under CAF-conditioned culture, our cytokine data support the view that ATF4/FGFR4-related programs are linked to reduced inflammatory output and a weaker tumor-promoting context.

The transcriptomic data provide a broader framework for these phenotypic observations. The enrichment of cell cycle- and apoptosis-related pathways is consistent with the reduced proliferation and increased cell death seen after ATF4 and/or FGFR4 silencing. At the same time, the enrichment of inflammatory signaling, including TNF/NF-κB-related programs, fits well with the decrease in IL-6 and TNF-α secretion. Changes in protein folding and endoplasmic reticulum-associated processes are also notable, given that ATF4 is a central effector of the integrated stress response and can be induced by hypoxia, nutrient deprivation, oxidative stress, and endoplasmic reticulum stress within the tumor microenvironment [12–14]. Previous studies have further shown that stromal signals can elevate ATF4 expression in cancer cells, and CAF-mediated ATF4 activation has been implicated in malignant progression and therapy resistance in pancreatic cancer [15, 16]. Taken together, these transcriptomic changes suggest that ATF4 and FGFR4 are linked to a broader regulatory state that integrates proliferation, survival, inflammatory output, and stress tolerance, rather than to a single isolated downstream event.

The in vivo findings reinforce this interpretation. Silencing of ATF4 and/or FGFR4 suppressed xenograft growth and increased tumor apoptosis, indicating that the growth-promoting role of these factors is maintained in vivo. In parallel, the reduction in α-SMA, Vimentin, and FAP staining suggests attenuation of CAF-associated activation within the tumor context. Previous studies have reported that FGFR4-related signaling may participate in tumor–stroma communication and CAF activation [17], while FGF19/FGFR4 signaling has been implicated in malignant progression in several cancers [18, 19]. Therefore, although our study does not establish a direct ATF4–FGFR4 molecular axis, the reciprocal expression changes observed after single-gene silencing and the reduction in CAF-associated marker expression support a functional association between these molecules and the tumor-supportive stromal context.

Several limitations should be acknowledged. First, the RNA-seq cohort did not include an sh-NC group, and transcriptomic profiling was therefore limited to comparisons among the sh-ATF4, sh-FGFR4, and sh-ATF4 + sh-FGFR4 conditions. Accordingly, these results reflect relative transcriptomic differences among the three knockdown groups but do not define changes induced by gene silencing relative to a negative-control baseline. Second, although the present study supports a functional association between ATF4 and FGFR4 and links both factors to CAF-related tumor-promoting phenotypes, it does not define the molecular mechanism connecting them. Third, stromal involvement was inferred mainly from CAF-conditioned medium experiments and changes in CAF-associated markers in vivo, rather than from direct functional investigation of CAFs themselves. Fourth, the current work was performed primarily in a single thyroid cancer cell line, and broader validation in additional thyroid cancer models and clinical samples will be needed to strengthen the generalizability of the findings. These limitations highlight the need for transcriptomic validation using an appropriate negative-control baseline, further clarification of the regulatory relationship between ATF4 and FGFR4, direct investigation of CAF-mediated tumor–stroma interactions, and validation in more clinically relevant models.

In conclusion, this study supports a functionally important role for ATF4 and FGFR4 in thyroid cancer progression and suggests that their impact extends from malignant cellular behavior to the maintenance of a CAF-associated tumor-promoting context. By linking tumor growth, apoptotic control, inflammatory output, and stromal association within one framework, our findings provide a basis for considering ATF4/FGFR4-related programs as potential targets for therapeutic intervention in thyroid cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Z: Conceptualization, Writing – review and editing. C: Validation, Investigation, Writing – review and editing. R: Writing – original draft, Writing – review and editing.

Funding

This work was supported by the Xiamen Municipal Healthcare Guidance Project (No. 3502Z20209133).

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval

All animal experiments were approved by the Animal Ethical and Welfare Committee (AEWC) of Guangzhou Miles Biosciences (Approval No. MIS2026006) and were performed in accordance with institutional guidelines for the care and use of laboratory animals.

Conflict of interests

The authors have no relevant financial or non-financial interests to disclose.