NFE2L3 drives hepatocellular carcinoma cell proliferation by regulating the proteasome-dependent degradation of ISGylated p53

Abstract

Nuclear factor erythroid 2-like 3 (NFE2L3) is a member of the cap 'n' collar basic-region leucine zipper (CNC-bZIP) transcription factor family that plays a vital role in modulating oxidation-reduction steady-state and proteolysis. Accumulating evidence suggests that NFE2L3 participates in cancer development; however, little is known about the mechanism by which NFE2L3 regulates hepatocellular carcinoma (HCC) cell growth. Here, we confirmed that NFE2L3 promotes HCC cell proliferation by acting as a transcription factor, which directly induces the expression of proteasome and interferon-stimulated gene 15 (ISG15) to enhance the proteasome-dependent degradation of ISGylated p53. Post-translational ISGylation abated the stability of p53 and facilitated HCC cell growth. In summary, we uncovered the pivotal role of NFE2L3 in promoting HCC cell proliferation during proteostasis. This finding may provide a new target for the clinical treatment of HCC.

Keywords: ISGylation; NFE2L3; cell proliferation; p53; proteasome.

FIGURE 1

NFE2L3 is overexpressed in HCC. (A, B) NFE2L3 mRNA levels are increased in HCC samples in The Cancer Genome Atlas (TCGA) database. (C, D) HCC tissues were immunostained for NFE2L3. IHC score represents the expression level of NFE2L3 (LSBio, Cat#LS‐B8066, 1:50). Bars, 50 μm (left) and 20 μm (right). (E) Diagram of N3D (NFE2L3^{ΔC(577–694aa)}): 138 amino acid residues (DBD region) are deleted, the DBD region is highly homologous in the CNC family. (F, G) Expression levels of NFE2L3 in HepG2 and MHCC97H cells after lentivirus overexpression of NFE2L3 and NFE2L3^{ΔC(577–694aa)} or transfection with plasmids (pCMV expression vector used to validate the expression and molecular weight of NFE2L3; Abcepta, Cat#AP50594, 1:1000). NFE2L3–1: NFE2L3 qPCR primer‐1 (DBD region), NFE2L3–2: NFE2L3 qPCR primer‐2 (N‐terminal region); lvNC: negative control lentivirus, lvN3: NFE2L3 overexpression lentivirus, lvN3D: NFE2L3^{ΔC(577–694aa)} overexpression lentivirus. (H) Dual luciferase activity was detected in HepG2 and HEK‐293T cells. *, p < 0.05; ***, p < 0.001.

FIGURE 2

NFE2L3 regulates HCC cell proliferation depending on its transcription factor activity. (A–D) Cell lines were established and were used for CCK‐8, colony‐formation and EdU incorporation assays. Fluorescence immunostaining showed nuclei (blue) and EdU (red). Bar, 200 μm. (E, F) In total, 5 × 10⁶ cells/mouse experimental cells were inoculated subcutaneously into nude mice (n = 3/group) and tumor size was measured. After 35 days, the animals were sacrificed and tumors were excised. (G) The expression of NFE2L3 (Abcepta, Cat#AP50594, 1:1000), p21, PCNA, CDK2 and CCND1 was detected. *, p < 0.05; **, p < 0.01.

FIGURE 3

NFE2L3 directly induces proteasome gene expression depending on its transcription factor activity. (A) Enrichment analysis of transcriptomic sequencing data obtained by overexpression of N3 and N3D (−log10 p‐value). (B) Association between N3 or N3D expression and proteasome, based on GSEA. (C–E) The expression of proteasome in lvNC, lvN3 and lvN3D cells was detected. (F, G) The expression of NFE2L3 and proteasome in shNC, shN3‐1 and shN3‐2 cells was detected. shNC: negative control short‐hairpin lentivirus; shN3‐1: NFE2L3 short‐hairpin lentivirus‐1; shN3‐2: NFE2L3 short‐hairpin lentivirus‐2. (H, I) The protein levels of NFE2L3 (Abcepta, Cat#AP19864B, 1:1000) and p53 in the established cells were detected. *, p < 0.05; **, p < 0.01.

