Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes

Contribution of KITLG-activated KIT signaling to baseline proliferation of GIST and LX-2 cells.

<p><i>A</i>, KIT Y721 phosphorylation was activated by exogenous rhKITLG (100 ng/mL for 10 min following 2 h serum deprivation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#B13" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#B35" target="_blank">35</a>]) in GIST-T1 cells containing a heterozygous activating <i>KIT</i> mutation (n=4/group) and in GIST-T1-5R cells containing an additional, imatinib-resistant <i>KIT</i> mutation (n=6/group), but not in GIST882 cells lacking a WT <i>KIT</i> allele (n=5/group). <i>B</i>, Culturing with anti-human KITLG neutralizing antibody for 4 days inhibited baseline proliferation of GIST-T1 cells (<i>P</i><0.001; n=3; regression and 95% confidence band are shown as solid and dashed lines, respectively) and GIST-T1-5R cells (<i>P</i><0.001; n=6). The effect of KITLG immunoneutralization on GIST-T1 cells was blocked by preabsorbing the antibody with 10-fold molar excess of rhKITLG (see open symbols in the left panel, second row; n=3/group). KITLG immunoneutralization did not inhibit the proliferation of GIST882 or GIST48B cells lacking a WT <i>KIT</i> allele (note that GIST48B cells also express very low to undetectable levels of KIT protein, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone.0076822.s001" target="_blank">Figure S1E</a>) and of LX-2 cells lacking KIT protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone.0076822.s001" target="_blank">Figure S1E</a>) (n=3/cell line). <i>C</i>, Inhibition of the proliferation of GIST-T1 and GIST-T1-5R cells by siRNA-mediated knock-down of <i>KITLG</i> (n=4/cell line/group; <i>P</i>_GIST-T1: day 2: 0.008, days 4, 6 and 8: <0.001; <i>P</i>_GIST-T1-5R: day 2: <0.02, day 4: 0.004, days 6 and 8: <0.001).</p

GSK3i stimulates Kitl expression without activating cyclin D1 expression and p70S6K and ERK1/2 phosphorylation.

<p>The GSK3α/β inhibitor SB415286 was applied to organotypic cultures of gastric corpus+antrum muscles from of 14-16-day-old BALB/c mice at 30 µM. <i>A</i>, Effect of 18-h application of SB415286 on Kitl expression; n=5/group. <i>B</i>, Effect of the same treatment on cyclin D1 expression; n=3/group. <i>C</i>, Effects of 1-h exposure to SB415286 on p70S6K and ERK1/2 phosphorylation; n=3/group. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone-0076822-g002" target="_blank">Figure 2</a> for further details.</p

Kitl protein expression is stimulated by IGF1 via multiple IGF1R-activated pathways.

<p><i>A</i>, Time-dependent stimulation of Kitl expression in the gastric <i>tunica </i><i>muscularis</i> of 14-16-day-old BALB/c mice by the potent IGF1 analog LR3-rhIGF1 administered in a single 150 µg/kg dose i.p.; n=5 mice/group. <i>B</i>, Time-dependent stimulation of Kitl expression by 100 ng/mL rhIGF1 in gastric corpus+antrum <i>tunica </i><i>muscularis</i> organotypic cultures from 14-16-day-old BALB/c mice; n=4-5/group. <i>C</i>, Concentration-dependent stimulation of Kitl protein expression by 18-h rhIGF1 treatment; n=7 organotypic cultures/group. <i>D</i>, Blockade of the rhIGF1-induced Kitl expression in organotypic cultures by AG1024 (1 µM), a specific inhibitor of IGF1/insulin receptor tyrosine kinase activity; n=4/group. <i>E</i>-<i>F</i>, Effects of 1-h (<i>E</i>) and 3-h (<i>F</i>) rhIGF1treatment on AKT (S473/S474/S472), GSK3β (S9), p70S6K (T389) and ERK1/2 (T202/Y204 and T185/Y187) phosphorylation; n=5 organotypic cultures/group. <i>G</i>, Blockade of rhIGF1-induced Kitl expression in organotypic cultures by specific inhibitors of AKT (AKT Inhibitor X; 150 µM), MTOR–p70S6K (rapamycin; 1.5 nM) and MEK–ERK (PD98059; 50 µM); n=5-16/group. The GSK-3 inhibitor SB415286 (30 µM), which is expected to mimic, rather than inhibit, the effect of the IGF1-induced inhibitory phosphorylation of GSK-3, had no significant effect. Kitl and Gapdh or total (T) and phosphorylated proteins (P) were simultaneously detected in the same samples by two-color immunofluorescence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone.0076822.s001" target="_blank">Figure S1</a>). Representative immunoblots show identical areas of the blots imaged at different wavelengths. Box plots show medians and interquartile ranges; bar graphs indicate means±SEM. Data were normalized to the control groups indicated in the panels (dashed lines). Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other (<i>P</i><0.05 by post-hoc multiple comparisons). IGF1 stimulated Kitl expression in gastric smooth muscles in vivo and in vitro in a time- and concentration-dependent manner by activating IGFR1 and the AKT–GSK3, MTOR–p70S6K and ERK MAPK pathways.</p

IGF1 and GSK3i stimulate <i>Kitl/KITLG</i> transcription.

