TRIM21 induces selective autophagic degradation of c-Myc and sensitizes regorafenib therapy in colorectal cancer

Abstract

Kirsten rat sarcoma virus (KRAS) mutation is associated with malignant tumor transformation and drug resistance. However, the development of clinically effective targeted therapies for KRAS-mutant cancer has proven to be a formidable challenge. Here, we report that tripartite motif-containing protein 21 (TRIM21) functions as a target of extracellular signal-regulated kinase 2 (ERK2) in KRAS-mutant colorectal cancer (CRC), contributing to regorafenib therapy resistance. Mechanistically, TRIM21 directly interacts with and ubiquitinates v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) at lysine 148 (K148) via K63-linkage, enabling c-Myc to be targeted to the autophagy machinery for degradation, ultimately resulting in the downregulation of enolase 2 expression and inhibition of glycolysis. However, mutant KRAS (KRAS/MT)-driven mitogen-activated protein kinase (MAPK) signaling leads to the phosphorylation of TRIM21 (p-TRIM21) at Threonine 396 (T396) by ERK2, disrupting the interaction between TRIM21 and c-Myc and thereby preventing c-Myc from targeting autophagy for degradation. This enhances glycolysis and contributes to regorafenib resistance. Clinically, high p-TRIM21 (T396) is associated with an unfavorable prognosis. Targeting TRIM21 to disrupt KRAS/MT-driven phosphorylation using the antidepressant vilazodone shows potential for enhancing the efficacy of regorafenib in treating KRAS-mutant CRC in preclinical models. These findings are instrumental for KRAS-mutant CRC treatment aiming at activating TRIM21-mediated selective autophagic degradation of c-Myc.

Keywords: KRAS; MYC; TRIM21; drug resistance; regorafenib.

Fig. 1.

TRIM21 inhibits c-Myc signaling by destabilizing the c-Myc protein. (A) Volcano plot illustrating the differentially expressed proteins identified through proteomic analysis in HCT116 cells (shRNA/TRIM21 versus shRNA/Control). (B) Enrichment analysis of biological processes and pathways for the up-regulated proteins in HCT116 cells (shRNA/TRIM21 versus shRNA/Control). (C and D) WB analysis of c-Myc expression in the CRC cells overexpressing TRIM21 (C) or stably silencing TRIM21 (D). (E) Representative images (Top) and correlation analysis (Bottom) of the indicated protein in 150 cases of CRC tissues. (F and G) The effect of overexpressing (F) or silencing (G) TRIM21 on the half-life of c-Myc protein was analyzed in HCT116 or RKO cells, respectively, treated with cycloheximide (CHX).

Fig. 2.

TRIM21 interacts with c-Myc to catalyze K63-linked ubiquitination at lysine 148. (A and B) Co-IP analysis of the interaction between TRIM21 and c-Myc at both the endogenous levels (A) and exogenous levels (B). (C) Confocal microscope images show PLA spots (red) in RKO cells, with each spot representing a single protein interaction. Nuclei were stained with DAPI (blue). (D) His pull-down assays were conducted using purified TRIM21 and c-Myc proteins. (E and F) Ubiquitination assays of endogenous c-Myc were performed using lysates from TRIM21-overexpressing (E) or TRIM21-silencing (F) RKO cells. (G) Ubiquitination assays of endogenous c-Myc in the lysates from RKO cells, overexpressing TRIM21 or the TRIM21CA mutant. (H) In vitro ubiquitination assays were performed by incubating Myc-TRIM21 and His-c-Myc in the presence of E1, E2, and ubiquitin. (I) Ubiquitination assays of exogenous c-Myc were conducted using lysates from HEK293T cells transfected with HA-Ub or HA-Ub/K63R. (J) An illustration is presented to depict c-Myc ubiquitination at K148 identified by Mass-spectrometry (MS). (K) An illustration displays the alignment of various c-Myc amino acid sequences, with the location of K148 depicted in red in the indicated species. (L) Co-IP analysis was performed to assess the interaction between TRIM21 and c-Myc in RKO cells expressing the indicated constructs. (M) Ubiquitination assays of exogenous c-Myc were carried out using lysates from RKO cells expressing the indicated constructs.

