Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells

Vad-Nielsen, Johan; Staunstrup, Nicklas Heine; Kjeldsen, Magnus Lindkvist; Dybdal, Nina; Flandin, Guillaume; Stradis, Claudia De; Daugaard, Tina Fuglsang; Vilsbøll-Larsen, Trine; Maansson, Christoffer Trier; Doktor, Thomas Koed; Sørensen, Boe Sandahl; Nielsen, Anders Lade

doi:10.21037/tlcr-22-507

Cited by 4 publications

(5 citation statements)

References 65 publications

(177 reference statements)

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“…In addition, the cell shape morphology of the HCC2827dRGMB cells was more elongated than the HCC827 sgRNA control cells and presented with less condensed cell‐cell contacts (Figure 2c). Altogether, data resembled the previously described mRNA expression and phenotypic profile associated with partial EMT in HCC827 cells (Bakir et al, 2020; Jakobsen et al, 2017; Vad‐Nielsen et al, 2023).…”

Section: Resultssupporting

confidence: 87%

“…Altogether, data resembled the previously described mRNA expression and phenotypic profile associated with partial EMT in HCC827 cells (Bakir et al, 2020;Jakobsen et al, 2017;Vad-Nielsen et al, 2023).…”

Section: Crispr/cas9-mediated Rgmb Depletion In Hcc827 Cells Induces ...supporting

confidence: 86%

“…For retrospective mRNA expression analyses in HCC827 and A549 NSCLC cells, we used the depmap portal https://depmap.org. mRNA expression data for HCC827 cells possessing EMT were previously described (Vad‐Nielsen et al, 2023). These mRNA expression data are presented in Supporting Information: Table S2.…”

Section: Methodsmentioning

confidence: 99%

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RGMb impacts partial epithelial‐mesenchymal transition and BMP2‐Induced ID mRNA expression independent of PD‐L2 in nonsmall cell lung cancer cells

Dorset,

Daugaard,

Larsen

et al. 2023

Cell Biology International

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PD‐1/PD‐ligand‐axis immunotherapy‐mediated activation of T‐cells for cancer cell elimination is a promising treatment of nonsmall cell lung cancer (NSCLC). However, the effect of immunotherapy on intracellular signaling pathways in cancer cells still needs further delineation. Repulsive Guidance Molecule b (RGMb), a regulator of Bone Morphogenetic Proteins (BMPs) signaling, interacts with the PD‐ligand, PD‐L2, at cancer cell membranes. Accordingly, a clarification of the functions of RGMb and its relation to PD‐L2 might provide insight into NSCLC cell signaling responses to PD‐1/PD‐ligand‐axis immunotherapy. In this study, the functions of RGMb and PD‐L2 were examined using the two NSCLC cell lines HCC827 and A549. CRISPR/Cas9 was used to decrease the expression of RGMb and PD‐L2, while lentiviral vectors were used to increase their expression. Downstream effects were examined by RT‐qPCR and immunoassays. Ectopic expression of RGMb impacted BMP2‐induced expression of ID1 and ID2 messenger RNA (mRNA) independently of PD‐L2, while RGMb depletion by CRISPR/Cas9 did not affect the BMP2‐mediated induction of ID1, ID2, and ID3 mRNA. However, depletion of RGMb resulted in a partial epithelial‐mesenchymal transition (EMT) gene expression profile in HCC827 cells, which was not mimicked by PD‐L2 depletion. The results show that RGMb is a coregulator of BMP signaling and hence, ID mRNA expression and that RGMb can control the EMT balance in NSCLC cells. However, RGMb appears to exert these functions independently of PD‐L2, and accordingly, the PD‐1/PD‐ligand axis for immune surveillance in NSCLC cells.

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Section: Resultssupporting

confidence: 87%

Section: Crispr/cas9-mediated Rgmb Depletion In Hcc827 Cells Induces ...supporting

confidence: 86%

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RGMb impacts partial epithelial‐mesenchymal transition and BMP2‐Induced ID mRNA expression independent of PD‐L2 in nonsmall cell lung cancer cells

Dorset,

Daugaard,

Larsen

et al. 2023

Cell Biology International

Self Cite

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“…To address the eventual existence of additional PD-L1 CpG sites with methylation levels correlating with the mRNA expression observed in NSCLC, we screened a previously generated dataset including genome-wide information on DNA methylation (Infinium MethylationEPIC 850K array) and mRNA expression (RNA-sequencing) from a group of five isogenic HCC827 cell clones [ 49 ]. These included parental HCC827 cells, as well as erlotinib TKI-resistant cell clones, with the amplification of c- MET (HCC827ER2 and HCC827ER3) and EMT (HCC827ER4 and HCC827ER10) [ 47 ].…”

Section: Resultsmentioning

confidence: 99%

“…Infinium MethylationEPIC 850K Array (Illumina, San Diego, CA, USA) analyses were performed for the HCC827 parental and erlotinib-resistant cell clones as previously described [ 49 ]. For bisulfite-pyrosequencing, genomic DNA was processed with the EpiTect Bisulfite kit (Qiagen, Venlo, Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Larsen

Dybdal

Daugaard

et al. 2023

Cancers

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Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

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Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells

Bui,

Daugaard,

Sorensen

et al. 2024

Biochemical and Biophysical Research Communications

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Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells

Cited by 4 publications

References 65 publications

RGMb impacts partial epithelial‐mesenchymal transition and BMP2‐Induced ID mRNA expression independent of PD‐L2 in nonsmall cell lung cancer cells

RGMb impacts partial epithelial‐mesenchymal transition and BMP2‐Induced ID mRNA expression independent of PD‐L2 in nonsmall cell lung cancer cells

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Expression of the non-coding RNA nc886 facilitates the development of tyrosine kinase inhibitor resistance in EGFR-mutated non-small-cell lung cancer cells

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