Expression data from Tsc2-null cell line ELT3, and the ELT3-derivative rapamycin-resistant cell line ELT3-245

Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM) are caused by inactivating mutations in TSC1 or TSC2, leading to mTORC1 hyperactivation. The mTORC1 inhibitors rapamycin and analogs (rapalogs) are approved for treating of TSC and LAM. Due to their cytostatic and not cytocidal action, discontinuation of treatment leads to tumor regrowth and decline in pulmonary function. Therefore, life-long rapalog treatment is proposed for the control of TSC and LAM lesions, which increases the chances for the development of acquired drug resistance. Understanding the signaling perturbations leading to rapalog resistance is critical for the development of better therapeutic strategies. We developed the first Tsc2-null rapamycin-resistant cell line, ELT3-245, which is highly tumorigenic in mice, and refractory to rapamycin treatment. In vitro ELT3-245 cells exhibit enhanced anchorage-independent cell survival, resistance to anoikis, and loss of epithelial markers. A key alteration in ELT3-245 is increased β-catenin signaling. We propose that a subset of cells in TSC and LAM lesions have additional signaling aberrations, thus possess the potential to become resistant to rapalogs. Alternatively, when challenged with rapalogs TSC-null cells are reprogrammed to express mesenchymal-like markers. These signaling changes could be further exploited to induce clinically-relevant long-term remissions.结节性硬化症（Tuberous Sclerosis Complex, TSC）与淋巴管肌瘤病（Lymphangioleiomyomatosis, LAM）均由TSC1或TSC2基因的失活突变引发，进而导致雷帕霉素靶蛋白复合物1（mTORC1）过度激活。雷帕霉素及其类似物（rapalogs）已被批准用于治疗TSC与LAM。鉴于此类药物仅具有细胞生长抑制活性而非细胞杀伤活性，停药后可引发肿瘤再生与肺功能下降。因此有研究提出需通过终身雷帕霉素类药物治疗以控制TSC与LAM病灶，但此举会增加获得性耐药的发生风险。明确介导雷帕霉素类药物耐药的信号通路扰动机制，对开发更优治疗策略至关重要。本研究构建了首个Tsc2基因敲除的雷帕霉素耐药细胞系ELT3-245，该细胞系在小鼠体内具有高致瘤性，且对雷帕霉素治疗难治。体外实验显示，ELT3-245细胞可增强非锚定依赖性细胞存活能力、抵抗失巢凋亡，并丢失上皮标志物。ELT3-245细胞的关键分子改变为β-连环蛋白信号通路活性升高。我们推测，TSC与LAM病灶中的部分细胞存在额外的信号通路异常，因此具备对雷帕霉素类药物产生耐药的潜力；此外，在雷帕霉素类药物的选择压力下，TSC基因缺失的细胞会被重编程以表达间充质样标志物。上述信号通路改变可被进一步利用，以实现临床相关的长期疾病缓解。