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. 2020 Apr 28;40(10):e00597-19.
doi: 10.1128/MCB.00597-19. Print 2020 Apr 28.

NRF3-POMP-20S Proteasome Assembly Axis Promotes Cancer Development via Ubiquitin-Independent Proteolysis of p53 and Retinoblastoma Protein

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NRF3-POMP-20S Proteasome Assembly Axis Promotes Cancer Development via Ubiquitin-Independent Proteolysis of p53 and Retinoblastoma Protein

Tsuyoshi Waku et al. Mol Cell Biol. .
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Abstract

Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap 'n' collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

Keywords: 20S proteasome assembly; NFE2L3; NRF3; POMP; Rb; TP53; cancer development; colorectal cancer; p53; retinoblastoma; ubiquitin-independent proteolysis.

Figures

FIG 1
FIG 1
NRF3 sustains cancer cell growth and enhances 20S proteasome activity. (A) Dot plots showing NRF3 (top) and NRF1 (bottom) gene expression levels across multiple cancer types and paired normal samples. Red and green dots represent RNA sequencing expression values of patient-matched tumors and adjacent normal tissue archived at TCGA and the GTEx database. Red and blue abbreviations at the top of each graph indicate cancer types with significantly high and low expression levels of each NRF gene compared to normal samples, respectively (TPM, transcripts per million) (q value cutoff of 0.01 by ANOVA). The numbers of specimens are summarized in Table S3 in the supplemental material. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangial carcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower-grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectal adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma. (B) Endogenous NRF3 mRNA levels in HCT116 (colorectal carcinoma), H1299 (non-small-cell lung cancer), LNCaP (prostate adenocarcinoma), A-172 (glioblastoma), T98G (glioblastoma multiforme), U2OS (bone osteosarcoma), and HeLa (cervical adenocarcinoma) cell lines. NRF3 mRNA levels were assessed by RT-qPCR (n = 3; means + standard deviations [SD]). (C) Impact of NRF3 knockdown on cell viability. NRF3 or control siRNA (siCont) was transfected into the indicated cells. After 48 h, the cells were counted using a hemocytometer. *, P < 0.05; ‡, P < 0.005; n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (D) Impact of NRF3 overexpression [NRF3(OE)] or knockdown [NRF3(KD)] on proteasome activity. The indicated cell extracts were fractionated into 20 fractions using 10% to 40% glycerol gradient centrifugation and assayed for succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC; chymotrypsin-like)-hydrolyzing activity of 20S proteasomes (+SDS/−ATP) (top) or 26S proteasomes (−SDS/+ATP) (bottom). The mean and individual values are represented as lines and marks, respectively (n = 2). The activity in fractions 1 to 5 was derived from nonproteasomal proteases. The NRF3 overexpression and shNRF3 stable-expression cell lines were represented as oeNRF3 and shNRF3, respectively. GFP overexpression (oeGFP) and control shRNA stable-expression (shCont) cell lines were used as controls.
FIG 2
FIG 2
NRF3 overexpression induces POMP gene expression and 20S proteasome assembly. (A and B) Impact of NRF3 overexpression or knockdown on mRNA levels of 33 proteasome subunits (A) or a 20S proteasome assembly chaperone, POMP (B). The indicated mRNA levels in the H1299-NRF3(OE), HCT16-NRF3(OE), or HCT116-NRF3(KD) cell line were assessed by RT-qPCR. *, P < 0.05; †, P < 0.01; ‡, P < 0.005; n.s., not significant (n = 3; means + SD) (determined by t tests). (C) Impact of NRF3 overexpression on protein levels of proteasome subunits or a 20S proteasome assembly chaperone. The indicated proteins in H1299-oeNRF3#2 or -oeGFP#2 cells were detected by immunoblotting. (D and E) Impact of NRF3 overexpression on proteasome assembly. Fractions of H1299-oeNRF3#2 or -oeGFP#2 cells in Fig. 1D were immunoblotted for the indicated proteins in distinct SDS-PAGE gels (D) or in a single SDS-PAGE gel (E). In panel E, the expression values of the indicated proteins in H1299-oeNRF3#2 or -oeGFP#2 cells were assessed by immunoblotting and are presented in bar graphs. *, P < 0.05; †, P < 0.01; n.s., not significant (n = 3; means + SD) (determined by t tests).
FIG 3
FIG 3
