Epiregulin reprograms cancer-associated fibroblasts and facilitates oral squamous cell carcinoma invasion via JAK2-STAT3 pathway

Abstract

Background: Local resident normal fibroblasts (NFs) are the major source of cancer-associated fibroblasts (CAFs), which are distinguishable from NFs by their tumor-supportive properties. However, the mechanism and the effects underlying the transition of NFs to CAFs in oral squamous cell carcinoma (OSCC) remain unclear.

Methods: Five pairs of matching primary NFs and CAFs derived from OSCC patients were sent for RNA sequencing. Epiregulin (EREG) expression was analyzed by IHC in fibroblasts from OSCC patients. The role of EREG in the NF-CAF transition and the consequential effects on OSCC progression were examined by upregulation/downregulation of EREG in NFs/CAFs both in vitro and in vivo.

Results: Here, we identified epiregulin (EREG) as the most remarkably upregulated gene in CAFs. High EREG expression in CAFs correlated with higher T stage, deeper invasion and inferior worst pattern of invasion (WPOI) in OSCC patients and predicted shorter overall survival. Overexpression of EREG in NFs activated the CAF phenotype. Mechanistically, the JAK2/STAT3 pathway was enhanced by EREG in parallel with increased IL-6 expression, which could be inhibited by the JAK2 inhibitor AG490. Recombinant IL-6 upregulated the JAK2/STAT3/EREG pathway in a feedback loop. Moreover, EREG-induced CAF activation promoted the epithelial-mesenchymal transition (EMT) necessary for migration and invasion, which was dependent on JAK2/STAT3 signaling and IL-6. In vivo, EREG expression in stroma fibroblasts promoted tumor growth with high stromal α-SMA, phospho-JAK2/STAT3, and IL-6 expression and upregulated EMT in HSC3 cells.

Conclusions: EREG is essential for the NF-CAF transformation needed to induce EMT of tumor cells in a JAK2-STAT3- and IL-6-dependent manner in OSCC.

Keywords: Cancer-associated fibroblasts; Epiregulin; Invasion; Metastasis; Oral squamous cell carcinoma; Transition.

Fig. 1

EREG is highly expressed in CAFs. a, Gene expression differences between CAFs and NFs (vertical axis) and the average expression of genes in CAFs versus that in NFs (horizontal axis) are presented as a Bland-Altman plot. Data are from our RNA-seq analysis. b, Relative Ereg mRNA expression was tested in paired NFs/CAFs through quantitative RT-PCR, showing higher expression in CAFs than in NFs. c, Representative images and quantitative analysis of western blotting showing that EREG, N-cadherin, vimentin, and SMA were highly expressed in primary cultured CAFs. GAPDH was used as a loading control. d, Immunofluorescence staining showing the subcellular location and the expression of SMA and EREG in NFs and CAFs. Magnification: 200×. e, Immunofluorescence staining showing that EREG expression was significantly higher in tumor stroma than in the stroma of normal epithelium. Magnification: 200×. **: p < 0.01

Fig. 2

High EREG expression in CAFs of OSCC is related to poor overall survival (OS) and poor disease-free survival (DFS). a, Representative IHC images of EREG expression in CAFs from clinical samples. b, Survival analysis using Kaplan–Meier curves to compare patients with low or high EREG expression in CAFs from OSCC patients. The results revealed that high EREG expression in CAFs was closely related to inferior overall survival (OS, p = 0.0398), and disease-free survival (DFS, p = 0.0314)

