The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

From MDPI via Jisc Publications RouterHistory: accepted 2021-10-19, pub-electronic 2021-10-22Publication status: PublishedFunder: Instituto de Salud Carlos III; Grant(s): PI18/00442Funder: European Commission; Grant(s): ITN-308 2016 721532Funder: Breast Cancer Now; Grant(s): 2012NovSP033Funder: Ministry of Economy, Industry and Competitiveness; Grant(s): RTI2018-094130-B-100Background: Tribbles pseudokinase 3 (TRIB3) has been proposed to both promote and restrict cancer generation and progression. However, the precise mechanisms that determine this dual role of TRIB3 in cancer remain to be understood. In this study we aimed to investigate the role of TRIB3 in luminal breast cancer, the most frequent subtype of this malignancy. Methods: We genetically manipulated TRIB3 expression in a panel of luminal breast cancer cell lines and analyzed its impact on cell proliferation, and the phosphorylation, levels, or subcellular localization of TRIB3 and other protein regulators of key signaling pathways in luminal breast cancer. We also analyzed TRIB3 protein expression in samples from luminal breast cancer patients and performed bioinformatic analyses in public datasets. Results: TRIB3 enhanced the proliferation and AKT phosphorylation in luminal A (HER2-) but decreased them in luminal B (HER2+) breast cancer cell lines. TRIB3 negatively regulated the stability of HER2 in luminal B breast cancer cell lines. TRIB3 expression was associated with increased disease-free survival and a better response to therapy in luminal breast cancer patients. Conclusions: Our findings support the exploration of TRIB3 as a potential biomarker and therapeutic target in luminal breast cancer

Zbtb7a is a transducer for the control of promoter accessibility by NF-kappa B and multiple other transcription factors

<div><p>Gene expression in eukaryotes is controlled by DNA sequences at promoter and enhancer regions, whose accessibility for binding by regulatory proteins dictates their specific patterns of activity. Here, we identify the protein Zbtb7a as a factor required for inducible changes in accessibility driven by transcription factors (TFs). We show that Zbtb7a binds to a significant fraction of genomic promoters and enhancers, encompassing many target genes of nuclear factor kappa B (NFκB) p65 and a variety of other TFs. While Zbtb7a binding is not alone sufficient to directly activate promoters, it is required to enable TF-dependent control of accessibility and normal gene expression. Using p65 as a model TF, we show that Zbtb7a associates with promoters independently of client TF binding. Moreover, the presence of prebound Zbtb7a can specify promoters that are amenable to TF-induced changes in accessibility. Therefore, Zbtb7a represents a widely used promoter factor that transduces signals from other TFs to enable control of accessibility and regulation of gene expression.</p></div

Target genes of a diverse set of TFs exhibit Zbtb7a-dependent promoter accessibility and expression.

<p>(A) DNA sequence motifs that are consistently enriched at Zbtb7a ChIP-seq peaks (note that the binding motif for Zbtb7a itself is also highly enriched, but not shown here). Known TF families with specificities matching each motif are indicated. Grey and green bars display the log2 fold-enrichment of each motif among the subset of peaks exhibiting unchanged (grey) or disrupted (green) DNase-I hypersensitivity levels in <i>Zbtb7a</i>-knockdown fibroblasts. *Note that the motif TGANTCA belongs to a previously identified set of motifs that are commonly found to be enriched within unrelated genome-wide datasets [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.ref024" target="_blank">24</a>], suggesting caution in interpretation based on motif enrichment alone, in this instance. (B) Schematic illustration of the strategy for identifying candidate Zbtb7a-utilising TF target genes and for experimentally analysing Zbtb7a dependence of promoter accessibility and gene expression. (C) Percentages of all promoters, and of target promoters of selected TFs, that exhibit Zbtb7a-regulated accessibility (experimentally defined as significantly reduced DNase-I hypersensitivity in Zbtb7a-knockdown fibroblasts, using a statistical cutoff of <i>P <</i> 0.05). Candidate TFs were identified using the scheme in panel b, and only targets of individual TFs that exhibit enrichment for Zbtb7a-regulated accessibility are shown. Error bars indicate 95% CIs. Significance of enrichments: Runx2 q = 1.4 × 10⁻⁶; cJun <i>P =</i> 1.4 × 10⁻⁶; Tead2 q = 2.3 × 10⁻¹¹; Cebpd q = 1.7 × 10⁻⁸; p65 <i>P =</i> 1.9 × 10⁻²⁹. (D) Microarray-based analysis of changes in mRNA expression levels upon Zbtb7a knockdown in fibroblasts, among target genes of selected TFs. Left: all TF target genes; right: target genes that also exhibit Zbtb7a-regulated accessibility. Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown. Significance of expression differences: Runx2 <i>P =</i> 8.9 × 10⁻³; cJun <i>P =</i> 2.3 × 10⁻⁷; Tead2 <i>P =</i> 9.7 × 10⁻¹¹; Cebpd <i>P =</i> 4.9 × 10⁻⁶; p65 <i>P =</i> 1.8 × 10⁻¹⁴. Additional details of statistical analysis are provided as Supporting information, and numerical values underlying figures are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s001" target="_blank">S1 Data</a>. ChIP-seq, chromatin immunoprecipitation sequencing; IQR, interquartile range; TF, transcription factor.</p

Zbtb7a is a widespread promoter- and enhancer-associated factor.

