Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis

Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our <i>in vitro</i> data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events. <b>Abbreviations:</b> ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFβ: transforming growth factor beta

巨自噬（Macroautophagy），亦称自噬（autophagy），是一种进化上保守的细胞应激响应机制。肿瘤微环境（tumor microenvironment, stroma，肿瘤基质）中的自噬激活已被证实可支持肿瘤代谢。然而，能够启动与周围细胞通讯并刺激肿瘤微环境中自噬的癌细胞分泌因子，尚未得到充分阐明。我们鉴定出心肌营养素1（cardiotrophin 1, CTF1/CT-1）可作为成纤维细胞及乳腺癌来源的癌相关成纤维细胞（carcinoma-associated fibroblasts, CAFs）中自噬的激活因子。我们证实，CTF1可刺激信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）的磷酸化及核转位，从而启动关键自噬蛋白的转录激活。此外，经CTF1处理后，我们观察到AMP活化蛋白激酶（AMP-activated protein kinase, AMPK）及UNC-51样激酶1（UNC-51 like kinase 1, ULK1）的激活。我们提供证据表明，自噬对于CTF1依赖的平滑肌肌动蛋白α2（actin, alpha 2, smooth muscle, ACTA2/α-SMA）的积累、应力纤维形成及成纤维细胞激活至关重要。此外，活化成纤维细胞对乳腺癌细胞迁移及侵袭的促进作用，依赖于CTF1及自噬。对患者来源的乳腺癌样本中CTF1表达水平的分析，使我们确立了肿瘤基质中CTF1表达与自噬之间的相关性。与我们关于癌细胞迁移及侵袭的体外（in vitro）实验数据一致，乳腺肿瘤中较高的CTF1表达水平与患者的淋巴结转移显著相关。因此，CTF1是肿瘤-基质相互作用、成纤维细胞激活及癌症转移的重要介质，而自噬在所有这些癌症相关事件中发挥关键作用。

<b>缩写：</b>
ACTA2/α-SMA：平滑肌肌动蛋白α2（actin, alpha 2, smooth muscle）
CAFs：癌相关成纤维细胞（carcinoma-associated fibroblasts）
CNT Ab.：对照抗体（control antibody）
CNTF：睫状神经营养因子（ciliary neurotrophic factor）
CTF1：心肌营养素1（cardiotrophin 1）
CTF1 Neut. Ab.：CTF1特异性中和抗体（CTF1-specific neutralizing antibody）
GFP-LC3 MEF：融合GFP的MAP1LC3蛋白转基因小鼠胚胎成纤维细胞（GFP-fused to MAP1LC3 protein transgenic MEF）
LIF：白血病抑制因子（leukemia inhibitory factor）
IL6：白细胞介素6（interleukin 6）
MEFs：小鼠胚胎成纤维细胞（mouse embryonic fibroblasts）
MEF-WT：野生型小鼠胚胎成纤维细胞（wild-type MEFs）
OSM：制瘤素M（oncostatin M）
TGFB/TGFβ：转化生长因子β（transforming growth factor beta）