<p>Results obtained in murine and human smooth muscle cells (<i>A</i>-<i>E</i>), human LX-2 stellate cells (<i>F</i>-<i>G</i>) and human GIST-T1 cells (<i>H</i>-<i>I</i>) are shown. <i>A</i>, Hoffman modulation contrast image of primary human gastric smooth muscle cells. <i>B</i>, <i>KITLG</i> mRNA (total: soluble+membrane-bound) was readily detectable in primary human smooth muscle cells (passage 3) maintained with Smooth Muscle Growth Medium-2 containing insulin, hFGF-B, hEGF and 5% FBS (Lonza) but not in 24-h growth factor- and serum-deficient basal medium. <i>C</i>, Both IGF1 (100 ng/mL) and the GSK3α/β inhibitor SB415286 (30 µM) stimulated <i>Kitl</i> expression in murine gastric <i>tunica </i><i>muscularis</i> organotypic cultures (n=3/group). <i>D</i>-<i>E</i>, SB415286 stimulated endogenous <i>Kitl</i> expression in murine primary gastric smooth muscle cells (<i>D</i>; n=3/group) and <i>KITLG</i> transcriptional activity in the same cell type transfected with a <i>KITLG</i> promoter (-2120 bp to +407 bp)-pGL3 luciferase construct (<i>E</i>; n=3/group). IGF1 (100 ng/mL; n=3/group) and SB415286 (30 µM; n=6/group) also increased endogenous <i>KITLG</i> mRNA expression in LX-2 (<i>F</i>) and GIST-T1 cells (<i>H</i>) and stimulated <i>KITLG</i> promoter activity in a time-dependent fashion (LX-2: n=6-9/group; <i>G</i>; GIST-T1: n=3-11/group; <i>I</i>). Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other (<i>P</i><0.05 by post-hoc multiple comparisons).</p

Role of reduced H3K9 methylation in the activation of <i>Kitl/KITLG</i> transcription by IGF1 and GSK3i.

<p><i>A</i>-<i>B</i>, Reduced occupancy of the <i>Kitl</i> core promoter by the repressive H3K9me2 (<i>A</i>) and H3K9me3 (<i>B</i>) histone modifications in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. <i>C</i>-<i>D</i>, Probing the role of HP1 isoforms in transcriptional repression of <i>KITLG</i> in LX-2 cells by siRNA- (CBX5: HP1α; 25 nM, 72 h; <i>C</i>) or shRNA-mediated knock-down (CBX1: HP1β; CBX3: HP1γ; 30 µg plasmid, 72 h; <i>D</i>). Note activation of <i>KITLG</i> expression by shRNA-mediated knock-down of HP1γ (n=3/group). <i>E</i>-<i>F</i>, Stimulation of <i>KITLG</i> expression by the G9A/GLP H3K9me1/2 HKMT inhibitor BIX-01294 in LX-2 cells (<i>E</i>) and GIST-T1 cells (<i>F</i>) (n=3/group). <i>G</i>, Stimulation of <i>KITLG</i> expression by the H3K9 HKMT inhibitor chaetocin in LX-2 cells (n=3/group). Drugs were applied for 24 h following 24-h serum deprivation. See further details in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone-0076822-g005" target="_blank">Figure 5</a>.</p

Role of trithorax- and polycomb-mediated histone modifications in IGF1- and GSK3i-induced activation of <i>Kitl/KITLG</i> transcription.

<p><i>A</i>, Increased occupancy of the <i>Kitl</i> core promoter by the trithorax group-mediated, activating H3K4me2 histone modification in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. <i>B</i>-<i>C</i>, Reduced occupancy of the <i>Kitl</i> core promoter by the PRC2-mediated, repressive H3K27me3 histone modification (<i>B</i>) and by the PRC2 histone methyltransferase EZH2 (<i>C</i>) in response to rhIGF1 and SB415286 in the same tissues. <i>D</i>-<i>F</i>, Stimulation of <i>KITLG</i> expression by the indirect histone methyltransferase inhibitor Adox in murine gastric smooth muscles (<i>D</i>), LX-2 cells (<i>E</i>) and GIST-T1 cells (<i>F</i>) (n=3/group). Adox was applied for 72 h following 24-h serum deprivation. See further details in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076822#pone-0076822-g005" target="_blank">Figure 5</a>.</p

Role of histone acetylation in the activation of <i>Kitl/KITLG</i> transcription by IGF1 and GSK3i.

<p><i>A</i>, Increased occupancy of the <i>Kitl</i> core promoter region by H4ac in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. Only SB415286 increased occupancy by H3K9ac (<i>B</i>). Representatives of two independent experiments, each performed in triplicates, are shown. <i>C</i>, Dose-dependent stimulation of <i>KITLG</i> expression in LX-2 cells by 24-h treatment with the class I-II HDAC inhibitor SAHA (n=3/group). <i>D</i>, Dose-dependent inhibition of the SB415286-induced stimulation of <i>KITLG</i> expression by the p300/PCAF HAT inhibitor garcinol (24 h) in LX-2 cells (n=3/group). Drugs were applied following 24-h serum deprivation. Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other (<i>P</i><0.05 by post-hoc multiple comparisons).</p