Pattarozzi Alessandra, Carra Elisa, Favoni Roberto E, Würth Roberto, Marubbi Daniela, Filiberti Rosa Angela, Mutti Luciano, Florio Tullio, Barbieri Federica, Daga Antonio
Department of Internal Medicine (DiMI) and Centre of Excellence for Biomedical Research (CEBR), University of Genova, Viale Benedetto XV, 2, 16132, Genova, Italy.
Department of Experimental Medicine (DIMES), University of Genova, Via L.B. Alberti, 2, 16132, Genova, Italy.
Stem Cell Res Ther. 2017 May 25;8(1):119. doi: 10.1186/s13287-017-0573-7.
Malignant pleural mesothelioma is an aggressive cancer, characterized by rapid progression and high mortality. Persistence of tumor-initiating cells (TICs, or cancer stem cells) after cytotoxic drug treatment is responsible for tumor relapse, and represents one of the main reasons for the poor prognosis of mesothelioma. In fact, identification of the molecules affecting TIC viability is still a significant challenge.
TIC-enriched cultures were obtained from 10 human malignant pleural mesotheliomas and cultured in vitro. Three fully characterized tumorigenic cultures, named MM1, MM3, and MM4, were selected and used to assess antiproliferative effects of the multi-kinase inhibitor sorafenib. Cell viability was investigated by MTT assay, and cell cycle analysis as well as induction of apoptosis were determined by flow cytometry. Western blotting was performed to reveal the modulation of protein expression and the phosphorylation status of pathways associated with sorafenib treatment.
We analyzed the molecular mechanisms of the antiproliferative effects of sorafenib in mesothelioma TIC cultures. Sorafenib inhibited cell cycle progression in all cultures, but only in MM3 and MM4 cells was this effect associated with Mcl-1-dependent apoptosis. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt, and STAT3 phosphorylation. These effects were abolished by sorafenib only in bFGF-treated cells, while a modest inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGF receptor (FGFR) inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. Moreover, in MM1 cells, which release high levels of bFGF and showed autocrine activation of FGFR1 and constitutive phosphorylation/activation of MEK-ERK1/2, sorafenib induced a more effective antiproliferative response, confirming that the main target of the drug is the inhibition of FGFR1 activity.
These results suggest that, in malignant pleural mesothelioma TICs, bFGF signaling is the main target of the antiproliferative response of sorafenib, acting directly on the FGFR1 activation. Patients with constitutive FGFR1 activation via an autocrine loop may be more sensitive to sorafenib treatment and the analysis of this possibility warrants further clinical investigation.
恶性胸膜间皮瘤是一种侵袭性癌症，其特点是进展迅速且死亡率高。细胞毒性药物治疗后肿瘤起始细胞（TICs，即癌症干细胞）的持续存在导致肿瘤复发，这也是间皮瘤预后不良的主要原因之一。事实上，鉴定影响TIC生存能力的分子仍然是一项重大挑战。
从10例人类恶性胸膜间皮瘤中获取富含TIC的培养物并进行体外培养。选择三种特征明确的致瘤培养物，命名为MM1、MM3和MM4，用于评估多激酶抑制剂索拉非尼的抗增殖作用。通过MTT法研究细胞活力，通过流式细胞术确定细胞周期分析以及凋亡诱导情况。进行蛋白质印迹分析以揭示与索拉非尼治疗相关的蛋白质表达调节和信号通路的磷酸化状态。
我们分析了索拉非尼在间皮瘤TIC培养物中抗增殖作用的分子机制。索拉非尼在所有培养物中均抑制细胞周期进程，但仅在MM3和MM4细胞中，这种作用与Mcl-1依赖性凋亡相关。为了研究索拉非尼介导的抗增殖活性机制，用表皮生长因子（EGF）或碱性成纤维细胞生长因子（bFGF）处理TICs，导致MM3和MM4细胞中MEK、ERK1/2、Akt和STAT3磷酸化。索拉非尼仅在bFGF处理的细胞中消除了这些作用，而在EGF刺激后出现适度抑制，这表明索拉非尼的作用主要归因于对成纤维细胞生长因子受体（FGFR）的抑制。事实上，索拉非尼抑制了FGFR1磷酸化。此外，在释放高水平bFGF并显示FGFR1自分泌激活以及MEK-ERK1/2组成性磷酸化/激活的MM1细胞中，索拉非尼诱导了更有效的抗增殖反应，证实该药物的主要靶点是抑制FGFR1活性。
这些结果表明，在恶性胸膜间皮瘤TICs中，bFGF信号是索拉非尼抗增殖反应的主要靶点，直接作用于FGFR1激活。通过自分泌环组成性激活FGFR1的患者可能对索拉非尼治疗更敏感，对此可能性的分析值得进一步的临床研究。