Fig. 3.

TRIM21 promotes c-Myc targeting for autolysosomal degradation. (A) WB analysis of c-Myc expression in RKO cells transfected with TRIM21 and treated with 10 μM MG132 or 10 mM CQ for 6 h prior to harvest. (B) WB analysis of c-Myc expression in RKO cells with stable silencing ATG5 or ATG7. (C) Co-IP analysis of the interaction between TRIM21 and c-Myc, TRIM21 and Beclin1, or TRIM21 and p62 in RKO cells. (D) Co-IP analysis was conducted to assess the interaction between TRIM21 or its TRIM21/MT (1 to 267) with c-Myc or p62 in RKO cells. (E) The PLA was used to detect the interaction between TRIM21 or its TRIM21/MT (1 to 267) and c-Myc in RKO cells. Confocal microscope images display PLA spots (in red), with each spot representing a single protein interaction. Nuclei were stained with DAPI (in blue). (F and G) The PLA was employed to detect the interaction between p62 and c-Myc in RKO cells, with TRIM21-silenced (F) or overexpressed (G). Each data point represents the number of interacting signals observed per cell. (H and I) The PLA was employed to detect the interaction between LC3B and c-Myc in RKO cells, with TRIM21-silenced (H) or overexpressed (I), during growth in normal medium with or without fetal bovine serum (FBS). (J) Co-IP analysis was performed to assess the interaction between p62 and c-Myc or its mutant c-Myc/K148R in RKO cells expressing the indicated constructs. (K and L) The PLA was used to detect the interaction between p62 and c-Myc or its mutant c-Myc/K148R (K), as well as between LC3B and c-Myc or its mutant c-Myc/K148R (L) in TRIM21 stably overexpressed HCT116 cells during growth in normal medium (RPMI-1640) without FBS.

Fig. 4.

TRIM21 suppresses glycolysis by regulating c-Myc-mediated ENO2 expression. (A) GSEA of glycolysis-associated genes in HCT116 cells stably expressing shRNA/TRIM21 or shRNA/control. (B) A heatmap illustrates the expression of specific glycolysis-related genes. (C) WB analysis of the indicated protein in HCT116 and RKO cells. (D and E) qPCR (D) and WB (E) analysis were carried out to assess ENO2 expression in TRIM21-overexpressing HCT116 cells. (F) Representative images (Left) and correlation analysis (Right) of TRIM21 and ENO2 protein levels in 150 cases of CRC tissues. (G and H) Enolase activity was determined using ELISA in TRIM21-overexpressing (G) or –silencing (H) HCT116 cells. (I) Enolase activity was determined using ELISA in HCT116 cells coexpressing shRNA/TRIM21 and shRNA/ENO2. (J and K) ELISA analysis of glucose uptake, lactate production, and ATP levels in TRIM21-overexpressing (J) or –silencing (K) HCT116 cells. (L and M) qPCR (L) and WB (M) were used to measure ENO2 levels in HCT116 cells coexpressing shRNA/TRIM21 and shRNA/c-Myc. (N and O) ChIP assays were performed to assess the ability of c-Myc to bind the Eno2 promoter in TRIM21-overexpressing HCT116 cells (N) and TRIM21-silencing RKO cells (O). (P) An illustration showing the predicted MYC binding site in the Eno2 promoter. (Q) Mutation of the potential MYC binding site in Eno2 promotor and analysis of the promoter activity after transfection of c-Myc.

Fig. 5.

