Interaction between STAT3 and GLI1/tGLI1 oncogenic transcription factors promotes the aggressiveness of triple-negative breast cancers and HER2-enriched breast cancer

Abstract

Signal transducer and activator of transcription 3 (STAT3), glioma oncogene homolog 1 (GLI1), and truncated GLI1 (tGLI1) are oncogenic transcription factors playing important roles in breast cancer. tGLI1 is a gain-of-function GLI1 isoform. Whether STAT3 physically and/or functionally interacts with GLI1/tGLI1 has not been explored. To address this knowledge gap, we analyzed 47 node-positive breast cancer specimens using immunohistochemical staining and found that phosphorylated-STAT3 (Y705), GLI1, and tGLI1 are co-overexpressed in the majority of triple-negative breast carcinomas (64%) and HER2-enriched (68%) breast carcinomas, and in lymph node metastases (65%). Using gene set enrichment analysis, we analyzed 710 breast tumors and found that STAT3 activation and GLI1/tGLI1 activation signatures are co-enriched in triple-negative subtypes of breast cancers and HER2-enriched subtypes of breast cancers, but not in luminal subtypes of breast cancers. Patients with high levels of STAT3 and GLI1/tGLI1 co-activation in their breast tumors had worse metastasis-free survival compared to those with low levels. Since these proteins co-overexpress in breast tumors, we examined whether they form complexes and observed that STAT3 interacted with both GLI1 and tGLI1. We further found that the STAT3-GLI1 and STAT3-tGLI1 complexes bind to both consensus GLI1-binding and STAT3-binding sites using chromatin immunoprecipitation (ChIP) assay, and that the co-overexpression markedly activated a promoter controlled by GLI1-binding sites. To identify genes that can be directly co-activated by STAT3 and GLI1/tGLI1, we analyzed three ChIP-seq datasets and identified 34 potential target genes. Following validations using reverse transcription polymerase chain reaction and survival analysis, we identified three genes as novel transcriptional targets of STAT3 and GLI1/tGLI1, R-Ras2, Cep70, and UPF3A. Finally, we observed that co-overexpression of STAT3 with GLI1/tGLI1 promoted the ability of breast cancer cells to form mammospheres and that STAT3 only cooperates with tGLI1 in immortalized mammary epithelial cells. In summary, our study identified novel physical and functional cooperation between two families of oncogenic transcription factors, and the interaction contributes to aggressiveness of breast cancer cells and poor prognosis of triple-negative breast cancers and HER2-enriched breast cancers.

Figure 1. Significant co-expression of p-STAT3 (Y705), GLI1 and tGLI1 in triple-negative and HER2-enriched node-positive breast carcinomas and in lymph node metastases

A) A TMA of 47 node-positive breast carcinomas tumors with subtype information was analyzed for expression levels of p-STAT3 (Y705), GLI1, and tGLI1 using IHC. >20% nuclear staining denotes positivity. *,** denote p<0.05. B) Representative images. C) Sixty lymph node metastases without subtype information were analyzed via IHC for p-STAT3 (Y705), GLI1, and tGLI1. All slides were scored by a pathologist.

Figure 2. Co-activation of the STAT3 and GLI1/tGLI1 pathways is enriched in triple-negative and HER2-enriched breast cancers

GSEA was used to determine if activated STAT3 (A) and GLI1 (B) signatures were enriched in 710 breast tumors. Expression profiles of 710 tumors were retrieved from the GEO database (GSE12276/2034/2603/5327/14020). NES, normalized enrichment score. C) Heat maps were generated via GraphPad Prism. D) Triple-negative and HER2-enriched breast cancers have significantly higher levels of STAT3 activation and GLI1/tGLI1 activation than the ER+ liminal tumors.

Figure 3. Co-activation of the STAT3 and GLI1/tGLI1 is associated with shortened metastasis-free survival of triple-negative and HER2-enriched breast cancers

Using the Kaplan-Meier analysis, log-rank analyses, univariate and multivariate analyses, and the STAT3 and GLI1/tGLI1 activation signatures, we analyzed 672 patients whose expression profiles and survival data were obtained from the GEO database (GSE12276/2034/2603/5327/14020). The cohort was divided into four groups according to the extent of activation for both pathways. Median survival years are indicated. A) Triple-negative breast cancer cohort. B) HER2-enriched breast cancer cohort. C) Univariate and multivariate analyses of triple-negative breast cancer cohort. D) Univariate and multivariate analyses of Her2-enriched breast cancer cohort.

