NRF2 Enables EGFR Signaling in Melanoma Cells

Abstract

Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.

Keywords: EGF; EGFR; HNSC; KEAP1; MITF-low; NFE2L2; NRF2; NSCLC; TGF-alpha.

Figure 1

Regulation of EGFR and EGFR ligands by NRF2. (A) Kaplan–Meier plot of cumulative survival in SKCM melanomas, split into two groups with the 10% highest and the 10% lowest EGFR expression (https://cistrome.shinyapps.io/timer/ (accessed on 22 March 2021)). (B) Real-time PCR of EGFR gene expression after siRNA-dependent knockdown of NFE2L2 in UACC-62 cells (3 d). Two independent siRNAs against NFE2L2 are termed “N1” and “N2”. One-way ANOVA with Dunnett’s multiple comparisons test was carried out to calculate significant differences. Error bars represent SD. (C) Immunoblot showing NRF2 and EGFR expression after siRNA-dependent knockdown of NFE2L2 in UACC-62 cells (3 d). Vinculin served as loading control. (D) Immunoblot showing NRF2 and EGFR expression after siRNA-dependent knockdown of NFE2L2 in A375 and M14 cells. Actin served as loading control. (E) Immunoblot of NRF2 and EGFR expression in response to H₂O₂ treatment (200 or 400 µM, 5 h). Actin served as loading control. (F) Differential expression of EGFR pathway target genes in UACC-62 melanomas cells treated with control or NFE2L2-specific siRNA. Data are derived from previously published transcriptome data [23] (Bioproject accession number PRJNA601317), and normalized read counts were analyzed in GraphPadPrism (padj < 0.05). (G) EGF and TGFA expression, derived from the same dataset as subfigure (F). Two-tailed Student’s t-test was carried out to calculate significant differences. (H) Real-time PCR of EGF and TGFA gene expression after siRNA-dependent knockdown of NFE2L2 in UACC-62 cells (3 d). One-way ANOVA with Dunnett’s multiple comparisons test was carried out to calculate significant differences. (I) TGFα and EGF ELISA done with UACC-62 cells after siRNA-dependent knockdown of NFE2L2 and compared to control siRNA-treated cells. One-way ANOVA with Dunnett’s multiple comparisons test was carried out to calculate significant differences. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Direct binding of NRF2 to the EGF promoter. (A) Genome browser tracks of the EGF promotor region bound by NRF2, evaluated by ChIP-Seq analysis [23]. NRF2 was stabilized by treatment with sulforaphane (7.5 µM SFN, 24 h). (B) Left: Consensus NRF2 binding ARE sequence, according to the matrix profile from JASPAR 2020 (http://jaspar.genereg.net (accessed on 22 March 2021)) [38]. Right: Comparison of ARE sequences in the promoter region of the indicated human genes. Information about the EGF promoter is from the present study, while NQO1, HMOX1, and SLC7A11 information is from [39]. (C) Real-time PCR of EGF expression in response to H₂O₂ (400 µM, 5 h). Two-tailed Student’s t-test with Mann–Whitney test was carried out to calculate significant differences (* p < 0.05). (D) RNA expression of EGF in TCGA lung adenocarcinoma (LUAD) (Pan-Cancer Atlas) and TCGA head and neck squamous cell carcinoma (HNSC) (Pan-Cancer Atlas) divided into KEAP1/NFE2L2 wild type (LUAD: n = 417; HNSC: n = 449) and KEAP1/NFE2L2 mutant tumors (LUAD: n = 109; HNSC: n = 46). Data were downloaded from the GDC portal (https://portal.gdc.cancer.gov (accessed on 8 March 2021)). Statistical analysis was done using the Kruskal–Wallis test (** p < 0.01, **** p < 0.0001).

