The Axin2-snail axis promotes bone invasion by activating cancer-associated fibroblasts in oral squamous cell carcinoma

Abstract

Background In bone-invasive oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAFs) infiltrate into bony tissue ahead of OSCC cells. In the present study, we aimed to investigate the role of the Axin2-Snail axis in the biological behaviour of CAFs and bone invasion in OSCC. Methods The clinicopathological significance of Axin2 and Snail expression was investigated by immunohistochemistry in an OSCC cohort containing 217 tissue samples from patients with long-term follow-up. The influence of the Axin2-Snail axis on the biological behaviour of OSCC cells and CAFs was further investigated both in vitro and in vivo. Results Axin2 expression was significantly associated with Snail expression, the desmoplasia status, and bone invasion in patients with OSCC. In multivariate analysis, lymph node metastasis, desmoplasia, Axin2 expression, and Snail expression were independent poor prognostic factors in our cohort. Consistent with these findings, OSCC cells demonstrated attenuated oncogenic activity as well as decreased expression of Snail and various cytokines after Axin2 knockdown in vitro. Among the related cytokines, C-C motif chemokine ligand 5 (CCL5) and interleukin 8 (IL8) demonstrated a strong influence on the biological behaviour of CAFs in vitro. Moreover, both the desmoplastic reaction and osteolytic lesions in the calvaria were predominantly decreased after Axin2 knockdown in OSCC cells in vivo using a BALB/c athymic nude mouse xenograft model. Conclusions Oncogenic activities of the Axin2-Snail axis are not limited to the cancer cells themselves but rather extend to CAFs via regulation of the cytokine-mediated cancer-stromal interaction, with further implications for bone invasion as well as a poor prognosis in OSCC.

Risk Factors of Microscopically Tumor-Free Surgical Margins for Recurrence and Survival of Oral Squamous Cell Carcinoma Patients

“…Snail, a transcriptional repressor of E-cadherin, is overexpressed in a variety of cancers and promotes the proliferation, invasion, and tumorigenesis of cancer cells ( 46 , 47 ). Moreover, Axin2 and Snail expression were significantly associated with some of the classic poor prognostic indicators, such as lymph node metastasis, vascular invasion, and bone invasion in various types of cancers including OSCC ( 48 – 50 ). In our previous studies, we found that the Axin2–Snail axis is significantly related to malignant transformation of oral leukoplakia ( 23 ).…”

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line‑derived xenograft model

“…Bone invasion of OSCC is classified into three types: Erosive, infiltrative and mixed ( 31 , 32 ). A previous study reported that the interaction between cancer-associated fibroblasts and cancer cells promoted bone invasion and prognosis of OSCC ( 27 ). The present study demonstrated that bone invasion of the HSC-3 + G-SCs group was of the mixed type, whereas the bone invasion of the HSC-3 + P-SCs group was erosive.…”

“…Anesthetization of mice was confirmed by assessing whether mice returned to a prone position when placed on their backs. Following intraperitoneal anesthesia, 200 µl mixed cells (HSC-3, 1×10 6 , 100 µl mixed with G-SCs, P-SCs or HDFs, 3×10 6 , 100 µl) were injected into subcutaneous tissue on the top of the head of 20 healthy female BALB-nu-nu mice (age, 4 weeks; mean weight, 15 g; Shimizu Laboratory Supplies Co., Ltd.), as previously described ( 27 ). All mice were kept in the animal center of Okayama University, Okayama, Japan (25°C; 50–60% humidity; 12/12-h light/dark cycle) and had free access to food and water.…”

Stromal cells in the tumor microenvironment promote the progression of oral squamous cell carcinoma

“…All animal experiments were conducted according to the relevant guidelines and regulations, and were approved by the institutional committees at Okayama University (OKU-2017406). Following intraperitoneal anesthesia with ketamine hydrochloride (75 mg/kg body weight) and medetomidine hydrochloride (0.5 mg/kg body weight), 200 µ l mixed cells including HSC-3 (1×10 6 , 100 µ l) and stromal cells (VSCC-SCs, SCC-SCs and HDFs, 3×10 6 , 100 µ l) were injected into the central region of the top of the head (in the lamina propria) of 12 BALB/c nu-nu mice (4-week-old healthy females) obtained from SHIMIZU Laboratory Supplies Co., Ltd, as previously described ( 18 , 19 ). All mice were reared in an animal room at a temperature of 25°C and 50-60% humidity under a 12-h light/dark cycle.…”