Transforming growth factor-β1 treatment of oral cancer induces epithelial-mesenchymal transition and promotes bone invasion via enhanced activity of osteoclasts

Abstract

This study investigates relationships between EMT and bone invasion by OSCC. Three OSCC cell lines, SCC25, HN5, and Tca8113 were artificially induced to display EMT by adding 5 ng/mL of TGF-β1 to culture media for 1–3 days. Cell morphology and phenotypic changes was examined by immunocytochemical staining of CK and VIM. EMT markers, cell-invasion factors, and osteoclast-related molecules were analysed at mRNA, gelatine and protein levels by real-time PCR, gelatine zymography and Western blotting respectively. Mature osteoclasts differentiated from Raw264.7 cells were treated by conditioned medium (CM) of OSCC cells with/without TGF-β1. Immunohistochemistry was performed to validate proteins of CK, VIM, E-cad and Snail1 in OSCC tissue samples with bone invasion. Results showed minimal staining of VIM was found in SCC25 and HN5, while Tca8113 cells stained strongly. EMT markers Twist1 and N-cad were up-regulated; Snail1 and E-cad down-regulated in all cells. Of factors associated with invasion, MMP-2 was unchanged and MMP-9 increased in SCC25 and Tca8113, while MMP-2 was increased and MMP-9 unchanged in HN5. For osteoclast-related molecules, both MT1-MMP and RANKL were up-regulated, while OPG was down-regulated in all cells. CM of OSCC cells pre-treated with TGF-β1 showed to prolong survival of osteoclasts up to 4 days. All target molecules were validated in OSCC samples of bone invasion. These findings suggest that TGF-β1 not only induces EMT to increase the capacity of OSCC for invasion, but also promotes factors which prolong osteoclast survival. TGF-β1 may enhance the ability of MMP2/9 in resorbing bone and favouring invasion of cancer cells.

Mechanisms of Invasion in Head and Neck Cancer

“…66,67 Transforming growth factor b (TGFb) can be produced by tumor-associated fibroblasts 68 and induce NF-jB activation 69 and SMAD-dependent MMP production, 70 as well as epithelial to mesenchymal transition (EMT) factors, such as Snail and Slug. [71][72][73][74][75][76] Invasion is stimulated by hypoxia in a Notch-dependent fashion. 77 Inhibition of Notch can also suppress MMP production.…”

TGF-ß alterations in oral squamous cell carcinoma. Narrative review

“…The mechanism that allows the invasion of neoplastic epithelial cells into adjacent connective tissue has been associated with the expression of cytokines, such TGF-ß 28,45 . Hwang et al 46 suggested that TGF-ß1…”

“…This facilitates neoplastic cellular invasion with a more aggressive growth phenotype. In oral cancer, TGF-ß1 deregulation promotes EMT 28,45,47,48 . This is why studies focused on EMT and its relationship in carcinogenesis have increased recently.…”

“…It is assumed that this mechanism could occur because TGF-ß1 promotes the differentiation of stromal cells to osteaclast cells. This suggests that TGF-ß1 induces EMT not only to increase the capacity of invasion of OSCC, but it could also promote factors that prolong the survival of osteoclasts 45 .…”

Exogenous expression ofIL-1RaandTGF-β1promotes in vivo repair in experimental rabbit osteoarthritis

“…Transforming growth factor-β1 (TGF-β1) is a potent cytokine and bone-derived factor that is the founding member of the TGF-β superfamily (11). TGF-β1 is secreted by platelets, macrophages, and many other cell types, and exerts various biological effects, including cell cycle regulation, proliferation, differentiation, maturation, and apoptosis or immune activity, through binding to cell surface receptors and initiating intracellular signalling cascades (12).…”