Gli1 mediates lung cancer cell proliferation and sonic hedgehog-dependent mesenchymal cell activation.

Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options

SYNCYTIOTROPHOBLASTIC LOCALIZATION OF THE HUMAN GROWTH-HORMONE VARIANT MESSENGER-RNA IN THE PLACENTA

The hGH/hCS genes, clustered on chromosome 17 in the 5' to 3' order GH-N, CS-L, CS-A, GH-V and CS-B, show a high degree of sequence identity. The expression product of the GH-V gene is the placental growth hormone, which replaces pituitary GH in maternal blood throughout pregnancy. By means of mRNA competitive hybridization using P-32-labelled and unlabelled 30 bases long oligonucleotides, we first optimized specific hybridization conditions. In situ hybridization was then performed to locate the GH-V mRNA encoding placental growth hormone. The hGH-V gene appears expressed in the placental syncytiotrophoblast. Unlike the CS-A and CS-B genes (both encoding hPL) which are expressed uniformly in the syncytiotrophoblast, the GH-V mRNA is located in a few syncytiotrophoblast cells only

NSCLC cells do not respond to exogenous Shh but can secrete Shh ligand.

<p>NSCLC cells were treated or not with recombinant Shh (500 ng/ml) at the indicated times. RT-qPCR was performed to evaluate Gli1 and Ptch1 mRNA levels upon treatment in A549 cells (<b>A</b>) and H520 cells (<b>C</b>). Results are presented as fold differences of mRNA levels (2^∧∧Ct) compared with non-treated cells for each time point. Western blot was performed to evaluate Gli1 and Ptch1 protein levels in A549 cells (<b>B</b>) and in H520 cells (<b>D</b>) treated or not with Shh. ß-actin was used as a loading control. Secretion of human Shh was evaluated in the supernatants of A549 and H520 cells by ELISA (<b>E</b>) and confirmed by western blot using an antibody recognizing the secreted active form of Shh (<b>inset E</b>). The western blot was performed with H520 supernatant (H520 sup.) and with recombinant Shh (Rec. Shh) used as a positive control. <b>(F</b>) The knockdown of Shh gene by siRNA was realized in H520 cells. Shh secretion was evaluated by ELISA and expressed in percentage as relative secretion compared with cells transfected with a negative control siRNA (NC) having no homology in vertebrate transcriptome. **p<0,05 (G) After silencing of Shh, NSCLC cells were treated or not with recombinant Shh (500 ng/ml). RT-qPCR was performed to evaluate Gli and Ptch1 mRNA levels. Results are presented as fold of mRNA differences (2^∧∧Ct) in treated cells compared with non-treated cells.</p

Shh mediates NSCLC/lung fibroblast reciprocal crosstalk.

<p>CCL206 lung fibroblasts were cultured or not with the supernatant of H520 transfected with a NC siRNA(Sup) or with Shh siRNA (Sup siShh) for 48 h. (A) Levels of secreted Leukemia Inhibitory Factor (LIF) were evaluated in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. *p<0,1; **p<0,05 (B) Levels of secreted Vascular Endothelial Growth Factor (VEGF were assessed in CCL206 supernatant by multiplex biometric ELISA-based immunoassay (Bioplex system). Results are presented in percentage as relative secretion compared with cells cultured without H520 supernatant. **p<0,05 (C) A549 and H520 cells were pre-stained with Hoechst (1 µg/ml) and then cultured for 48 h with CCL206 lung fibroblasts pre-treated with Shh (500 ng/ml) or with SAG (3 nM). The number of Hoechst positive cells was evaluated by fluorescent microscopy and is presented in percentage as relative number of cells compared with the number of cancer cells cultured alone. (D) NSCLC cells A549 and H520 were pre-stained with Hoechst (1 µg/ml) and then co-cultured for 72 h either alone or with CCL206 pre-treated with Shh (500 ng/ml) or with 3 nM SAG. The number of NSCLC cells transmigrating is expressed in percentage as the relative number of cells migrating compared with the total number of cancer cells per transwell. *p<0,1; ***p<0,01.</p

Inhibition of Hedgehog signaling reduces the proliferation of NSCLC cells.

<p>Lung adenocarcinoma A549 cells (<b>A</b>) and lung squamous carcinoma H520 cells (<b>B)</b> were cultured in presence or absence of 10 µM cyclopamine for 5 days. Proliferation was assessed by cell counting and cell survival by MTT assay. *p<0,1; **p<0,05. (<b>C</b>) The Hedgehog-responsive transcription factors Gli1, Gli2 or Gli3 were knocked down with siRNA in A549 cells. RT-qPCR was performed to confirm the specific silencing of each Gli and to assess the expression of the Hedgehog receptor Ptch1. Results are presented as fold differences of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. **p<0,05, ***p<0,01. The impact of silencing Gli1, Gli2 or Gli3 in A549 cell proliferation was assessed by cell counting (<b>D</b>) and in cell survival by MTT assay (<b>E</b>)<b>.</b> Results are presented in percentage as relative proliferation and relative survival compared with cells transfected with the negative control siRNA (NC). *p<0,1<b>.</b> (<b>F</b>) Representative phase-contrast microscopic pictures after 72 hours of siRNA are presented<b>.</b> (<b>G</b>) RT-qPCR was performed to evaluate the effect of the siRNA of Gli1, Gli2 and Gli3 in the expression of the G1/S phase cyclins D (Cyc D1, Cyc D2, Cyc D3) and cyclin E (Cyc E1)<b>.</b> Results are presented as fold of mRNA levels (2^∧∧Ct) compared with cells transfected with a negative control siRNA (NC siRNA) having no homology in vertebrate transcriptome. *p<0,1; **p<0,05. (<b>H</b>) Western blot of cyclin D2 in A549 cells transfected with Gli siRNA or with a negative control siRNA (NC). Blotting of ß-actin was used as loading control.</p

Shh pathway affects lung fibroblast migration, invasion and collagen synthesis.

<p>(<b>A</b>) Accumulated distance of migration of primary human lung fibroblasts treated with Shh (500 ng/ml) or cyclopamine (10 µM) and monitored by live cell microscopy for 48 hours. The accumulated distance of migration of each cell was determined using ImageJ. ***p<0,01. (<b>B</b>) Scratch wound assay was performed in lung fibroblasts CCL206 treated or not with Shh at the doses indicated or with 10 µM cyclopamine (cyclop) for up to 48 hours. Migration of the cells was recorded using live cell microscopy and representative pictures at 1,5, 12,5 and 26 hours are shown. The colored lines indicate the border of cell migration in each case. (<b>C</b>) The area of the wound was quantified after 26 hours and the percentage of wound closure, relative to the initial area of scratch for each case, was determined. (<b>D</b>) Transmembrane invasion assay was performed in lung fibroblasts treated with Shh (500 ng/ml) or with cyclopamine (10 µM). (<b>E</b>) RT-qPCR was performed to assess MMP2 and MMP9 expression in fibroblasts treated or not with Shh (500 ng/ml) for 48hr. Results are presented as fold of mRNA levels in treated cells compared with non-treated cells. *p<0,1. (<b>F</b>) The total collagen content of lung fibroblasts treated with Shh (500 ng/ml) or TGF-ß1 (5 ng/ml) for the indicated times was quantified using the Sircol collagen assay. *p<0,1, **p<0,05.</p