Integrin α6 signaling induces STAT3-TET3-mediated hydroxymethylation of genes critical for maintenance of glioma stem cells

Abstract

Both the extracellular matrix (ECM) and DNA epigenetic regulation are critical for maintaining stem cell phenotype and cancer progression. Whether and how ECM regulates epigenetic alterations to influence cancer stem cells (CSCs) remain to be explored. Here we report that ECM through laminin-integrin α6 upregulates ten-eleven translocation enzyme 3 (TET3) dioxygenase. TET3 in turn mediates DNA cytosine 5'-hydroxymethylation (5hmC) and upregulates genes critical for maintenance of glioma stem cells (GSCs). Activating integrin α6-FAK pathway increases STAT3 activity, TET3 expression and 5hmC levels in GSCs. Moreover, targeting STAT3 disrupts integrin α6-FAK signaling and inhibits TET3⁺ GSC maturation in vivo. STAT3 directly regulates TET3 expression and the two proteins are co-localized with 5hmC in GSC clusters. 5hmC is upregulated by STAT3 at the promoters of several tumorigenic genes, including c-Myc, known to be critical for GSCs. In vivo silencing of TET3 in GSC-enriched tumors reduces 5hmC accumulation and expression of the GSC critical genes, leading to tumor growth inhibition. TET3 expression and 5hmC accumulation also co-segregate with integrin α6 in patient malignant glioma. Thus, ECM- integrin α6-STAT3-TET3 axis regulates hydroxymethylation of genes important for GSCs, thereby increasing GSC tumorigenicity and resistance to therapies.

Fig. 1

TET3 critical for characteristic tumor sphere formation is overexpressed in glioma stem cells favoring 5hmC accumulation. a TET3 protein expression was assessed by Western blotting compared in differentiated non-stem counterparts and primary human GSCs. Actin was re-probed and shown as a loading control. b Reduced tumor sphere formation by TET3 silencing was shown in primary human GSCs, GSC008, GSC009, GSC030 and GSC106. GSC008, GSC009, and GSC030 cells were treated with CpG-luciferase-siRNA or CpG-TET3-siRNAs every other day (n = 6) (upper panels). GSC030 and GSC106 were stably transduced with non-targeting shRNA or inducible TET3 shRNAs, and were treated with 5 μg/ml of doxycycline for shRNA induction (n = 6) (lower panels). SD shown, T-test: *) P < 0.05, **) P < 0.01, ***) P < 0.001. c Increased tumor sphere formation by TET3 overexpression was shown in primary human GSCs, GSC030 and GSC106. GSC030 and GSC106 were stably transduced with full length human TET3 cDNA (n = 6). SD shown, T-test: *) P < 0.05, **) P < 0.01. d Decreased tumorigenicity of primary human GSCs upon TET3 silencing was assessed by LDA. GSCs were stably transduced with non-targeting shRNA or inducible shTET3s, and treated with 5 μg/ml of doxycycline for shRNA induction. e 5hmC accumulation in primary human GSCs, GSC008, GSC030, and GSC106 were confirmed by flow cytometry upon digestion of RNA species. 5hmU accumulation as a TET3 independent DNA deamination alternative to TET3 dependent 5hmC-5fC conversion was included. f 5hmC in GSCs were compared to their non-stem counterparts by flow cytometry. g 5hmC (green) accumulation restricted to pSTAT3⁺MSI-1⁺ and pSTAT3⁺SOX2⁺ GBM cells but not to single positive pSTAT3⁺ GBM cells shown by confocal microscopy upon staining glioma patient tissue sections (left). Scale, 20 μm. Tissue profiles showing 5hmC accumulation confined to GSC-marker⁺ cells (right). a-g Representative data from 3 independent experiments are shown.

Fig. 2

Integrin α6/FAK signaling feeds STAT3 dependent TET3 expression and 5hmC accumulation. a Integrin α6 protein expressions in GSCs were compared to those of differentiated non-stem counterparts by Western blotting. Actin was re-probed and shown as a loading control. b Integrin α6 signaling mediated TET3 expression was assessed by flow cytometry and Western blotting in primary human stem-like GSC030 upon engagement with laminin compared to other components of the ECM (upper panels). Characteristic sphere formation upon exposure of a GSC single cell suspension to laminin and other ECM components is shown by bright field microscopy (lower panels). Scale, 100 μm. c Ectopic stimulation of stem-like GSC030 with laminin triggers activation of FAK/STAT3, expression of TET3 and 5hmC accumulation as shown by flow cytometry (top to bottom). d Blocking FAK activity with FAK inhibitor during stimulation of GSCs with laminin reduced activation of FAK/STAT3, expression of TET3 and 5hmC accumulation as shown by flow cytometry (top to bottom). e Silencing integrin α6 with inducible shRNA during stimulation of GSCs with laminin reduced activation of FAK/STAT3, expression of TET3 and 5hmC accumulation as shown by flow cytometry (top to bottom).f Silencing TET3 with inducible shRNA during stimulation of GSCs with laminin reduces activation of FAK/STAT3, expression of TET3 and 5hmC accumulation as shown by flow cytometry (top to bottom). g STAT3 silencing reducing TET3 expression was confirmed in vivo using human glioma U251 cells. GFP expression was controlled by the SOX2 promoter. GFP⁺ glioma cells considered stem-like phenocopies were analyzed for TET3 expression by flow cytometry once tumors were dissected and a single cell suspension was prepared. h STAT3 contributing to TET3 expression was validated by ChIP assay in two independent primary human GSCs, GSC009 and GSC030, and quantified. SD shown, T-test: *) P < 0.01, **) P < 0.01, **) P < 0.001. a-h Representative data from 3 independent experiments are shown.

