Cancer Res Treat.  2023 Jul;55(3):737-745. 10.4143/crt.2022.1414.

Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers

• Kim JS
• Lee JH
• Jeon SR
• Kim Y
• Jeon SH
• Wu HG

• KMID: 2544157
• DOI: http://doi.org/10.4143/crt.2022.1414

Abstract

Purpose
Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)–overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen.
Materials and Methods
Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription–quantitative polymerase chain reaction.
Results
We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment.
Conclusion
The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

Keyword

Apoptosis; CRISPR screen; DUSP1; Epidermal growth factor

Figure

• 

  Fig. 1 CRISPR/Cas9 knockout screening using A431 cells. (A) The surviving fraction of cells at varying concentrations of EGF treatment at 3 days (n=3); the EGF treatment range was 0–1,000 nM and 0–100 nM in A431 and HEK293FT cells, respectively. Data are presented as the mean±SEM from one-way ANOVA followed by Dunnett’s multiple comparisons test or Student’s t test, ****p < 0.0001. (B) After sequential transduction of GeCKO library v2 components into A431 cells, A431 cells were split in two groups. The experimental group was treated with EGF (100 nM) and the control group was treated with an equivalent volume of distilled water. To achieve enough EGF-resistant cells, this procedure was repeated four times after every cell confluency recovery to maintain the sgRNA coverage. The screening was performed in duplicates (created with BioRender.com). (C) Volcano plot demonstrating enriched or depleted genes in the EGF screen. Blue dots indicate genes conferring sensitivity and red dots indicate genes conferring resistance to EGF in the gene knockouts. (D) Top five enriched genes according to the p-value and FDR. ANOVA, analysis of variance; Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeats; EGF, epidermal growth factor; FDR, false discovery rate; SEM, standard error of mean; sgRNA, single guide RNA.

• 

  Fig. 2 Validation of CRISPR/Cas9 knockout screening results. (A) Validation of the top five genes using shRNA transfection. The cell surviving fraction was calculated 3 days after EGF treatment (n=3). Each gene was validated with three distinct shRNAs and the graph shows the most efficient shRNA. Data are presented as the mean±SEM from one-way ANOVA followed by Dunnett’s multiple comparisons tests, ****p < 0.0001. (B) The graph showing mRNA levels of DUSP1 quantified using RT-qPCR at three-time intervals: 3, 6, and 24 hours in the control and EGF-treated A459 cells (n=3). (C) The graph showing mRNA levels of DUSP1 quantified using RT-qPCR in the indicated cell lines at 24 h after EGF treatment (n=3). GAPDH was used as a loading control for RT-qPCR. Data are presented as the mean±SEM from Student’s t test, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Western blot demonstrating the levels of EGFR, DUSP1, ERK, p-ERK, and caspase-3 in A549 cells. The control and EGF-treated cells were split in two groups: the shRNA-transfected and control (not transfected) groups (n=1). Actin was used as a loading control for western blotting. (E) DUSP1 mRNA levels according to the EGFR-activating mutations in patients with head and neck cancer or lung cancer using the cBioPortal for Cancer Genomics database. Wild type (WT, n=84) indicates both no mutations and no CNV of EGFR, and mutation (n=39) indicates patients with EGFR-activating mutations regardless of the CNV status. Data are presented as the mean±SEM from Student’s t test, ****p < 0.0001. ANOVA, analysis of variance; Cas9, CRISPR-associated protein 9; CNV, copy number variation; CRISPR, clustered regularly interspaced short palindromic repeats; DUSP1, dual-specificity phosphatase 1; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated protein kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC-shRNA, negative control shRNA; RPKM, reads per kilobase of transcript per million mapped reads; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; SEM, standard error of mean; shRNA, short hairpin RNA.

• 

  Fig. 3 Proposed mechanism of EGF-induced apoptosis. EGF activates EGFR signaling pathway. EGFR dimerization activates STAT1 dimerization, which then increases DUSP1 expression. DUSP1 inhibits the ERK signaling pathway also activated by EGFR. As a negative feedback, activated ERK increases the expression of its inhibitor, DUSP1. Due to EGFR overexpression and high concentration of EGF, EGFR endocytosis is unable to stop the signaling. Finally, DUSP1 activity overwhelms ERK activity, leading to apoptosis (Created with BioRender.com). DUSP1, dual-specificity phosphatase 1; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated protein kinase; STAT1, signal transducer and activator of transcription 1.

Reference

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