Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 27;11(1):9011.
doi: 10.1038/s41598-021-87966-6.

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells

Anna M Schläfli  1 , Igor Tokarchuk  2   3 , Sarah Parejo  2 , Susanne Jutzi  2 , Sabina Berezowska  2   4 , Nikolai Engedal #  5   6 , Mario P Tschan #  2   3
Affiliations

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells

Anna M Schläfli et al. Sci Rep. .

Abstract

ALK inhibitors effectively target EML4-ALK positive non-small cell lung cancer, but their effects are hampered by treatment resistance. In the present study, we asked whether ALK inhibition affects autophagy, and whether this may influence treatment response. Whereas the impact of targeted therapies on autophagic activity previously have been assessed by surrogate marker proteins such as LC3B, we here thoroughly examined effects on functional autophagic activity, i.e. on the sequestration and degradation of autophagic cargo, in addition to autophagic markers. Interestingly, the ALK inhibitor Ceritinib decreased mTOR activity and increased GFP-WIPI1 dot formation in H3122 and H2228 EML4-ALK+ lung cancer cells, suggesting autophagy activation. Moreover, an mCherry-EGFP-LC3B based assay indicated elevated LC3B carrier flux upon ALK inhibition. In accordance, autophagic cargo sequestration and long-lived protein degradation significantly increased upon ALK inhibition. Intriguingly, autophagic cargo flux was dependent on VPS34 and ULK1, but not LC3B. Co-treating H3122 cells with Ceritinib and a VPS34 inhibitor or Bafilomycin A1 resulted in reduced cell numbers. Moreover, VPS34 inhibition reduced clonogenic recovery of Ceritinib-treated cells. In summary, our results indicate that ALK inhibition triggers LC3B-independent macroautophagic flux in EML4-ALK+ cells to support cancer cell survival and clonogenic growth.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Ceritinib decreases cell viability, abolishes ALK phosphorylation and activates autophagy in EML4-ALK positive NSCLC cells (A) H3122 and H2228 cells were treated with Crizotinib or Ceritinib with indicated concentrations for 3 days before metabolic activity was determined using Alamarblue assay (n = 2). Two-Way ANOVA was applied to compare the two drugs. ****p < 0.0001, **p < 0.01. (B) Western Blot for p-mTOR, p-ULK1, p-EML4-ALK and p-p70S6K is shown of protein lysate from H3122 and H2228 cells treated with 0, 0.1 or 1 µM Ceritinib for 24 h. Total protein serves as a loading control. (C) Confocal microscopy pictures of GFP-WIPI1 transiently over-expressed in H3122 cells treated with DMSO (Ctrl) or Ceritinib for 18 h. (D) Quantification of GFP-WIPI1 dots shown in C. Dot plot includes data from 3 independent experiments with 6–7 pictures per experiment (n = 20/21). Mann–Whitney U, **p < 0.01. (E) Histograms representing the mCherry to GFP ratio of H3122 and H2228 cells stably expressing the mCherry-EGFP-LC3B construct after Ceritinib treatment (0–1 µM for 24 and 48 h, n = 3). (F) Quantification of the ratiometric FACS analysis performed in H. Kruskal–Wallis followed by Dunn`s multiple comparison was applied for statistical testing *p < 0.05, **p < 0.01, ***p < 0.001. (G) p62 dots were analyzed by immunofluorescence after H3122 cells were treated with Ceritinib (18 h) ± BafA (last 2 h). Nuclei were counterstained with DAPI. (H) Left panel: Bar plot shows quantification of p62 dots shown in G. Experiment has been performed 3 times. Right panel: p62 flux calculated based on the p62 dot quantification by subtracting the BafA untreated sample from its respective BafA treated condition. Mann–Whitney U, ***p < 0.001.
Figure 2
Figure 2
Ceritinib triggers ULK1- and VPS34-dependent autophagy (A) Bar plot represents percent proteolysis of H3122 cells after treatment with DMSO or Ceritinib for 24 h in the presence or absence of BafA during the last 5 h (n = 3). (B) Quantification of BafA-sensitive and SAR-405-sensitive proteolysis in H3122 cells after treatment with 1 µM Ceritinib for 24 h (n = 3). Percent proteolysis was determined and calculated as described in the methods section. (C) H3122 cells were treated with DMSO or 1 µM Ceritinib (24 h) ± BafA and ± SAR405 (during the last 3 h) before LDH sequestration was determined (n = 3). (D) H3122 and A549 cells were treated with DMSO or 1 µM Ceritinib/Alectinib/Lorlatinib (24 h) ± BafA and ± MRT68921 (during the last 3 h) before LDH sequestration was determined (n = 3). (E) H3122 control (shCtrl) and two ULK1 knock down (shULK1#1 and shULK1#2) cell lines were subjected to LDH sequestration assay after 18 h of Ceritinib treatment. BafA was added during the last 3 h (n = 3). (F) Experiment as in E, but with H3122 shCtrl, shLC3B#1 and shLC3B#2 cells (n = 3). Mann–Whitney U was applied to compare two groups; *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
Blocking autophagy via VPS34 inhibition sensitizes cells to Ceritinib (A) H3122 cells were treated with DMSO or 1 µM Ceritinib in the presence or absence of VPS34-IN1 for 2 days before living cells were counted and re-seeded for clonogenic assays. Bar plot represents percent of living cells compared to control treated cells (n = 4). (B) Clonogenic assay of experiments as described in A. After 2 days of treatment, 5 × 103 living cells were re-seeded and kept for 10 days without treatment. Thereafter, colonies were stained with crystal violet and counted. (C) Quantification of the clonogenic assays described in A (n = 4). (D) H3122 cells were treated with DMSO or 1 µM Ceritinib in the presence or absence of increasing concentrations of BafA (40, 80 and 120 nM) for 2 days before living cells were counted and quantified from 5 independent experiments as in A. (E) Clonogenic assay of cells as described in D. Living cells were re-seeded at low density (5 × 103 cells per well in a 6-well plate) and cultured in the absence of drugs for 10 days. Colonies were counterstained using crystal violet and counted. (F) Quantification of colonies shown in E (n = 5). (G) H3122 cells were treated with DMSO or 1 µM Ceritinib in the presence or absence of 15 µM chloroquine and asssessed as in A (n = 3). (H) Clonogenic assay of chloroquine (CQ) treated cells as described in G and assessed as in B. (I) Quantification of colonies shown in H (n = 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sabir, S. R., Yeoh, S., Jackson, G. & Bayliss, R. EML4-ALK Variants: Biological and Molecular Properties, and the Implications for Patients. Cancers (Basel)9, (2017). - PMC - PubMed
    1. Gelatti ACZ, Drilon A, Santini FC. Optimizing the sequencing of tyrosine kinase inhibitors (TKIs) in epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) Lung Cancer. 2019;137:113–122. doi: 10.1016/j.lungcan.2019.09.017. - DOI - PMC - PubMed
    1. Takeuchi K, et al. RET, ROS1 and ALK fusions in lung cancer. Nat. Med. 2012;18:378–381. doi: 10.1038/nm.2658. - DOI - PubMed
    1. Ricciuti B, et al. Precision medicine against ALK-positive non-small cell lung cancer: beyond crizotinib. Med. Oncol. 2018;35:72. doi: 10.1007/s12032-018-1133-4. - DOI - PubMed
    1. Fry AM, O’Regan L, Montgomery J, Adib R, Bayliss R. EML proteins in microtubule regulation and human disease. Biochem. Soc. Trans. 2016;44:1281–1288. doi: 10.1042/BST20160125. - DOI - PubMed

Publication types

MeSH terms