Fig. 3

STAT3 and TET3 physically interact and cooperate in binding to methylated STAT3 target DNA sequences. a STAT3 and TET3 subcellular co-localization was assessed in four independent primary human GSC lines GSC008, GSC009, GSC030, and GSC106 by confocal microscopy. Scale, 50 μm. b Physical interaction of STAT3 and TET3 was shown by co-IP pulling down TET3 and detecting STAT3 and vice versa using whole cell lysates prepared from primary human GSCs, GSC030 and GSC106. c Physical interaction of STAT3 and TET3 was reduced by silencing integrin α6 with inducible shITGA6 as shown by co-IP pulling down TET3 and detecting STAT3 and vice versa using whole cell lysates prepared from primary human GSC106. d Nuclear co-localization of activated pY⁷⁰⁵STAT3 with TET3 and 5hmC was assessed by confocal STED microscopy in glioma patient tissue. Scale, 5 μm. e Enhanced cooperative binding of STAT3 and TET3 to methylated STAT3 target DNA sequence (SIE^5mC) was shown by oligo-pull-down assay using unmethylated or methylated SIE oligo. Protein complexes were detected in Western blot procedure after electrophoretic protein separation on SDS-PAGE. a-e Representative data from 3 independent experiments are shown.

Fig. 4

STAT3 contributes to oncogene promoter hydroxymethylation in GSCs in vitro and STAT3 or TET3 knockdown prolongs mouse survival in vivo. a Hydroxymetylation of oncogene promoters upon STAT3 knockdown (shSTAT3) or TET3 knockdown (shTET3) was assessed by PCR using mRNA harvested from human primary GSCs, GSC030 and GSC106. Amplified cDNA was treated to sensitize non-hydroxymethylated cDNA for restriction enzyme digestion and products were separated on DNA gel (upper panels). Decreased oncogene promoter hydroxymetylation upon STAT3 knockdown was quantified by qPCR (lower panels). SD shown; T-test: *) P < 0.05, **) P < 0.01, ***) P < 0.001. Representative data from 3 independent experiments are shown. b Tumors grown from primary human GSCs stably expressing non-targeting shRNA or inducible shTET3 were treated with 2 mg/ml doxycycline water. Mouse survival was plotted out by Kaplan-Meier survival curves. c Tumors grown from primary human GSC, GSC030 were treated with CpG-siRNA conjugates as indicated including control CpG-luciferase-siRNA. Tumor growth kinetics were assessed. SD shown; T-test: ***) P < 0.001. d STAT3 and TET3 sensitive c-Myc oncogene promoter hydroxymetylation of GSC tumors treated as indicated was determined upon isolation of gDNA and treatment of gDNA to sensitize non-hydroxymethylated cDNA for restriction enzyme digestion. SD shown; T-test: *) P < 0.05, ***) P < 0.001.

Fig. 5

STAT3 critically contributes to oncogene promoter hydroxymethylation in GSCs in vivo. a STAT3- and TET3-sensitive protein expressions of integrin α6 as well as 5hmC accumulation in GSC tumors treated as indicated were assessed by confocal microscopy and b quantified. Scale, 50 μm. SD shown; T-test: *) P < 0.05, **) P < 0.01, ***) P < 0.001. c FAK protein expression in GSC tumors treated as indicated were assessed by confocal microscopy. Scale, 50 μm. d Reduced oncogene expression upon treatment of GSC tumors as indicated as determined by confocal microscopy and quantified. SD shown; T-test: **) P < 0.01, ***) P < 0.001.

Fig. 6

Integrin α6 and TET3 expression resulting in 5hmC accumulation is confined to high grade glioma. a Integrin α6 and 5hmC (upper panels) or TET3 (lower panels) expression was assessed by immunohistochemistry followed by confocal microscopy of human brain tumor patient biopsies. Scale 20 μm (upper panels) and 10 μm (lower panels). b Quantitative case distribution of integrin α6/5hmC (upper panels) or integrin α6/TET3 (lower panels) expression. A total of 19 brain tumor cases were analyzed. c High frequency occurrence of integrin α6+/5hmC+ (left panel) or integrin α6+/TET3+ (right panel) expression is restricted to high grade glioblastoma as assessed from 19 brain tumor cases analyzed. a-c Total 19 patient cases were analyzed, and 5 to 10 images were acquired per slide.