Anne M. Vrabel

The Transcription Factor GLI1 Mediates TGFβ1 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-β1 (TGFβ1) 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 TGFβ1 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.

GLI1 Inhibition Promotes Epithelial-to-Mesenchymal Transition in Pancreatic Cancer Cells

The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here, we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility, and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.

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.

2-Methoxyestradiol Suppresses Osteolytic Breast Cancer Tumor Progression In vivo

2-Methoxyestradiol (2ME(2)), a physiologic metabolite of 17beta-estradiol (estrogen), has emerged as a promising cancer therapy because of its potent growth-inhibitory and proapoptotic effects on both endothelial and tumor cells. 2ME(2) also suppresses osteoclast differentiation and induces apoptosis of mature osteoclasts, and has been shown to effectively repress bone loss in an animal model of postmenopausal osteoporosis. Given these observations, we have examined whether 2ME(2) could effectively target metastasis to bone, osteolytic tumors, and soft tissue tumors. A 4T1 murine metastatic breast cancer cell line was generated that stably expressed Far Red fluorescence protein (4T1/Red) to visualize tumor development and metastasis to bone. In an intervention study, 4T1/Red cells were injected into bone marrow of the left femur and the mammary pad. In the latter study, 2ME(2) (10, 25, and 50 mg/kg/d) treatment began on the same day as surgery and was continued for the 16-day duration of study. Tumor cell growth and metastasis to bone were monitored and bone volume was determined by micro-computed tomography. 2ME(2) inhibited tumor growth in soft tissue, metastasis to bone, osteolysis, and tumor growth in bone, with maximum effects at 50 mg/kg/d. Furthermore, tumor-induced osteolysis was significantly reduced in mice receiving 2ME(2). In vitro, 2ME(2) repressed osteoclast number by inducing apoptosis of osteoclast precursors as well as mature osteoclasts. Our data support the conclusion that 2ME(2) could be an important new therapy in the arsenal to fight metastatic breast cancer.

Characterization of the mouse TGFβ-inducible early gene (TIEG): conservation of exon and transcriptional regulatory sequences with evidence of additional transcripts

The humanTGFb InducibleEarly Gene (TIEG) cDNA was originally isolated as a transcript that was rapidly induced by transforming growth factor beta (TGF b) in normal human osteoblasts (Subramaniam et al. 1995). Further studies demonstrated that the accumulation of TIEG cDNA was also regulated by bone morphogenetic protein-2 (BMP-2), epidermal growth factor (EGF), and estrogen (Subramaniam et al. 1995; Tau et al. 1998). The existence of three zinc finger domains in the carboxyl terminus of the predicted protein led to the hypothesis that TIEG was an inducible transcription factor with a possible role in mediating the effects of some growth factors on target cells. Isolation and characterization of TIEG has recently demonstrated that both the TIEG protein and a related protein called Early Growth Response alpha (EGR a) are the products of a single gene located on human Chromosome (Chr) 8q22.2 (Subramaniam et al. 1998; Fautsch et al. 1998). The EGR a cDNA was first cloned from a human prostate cDNA library on the basis of its differential expression in androgen-dependent and androgen-independent human prostate cell lines (Blok et al. 1995). Recently we have demonstrated that TIEG and EGR a are transcribed from differentially regulated alternative promoters but use common exons for almost all of their coding regions (Fautsch et al. 1998). Thus, TIEG and EGRa differ in sequence only at their amino termini. The functional consequences of this difference in structure are not yet known, but the interpretation of studies on TIEG and EGR a regulation have been complicated by the use of cDNA probes that span the common exons. The protein sequence of human TIEG (hTIEG) is closely related to two different mouse sequences. The predicted sequence of the murine mGIF protein is 88% identical to hTIEG. The mGIF cDNA was recently characterized as a negative-acting transcription factor that can be induced by Glial cell-DerivedNeurotrophic Factor (GDNF) in murine neuroblastoma cells (Yajima et al. 1997). Despite the high amino acid conservation between mGIF and hTIEG, significant differences were noted in the tissue distribution of these mRNAs, leading to the hypothesis that mGIF is not the mouse TIEG homolog (Yajima et al. 1997). The nucleotide sequence of the hTIEG cDNA is also highly related to the sequence of an unpublished murine GC-binding protein, displaying 82% identity to hTIEG over 3 kb. The predicted protein sequences of mGIF and the GC-binding protein are identical at 419 of 481 residues. To test the hypothesis that the murine GC-binding protein was the mouse TIEG homolog, we used oligonucleotides corresponding to the GC-binding protein sequence in low stringency PCR reactions to isolate a 3-kb cDNA called mTIEG from a mouse lens epithelium plasmid library (Fig. 1). The 5 8 end of the mTIEG sequence was determined by 5 8 RACE analysis with mRNA isolated from mouse AKR2B cells. Analysis of several clones revealed that transcription starts at the analogous position in both mTIEG and hTIEG (Fautsch et al. 1998). The nucleotide sequence of mTIEG was originally found to be distinct from that of GCbinding protein as reported in Genbank. However, further sequencing of the original cDNA isolates for GC-binding protein (kindly provided by Eric Barklis) revealed that these two sequences are identical. The predicted protein sequence of mTIEG is also identical to that of mGIF. However, previous studies of mGIF expression revealed a tissue distribution pattern different from that previously reported for human TIEG (Fautsch et al. 1998). With a 36-bp oligonucleotide identical to a segment of the mTIEG cDNA sequence (see Fig. 1 for location), mGIF was found to be highly expressed in kidney, lung, brain, liver, and heart (Yajima et al. 1997). Low expression was seen in testis, while no expression of the 3.6-kb mGIF mRNA was detected in spleen or skeletal muscle. To determine whether mTIEG and mGIF have similar tissue distribution patterns, a mouse multiple tissue Northern was probed with a 2.8-kb fragment isolated from mTIEG cDNA (Fig. 2a). Our results show that mTIEG is expressed in all tissues tested, including skeletal muscle and spleen. The accumulation of mTIEG mRNA is also high in liver, lung, heart, and testis, but is lower in the kidney. All of these results are consistent with those previously reported for hTIEG. However, in contrast to mGIF expression, mTIEG is expressed at high levels in skeletal muscle, whereas lower levels are seen in kidney. Essentially identical results, including high levels of mTIEG mRNA in skeletal muscle, were obtained by probing the mouse multiple tissue blot with a TIEGspecific probe corresponding to the 5 8 end of the TIEG cDNA (data not shown). The expression of mTIEG in skeletal muscle was also verified by Northern analysis of mRNA isolated from the mouse skeletal muscle cell lines C2C12 and Sol8, with both the 2.8-kb mTIEG cDNA fragment (Fig. 2b) and the TIEG-specific 5 8 probe (Fig. 2c). These discrepancies between the expression patterns of mTIEG and mGIF mRNA are difficult to reconcile. They could reflect differences in TIEG expression in different mouse strains, and it remains possible that the oligonucleotide probe used in the mGIF study could be detecting the products of a separate gene. Long exposures of the mTIEG multiple tissue Northern blot reveal a faint hybridizing band approximately 400–500 nucleotides larger than that for mTIEG in several of the tissues. This larger band is not present in either C2C12 or Sol8 cells, suggesting it is not present in skeletal muscle. These observations are consistent with those reported for mGIF mRNA expression. Correspondence to: E.D. Wieben