<p>(A, B) Genome browser example tracks of a Zbtb7a ChIP-seq and input coverage (top) across representative 1 Mb genomic intervals, illustrating the prominent overlap between Zbtb7a peaks and TSSs, and (bottom) zoomed-in on 25 kb regions including the Zbtb7a-bound <i>Camp</i> (A) and <i>Col4a1/2</i> (B) gene promoters. Lower tracks indicate predicted Zbtb7a binding peaks and RefSeq genes. (C) Fractions of the genome (left pie) and of Zbtb7a ChIP-seq peaks (right pie) that overlap with selected genomic features. (D) Fractions of the genome, of all promoters, and of p65 target promoters that are associated with predicted Zbtb7a ChIP-seq peaks. (E) DNA sequence motifs matching the described binding specificity of Zbtb7a, enriched among all (top) and promoter-associated (bottom) Zbtb7a ChIP-seq peaks, identified by de novo motif prediction. (F) Fractions of all promoters (top) or of promoters overlapping (bottom) or within 15 kb of (middle) predicted Zbtb7a ChIP-seq peaks that are expressed in fibroblasts at the indicated mRNA levels. (G) Enrichment of GO annotations among genes whose promoters are associated with Zbtb7a peaks (first column, “Zbtb7a promoter peak”) or with increased or decreased mRNA expression in control fibroblasts, compared to Zbtb7a-knockdown fibroblasts (second and third columns). Dots indicate statistically enriched annotations (<i>P <</i> 5 × 10⁻⁶; corrected q < 0.05). The most-significantly enriched annotations among genes with Zbtb7a-associated promoters are shown, of which some are also enriched among genes exhibiting Zbtb7a-regulated expression. Bars indicate levels of enrichment compared to all annotated genes. Statistical analysis is provided as Supporting information, and numerical values underlying figures are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s001" target="_blank">S1 Data</a>. ChIP-seq, chromatin immunoprecipitation sequencing; FPKM, RNA-sequencing fragments per kilobase transcript per million reads; GO, gene ontology; RefSeq, NCBI reference sequence database; TSS, transcription start site.</p

Zbtb7a is required for ongoing regulation of accessibility at a subset of its genomic binding sites.

<p>(A–D) Genome browser example tracks of DNase-I hypersensitivity surrounding (A, B) the <i>Camp</i> and <i>Col4a1/2</i> gene promoters (Zbtb7a-bound and Zbtb7a-dependent accessibility), (C) the promoter region of the <i>JmjD2d</i> and <i>Cwc15</i> genes (Zbtb7a-bound but ongoing accessibility is Zbtb7a-independent), and (D) the control <i>Hprt</i> promoter (non Zbtb7a-bound), in control or Zbtb7a-knockdown fibroblasts. Lower tracks indicate predicted Zbtb7a binding peaks and RefSeq genes. (E–G) Fractions of (E) promoters, (F) enhancers, and (G) TA3-responsive p65 target promoters that exhibit significant evidence for Zbtb7a-regulated accessibility in fibroblasts. Left (E, F): regions without any associated Zbtb7a peak (“Zbtb7a-negative”); right (E, F): regions with associated Zbtb7a peaks. Yellow/cyan slices indicate promoters with increased/decreased DNase-I hypersensitivity in control fibroblasts compared to Zbtb7a-knockdown fibroblasts, at the indicated <i>P</i> value cutoffs. Statistical analysis is provided as Supporting information, and numerical values underlying figures are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s001" target="_blank">S1 Data</a>. RefSeq, NCBI reference sequence database; TNF-α, tumour necrosis factor alpha.</p

Zbtb7a transduces regulation of accessibility upstream and independently of transcriptional activation.

