Lung cancer models reveal SARS-CoV-2-induced EMT contributes to COVID-19 pathophysiology

  1. C. Allison Stewart
  2. Carl M. Gay
  3. Kavya Ramkumar
  4. Kasey R. Cargill
  5. Robert J. Cardnell
  6. Monique B. Nilsson
  7. Simon Heeke
  8. Elizabeth M. Park
  9. Samrat T. Kundu
  10. Lixia Diao
  11. Qi Wang
  12. Li Shen
  13. Yuanxin Xi
  14. Bingnan Zhang
  15. Carminia Maria Della Corte
  16. Youhong Fan
  17. Kiran Kundu
  18. Boning Gao
  19. Kimberley Avila
  20. Curtis R. Pickering
  21. Faye M. Johnson
  22. Jianjun Zhang
  23. Humam Kadara
  24. John D. Minna
  25. Don L. Gibbons
  26. Jing Wang
  27. John V. Heymach
  28. Lauren Averett Byers

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Abstract

COVID-19 is an infectious disease caused by SARS-CoV-2, which enters host cells via the cell surface proteins ACE2 and TMPRSS2. Using a variety of normal and malignant models and tissues from the aerodigestive and respiratory tracts, we investigated the expression and regulation of ACE2 and TMPRSS2 . We find that ACE2 expression is restricted to a select population of highly epithelial cells. Notably, infection with SARS-CoV-2 in cancer cell lines, bronchial organoids, and patient nasal epithelium, induces metabolic and transcriptional changes consistent with epithelial to mesenchymal transition (EMT), including upregulation of ZEB1 and AXL , resulting in an increased EMT score. Additionally, a transcriptional loss of genes associated with tight junction function occurs with SARS-CoV-2 infection. The SARS-CoV-2 receptor, ACE2, is repressed by EMT via TGFbeta, ZEB1 overexpression and onset of EGFR TKI inhibitor resistance. This suggests a novel model of SARS-CoV-2 pathogenesis in which infected cells shift toward an increasingly mesenchymal state, associated with a loss of tight junction components with acute respiratory distress syndrome-protective effects. AXL-inhibition and ZEB1-reduction, as with bemcentinib, offers a potential strategy to reverse this effect. These observations highlight the utility of aerodigestive and, especially, lung cancer model systems in exploring the pathogenesis of SARS-CoV-2 and other respiratory viruses, and offer important insights into the potential mechanisms underlying the morbidity and mortality of COVID-19 in healthy patients and cancer patients alike.

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  1. Version published to 10.1101/2020.05.28.122291v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.05.28.122291: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies used for western analysis include ACE2 (MA5-32307, ThermoFisher), GLUL (80636, Cell Signaling Technology), Vimentin (3932, Cell Signaling Technology), ZEB1 (3396, Cell Signaling Technology), and vinculin (Sigma, V9131) as a loading control.
    ACE2
    suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)
    GLUL (80636, Cell Signaling Technology), Vimentin (3932, Cell Signaling Technology), ZEB1 (3396, Cell Signaling Technology), and vinculin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Transcriptional data from experimental datasets were publicly available, including EMT induction by three weeks of TGFβ71, 393P murine cells with Zeb1 overexpression68, erlotinib-resistance via EMT in EGFR-mutated HCC4006 and HCC827 NSCLC cell lines76, gefitinib-resistant PC-9 cells via EMT77, T790M-mediated gefitinib-resistance in PC-9 cells78, Calu-3 or A549 transduced with a vector expressing human ACE2 were mock infected or infected with SARS-CoV-2 (USA-WA1/2020)60, forced miR-200 expression in 344SQ lung adenocarcinoma cells with high metastatic potential70, human bronchial organoids were infected with SARS-CoV-2 for five days65, and nasopharangeal swabs from patients with positive or negative SARS-CoV-2 PCR results66.
    PC-9
    suggested: None
    Calu-3
    suggested: None
    ChIPseq analysis of ZEB1 binding in the ACE2 promoter of HepG2, human hepatocyte carcinoma cells, as previously described97.
    HepG2
    suggested: None
    Flow Cytometry: One million cells each for HCC2302, H3255, HCC827, H1944, H441, H2023, HCC364, A549, H1355, HCC827 vector, and HCC827 ZEB1 were surfaced stained with ACE2 (Santa Cruz; sc-390851) then fixed in 2% PFA and permeabilized using 1X Intracellular Staining Perm Wash Buffer (BioLegend; 421002) prior to intracellular staining with Vimentin (BD Pharmingen; 562338).
    HCC364
    suggested: RRID:CVCL_5134)
    A549
    suggested: None
    HCC827 ZEB1
    suggested: None
    Metabolite Assay: One million cells each for 393P ZEB1 (DMSO 16h), 393P ZEB1 (DOX 16h), H441, H1944, HCC2302, HCC827, H2023, and H1355 were harvested.
    HCC2302
    suggested: RRID:CVCL_V636)
    HCC827
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    The expression data of a curated list of miR-200 family members (hsa-miR-200b-5p, hsa-miR-200b-3p, hsa-miR-200c-5p, hsa-miR-200c-3p, hsa-miR-200a-5p, hsa-miR-200a-3p, hsa-miR-429, hsa-miR-141-5p, hsa-miR-141-3p) known to be involved with EMT in NSCLC67 were compared with ACE2 expression data.
    hsa-miR-200b-5p, hsa-miR-200b-3p, hsa-miR-200c-5p, hsa-miR-200c-3p, hsa-miR-200a-5p, hsa-miR-200a-3p, hsa-miR-429, hsa-miR-141-5p
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were analyzed on a BD LSRFortessa Flow Cytometer and data was analyzed using FlowJo 10.7.1.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Graphs and statistical analysis were done using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Images were quantified with MicroVigene 4.0 (VigeneTech, Carlisle, MA).
    MicroVigene
    suggested: (MicroVigene, RRID:SCR_002820)
    Computational prediction of binding sites: To generate the predicted ZEB1 (E-Box) binding sites on the ACE2 promoter, the promoter sequence for human ACE2 was downloaded and used in the matrix profile search on the JASPAR web portal72.
    JASPAR
    suggested: (JASPAR, RRID:SCR_003030)
    The resulting sites were ranked and accordingly the highest-scoring motifs were annotated on the promoter segment using Snapgene.
    Snapgene
    suggested: (SnapGene, RRID:SCR_015052)
    The drug-target constellation map (DTECT map) was generated based on drug screening data using a suite of R packages, including ggplot2, ggraph and igraph.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 25, 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  3. Version published to 10.1101/2020.05.28.122291v1 on bioRxiv