Activation of the Transcription Factor GLI1 by WNT Signaling Underlies the Role of SULFATASE 2 as a Regulator of Tissue Regeneration

Background: Tissue regeneration is a complex process involving a network of ligand-activated pathways. Results: The sulfatase SULF2 modulates cell proliferation and organ growth through a WNT-dependent activation of the transcription factor GLI1. Conclusion: Together, these data define a novel cascade regulating tissue regeneration. Significance: The knowledge derived from this study will contribute to the understanding of the molecular mechanisms modulating regeneration and organogenesis. Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced β-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and β-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.

The Transcription Factor GLI1 Interacts with SMAD Proteins to Modulate Transforming Growth Factor β-Induced Gene Expression in a p300/CREB-binding Protein-associated Factor (PCAF)-dependent Manner

Background: The molecular mechanisms mediating the oncogenic activity of the transcription factor GLI1 remain elusive. Results: GLI1 interacts with SMAD factors and PCAF to regulate TGFβ-induced gene expression. Conclusion: These results define a novel epigenetic mechanism underlying the role of GLI1 as an oncogene. Significance: This study increases our understanding of gene expression regulation in cancer cells and its potential impact in tumor development. The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFβ signaling in the regulation of gene expression in cancer cells. TGFβ stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFβ-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFβ pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFβ- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFβ targets. We found that two additional TGFβ-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells.

MYOCILIN LEVELS IN PRIMARY OPEN-ANGLE GLAUCOMA AND PSEUDOEXFOLIATION GLAUCOMA HUMAN AQUEOUS HUMOR

PurposeTo determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. MethodsAqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor &bgr; 2 levels were assessed by enzyme-linked immunosorbent assay. ResultsMyocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/&mgr;L vs. 0.23±0.20 ng/&mgr;L, P<0.001; AB2: 0.98±0.59 ng/&mgr;L vs. 0.65±0.5 ng/&mgr;L, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor &bgr; 2 levels was observed. ConclusionsMyocilin is elevated in POAG, but not in PEXG aqueous humor.

Modulation of the IL-6 Receptor α Underlies GLI2-Mediated Regulation of Ig Secretion in Waldenström Macroglobulinemia Cells

Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.