TRIB3 Modulates PPARγ-Mediated Growth Inhibition by Interfering with the MLL Complex in Breast Cancer Cells

Abstract

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer.

Keywords: MLL–WRAD complex; PPARγ; Tribbles; breast cancer; epigenetics.

Figure 1

TRIB3 regulates PPARγ expression in MCF7 cells. (A) Schematic representation of the RNA-seq experiment and volcano plot showing differentially expressed genes between Sh-TRIB3 and Sh-control cells. (B) Up- and downregulated genes (p-adjusted value > 0.05). (C) Counts per million of PPARG in Sh-control and Sh-TRIB3 cells. (D) Western blot of endogenous TRIB3 expression and PPARG in Sh-control and Sh-TRIB3 in MCF7 cells and Tubulin as loading control. (E) Western blot of TRIB3-tGFP using anti-tGFP antibody and endogenous PPARG in inducible TRIB3-tGFP MCF7 cells. (F) Relative mRNA expression of PPARG in inducible TRIB3-tGFP MCF7 cells treated with and without doxycycline. * p < 0.05.

Figure 2

The phosphoproteome of TRIB3 knock-down cells reveals downstream targets of TRIB3. (A) Schematic representation of phosphoproteomics experiment using SILAC. (B) Pathway analysis using Metascape [41] of phospho-peptides found differentially phosphorylated in Sh-TRIB3 compared to Sh-control in MCF7 cells. (C) Schematic representation of phosphorylation of T916 in SET1A. (D) Post-translational modifications in SET1A according to Phosphositeplus^® [42].

Figure 3

TRIB3 binds to WDR5 and ASHL2, subunits of the WRAD complex and to the SET domain of MLL/SET1 proteins. (A) Co-immunoprecipitation (IP) of TRIB3-GFP together with wild-type Flag-WDR5, Flag-WDR5-S91K and Flag-WDR5-F133A, as well as TRIB3-R36A-GFP and TRIB3-R58A-GFP mutants. (B) Co-IP of TRIB3-GFP with Flag-RBBP5, Flag-DPY30 and Flag-ASHL2. (C) Co-IP of TRIB3-GFP, MLL-Flag and WDR5-MYC. All Co-IPs were performed using HEK293T cells.

Figure 4

TRIB3 inhibits WDR5–MLL complex formation. (A) Co-IP of MLL-Flag together with WDR5-MYC and different concentrations of TRIB3-GFP in HEK293T cells. Different expression levels of TRIB3 were achieved by co-transfecting TRIB3, WDR5 and MLL in a 1:1:1 ratio (lane 5) or 2:1:1 (lane 6). GFP plasmid was used to compensate for the total amount of DNA transfected per condition. (B) Co-IP of TRIB3-GFP and TRIB3-ΔN-terminal-GFP with MLL-Flag and MLL-R449A mutant in HEK293T cells. (C) H3K4me³ ELISA in inducible TRIB3-tGFP MCF7 cells treated with and without doxycycline and inducible TRIB3-tGFP cells without doxycycline and treated with the WDR5 inhibitor OICR-9429. Data are indicated as mean ± SEM. p-values were calculated using two-tailed Student’s t-test (* p < 0.05; ** p < 0.01) (D) Western blot of TRIB3-tGFP in cytoplasmatic, nuclear and chromatin-bond fractions in inducible TRIB3-tGFP MCF7 cells with and without doxycycline.

Figure 5

TRIB3 expression influences H3K4me³ mark in the PPARG locus in MCF7 cells. (A) ChIP-seq data of WDR5 (GSM1493030), RBBP5 (GSM1037511), KMT2A (GSM2373702), KMT2D (GSM3444924) and H3K4me³ (GSM3444908) in HEK293T cells as well as KMT2C (GSM3414777) and H3K4me³ (GSM2813049) in MCF7 cells with 7 kilo-bases around PPARG locus shown. (B) ChIP-RT-qPCR of H3K4me³ in Sh-TRIB3 and Sh-control cells in MCF7 using 2 different sets of primer pairs (B,C) designed around the PPARG transcription start site (TSS) shown in A. (D) ChIP-RT-qPCR of H3K4me³ around TSS of HSCB gene.

Figure 6

TRIB3 levels affects the sensitivity of MCF7 cells to TZD treatment. (A) Sh-control, shTRIB3 and TRIB3-tGFP-inducible MCF7 cells were treated for 72 h with rosiglitazone at 40 μm. TRIB3-tGFP-inducible cells were treated with or without doxycycline for 24 h before cell viability was measured. Data are indicated as mean ± SEM. p-values were calculated using two-tailed Student’s t-test *** p < 0.001) (B) Schematic representation of the role of TRIB3 as an epigenetic regulator.