Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis, and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1 and 4-1BB, in AA-treated cells. We demonstrated that downregulation of the transcription factor GLI1 in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1 and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules, and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and downregulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating NFATc1 nuclear translocation, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants reduced the AA-induced apoptosis, downregulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for the future PUFA-based therapeutic approaches

The Transcription Factor GLI1 Mediates TGFb1 Driven EMT in Hepatocellular Carcinoma via a SNAI1-Dependent Mechanism

The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-b1 (TGFb1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFb1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.National Institutes of Health Grants CA100882 and CA128633 (to LRR) and CA165076; the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567) (to MEFZ); the Mayo Clinic Cancer Center (CA15083), the Mayo Clinic Center for Translational Science Activities (NIH/NCRR CTSA Grant Number KL2 RR024151), and an American Gastroenterological Association Foundation for Digestive Health and Nutrition Bridging Grant (to LRR)

TGF-β Inducible Early Gene 1 Regulates Osteoclast Differentiation and Survival by Mediating the NFATc1, AKT, and MEK/ERK Signaling Pathways

TGF-β Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-β treatment. As reported previously, TIEG1−/− mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1−/− osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1−/− precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1−/− osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1−/− osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1−/− cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1−/− precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling

Nuclear factor of activated T cells-dependent downregulation of the transcription factor glioma-associated protein 1 (GLI1) underlies the growth inhibitory properties of arachidonic acid

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells.Wedemonstrated that down-regulation of the transcription factor gliomaassociated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed thatAArepresses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AAinduced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches.Fil: Comba, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Almada, Luciana Victoria. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Tolosa, Ezequiel J.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Iguchi, Eriko. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Marks, David L.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Vara Messler, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Silva, Renata Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Fernandez-Barrena, Maite G.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Enriquez-Hesles, Elisa. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Vrabel, Anne L.. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados UnidosFil: Botta, Bruno. Sapienza University; ItaliaFil: Di Marcotulio, Lucia. Sapienza University; ItaliaFil: Ellenrieder, Volker. University Medical Center Göttingen; AlemaniaFil: Eynard, Aldo Renato. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Pasqualini, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: Fernandez Zapico, Martin Ernesto. Mayo Clinic - Schuze Center Of Novel Therapeutic; Estados Unido

GLI1 promotes cell proliferation, viability, migration, invasion and colony formation of HCC cells.

<p>(A) Overexpression of GLI1 increases BrdU incorporation of Huh7 cells and silencing GLI1 expression in SNU398 cells decreases BrdU incorporation. (B) As assessed by the MTT assay, cell viability is increased at 48, 72 and 96 hours by overexpression of GLI1 in Huh7 cells and decreased at 24, 48, 72 and 96 hours by knockdown of GLI1 in SNU398 cells. (C) As assessed by the wound-healing assay, at both 24 and 48 hours, cell migration rate is increased in Huh7 cells by overexpression of GLI1 and decreased in SNU398 cells by silencing GLI1 expression. (D) Cell invasion is increased by overexpression of GLI1 in Huh7 cells and decreased by knockdown of GLI1 in SNU398 cells. (E) Overexpression of GLI1 promotes colony formation of Huh7 cells; in contrast, knockdown of GLI1 represses colony formation of SNU398 cells.</p