FIGURE 4

NFE2L3 enhances p53 degradation through proteasome. (A–F) The protein levels of NFE2L3 (Abcepta, Cat#AP50594, 1:1000) and p53 in experimental cells treated with CHX (50 μg/mL), CHX (50 μg/mL) and CQ (10 μM), CHX (50 μg/mL) and MG132 (10 μM).

FIGURE 5

NFE2L3 enhances ISGylation of p53 and promotes its degradation. (A, B) The ubiquitination of p53 was detected in experimental cells treated with MG132. (C, D) Ubiquitin and p53 were detected after treatment with 10 μM TAK‐243. (E–G) The expression of ISG15 and NFE2L3 (Abcepta, Cat#AP19864B, 1:1000) in established cells was detected. (H) ChIP‐qPCR was used to analyze the combination of NFE2L3 and ISG15 promoter. *, p < 0.05; **, p < 0.01.

FIGURE 6

NFE2L3 enhances ISGylation of p53 and promotes its degradation. (A, B) The ISGylation of p53 was detected in experimental cells treated with MG132. (C, D) Immunoprecipitation was performed with p53 antibody followed by detection of p53, ISGylation, and HA. MG132, 10 μM. (E) Immunoprecipitation was performed with p53 antibody followed by detection of p53 and ISGylation. MG132, 10 μM. (F, G) Expression and subcellular localization of p53 (green) and ISG15 (red). Nuclei were stained with DAPI (blue) for reference. Bars, 20 μm.

FIGURE 7

ISG15 knockdown inhibits NFE2L3‐mediated cell proliferation. (A, B) CCK‐8, (C, D) EdU incorporation and (E) colony‐formation assays were used to analyze cell proliferation with ISG15 knockdown (siRNA, 150 nM). Bar, 200 μm. (F) The protein levels of ISG15, p53, p21, CDK2, CCND1 and PCNA in NFE2L3‐overexpression cells with ISG15 knockdown. (G) The protein level of proteasome were detected. (H, I) Total ubiquitination and ISGylation were detected. (J) Relative mRNA fold change of p53 was detected. (K, L) The expression of p53, NFE2L3 (Abcepta, Cat#AP50594, 1:1000) and ISG15 was detected after treatment with siISG15 and CHX. *, p < 0.05.

FIGURE 8

Nfe2l3 knockout inhibits ISGylation in the liver. (A–C) The expression of Nfe2l3 (A), p53 (B), Isg15 (C) and the proteasome (D, E) in the liver of Nfe2l3 ^−/− mice. (F) The protein levels (LSBio, Cat#LS‐B8066, 1:1000), and (G) the total ubiquitination and ISGylation in the liver of Nfe2l3 ^−/− mice. *, p < 0.05; **, p < 0.01.

FIGURE 9

NFE2L3 is positively correlated with ISG15 expression and is associated with overall survival in multiple cancer types. (A) ISG15 protein expression in HCC tissue microarray. Bars, 200 μm. (B) IHC score of ISG15 in HCC tissue microarray. (C) The expression correlation of NFE2L3 with ISG15 was examined in an HCC tissue microarray by Spearman's correlation (n = 93). The overall survival of HCC (D, E), PAAD (G, H) and LGG (J, K) patients with NFE2L3 and ISG15 levels in TCGA using TIMER2.0. The expression correlation of NFE2L3 with ISG15 was examined in the PAAD (F) and LGG (I) based on TCGA using TIMER2.0. *, p < 0.05.

FIGURE 10

Diagram of the multidimensional regulation of NFE2L3 on the proteasome‐dependent degradation of ISGylated p53 to drive HCC cell proliferation.