NRF3 overexpression enhances 20S proteasome activity by directly inducing POMP gene expression. (A) Genomic locus of the POMP gene with an NRF3 ChIP sequencing peak. Multiple sequences of a candidate ARE within the POMP promoter are indicated for different species. (B) ChIP-qPCR validation of NRF3 recruitment to the POMP promoter. H1299-oeNRF3#2 or -oeGFP#2 cells were subjected to ChIP assays using anti-NRF3 antibodies. Immunoprecipitated DNA was assessed by RT-qPCR assays using primers specific for each genomic region, as indicated in panel A. †, P < 0.01; n.s., not significant (n = 3; means ± SD) (determined by a t test). (C) CRISPR/Cas9-based mutagenesis of the ARE within the POMP promoter. POMP-ARE mutant cells (mtPOMP) were generated from H1299-oeNRF3#2 cells (parental). The protospacer-adjacent motif (PAM), CRISPR target, and POMP-ARE region are indicated. (D) Impact of POMP-ARE mutation on NRF3 recruitment. The indicated cells were subjected to ChIP assays using anti-NRF3 antibodies. Immunoprecipitated DNA was assessed by RT-qPCR assays using primers specific for the POMP-ARE region. ‡, P < 0.005; n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (E and F) Impact of POMP-ARE mutation on mRNA and protein levels of POMP. mRNA and protein levels of POMP were assessed by RT-qPCR (E) and by immunoblotting (F), respectively. *, P < 0.05; n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test [E]). (G) Impact of POMP-ARE mutation on proteasome activity. The indicated cell extracts were fractionated into 20 fractions by 10% to 40% glycerol gradient centrifugation and assayed for Suc-LLVY-AMC (chymotrypsin-like)-hydrolyzing activity of 20S proteasomes (+SDS/−ATP) (top) or 26S proteasomes (−SDS/+ATP) (bottom). The mean and individual values are represented as lines and marks, respectively (n = 2). The activity in fractions 1 to 5 was derived from nonproteasomal proteases.
FIG 4
FIG 4
NRF3 knockdown increases Rb and p53 protein levels and induces p53-mediated cell cycle arrest and apoptosis. (A) Impact of NRF3 knockdown on Rb and p53 protein levels. HCT116 cells were transfected with the indicated siRNAs. After 2 days, the Rb and p53 proteins were detected by immunoblotting. (B) Impact of NRF3 knockdown on mRNA levels of Rb and p53. HCT116 cells were transfected with the indicated siRNAs. After 2 days, mRNA levels of the indicated genes were assessed by RT-qPCR. HCT116 p53KO cells were used as controls. ‡, P < 0.005; n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (C) Impact of NRF3 knockdown on Rb and p53 protein levels under cycloheximide (CHX) treatment. HCT116 cells were transfected with the indicated siRNAs. Two days after transfection, the cells were treated with 50 μg/ml cycloheximide, and the whole-cell extracts were prepared at the indicated time points. (D) Impact of NRF3 knockdown on mRNA levels of two p53 target genes, p21 and PUMA. HCT116 cells were transfected with the indicated siRNAs. After 2 days, mRNA levels of the indicated genes were assessed by RT-qPCR. HCT116 p53KO cells were used as controls. *, P < 0.05; 0.17, P = 0.17; n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (E) Impact of NRF3 knockdown on p53 recruitment to the p21 and PUMA promoters. HCT116 cells were transfected with the indicated siRNAs. After 2 days, ChIP assays were performed using anti-p53 antibodies, and immunoprecipitated DNA was assessed by RT-qPCR assays with primers specific for each p53 response element (p53RE). Normal mouse IgG was used as a control. *, P < 0.05; n.s., not significant (n = 3; means + SD) (determined by a t test). (F) Impact of NRF3 knockdown on p53-mediated cell cycle arrest. HCT116 cells were transfected with the indicated siRNAs. After 2 days, the cells were cultured with EdU for 2 h and stained with the Click-iT reaction mixture and PI following flow cytometry (n = 3; means + SD). HCT116 p53KO cells were used as controls. Representative contour plots are shown in Fig. S3A in the supplemental material. (G) Impact of NRF3 knockdown on p53-mediated apoptosis. HCT116 cells were transfected with the indicated siRNAs. After 2 days, the cells were stained with annexin V and PI, followed by flow cytometry (n = 3; means + SD). HCT116 p53KO cells were used as controls. The populations of annexin V-single-positive and annexin V-PI-double-positive cells are represented as “Early apoptosis” and “Late apoptosis + Necrosis,” respectively (Fig. S3C). (H) Impact of NRF3 knockdown on cell viability. NRF3 or control siRNA was transfected into the indicated cells. After 48 h, the cells were counted using a hemocytometer (n = 3; means + SD).
