Role of interactions among Arg tyrosine kinase, TGFβ1 and Lysyl oxidase in ccRCC progression
Bone metastases develop in about 30% of ccRCC patients and an involvement of TGFβ1 in promoting the development of these metastases has been described (1). In breast cancer the extracellular matrix modifying enzyme Lysyl oxidase (Lox) has a key role in formation of pre-metastatic bone lesions through osteoclast activation and osteoblast inhibition (2). Our recent data evidenced that Lox is overexpressed in ccRCC (3) and TGFβ1 production is modulated by Arg tyrosine kinase in human renal tubular cells under high glucose conditions (4). Arg is also involved in modulation of invasion and metastasis in breast and prostate cancer through the induction of invadopodia, the cytoskeletal protrusions used by carcinoma cells to invade matrix during metastatic process (5). All these data suggest that Arg, TGFβ1 and Lox might be molecularly and functionally related to promote tumor invasion and metastasis. The aim of our study was to analyse the molecular and functional interactions among Arg, TGFβ1 and Lox in ccRCC and to evaluate their impact on osteoclast- and osteoblast- dependent formation of pre-metastatic bone lesions. Our study took advantage of an in vitro model of ccRCC primary cell cultures and cell lines.
Materials and Methods
Primary cell cultures, obtained from specimens of ccRCC and matched normal renal cortex, were characterized by FACS analysis. 786-O and Caki-1 ccRCC, Raw264 macrophage and MC3T3-E1 osteoblast cell lines were also used. ccRCC cell lines were treated with 5-10 ng/ml TGFβ1 or with Arg siRNA with or without the TGFβ-receptor inhibitor SB431542. The osteoclastic differentiation/activation of Raw264 cells and the proliferation of MC3T3-E1 osteoblasts was evaluated after treatment with conditioned media of Arg-silenced ccRCC cells by TRAP staining and MTT assay, respectively. Arg, TGFβ1-precursor, phospho-Smad2 and Smad2/3 protein expression was evaluated by Western blot, Lox and TGFβ1 in culture media by ELISA.
TGFβ1 and Lox were significantly more abundant in media of high-grade ccRCC primary cultures than in those of low-grade ccRCC and normal cortex. Arg protein was upregulated in low-grade with respect to high-grade ccRCC primary cultures. Even in ccRCC cell lines the expression of Arg and TGFβ1 were inversely correlated. Treatment of ccRCC cell lines with TGFβ1 induced an activation of Smad pathway and an increase of Lox secretion. Moreover, Arg silencing in ccRCC cells increased TGFβ1 secretion and activated Smad pathway. We are now evaluating Lox secretion after treatment of Arg-silenced ccRCC cells with SB431542 as well as Raw264 cell activation and MC3T3-E1 cell proliferation after treatment with Arg-silenced ccRCC cell conditioned media.
Our data highlighted that Arg expression is inversely correlated with TGFβ1 and Lox production in high-grade ccRCC cultures and cell lines accordingly with an expected more invasive and metastatic behaviour. Moreover, the effects of TGFβ1 treatment and Arg silencing in ccRCC cell lines suggest an involvement of Arg in regulation of TGFβ1 secretion that, through Smad pathway activation, induced Lox secretion. The eventual osteoclast activation and osteoblast inhibition induced by Arg-silenced ccRCC cell conditioned media, if confirmed by our ongoing analyses, might suggest that the complex interactions among Arg, TGFβ1 and Lox are important also for bone pre-metastatic niche formation.
The evidence of complex functional interactions among Arg, TGFβ1 and Lox may help to shed light on the molecular mechanisms responsible for ccRCC progression and bone pre-metastatic niche formation.
1. Kominsky et al. J Bone Miner Res, 2007
2. Coxe et al. Nature, 2015
3. Di Stefano et al. Am J Pathol, 2016
4. Torsello et al. J Cell Sci, 2016
5. Beaty et al. Mol Biol Cell, 2013