<p>(A) Left: mRNA expression levels of 153 direct p65 target genes (73 “TA3-responsive” plus 80 “non-TA3-responsive”), in TNF-α-treated p65-knockout fibroblasts and in fibroblasts reconstituted with p65 or p65 TA3. Middle: p65 ChIP signals, and right: Zbtb7a ChIP signals, at promoters of p65 target genes in TNF-α-treated normal fibroblasts. mRNA levels are log2 microarray signal differences to non-reconstituted p65-knockout fibroblasts, for 3 biological replicates. (B) mRNA expression differences between TNF-α-treated control and Zbtb7a-knockdown fibroblasts expressing p65 TA3, at distinct gene sets. Dots in violins indicate mean values. Significance of expression difference between TA3-responsive and control genes: <i>P =</i> 3.0 × 10⁻¹⁴. (C) GFP reporter expression in TNF-α-treated control (left) or Zbtb7a-knockout (right) fibroblasts, expressing p65 C-terminal regions fused to the DBD of Gal4 and cotransfected with plasmids carrying 1 kb promoter sequences from the <i>Cxcl2</i> gene, in which NFκB binding motifs are replaced by the Gal4-UAS. Zbtb7a-knockout and congenic-control fibroblasts are both derived on a p53-knockout background, to prevent premature senescence [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.ref020" target="_blank">20</a>]. Error bars indicate SEM. (D) Mean TA3-induced DNase hypersensitivity levels across TA3-responsive (left) or non-TA3-responsive (right) p65 target promoters, in TNF-α-treated control and Zbtb7a-knockdown fibroblasts. (E) DNase hypersensitivity levels induced by p65 TA3 at TA3-responsive, non-TA3-responsive, or control (non-NFκB target) promoters, in TNF-α-treated control and Zbtb7a-knockdown fibroblasts. Induced levels represent differences in mean cut frequencies within ±600 bp surrounding the TSS, compared to p65-knockout fibroblasts. Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown. Significance of difference to Zbtb7a knockdown at non-TA3-responsive <i>P =</i> 4.7 × 10⁻². (F) Mean induced DNase hypersensitivity levels across p65 target promoters, in TNF-α-treated fibroblasts expressing p65 TA3 or a loss-of-function TA3 mutant that does not interact with Zbtb7a (“p65 TA3 mutant”). Additional details of statistical analysis are provided as Supporting information, and numerical values underlying figures are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s001" target="_blank">S1 Data</a>. ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; GFP, green fluorescent protein; IQR, interquartile range; NFκB, nuclear factor kappa B; SEM, standard error of the mean; TNF-α, tumour necrosis factor alpha; TSS, transcription start site.</p

A SILAC-based screen identifies the protein Zbtb7a as a factor associated with context-dependent gene regulation.

<p>(A) Schematic illustration of p65 variants. (B) GFP reporter expression from a plasmid containing 3 tandem NFκB-binding motifs linked to a minimal promoter, in transfected p65-knockout fibroblasts expressing p65 variants and treated with TNF-α. Error bars indicate SEM. (C) Expression of endogenous <i>Cxcl2</i> mRNA in TNF-α-treated p65-knockout fibroblasts expressing p65 or p65 TA3, or in untransduced p65-knockout fibroblasts. mRNA levels are expressed relative to the level in unstimulated p65-knockout cells. (D) DNase-I hypersensitivity levels induced by p65 or by p65 TA3, at TA3-responsive or control (non-NFκB target) promoters in transduced p65-knockout fibroblasts. Induced levels represent differences in mean cut frequencies within ±600 bp surrounding the TSS, compared to p65-knockout fibroblasts. TA3-responsive promoters are defined as promoters of genes whose expression is activated by p65 TA3 and that are associated with p65 ChIP-seq peaks (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#sec010" target="_blank">Materials and methods</a>). Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown. (E) GFP reporter expression from plasmids containing 1 kb promoter sequences from the TA3-responsive <i>Cxcl2</i> (left) and <i>Saa3</i> (right) genes, or the <i>Cxcl2</i> promoter with targeted mutations that delete the 2 consensus NFκB-binding motifs (centre), in transfected p65-knockout fibroblasts expressing p65 variants and treated with TNF-α. Error bars indicate SEM. (F) Expression of endogenous <i>Cxcl2</i> mRNA in TNF-α-treated p65-knockout fibroblasts expressing p65 variants (the TA3 deletion corresponds to removal of amino acids 361–396) (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s003" target="_blank">S1E Fig</a>). mRNA levels are expressed as the percentage of those in cells reconstituted with p65 TA3. Error bars indicate SEM. (G) Cartoon of the SILAC-based pull-down strategy to identify proteins that specifically interact with the functional TA3 region. (H) Combined log2 SILAC ratios of 701 proteins identified in GST-TA3 pull-down using NEs from TNF-α-treated HeLa cells. Ratios represent the means of heavy/light ratios derived from separate pull-downs with labels swapped between samples. (I) Validation of Zbtb7a as a functional TA3 interactor by pull-down from transfected HEK-293 cells overexpressing Zbtb7a, using GST alone, GST-TA3, or GST-TA3mut. Zbtb7a was detected by immunoblotting. Arrowhead indicates Zbtb7a. (J) Measurement of in vivo Zbtb7a interaction with p65 using BiFC. BiFC fluorescence values represent the GMFI of cells co-expressing the indicated proteins tagged with V1 (carboxy-terminal) or V2 (amino-terminal) fragments of Venus fluorescent protein, expressed as a percentage of the GMFI of cells expressing Zbtb7a-V1 plus p65-V2. Error bars indicate SEM. (K) Expression of endogenous <i>Cxcl2</i> mRNA in TNF-α-treated p65-knockout fibroblasts expressing p65 TA3, with or without shRNA knockdown of <i>Zbtb7a</i>. mRNA levels are expressed relative to the level in unstimulated cells without <i>Zbtb7a</i> knockdown. Statistical analysis of all experiments is provided as Supporting information, and numerical values underlying figures are reported in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s001" target="_blank">S1 Data</a>. BiFC, bimolecular fluorescence complementation; ChIP-seq, chromatin immunoprecipitation sequencing; CT, carboxy terminal domain; GFP, green fluorescent protein; GMFI, geometric mean fluorescence intensity; GST, glutathione S-transferase; HEK-293, human embryonic kidney cells 293; IQR, interquartile range; m/z, mass to charge ratio; NE, nuclear extract; NFκB, nuclear factor kappa B; SEM, standard error of the mean; shRNA, short hairpin RNA; SILAC, stable isotope labelling of amino acids in cell culture; TNF-α, tumour necrosis factor alpha; TSS, transcription start site; Zip, control self-interacting leucine zipper sequence derived from yeast GCN4.</p

