Creative Research Initiative Center for concurrent control of emphysema and lung cancer, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea.
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea.
Theranostics. 2022 Jan 1;12(1):105-125. doi: 10.7150/thno.63788. eCollection 2022.
The heat shock protein (Hsp) system plays important roles in cancer stem cell (CSC) and non-CSC populations. However, limited efficacy due to drug resistance and toxicity are obstacles to clinical use of Hsp90 inhibitors, suggesting the necessity to develop novel Hsp90 inhibitors overcoming these limitations. The underlying mechanism of resistance to Hsp90 inhibitors was investigated by colony formation assay, sphere formation assay, western blot analysis, and real-time PCR. To develop anticancer Hsp90 inhibitors that overcome the signal transducer and activator of transcription 3 (STAT3)-mediated resistance, we synthesized and screened a series of synthetic deguelin-based compounds in terms of inhibition of colony formation, migration, and viability of non-small cell lung cancer (NSCLC) cells and toxicity to normal cells. Regulation of Hsp90 by the selected compound NCT-80 [5-methoxy-N-(3-methoxy-4-(2-(pyridin-3-yl)ethoxy)phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide] was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. The antitumor, antimetastatic, and anti-CSC effects of NCT-80 were examined and using various assays such as MTT, colony formation, and migration assays and flow cytometric analysis and tumor xenograft models. We demonstrated a distinct mechanism in which Hsp90 inhibitors that block N-terminal ATP-binding pocket causes transcriptional upregulation of Wnt ligands through Akt- and ERK-mediated activation of STAT3, resulting in NSCLC cell survival in an autocrine or paracrine manner. In addition, NCT-80 effectively reduced viability, colony formation, migration, and CSC-like phenotypes of NSCLC cells and their sublines with acquired resistance to anticancer drugs by inducing apoptosis and inhibiting epithelial-mesenchymal transition and the growth of NSCLC patient-derived xenograft tumors without overt toxicity. With regards to mechanism, NCT-80 directly bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the interaction between Hsp90 and STAT3 and degrading STAT3 protein. Moreover, NCT-80 inhibited chemotherapy- and EGFR TKI-induced programmed cell death ligand 1 expression and potentiated the antitumor effect of chemotherapy in the LLC-Luc allograft model. These data indicate the potential of STAT3/Wnt signaling pathway as a target to overcome resistance to Hsp90 inhibitors and NCT-80 as a novel Hsp90 inhibitor that targets both CSCs and non-CSCs in NSCLC.
热休克蛋白（Hsp）系统在癌症干细胞（CSC）和非 CSC 群体中发挥重要作用。然而，由于耐药性和毒性导致的有限疗效是 HSP90 抑制剂临床应用的障碍，这表明有必要开发新型 HSP90 抑制剂来克服这些限制。通过集落形成试验、球体形成试验、Western blot 分析和实时 PCR 研究了 HSP90 抑制剂耐药的潜在机制。为了开发克服信号转导和转录激活因子 3（STAT3）介导的耐药性的抗癌 HSP90 抑制剂，我们根据抑制非小细胞肺癌（NSCLC）细胞集落形成、迁移和活力以及对正常细胞的毒性，合成并筛选了一系列基于去氢骆驼蓬碱的化合物。通过免疫沉淀、药物亲和反应靶标稳定性测定、ATP-琼脂糖珠和生物素化药物结合实验以及对接分析研究了选定化合物 NCT-80[5-甲氧基-N-(3-甲氧基-4-(2-(吡啶-3-基)乙氧基)苯基)-2,2-二甲基-2H-色烯-6-甲酰胺]对 HSP90 的调节作用。使用 MTT、集落形成和迁移试验以及流式细胞术分析和肿瘤异种移植模型等各种试验，研究了 NCT-80 的抗肿瘤、抗转移和抗 CSC 作用。我们证明了一种独特的机制，即阻断 N 端 ATP 结合口袋的 HSP90 抑制剂通过 Akt 和 ERK 介导的 STAT3 激活引起 Wnt 配体的转录上调，从而导致 NSCLC 细胞以自分泌或旁分泌方式存活。此外，NCT-80 通过诱导细胞凋亡、抑制上皮-间充质转化和 NSCLC 患者来源异种移植肿瘤的生长，有效降低获得性耐药的 NSCLC 细胞及其亚系的活力、集落形成、迁移和 CSC 样表型，且无明显毒性。关于机制，NCT-80 直接与 HSP90 的 C 端 ATP 结合口袋结合，破坏 HSP90 与 STAT3 之间的相互作用并降解 STAT3 蛋白。此外，NCT-80 抑制化疗和 EGFR TKI 诱导的程序性细胞死亡配体 1 表达，并增强 LLC-Luc 同种异体移植模型中的化疗抗肿瘤作用。这些数据表明，STAT3/Wnt 信号通路作为克服 HSP90 抑制剂耐药性的靶点具有潜力，NCT-80 作为一种新型 HSP90 抑制剂，可针对 NSCLC 中的 CSCs 和非 CSCs。