FIG 5
FIG 5
NRF3 contributes to the ubiquitin-dependent degradation of the Rb and p53 proteins and resistance to BTZ in a POMP gene expression-dependent manner. (A) Impacts of NRF3 and POMP on the ubiquitin (Ub)-independent degradation of the Rb and p53 proteins. Each protein was detected by immunoblotting after 24 h of treatment with 10 μM TAK-243, a ubiquitin-activating enzyme E1 inhibitor. DMSO was used as a control. POMP-ARE mutant mtPOMP-oeNRF3#2 cells were also used to check the impact of the defect on the NRF3-increased 20S proteasome. (B) Impacts of NRF3 and POMP on mRNA levels of Rb and p53. Rb and p53 mRNA levels of the indicated stable cells were assessed by RT-qPCR. POMP-ARE mutant mtPOMP-oeNRF3#2 cells were also used to check the impact of the defect on the NRF3-increased 20S proteasome. n.s., not significant (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (C) Impacts of NRF3 and POMP on resistance to a proteasome inhibitor, BTZ, or a ubiquitin-activating enzyme E1 inhibitor, TAK-243. Viabilities of NRF3 overexpression HCT116 or H1299 cells were assessed by WST-1 assays after 24 h of treatment with the indicated concentrations of BTZ (top) or TAK-243 (bottom). POMP-ARE mutant mtPOMP-oeNRF3#2 cells were also used to check the defect’s impact on the NRF3-POMP axis (n = 3; means ± SD).
FIG 6
FIG 6
POMP-ARE mutation abolishes NRF3-induced tumor growth and hepatic metastasis. (A and B) Impacts of NRF3 and POMP on tumorigenesis. Mice were subcutaneously injected with the indicated H1299 cells in each flank. (A and B) Tumor growth curves (A) and photographs and weights of tumors 28 days after injection (B). POMP-ARE mutant mtPOMP-oeNRF3#2 cells were also used to check the defect’s impact on the NRF3-POMP axis. Bar, 10 mm. *, P < 0.05; ‡, P < 0.005 (n = 6; means + SD) (determined by ANOVA followed by a Tukey test [A]). (C and D) Impact of NRF3 overexpression on metastasis in vitro. The invasion and migration abilities of the indicated H1299 cells were assessed by transwell (C) and scratch (D) assays, respectively. GFP#2 cells were used as controls. ‡, P < 0.005 (n = 3; means + SD) (determined by ANOVA followed by a Tukey test). (E and F) Impacts of NRF3 and POMP on metastasis in vivo. Mice were injected in the spleen with the indicated H1299 cells. After 28 days, their livers were removed. Representative images (E) and RT-qPCR-based quantification (F) of hepatic metastasis are shown. Bar, 10 mm. Arrowheads indicate metastatic nodules. GFP-overexpressing cells were used as controls. POMP-ARE mutant mtPOMP-oeNRF3#2 cells were also used to check the defect’s impact on the NRF3-POMP axis. ‡, P < 0.005 (n = 5; means + SD) (determined by ANOVA followed by a Tukey test).
FIG 7
FIG 7
Colorectal cancer patients with higher POMP/NRF3 expression levels exhibit poor prognoses. (A to C) Clinical association of NRF3 and POMP genes with the prognoses of patients with colorectal adenocarcinoma (COAD) or rectal adenocarcinoma (READ). A Spearman correlation plot of these genes (A) and Kaplan-Meier analysis comparing overall (B) and disease-free (C) survival were analyzed using TCGA and GTEx data sets. The hazard ratio (HR) was calculated based on Cox’s proportional-hazards model. (D) Schematic model of cancer development through the NRF3-POMP-20S proteasome assembly axis. The NRF3 gene is highly expressed in cancer cells, e.g., in colorectal adenocarcinoma. In these cancer cells, NRF3 transcribes the POMP gene and indirectly assembles the 20S proteasome. Upregulation of the NRF3-POMP-20S proteasome assembly axis degrades Rb and p53 in a ubiquitin-independent manner, thereby abrogating the tumor suppression signals, including cell cycle arrest and apoptosis. Cancer patients with tumors expressing higher levels of POMP/NEF3 exhibit lower overall and disease-free survival rates. NRF3-expressing cancer cells also develop resistance to BTZ-type proteasome inhibitor anticancer drugs, which directly bind to the catalytic sites within the 20S proteasome subcomplex.

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