Fig. 3

High EREG expression promotes NF-CAF transition through the JAK2-STAT3 pathway. a&b, Representative images and quantitative analysis of western blotting showing that EREG overexpression in NFs and EREG interference in CAFs were successful. Moreover, the expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression and downregulated after EREG interference. GAPDH was used as a loading control. c & d& e, Both phosphorylated forms of JAK2 and STAT3 and the p-JAK2/JAK2 and p-STAT3/STAT3 ratios were significantly augmented after EREG overexpression. The change in expression of major participants of other pathways after EREG overexpression was not as significant. GAPDH was used as a loading control. f & g& h, Representative images and quantification of western blotting showing that expression of CAF markers, including N-cadherin, vimentin and SMA, was upregulated after EREG overexpression but was reduced after treatment with the JAK2 inhibitor AG490, indicating that AG490 antagonizes EREG-mediated NF activation. CAF markers, as well as (p-)JAK2 and (p-)STAT3 expression and p-JAK2/JAK2 and p-STAT3/STAT3 ratios decreased after EREG interference in CAFs but were restored by IL-6 treatment. GAPDH was used as a loading control. i, ELISA showing the IL-6 level in the CM of NFs/CAFs after the indicated treatment. J&K, Representative images and quantification of immunohistochemical staining in clinical samples revealed that high EREG expression was correlated with high phospho-JAK2 and phospho-STAT3 expression, while low EREG expression was correlated with low phospho-JAK2 and phospho-STAT3 expression. Magnification: 200×. *: p < 0.05, **: p < 0.01, ***: p < 0.001

Fig. 4

NFs/CAFs with modulated EREG expression regulate the migration and invasion of oral squamous cell carcinoma cell lines. a, CCK8 assay showing that CAF CM significantly promoted HSC3 cell viability compared with NF CM. However, alteration of EREG expression in the same type of fibroblasts did not significantly affect their influence on HSC3 cell viability. b, Quantification of colony formation revealed that CAF-siNC CM significantly promoted HSC3 proliferation compared with NF-pcDNA3.1 CM. However, alteration of EREG expression in the same type of fibroblasts did not significantly affect their influence on HSC3 proliferation. c, Representative images and transwell migration and invasion assays with the HSC3 cell line. EREG upregulation induced migration/invasion-promoting CAF-like functions in NFs, but this effect was attenuated by AG490 treatment. On the other hand, EREG downregulation in CAFs interfered with their migration/invasion-promoting ability but was restored by rIL-6 treatment. d, Representative HE staining images of a 3D invasion model using tissue engineering. The results show that CAFs significantly promoted HSC3 invasion compared with NFs, but their invasion-supportive ability was attenuated after EREG knockdown. Treatment with rIL-6 restored the invasion-promoting abilities of EREG-low CAFs. On the other hand, EREG overexpression in NFs led to an increased invasion-promoting ability, but this effect was attenuated by AG490 treatment. e-g, Quantification of transwell assays and 3D invasion assays. H&I, Representative images and quantification of EMT marker (E-cadherin, N-cadherin, vimentin, SMA, and Snail) expression in HSC3 cells after the indicated treatment. GAPDH was used as a loading control. *: p < 0.05, **: p < 0.01

Fig. 5

High EREG expression in fibroblasts promotes tumor growth in vivo. a, Photographs of tumor formation in nude mice and tumor xenografts 4 weeks after inoculation. b & c, Tumor growth curves measured after injection of HSC3 cells or conditioned fibroblasts as indicated. The tumor volume was calculated every 7 days until termination. d-g, RT-PCR analysis of Ereg, Jak2, Stat3 and Il6 expression in tissues of resected tumors, revealing successful overexpression/interference of Ereg and associated upregulation or downregulation of Jak2, Stat3 and Il6. h, Immunohistochemical staining showed that tumors that developed from EREG-overexpressing NFs had a higher level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors that developed from control NFs. Tumors that developed from siEREG-transfected CAFs showed a lower level of EREG, phospho-JAK2, phospho-STAT3, and IL-6 protein expression than tumors developed from siNC-transfected CAFs. S: stroma, T: tumor. Magnification: 200×. i & j, Western blotting showed that CAFs significantly promote EMT in vivo compared with NFs, with decreased E-cadherin expression and increased N-cadherin and vimentin expression. This EMT-promoting ability was attenuated after EREG knockdown. On the other hand, EREG overexpression in NFs gave NFs a CAF-like EMT-promoting ability. k, Increased EREG expression in NFs led to acquisition of the CAF phenotype in a JAK2-STAT3-dependent way and supported OSCC invasion through the promotion of EMT in tumor cells