Lung cancer models reveal SARS-CoV-2-induced EMT contributes to COVID-19 pathophysiology
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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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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 Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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. ACE2suggested: (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 vinculinSciScore 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 Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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. ACE2suggested: (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 vinculinsuggested: NoneExperimental Models: Cell Lines Sentences Resources 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-9suggested: NoneCalu-3suggested: NoneChIPseq analysis of ZEB1 binding in the ACE2 promoter of HepG2, human hepatocyte carcinoma cells, as previously described97. HepG2suggested: NoneFlow 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). HCC364suggested: RRID:CVCL_5134)A549suggested: NoneHCC827 ZEB1suggested: NoneMetabolite Assay: One million cells each for 393P ZEB1 (DMSO 16h), 393P ZEB1 (DOX 16h), H441, H1944, HCC2302, HCC827, H2023, and H1355 were harvested. HCC2302suggested: RRID:CVCL_V636)HCC827suggested: NoneExperimental Models: Organisms/Strains Sentences Resources 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-141-5psuggested: NoneSoftware and Algorithms Sentences Resources Samples were analyzed on a BD LSRFortessa Flow Cytometer and data was analyzed using FlowJo 10.7.1. FlowJosuggested: (FlowJo, RRID:SCR_008520)Graphs and statistical analysis were done using GraphPad Prism 8. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Images were quantified with MicroVigene 4.0 (VigeneTech, Carlisle, MA). MicroVigenesuggested: (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. JASPARsuggested: (JASPAR, RRID:SCR_003030)The resulting sites were ranked and accordingly the highest-scoring motifs were annotated on the promoter segment using Snapgene. Snapgenesuggested: (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. ggplot2suggested: (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.
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- No protocol registration statement was detected.
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