SOX13/TRIM11/YAP axis promotes the proliferation, migration and chemoresistance of anaplastic thyroid cancer

Abstract

Anaplastic thyroid cancer (ATC) is one of the most aggressive and virulent solid tumors. The ubiquitin proteasome system presents in all eukaryotic cells and is essential for cellular homeostasis. While its underlying role in ATC remains largely unclear. TRIM11 is an E3 ubiquitin ligase and has been reported to act as an oncogene in several human cancers. The present study aims to reveal the oncogenic function of TRIM11 in ATC. Western blot was used to measure the protein expression of TRIM11 and YAP, while the YAP target genes were measured by real-time PCR. CCK8 assay was used to detect cell viability; wound-healing assay and transwell assay were used to measure the migration ability of ATC. The xeno-graft tumor model was used for in vivo study. Immuno-precipitation assay was used to detect the interaction domain between YAP and TRIM11. And the ubiquitin-based Immuno-precipitation assays were used to detect the specific ubiquitination manner happened on YAP. TRIM11 depletion significantly decreases cell proliferation and migration capabilities of ATC cells, and elevates cell sensitivity to chemotherapy, which effect could be further rescued by YAP overexpression. TRIM11 depletion decreases YAP protein level and YAP/TEAD target genes, such as CTGF, ANKRD1 and CYR61 in ATC. Indicating that TRIM11 is a regulator of Hippo signaling pathway. Immuno-precipitation assay shows that the RING domain of TRIM11 is essential for the interaction with WW domain of YAP. Further mechanistic analysis suggests that TRIM11 promotes the mono-ubiquitination of YAP, thus prolongs its protein half. Furthermore, TRIM11 promoter analysis revealed that SOX13 activates TRIM11 transcription by binding to the promoter of TRIM11. In summary, our study describes the oncogenic function of TRIM11 in ATC, which acts as a post-translational modulating factor of Hippo pathway. Targeting TRIM11 may be a potential therapeutic method for ATC treatment.

Keywords: TRIM11; YAP; anaplastic thyroid cancer; mono-ubiquitination; stabilization.

Figure 1

TRIM11 depletion inhibits anaplastic thyroid cancer cell proliferation and migration. (A). TRIM11 depletion inhibits the cell proliferation in anaplastic thyroid cancer cells. (B). TRIM11 depletion induces G1 cell cycle arrest in anaplastic thyroid cancer cells. (C). TRIM11 depletion decreases clone formation capability of anaplastic thyroid cancer cells. (D, E). Representative images of EdU assay of anaplastic thyroid cancer cells. (F, G). Wound-healing assay of anaplastic thyroid cancer cells. (H). Transwell migration assay of anaplastic thyroid cancer cells. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001.

Figure 2

TRIM11 depletion decreases Hippo signaling activity in anaplastic thyroid cancer cells. (A). Heatmap of top 100 differentially expressed genes. (B). Pathway enrichment of differentially expressed genes. (C). TRIM11 depletion effect by two different siRNA oligos. Anaplastic thyroid cancer cells were transfected with two independent TRIM11 siRNAs or siControl. After 48 h, TRIM11 mRNA levels were determined by qRT-PCR. 36B4 was used as internal control. (D). TRIM11 depletion decreases YAP protein level. (E, F). TRIM11 depletion decreases YAP target genes using two different siRNA oligos. (G). TRIM11 depletion affects YAP-luciferase activity. CAL62 cells were transfected with SiTRIM11 or SiControl together with YAP-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection. (H).Immunofluorescence of YAP in KHM-5M cells. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

Figure 3

TRIM11 associates with YAP and increases its stability. (A) An immunofluorescence assay demonstrated that TRIM11 and YAP at least partially colocalized in CAL62 and KHM-5M cells. (B) Co-IP assay reveals association between endogenous TRIM11 and YAP in CAL62 cells. CAL62 cells were harvested with RIPA lysis buffer. Co-IP was performed using antibody as indicated. (C) In the presence of the proteasome inhibitor MG132, depletion of TRIM11 did not further decrease YAP protein levels. Anaplastic thyroid cancer cells were transfected with siTRIM11 or siControl. After 48 h, cells were treated with 10 nM MG132/vehicle for 6 h, cell lysates were prepared for western blot analysis. (D, E) TRIM11 depletion decreases YAP half-life in anaplastic thyroid cancer cells. Anaplastic thyroid cancer cells were transfected with siTRIM11 or siControl. After 48 h, cells were treated with 100 µM cycloheximide for indicated times. Cell lysates were prepared for western blot analysis.

Figure 4

TRIM11 associates with YAP WW domain through its RING domain. (A, B). YAP and TRIM11 domain structure and deletion mutants used in the study. (C). TRIM11 interacts with YAP through its WW domain. HEK293 cells were transfected with 2 µg Flag-TRIM11 together with Myc-YAP alpha full length or mutants. After 24 h, cells were harvested with NP-40 lysis buffer. Co-IP was performed using Flag antibody. The possible interacted YAP domains were detected by Myc antibody. (D). RING domain is required for TRIM11 to interaction with YAP. HEK293 cells were transfected with 2 µg Myc-YAP alpha together with Flag-TRIM11 full length or mutants. After 24 h, cells were harvested with NP-40 lysis buffer. Co-IP was performed using Myc antibody. The possible interacted TRIM11 domains were detected by Flag antibody.

Figure 5

TRIM11 stabilizes YAP though its RING domain. (A) Schematic diagram of TRIM11-2CA. (B) Increasing amounts of TRIM11 WT or 2CA were transfected into CAL62 cells and YAP expression was detected. (C). TRIM11 WT or 2CA was introduced into CAL62 cells together with TRIM11 siRNA. YAP levels were measured. (B). Interaction of Myc-TRIM11-2CA proteins with endogenous YAP in CAL62 cells was analyzed by co-IP assay. (E). HEK293 cells transfected with the indicated plasmids were treated with 100 µM cycloheximide, and collected at the indicated times for western blot. (F-I) TRIM11-2CA does not modulate poly/mono-ubiquitination of YAP protein.

Figure 6

Increased YAP expression recovered the effect of TRIM11 depletion. (A) Cell proliferation assay of CAL62. (B) Clone formation assay of CAL62. (C). Representative images of EdU assay of CAL62. (D) Wound-healing assay of CAL62. (E) Transwell migration assay of CAL62. (F) Overexpression of YAP in TRIM11-knockdown cells partly recovered tumor growth in vivo. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

Figure 7

SOX13 binds directly to the TRIM11 promoter. (A) The binding site of SOX13 provided by the JASPAR database. (B). A diagram shows the relative position of full-length and fragments of TRIM11 promoter reporters. (C, D) Serially truncated and mutated TRIM11 promoter constructs were transfected together with empty vector or SOX13 overexpression plasmids into CAL62 cells. Then a luciferase reporter assay was utilized. (E). A ChIP assay demonstrated the direct interactions between SOX13 and the TRIM11 promoter in the indicated anaplastic thyroid cancer cell lines. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

Figure 8

Mechanism diagram. Our study demonstrated that TRIM11could promote anaplastic thyroid cancer cells proliferation and migration both in vitro and in vivo. Furthermore, TRIM11 could increase YAP protein stability in ATC cells. SOX13 could activate transcription of TRIM11 in ATC cells by binding to the promoter region.