Figure 3

Negative correlation of MITF and EGFR/TGFA. (A–C) Genome browser tracks of the EGFR (A), TGFA (B), and NQO1 (C) genomic regions after NRF2 ChIP-Seq analysis [23]. NRF2 was stabilized by treatment with sulforaphane (7.5 µM SFN, 24 h). (D) Differential expression of MITF target genes in UACC-62 melanomas cells treated with control or NFE2L2-specific siRNA. Data are derived from previously published transcriptome data [23] (Bioproject accession number PRJNA601317) and normalized read counts were analyzed in GraphPadPrism (padj < 0.05). (E) Immunoblot of EGFR and MITF protein levels in indicated melanoma cell lines. Actin served as loading control. (F,G) Linear regression analysis of MITF and EGFR mRNA (F) or MITF and TGFA mRNA (G) (n = 470). The results shown here are based upon data derived from the TCGA dataset Skin Cutaneous Melanoma, and FPKM values were downloaded from the GDC portal (https://portal.gdc.cancer.gov (accessed on 8 March 2021)).

Figure 4

Suppression of TGFA by MITF. (A) Genome browser tracks of the TGFA genomic region after MITF ChIP-Seq analysis of SK-MEL-28 cells [40]. (B) Real-time PCR of TGFA gene expression in UACC-62 cells transgenic for the pSB control vector or the doxycycline-inducible pSB-MITF vector (500 ng/mL Dox, 3 d). (C) TGFα secretion, measured by ELISA, from UACC-62-pSB and pSB-MITF cells as described in subfigure (B). For subfigures (B,C), one-way ANOVA with Dunnett’s multiple comparisons test was carried out to calculate significant differences (* p < 0.05, *** p < 0.001). Error bars represent SD.

Figure 5

Suppression of EGFR by MITF. (A) Genome browser tracks of the EGFR genomic region after MITF ChIP-Seq analysis of indicated melanoma cell lines [40]. (B) Immunoblot of MITF and EGFR expression after Dox-inducible expression of MITF for 3 d. Vinculin served as loading control. (C) Real-time PCR of EGFR gene expression in UACC-62 cells transgenic for the pSB control vector or the doxycycline-inducible pSB-MITF vector (as in subfigure (B)). (D) Western blot, demonstrating EGFR and MLANA protein levels in UACC-62 cells treated, where indicated, with forskolin (“Fsk”, 20 µM, 24 h) and with H₂O₂ (400 µM, last 5 h before harvesting). MLANA served as indicator of MITF activity. Vinculin was used as loading control. For subfigure (C), one-way ANOVA with Dunnett’s multiple comparisons test was carried out to calculate significant differences (** p < 0.01). Error bars represent SD. (E) Overview of the proposed mechanism of NRF2-dependent EGFR pathway regulation in melanoma. Stress-induced NRF2 binds to the ARE in the EGF promoter and leads to elevation of soluble EGF, while simultaneously blocking MITF activity, resulting in derepression of EGFR and TGFA. This leads to EGFR activation on multiple levels, supporting the maintenance of EGFR^high/MITF^low/melanoma cells.

Figure 6

Cross-activation of NRF2 and EGFR pathways. (A) Protein blot, showing the activation of P-EGFR (Tyr1173), P-AKT (Ser473), and P-ERK1/2 (Thr202/Tyr204) as well as NRF2 and NQO1 expression in UACC-62 controls and UACC-62-NRF2-ko cells after indicated times of EGF stimulation (100 ng/mL). Actin served as loading control. (B) Western blot of NRF2 and P-AKT (Ser473) after transfection with control or NFE2L2-specific siRNA (3 d). Vinculin served as loading control. (C) Real-time PCR of NRF2 pathway target genes SLC7A11, HMOX1, and NQO1 in UACC-62 controls and UACC-62-NRF2-ko cells after indicated times of EGF stimulation (100 ng/mL). Two-tailed Student’s t-test test was carried out to calculate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent SD. (D) Immunofluorescence of NRF2 in UACC-62 cells and corresponding nuclear staining (Hoechst 33342) after indicated times of EGF induction (100 ng/mL).