Identification of pY654-β-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia

Abstract

Hypoxia is linked to epithelial-mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of β-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-β-catenin and HIF1α. All pY654-β-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for β-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-β-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated β-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-β-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-β-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

Figure 1

Multiple activated tyrosine kinases in human lung adenocarcinomas associated with formation of pY654-β-catenin complexes with HIF1α and p-Smad2. (a) Human lung tumors (T) and contiguous normal tissues (N) were lysed and the lysates immunoprecipitated for pY654-β-catenin and blotted for β-catenin and p-Smad2. Equal amount of protein from both normal and tumor lysates were mixed together as input for mouse IgG control. (b–d) The above lysates were blotted for EMT-related proteins listed in (b), immunoprecipitated for p-c-Met and blotted for total c-Met (c), and sequentially immunoprecipitated for pY654-β-catenin and total β-catenin and blotted for β-catenin, HIF1α, and Src (d). (e) Lysates were sequentially immunoprecipitated for two rounds of HIF1α and one round of pY654-β-catenin and blotted for β-catenin, HIF1α, and Src.

Figure 2

Hypoxia induces Src kinase-dependent pY654-β-catenin/HIF1α complexes and EMT in human lung adenocarcinoma cells. (a) pY654-β-catenin immunoprecipitation of H358 cells incubated in normoxia (21% O₂) (N) or hypoxia (1% O₂) (H) for 2 h or 12 h. (b) Immunoblots for HIF1α, pY416-Src, total Src, and Snail1 of H358 cells under normoxia or hypoxia for 24 h ± Src inhibitor. TGFβ1 stimulation-positive control; β-actin blot-loading control. (c, d) Sequential immunoprecipitation for pY654-β-catenin and total β-catenin (c) or two rounds of HIF1α and one round of pY654-β-catenin (d) of H358 cells under normoxia or hypoxia or hypoxia with Src inhibitor SU6656 (5 µM) (H+SU) for 4 h. (e) Myc immunoprecipitation of H358 cells expressing Myc-tagged wt (W) or Y654F mutant (F) β-catenin under normoxia or hypoxia for 4 h. (f) Fibronectin (orange) and E-cadherin (green) staining of H358 cells under normoxia or hypoxia for 56 h ± Src inhibitor. Scale bar, 100 µm.

Figure 3

Hypoxia-induced EMT requires both HIF1α and β-catenin expression. (a) HIF1α and pY654-β-catenin sequential immunoprecipitation of AECTs under normoxia or hypoxia ± Src inhibitor for 4 h. (b) Immortalized AECTs with floxed β-catenin were infected with Ad-Cre or Ad-GFP and cultured in hypoxia for 56 h. Cells were stained for E-cadherin (green) and β-catenin (orange). Scale bar, 50 µm. (c–e) AECTs were infected with Ad-Cre or Ad-GFP and incubated in hypoxia for 56 h ± ALK5 inhibitor SB431542 or Src inhibitor SU6656 (5 µM) before immunoblotting for proteins as indicated in panels (c) and (d). Conditioned medium was concentrated 10× for zymography with recombinant MMP-2 as positive control (e). (f, g) AECTs infected with Ad-Cre or Ad-GFP (f) or transfected with HIF1α or non-targeting control siRNA (g) were incubated in normoxia or hypoxia for 24 h before qRT-PCR analysis. Collagen I, Snail1, β-catenin, HIF1α mRNA levels were normalized to β-actin mRNA level. * indicates p<0.05 by t-test.

Figure 4

Y654-β-catenin phosphorylation is required for hypoxia-induced EMT and β-catenin promotion of HIF1α transcriptional activity. (a) E-cadherin (green) and α-SMA (orange) staining of AECTs expressing Y654E or Y654F-β-catenin. Scale bar, 100 µm. (b) Immunoblots for collagen I, α-SMA, Twist, and HIF1α of AECTs with β-catenin deletion (Cre), cells expressing wt (W) and Y654E (E) or Y654F (F) mutant in normoxia or hypoxia for 56 h. (c) Conditioned medium from the cells above were concentrated for zymography with recombinant MMP-2 as control. (d) EMT markers from B and C were quantified using Image J and the mean value normalized to β-catenin level from 3 independent experiments were shown. ns, not significant. * indicates p<0.05 by t-test. (e) HRE reporter activity of 293 cells expressing W, E or F mutant β-catenin under normoxia or hypoxia for 16 hours. Renilla activity was used as internal control. Mean value of hypoxia/normoxia ratio from 4 independent experiments was shown. * indicates p<0.05 by t-test. 293 cell lysates were also blotted for Myc-tagged β-catenin and β-actin.