Scheme of Zbtb7a function at gene-regulatory elements.

<p>Cartoon outlining role of Zbtb7a described in this paper. Principal experimental evidence for each depicted step is shown in the indicated figures. (A) Top row: Zbtb7a binds to many genomic regulatory sites, including promoters and enhancers (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g002" target="_blank">Fig 2C and 2D</a>). Zbtb7a binding is independent of the presence of client TFs, and binding may occur before client TF recruitment (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">Fig 6A</a>). Second row: Zbtb7a-utilising client TFs bind independently to neighbouring genomic sites. This may occur under normal, steady-state conditions or in response to stimulation (see Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g004" target="_blank">4A</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6B</a>). Third row: Zbtb7a transduces TF-dependent changes in local chromatin accessibility (see Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g004" target="_blank">4C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g005" target="_blank">5D</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6D</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6E and 6F</a>). In the case of p65, this is triggered by the interaction between Zbtb7a and the TA3 region of p65 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g005" target="_blank">Fig 5F</a>). Bottom row: Zbtb7a-transduced accessibility allows binding of secondary TFs and contributes to normal gene activation (see Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g004" target="_blank">4D</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6G</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.s005" target="_blank">S3L and S3M Fig</a>). (B) At genomic sites that lack Zbtb7a binding (which could occur due to the natural genomic distribution of Zbtb7 or through experimental manipulation), client TF binding is unimpaired (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">Fig 6B</a>), but Zbtb7a-dependent regulation of accessibility is abolished (see Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g004" target="_blank">4C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6D</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004526#pbio.2004526.g006" target="_blank">6E and 6F</a>). TF, transcription factor.</p

The Pseudokinase TRIB3 Negatively Regulates the HER2 Receptor Pathway and Is a Biomarker of Good Prognosis in Luminal Breast Cancer

Background: Tribbles pseudokinase 3 (TRIB3) has been proposed to both promote and restrict cancer generation and progression. However, the precise mechanisms that determine this dual role of TRIB3 in cancer remain to be understood. In this study we aimed to investigate the role of TRIB3 in luminal breast cancer, the most frequent subtype of this malignancy. Methods: We genetically manipulated TRIB3 expression in a panel of luminal breast cancer cell lines and analyzed its impact on cell proliferation, and the phosphorylation, levels, or subcellular localization of TRIB3 and other protein regulators of key signaling pathways in luminal breast cancer. We also analyzed TRIB3 protein expression in samples from luminal breast cancer patients and performed bioinformatic analyses in public datasets. Results: TRIB3 enhanced the proliferation and AKT phosphorylation in luminal A (HER2-) but decreased them in luminal B (HER2+) breast cancer cell lines. TRIB3 negatively regulated the stability of HER2 in luminal B breast cancer cell lines. TRIB3 expression was associated with increased disease-free survival and a better response to therapy in luminal breast cancer patients. Conclusions: Our findings support the exploration of TRIB3 as a potential biomarker and therapeutic target in luminal breast cancer