ENO2 plays a crucial role in TRIM21-associated tumor growth in the CRC mouse model. (A) The schematic shows the strategy to generate Trim21^−/−; Eno2^ΔIEC mice. (B) Design of the experimental AOM/DSS model. (C–E) Representative photographs (C) and H&E staining (D) of the colon, as well as the colon tumor number (E, Top) and tumor burden (E, Bottom) from Trim21^+/+; Eno2^fl/fl (n = 11), Trim21^−/−; Eno2^fl/fl (n = 12), Trim21^+/+; Eno2^ΔIEC (n = 8), and Trim21^−/−; Eno2^ΔIEC (n = 11) mice. (F) ELISA analysis of lactate (Top) and ATP levels (Bottom) in colon tumors of Trim21^+/+; Eno2^fl/fl (n = 11), Trim21^−/−; Eno2^fl/fl (n = 12), Trim21^+/+; Eno2^ΔIEC (n = 8), and Trim21^−/−; Eno2^ΔIEC (n = 11) mice. (G) Representative images (Top) and quantification (Bottom) of the indicated organoids formed by the colonic tumor from AOM/DSS-induced Trim21^+/+; Eno2^fl/fl, Trim21^−/−; Eno2^fl/fl, Trim21^+/+; Eno2^ΔIEC, and Trim21^−/−; Eno2^ΔIEC mice.

Fig. 6.

TRIM21 phosphorylation triggered by mutant KRAS enhances c-Myc expression. (A) RKO cells were transfected with KRAS/G12D, and whole-cell extracts were collected for immunoprecipitation with an anti-TRIM21 antibody, followed by WB analysis of TRIM21 phosphorylation using an anti-phospho-Ser/Thr antibody. (B) RKO cells transfected with KRAS/G12D were treated with various kinase inhibitors (PD98059, 10 μM; SB203580, 10 μM; SP600125, 10 μM), and the p-TRIM21 was determined. (C) The p-TRIM21 was analyzed in the ERK1 or ERK2 knockdown RKO cells transfected with KRAS/G12D. (D) An illustration displays that TRIM21 contains a phosphorylation consensus motif of ERK (P-X-S/T-P), ³⁹⁴PQTP³⁹⁷. (E) The dot-blot assay was performed to validate the antibody's specificity for T396-specific phosphorylation [p-TRIM21 (T396)]. (F and G) WB analysis of p-TRIM21 (T396) levels in the CRC cells overexpressing KRAS/G12D (F) or stably silencing KRAS (G). (H) IHC staining of p-TRIM21 (T396) in CRC tissues with and without mutant KRAS (Left) and Boxplot shows the p-TRIM21 (T396) levels in the CRC tissues (Right). (I) Kaplan–Meier survival curve of patients with CRC expressing high (n = 62) and low (n = 42) levels of p-TRIM21 (T396). (J) Interaction diagram between wild-type TRIM21 and c-Myc (Left). Interaction diagram following the mutation of TRIM21’s T396 to D396 (Right), illustrating the alterations in interactions with residue R424 on c-Myc resulting from the mutation of TRIM21’s T396 to D396. The hydrogen bond interaction is indicated by a black dashed line, while the nonpolar interactions are depicted by yellow dashed lines. (K) Co-IP analysis was performed to assess the interaction between TRIM21 and c-Myc in RKO cells transfected with KRAS/G12D (Left) or KRAS/G12V (Right).

Fig. 7.

Inhibition of TRIM21 phosphorylation sensitizes regorafenib therapy in CRC. (A) Graphical representation of a human PDO and PDX model. (B) Representative images depicting CRC PDOs with or without KRAS/MT in response to regorafenib treatment (Left). Regression curves and IC₅₀ values were depicted in the graph (Right). (C) CRC PDOs carrying KRAS/WT or KRAS/MT were treated with control vehicle, vilazodone (10 μM), regorafenib (5 μM), or a combination of treatments. Representative images of CRC PDOs (Left) and quantification (Right) are presented. (D) A schematic representation of the treatment plan for NOD/SCID mice bearing subcutaneous CRC tumors. (E) Depiction of tumor images (Left), tumor weight (Middle), and tumor growth curve (Right). (F) IHC staining and quantification of p-TRIM21 (T396) and PCNA in CRC PDX tumors in each group.