A non-genetic, cell cycle dependent mechanism of platinum resistance in lung adenocarcinoma
Abstract
We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.
Data availability
Raw RNAseq data has been uploaded to the NCBI Gene Expression Omnibus (GEO) data repository with the accession number GSE161800.
-
Pulsed cisplatin treatment of A549 lung adenocarcinoma cellsNCBI Gene Expression Omnibus, GSE161800.
Article and author information
Author details
Funding
National Breast Cancer Foundation (IIRS-18-103)
- Andrew Burgess
Tour de Cure (RSP-230-2020)
- Andrew Burgess
Cancer Institute NSW (10/FRL/3-02)
- Andrew Burgess
Cancer Institute NSW (2013/FRL102)
- David R Croucher
Cancer Institute NSW (15/REG/1-17)
- David R Croucher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experiments were carried out in compliance with the Australian code for the care and use of animals for scientific purposes and in compliance with Garvan Institute of Medical Research/St. Vincent's Hospital Animal Ethics Committee guidelines (ARA_18_17, ARA_16_13).
Copyright
© 2021, Gonzalez Rajal et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,481
- views
-
- 313
- downloads
-
- 19
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cancer Biology
For traditional laboratory microscopy observation, the multi-dimensional, real-time, in situ observation of three-dimensional (3D) tumor spheroids has always been the pain point in cell spheroid observation. In this study, we designed a side-view observation petri dish/device that reflects light, enabling in situ observation of the 3D morphology of cell spheroids using conventional inverted laboratory microscopes. We used a 3D-printed handle and frame to support a first-surface mirror, positioning the device within a cell culture petri dish to image cell spheroid samples. The imaging conditions, such as the distance between the mirror and the 3D spheroids, the light source, and the impact of the culture medium, were systematically studied to validate the in situ side-view observation. The results proved that placing the surface mirror adjacent to the spheroids enables non-destructive in situ real-time tracking of tumor spheroid formation, migration, and fusion dynamics. The correlation between spheroid thickness and dark core appearance under light microscopy and the therapeutic effects of chemotherapy doxorubicin and natural killer cells on spheroids’ 3D structure was investigated.
-
- Cancer Biology
Expression of NPRL2/TUSC4, a tumor-suppressor gene, is reduced in many cancers including NSCLC. Restoration of NPRL2 induces DNA damage, apoptosis, and cell-cycle arrest. We investigated NPRL2 antitumor immune responses in aPD1R/KRAS/STK11mt NSCLC in humanized-mice. Humanized-mice were generated by transplanting fresh human cord blood-derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung-metastases were developed from KRAS/STK11mt/aPD1R A549 cells and treated with NPRL2 w/wo pembrolizumab. NPRL2-treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. Antitumor effect was greater in humanized than non-humanized-mice. NPRL2 + pembrolizumab was not synergistic in KRAS/STK11mt/aPD1R tumors but was synergistic in KRASwt/aPD1S H1299. NPRL2 also showed a significant antitumor effect on KRASmt/aPD1R LLC2 syngeneic-tumors. The antitumor effect was correlated with increased infiltration of human cytotoxic-T, HLA-DR+DC, CD11c+DC, and downregulation of myeloid and regulatory-T cells in TME. Antitumor effect was abolished upon in-vivo depletion of CD8-T, macrophages, and CD4-T cells whereas remained unaffected upon NK-cell depletion. A distinctive protein-expression profile was found after NPRL2 treatment. IFNγ, CD8b, and TBX21 associated with T-cell functions were significantly increased, whereas FOXP3, TGFB1/B2, and IL-10RA were strongly inhibited by NPRL2. A list of T-cell co-inhibitory molecules was also downregulated. Restoration of NPRL2 exhibited significantly slower tumor growth in humanized-mice, which was associated with increased presence of human cytotoxic-T, and DC and decreased percentage of Treg, MDSC, and TAM in TME. NPRL2-stable cells showed a substantial increase in colony-formation inhibition and heightened sensitivity to carboplatin. Stable-expression of NPRL2 resulted in the downregulation of MAPK and AKT-mTOR signaling. Taken-together, NPRL2 gene-therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen-presentation and cytotoxic immune-cell activation.