Survivin, a novel target of the Hedgehog/GLI signaling pathway in human tumor cells

Abstract

Survivin, an important antiapoptotic protein, is expressed in tumors, whereas in normal tissues the expression of this protein is extremely low, defining a role for survivin as a cancer gene. Survivin exhibits multifunctional activity in tumor cells. However, why survivin expression is sharply and invariably restricted to tumor tissue remains unclear. Here, we identified 11 putative consensus binding sites for GLI transcription factors in the survivin promoter and characterized the promoter activity. Inhibitors of the Hedgehog/GLI pathway, cyclopamine and GANT61, decreased the promoter activity in reporter assays. ΔNGLI2 (which lacks the repressor domain) was the most potent vector in activating the survivin promoter–reporter. Moreover, GANT61, a GLI1/2 inhibitor, repressed endogenous survivin protein and mRNA expression in most cells across a large panel of tumor cell lines. Chromatin immunoprecipitation showed GLI2 binding to the survivin promoter. The ectopic GLI2-evoked expression of endogenous survivin was observed in normal human fibroblasts. GANT61 decreased survivin level in nude mice tumors, mimicking the activity of GANT61 in cultured cells. The immunohistochemistry and double immunofluorescence of human tumors revealed a correlation between the tissue regions showing high GLI2 and survivin positivity. Thus, these results demonstrated that survivin is a classical transcriptional target of GLI2, a Hedgehog pathway signaling effector. This potentially reflects the high expression of survivin in human tumor cells. As the Hedgehog pathway is upregulated in virtually all types of cancer cells, these findings substantially contribute to the explanation of uniform survivin expression in tumors as a potential target for the development of a more effective treatment of cancers through the inhibition of GLI2 to restrain survivin activity.

Insights into the Regulation of Survivin Expression in Tumors

“…GLI2 associated with survivin promoter in chromatin immunoprecipitation assays and the expression of endogenous survivin could be easily evoked by ectopic GLI2 in human fibroblasts IMR90, where both survivin and GLI2 were normally silent. These findings have been supported by immunohistochemical staining, revealing correlation of GLI2 and survivin reactivity in tumor areas with a strong expression of both proteins [35]. Thus, survivin is a transcriptional target of GLI2 in a high proportion of various cancer cell lines including melanoma, NSCLC, SCLC, pancreas, and colorectal cancer and these findings may constitute a more general mechanism of survivin upregulation in tumors.…”

“…However, it has been shown that these non-consensus GLI motifs are bound by GLI with similar affinity as consensus sites and lead to a strong transcriptional activation [36]. Across a wide panel of tumor cell lines, we found by using GANT61, a low molecular weight inhibitor of GLI factors, that survivin expression depends on GLI2 in at least one-half of tumor cell lines tested [35]. GLI2 associated with survivin promoter in chromatin immunoprecipitation assays and the expression of endogenous survivin could be easily evoked by ectopic GLI2 in human fibroblasts IMR90, where both survivin and GLI2 were normally silent.…”

“…We have identified many potential GLI-binding sites in the survivin promoter [35]. All sites contained 1-3 mismatched bases.…”

“…The data showing that survivin expression and Hedgehog signaling pathway are linked through the transcription factor GLI2, which activate endogenous survivin expression and can ectopically evoke survivin in normal, survivin non-expressing cells [35], could open new ways of tumor therapy. Chemical inhibitors of GLI2 and survivin are available and may act in synergy during treatment.…”

Impact of Sonic Hedgehog Pathway Expression on Outcome in HPV Negative Head and Neck Carcinoma Patients after Surgery and Adjuvant Radiotherapy

“…It has also been shown that Gli-2 expression was associated with cell death in mice that were treated with radiation therapy for intrahepatic carcinoma [36]. Nevertheless, a recent study has shown that overexpression of survivin, which is a classical transcriptional target of Gli-2, was associated with radiosensitivity and improved survival in patients with head and neck squamous cell carcinoma [37,38]. Thus, in our cohort, the enhanced survival outcomes of patients with of Gli-overexpression may be attributed to the Gli-induced up-regulation of survivin, which subsequently might cause a positive response to postoperative radiotherapy.…”