Localization Of Gli1 Research Articles

Gli1 promotes cell survival and is predictive of a poor outcome in ERα-negative breast cancer

Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERalpha mRNA was strongly correlated in ERalpha-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERalpha-positive cell lines; this increase was prevented by treatment with the ERalpha-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERalpha-negative cell lines, but caused only a modest reduction in ERalpha-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERalpha (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERalpha-negative, including triple negative, cancers (P = 0.005), but not ERalpha-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERalpha; however, our data indicate a greater functional effect of Gli1 in ERalpha-negative cancers.

Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway

The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.

Canonical and Non Canonical Activation of Hedgehog Pathway in Multiple Myeloma

To identify genes or Tumor-Associated Antigens (TAA) potentially involved in the transition from Monoclonal Gammopathy of Undetermined Significance (MGUS) to Multiple Myeloma (MM) and in the MM pathogenesis, we used SEREX and screened serum of MGUS and MM patients with a MM cDNA library. We identified 10 novel antigens and focused our attention on Oral-facial-digital syndrome 1 gene (Ofd1) due to a specific antibody response in MGUS (20.6%) as well as post-transplant MM patients (27.2%). Ofd1 is an embryonal antigen with an important role in the organogenesis and fetal survival; however, in the adult life its expression decreases significantly and is restricted to few tissues. Additionally it is a basal body/centrosome-related protein and a component of Tip60-HAT complex which is involved in cilia formation and functional regulation of Hedgehog (Hh)- and Wnt- pathways in mouse models. We first studied the expression of Ofd1 by western blotting analysis and observed that 11 MM cell lines and 4 primary MM patient cells express high levels compared with PBMCs. To define its role in MM pathogenesis, we specifically knocked-down Ofd1 using 100nM and 200nM of specific siRNA in MM1s cell line. Time-dependent but dose-independent inhibition of Ofd1 does not affect viability and proliferation of MM1s at 48h, as assayed by MTT and thymidine-uptake, respectively. Since mutations in proteins of cilia may block the Hh-signal transduction downstream to Smoothened (Smo) but upstream to Gli transcription factors, we next evaluated the effects of Ofd1 inhibition on the expression of Gli1 and Patched1 (Ptch1), hallmarks of Hh-activity. Western blotting analysis showed that specific knock-down of Ofd1 leads to down-regulation of Gli1 or Ptch1, and β-catenin. Therefore Ofd1 has a role in the functional regulation of Hh- and Wnt-pathway, and its overexpression might lead to constitutive activation of these tumorigenic pathways. These data strongly indicate the existence of an alternative and not canonical activation of Hh-pathway in MM. We also investigated if the canonical way is active in MM. To demonstrate that Hh-pathway is important in CD138+ MM patient cells, we used double staining with antibody anti-CD138 and anti-Sonic (Shh) to show that MM cells co-express Shh, the most common Hh-ligand, suggesting that MM cells might have autocrine and/or paracrine Hh-activity. We confirmed the expression of Gli1 and Ptch1, by western blotting, in 10 on 11 MM cell lines tested and using antibody anti-Gli1 we demonstrated, by immunofluorescence, predominant nuclear localization of Gli1 suggesting constitutive Hh-pathway activation. Treatment with 5μM of a specific Smo-inhibitor for 12–24 and 36 hours, induces down-regulation of Gli1 and Ptch1 in 50% MM cell lines. While down-regulation of Ptch1, a Gli-target gene, is an early event starting at 12 hours, down-regulation of Gli1 starts later at 36 hours. Therefore Gli1 is not the most important transducer of Hh-signaling in MM. In 50% MM cell lines, however, we didn't observe any down-regulation of Gli1 and Ptch1 after treatment with Smo-inhibitor; in these MM cell lines, the predominant nuclear localization of Gli1 is completely abrogated by treatment for 24 hours with 10μM of forskolin, a compound inhibiting the nuclear translocation of Gli1 via PKA. Taken together our data show canonical as well as alternative not canonical activation of Hh-pathway associated with overexpression of ciliary protein Ofd1. Further delineation of the role of primary cilia in regulating cellular response to Hh-signaling in MM will both define their role in disease pathogenesis and support novel therapies targeting Hh-signaling to improve patient outcome in MM.

Hedgehog signaling pathway is a possible therapeutic target for gastric cancer

It has been shown that the hedgehog (Hh) signaling pathway is activated in gastric cancer. To investigate the viability of the Hh pathway as a therapeutic target, we analyzed activation of the Hh pathway in gastric cancer. Surgically resected gastric carcinoma specimens and lymph nodes were analyzed immunohistochemically. We used the percentage of cancer cells with nuclear translocation of Gli1 as a marker of Hh pathway activation. Nuclear localization of Gli1 was higher in 28 undifferentiated-type tumors than in 30 differentiated-type tumors. Eighteen of the fifty-eight cancer specimens consisted of a mixture of a histologically predominant part and a small area with different histology. In these 18 tumors, the percentage of cells showing nuclear staining of Gli1 was higher in the undifferentiated-type part than in the differentiated-type part. Nuclear staining of Gli1 in primary tumors was positively correlated with lymph node metastasis. The Gli1 nuclear staining percentage of metastatic lymph nodes correlated closely with that of each primary carcinoma. Cyclopamine, a Hh pathway inhibitor, suppressed the growth of gastric cancer cells in vitro. The Hh pathway may be a useful therapeutic target for such as undifferentiated-type gastric cancer with lymph node metastasis.

Sonic Hedgehog Induces Capillary Morphogenesis by Endothelial Cells through Phosphoinositide 3-Kinase

Sonic hedgehog (Shh) acts as a morphogen in many cell types. Recent studies have shown that hedgehog signaling is involved in vascular development as well as postnatal angiogenesis. However, the direct action of Shh on cultured endothelial cells has not been clearly shown. To address this issue, we examined the effect of Shh on morphological changes by murine brain capillary endothelial cells (IBE cells) and human umbilical endothelial cells (HUVECs). Shh induced capillary morphogenesis by these cells. The effect was inhibited by cyclopamine or pertussis toxin. Shh-induced capillary morphogenesis was also blocked by LY294002, a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Shh rapidly increased PI3-kinase activity in IBE cells and HUVECs; this activity was inhibited by cyclopamine. Nuclear localization of Gli1 was increased in Shh-treated IBE cells, which was not affected by LY294002. Actinomycin D and cycloheximide inhibited Shh-induced capillary morphogenesis. In IBE cells expressing kinase-inactive c-Fes, Shh failed to stimulate PI3-kinase activity and capillary morphogenesis. Considered collectively, Shh induced capillary morphogenesis of endothelial cells through both rapid activation of c-Fes/PI3-kinase pathways and transcriptionally regulated pathways.

GLI1 localization in the germinal epithelial cells alternates between cytoplasm and nucleus: upregulation in transgenic mice blocks spermatogenesis in pachytene.

The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1.

Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.