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Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells.

ABSTRACT: The Hedgehog signaling pathway is one of the most dysregulated pathways in human cancers. The glioma-associated oncogene homolog 1 (GLI1) transcription factor is the terminal effector of the Hedgehog pathway, frequently activated in human breast cancer and an emerging target of breast cancer therapy. While somatic mutations in the human GLI1 gene have never been reported in any cell or tumor type, we recently uncovered the existence of a novel alternatively spliced, truncated GLI1 (tGLI1) that has an in-frame deletion of 41 codons spanning the entire exon 3 and part of exon 4 of the GLI1 gene. Using glioblastoma models, we showed that tGLI1 has gained the ability to promote glioblastoma migration and invasion via its gain-of-function transcriptional activity. However, the pathological impact of tGLI1 on breast cancer remains undefined. Here, we report that tGLI1 is frequently expressed in human breast cancer cell lines and primary specimens we have examined to date, but is undetectable in normal breast tissues. We found for the first time that tGLI1, but not GLI1, binds to and enhances the human vascular endothelial growth factor-A (VEGF-A) gene promoter, leading to its upregulation. Consequently, tGLI1-expressing MDA-MB-231 breast cancer cells secret higher levels of VEGF-A and contain a higher propensity, than the isogenic cells with control vector and GLI1, to stimulate in vitro angiogenesis of human vascular endothelial cells. We further showed that tGLI1 has gained the ability to enhance the motility and invasiveness of breast cancer cells in a proliferation-independent manner and that this functional gain is associated with increased expression of migration/invasion-associated genes, CD24, MMP-2 and MMP-9. tGLI1 has also acquired the property to facilitate anchorage-independent growth of breast cancer cells. Collectively, our results define tGLI1 as a gain-of-function GLI1 transcription factor and a novel mediator of the behavior of clinically more aggressive breast cancer.

SUBMITTER: Cao X

PROVIDER: S-EPMC3175334 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The function of the tumor suppressor RE1 silencing transcription factor (REST) is lost in colon and small cell lung cancers and is known to induce anchorage-independent growth in human mammary epithelial cells. However, nothing is currently known about the role of this tumor suppressor in breast cancer. Here, we test the hypothesis that loss of REST function plays a role in breast cancer. To assay breast tumors for REST function, we developed a 24-gene signature composed of direct targets of the transcriptional repressor. Using the 24- gene signature, we identified a previously undefined RESTless breast tumor subtype. Using gene set enrichment analysis, we confirmed the aberrant expression of REST target genes in the REST-less tumors, including neuronal gene targets of REST that are normally not expressed outside the nervous system. Examination of REST mRNA identified a truncated splice variant of REST present in the REST-less tumor population, but not other tumors. Histological analysis of 182 outcome-associated breast tumor tissues also identified a subpopulation of tumors that lack full-length, functional REST and over-express the neuroendocrine marker and REST target gene Chromogranin A. Importantly, patients whose tumors were found to be REST-less using either the 24-gene signature or histology had significantly poorer prognosis and were more than twice as likely to undergo disease recurrence within the first 3 years after diagnosis. We show here that REST function is lost in breast cancer, at least in part via an alternative splicing mechanism. Patients with REST-less breast cancer undergo significantly more early disease recurrence than those with fully functional REST, regardless of estrogen receptor or HER2 status. Importantly, REST status may serve as a predictor of poor prognosis, helping to untangle the heterogeneity inherent in disease course and response to treatment. Additionally, the alternative splicing observed in REST-less breast cancer is an attractive therapeutic target.

Project description: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.

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Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells.

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