Figure 5

Hypoxia-induced pY654-β-catenin formation and EMT are dependent on ROS. (a) H358 cells were cultured in normoxia or hypoxia for 2 h ± ROS scavenger NAC (10 mM). APF signal (green) indicates ROS activity and the fluorescence intensity was quantified by Image J. * indicates p< 0.05 by t-test. Scale bar, 50 µm. (b) H358 cells were cultured in normoxia or hypoxia for 4 h or treated with TGFβ1 (4 ng/ml) for 2 h ± EUK-134 or NAC. The lysates were immunoprecipitated for pY654-β-catenin and the lysates were blotted for β-catenin, pY416-Src, Src, HIF1α, and β-actin. (c) Immumoblots for Snail1 in H358 cells under normoxia or hypoxia for 24 h ± NAC. The numbers indicate relative Snail1 level normalized to β-actin. (d) H358 cells were incubated in normoxia or hypoxia for 24 h ± EUK-134 or NAC. The conditioned media were concentrated for zymography with recombinant MMP-9 as positive control.

Figure 6

pY654-β-catenin/HIF1α/Src complexes and EMT markers are increased in anti-VEGF antibody treated pancreatic tumors. (a) RIP-Tag2 tumor lysates from anti-VEGF- or control IgG-treated mice were immunoprecipitated for pY654-β-catenin and blotted for β-catenin and p-Smad2. (b) The above lysates were sequentially immunoprecipitated for two rounds of HIF1α and one round of pY654-β-catenin and blotted for β-catenin, HIF1α, and Src. (c) The above lysates were blotted for pY416-Src, Src, E-cadherin, N-cadherin, vimentin, Snail1, Twist, HIF1α, and β-actin.

Figure 7

pY654-β-catenin/HIF1α and p-c-Met accumulation, EMT markers, and tumor invasiveness induced by anti-VEGF are blocked by ROS inhibition. (a) Confocal images of anti-VEGF treated-RIP-Tag2 tumors ± NAC for 5 days stained for insulin (tumor cells, green) and E-cadherin (acinar cells and tumor cells, red). Scale bar, 200 µm. (b) Combination (anti-VEGF+NAC) or anti-VEGF alone (Anti-VEGF)-treated RIP-Tag2 tumor lysates were immunoprecipitated for pY654-β-catenin and blotted for β-catenin. (c–e) The above lysates were blotted for E-cadherin, N-cadherin, vimentin, Snail1, Twist, HIF1α, and β-actin (c) or pY416-Src, Src, pY845-EGFR, and EGFR (d) or immunoprecipitated with p-c-Met and blotted for c-Met (e).

Figure 8

Schematic diagram illustrating central role for ROS and Src in hypoxia-induced pY654-β-catenin/HIF1α formation and tumor EMT. Hypoxia exposure generates reactive oxygen species (ROS) and stabilizes HIF1α (10). Increased ROS activity results in the activation of Src family kinase(s) (p-Src) (45) that then promotes activation of tyrosine kinases such as EGFR and c-Met, further promoting p-Src. Active Src phosphorylates β-catenin at Y654, favoring β-catenin association with HIF1α over β-catenin degradation, binding to E-cadherin, or association with TCFs in the Wnt pathway. pY654-β-catenin/HIF1α complexes promote transcription of EMT genes as well as other hypoxia responsive genes. Although not shown, Src is also in complexes of pY654-β-catenin/HIF1α. TGFβ1 signaling is not enhanced by hypoxia but remains active within the tumor microenvironment and further promotes hypoxia-induced EMT.