Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial-mesenchymal transition is critical in conquering ALK-positive lung cancer

Abstract

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non–small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib)-resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy.

Molecular Chaperones in Cancer Stem Cells: Determinants of Stemness and Potential Targets for Antitumor Therapy

“…Such HSP90-inhibiting treatments resulted in depletion of the CSC fractions (CD44/ALDH double-positive cells) and downregulation of specific microRNAs associated with CSCs' resistance to chemotherapy [72]. Taken together, all these publications [65][66][67][68][69][70][71][72] testify the important role of HSP90 in forming the CSC phenotype through EMT, although there may be multiple HSP90-mediated mechanisms of triggering and promoting EMT in different types of cancer.…”

“…Using selective inhibitors of the HSP90 chaperone activity or techniques with HSP90 knockdown, it was shown that intracellular HSP90 can be conducive to EMT in carcinomas of different localization [65][66][67][68][69][70][71], while the mechanisms of such HSP90-dependent EMT may vary in different cases. So, intracellular HSP90 was reported to promote EMT via (i) activation of HIF1α and NFκB [65] and stabilization of an oncogenic nonhistone chromatin-binding protein high-mobility group AT-hook 2 (HMGA2) [66] in colorectal cancer cells, (ii) activation of the TGF-β1/Notch1 signaling [67] or anaplastic lymphoma kinase (ALK) signaling [68] in different variants of lung cancer, (iii) direct interactions with Twist1 to stabilize and activate the latter in hepatocellular carcinoma [69], (iv) stabilization of low-density lipoprotein receptor-related protein 5 (LRP5) followed by LRP5-mediated stimulation of Wnt/beta-catenin signaling in gastric cancer [70], and (v) activation of STAT3 followed by the Twist1 expression upregulation in ovarian, renal, and nasopharyngeal cancer [71].…”

“…In various models, some HSP90 activity inhibitors demonstrated quite hopeful results in suppressing EMT and/or repressing CSCs. So, experimental ways to prevent or reverse EMT have been described for inhibitors of HSP90 activity, such as NVP-AUY922 [66], ganetespib [65,68], KU711, and KU757 [72]. In many research groups, the CSC-repressing and/or CSC-sensitizing effects of the inhibition of intracellular HSP90 activity were observed with geldanamycin [63,76], AR-42 (a histone deacetylase inhibitor) [87], emodin [75], 17AAG [73,74,[92][93][94], 17DMAG [79,80], NVP-AUY922 [92], WGA-TA [74], KU711 [72,74], Ku757 [72], L80 [81], and panaxynol [95].…”

Hedgehog Signaling in Lung Cancer: From Oncogenesis to Cancer Treatment Resistance

“…Besides, ALK-EML4 translocation in NSCLC is associated to EMT and induces a CSC phenotype [ 132 ]. Several studies have also shown that EMT was associated with resistance to crizotinib (a first generation TKI targeting ALK and ROS1), through tumor hypoxia [ 133 ], activation of another tyrosine kinase receptor (AXL) [ 134 , 135 , 136 ] or TGF-β pathway [ 134 ]. In a cell line with ROS1 translocation, resistance to crizotinib was mediated by EMT and Twist1 activation [ 137 ].…”

Epithelial–mesenchymal transition: role in cancer progression and the perspectives of antitumor treatment

“…Additionally, ALK inhibition changes the cell phenotype to an epithelial one [ 209 ]. In a recent paper by Nakamichi et al [ 210 ], H2228 lines resistant to three different ALK inhibitors (crizotinib, alectinib, and ceritinib) were created. The obtained stable line was characterized by a reduced ALK expression and overexpression of another oncogenic protein, AXL, which is associated with EMT and stem cells.…”