Figure 4. Breast cancer cell lines and xenografts co-overexpress p-STAT3 (Y705), GLI1, and tGLI1

A) Breast cancer cell lines were analyzed by western blots for levels of p-STAT3, GLI1, and tGLI1. B) Cell line-derived xenografts were analyzed for p-STAT3 (Y705), GLI1, and tGLI1 via IHC. MDA-MB-231 triple-negative cells into the mammary fat pad (mfp; top panel) of female nude mice, and the brain-metastatic MDA-MB-231-BrM cells (bottom panel) into the mouse brain.

Figure 5. STAT3 physically interacts and functionally cooperates with GLI1 and tGLI1

A) STAT3 interacts with both GLI1 and tGLI1 as shown by IP-WB using MDA-MB-468 cells transfected with flag-tagged GLI1 or tGLI1 plasmids. A flag antibody (Ab) was used in IP. IgG was used as negative IP controls. B) STAT3 mutants (Constitutively Active (CA), S727A, and Y705F) similarly interacted with both GLI1 and tGLI1 as shown by IP-WB using isogenic HEK293-GLI1 or -tGLI1 cells transfected with flag-tagged STAT3 mutant plasmids. C) ChIP assay and sequential ChIP assay were used to examine the binding of STAT3, GLI1, and/or tGLI1 and their complexes to bind to the GLI1/tGLI1-binding sites in the PTCH1 and GLI1 gene promoters and/or the STAT3-binding sites (in the pGAS-Luc plasmid). D) Co-overexpression of STAT3CA with GLI1 or tGLI1 enhanced the activity of a promoter controlled by GLI1-binding sites in MDA-468 cells. 8x3′Gli-BS Luc was used with a control Renilla luciferase reporter. E) The STAT3-binding sites-containing reporter (pGAS-Luc) was activated by SHH, EGF, or in combination. Transfected cells were starved for 16 hrs and then treated with SHH (100 ng/ml) and/or EGF (100 ng/ml) for 4 hrs followed by determination of luciferase activity. F) Representative IF staining images depicting nuclear colocalization of ectopic STAT3 (GFP) and GLI1 or tGLI1 (RFP) in isogenic MDA-MB-231 cells. All experiments were done three times to derive means and standard deviations. Student t-test was used to compute p values (*, p<0.05).

Figure 6. Identification of genes co-activated by STAT3 and GLI1/tGLI1

A) Strategy to identify genes co-activated by STAT3 and GLI1/tGLI1. B) Three of the 18 potential gene targets were significantly upregulated by co-overexpression of STAT3-GLI1 and/or STAT3-tGLI1. qRT-PCR was used. tGLI1, TG. C) R-Ras2, Cep70, and UPF3A gene promoter and enhance regions that are bound to STAT3-GLI1 and/or STAT3-tGLI1 complexes. GLI1/tGLI1-bound regions are marked by green peaks (top panels). STAT3-bound regions are marked by blue peaks (bottom panels). D) High expression of R-Ras2, Cep70, or UPF3A genes is associated with worse metastasis-free survival. Gene expression profiles a survival data of 710 tumors were retrieved from the GEO database (GSE12276/2034/2603/5327/14020). E) STAT3-GLI1 and STAT3-tGLI1 complexes bind to the R-Ras2 gene promoter but not to the promoter of the internal reference control (Slug gene promoter) as shown by sequential ChIP assay with two antibodies sequentially.

Figure 7. STAT3 synergizes with GLI1/tGLI1 to promote mammosphere-forming ability of breast cancer cells and immortalized mammary epithelial cells

We transfected three cell lines with STAT3CA with and without GLI1/tGLI1 and then determine their ability to form mammospheres. A) Triple-negative MDA-MB-468 cells. B) Triple-negative BT20 cells. C) Immortalized human mammary epithelial MCF-10A cells. D) Immortalized human mammary epithelial HMLE cells. All experiments were done three times to derive means and standard deviations. Student t-test was used to compute p values (*, p<0.05).