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A novel HIF-1α-integrin-linked kinase regulatory loop that facilitates hypoxia-induced HIF-1α expression and epithelial-mesenchymal transition in cancer cells.

Chou CC, Chuang HC, Salunke SB, Kulp SK, Chen CS - Oncotarget (2015)

Bottom Line: We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3β phosphorylation.In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1.Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

ABSTRACT
Here, we described a novel regulatory feedback loop in which hypoxia induces integrin-linked kinase (ILK) expression through a HIF-1α-dependent mechanism and ILK, in turn, stimulates HIF-1α expression through cell type- and cell context-dependent pathways. HIF-1α increased ILK via transcriptional activation. ILK increased HIF-1α levels by promoting mTOR-mediated translation in PC-3 and MCF-7 cells, and by blocking GSK3β-mediated degradation in LNCaP cells, consistent with the cell line-/cellular context-specific functions of ILK as a Ser473-Akt kinase. We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3β phosphorylation. Also, ILK can de-repress HIF-1α signaling through the YB-1-mediated inhibition of Foxo3a expression. In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1. Thus, the ILK-HIF-1α regulatory loop could underlie the maintenance of high HIF-1α expression levels and the promotion of EMT under hypoxic conditions. Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.

No MeSH data available.


Related in: MedlinePlus

Evidence that ILK regulates HIF-1α expression through different mechanisms in PC-3 and MCF-7 cells versus LNCaP cells(A) Upper, Western blot analysis of the effects of T315 on hypoxia-induced changes in the expression/phosphorylation levels of HIF-1α, ILK, YB-1, GSK3β, and markers of EMT and Akt signaling. Lower, RT-PCR analysis of the corresponding effects on hypoxia-induced changes in the mRNA levels of HIF-1α and EMT markers. (B) Immunocytochemical analysis of the effect of T315 (1 μmol/L; 24 h) versus DMSO control on the cellular distribution of ectopically expressed GFP-Foxo3a under hypoxic conditions. Scale bar, 50 μm. (C) Left, effects of T315 (T; 2 μmol/L), SB-216763 (SB; 10 μmol/L), and rapamycin (Rap; 10 μmol/L) relative to DMSO control (Ctl) on hypoxia-induced HIF-1α expression. Nor, normoxia. Right, influence of shRNA-mediated depletion of FBW7 on the T315-induced suppression (1 and 2 μmol/L) of hypoxia-induced HIF-1α expression. Immunoblots and RT-PCR analyses are representative of three independent experiments.
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Figure 3: Evidence that ILK regulates HIF-1α expression through different mechanisms in PC-3 and MCF-7 cells versus LNCaP cells(A) Upper, Western blot analysis of the effects of T315 on hypoxia-induced changes in the expression/phosphorylation levels of HIF-1α, ILK, YB-1, GSK3β, and markers of EMT and Akt signaling. Lower, RT-PCR analysis of the corresponding effects on hypoxia-induced changes in the mRNA levels of HIF-1α and EMT markers. (B) Immunocytochemical analysis of the effect of T315 (1 μmol/L; 24 h) versus DMSO control on the cellular distribution of ectopically expressed GFP-Foxo3a under hypoxic conditions. Scale bar, 50 μm. (C) Left, effects of T315 (T; 2 μmol/L), SB-216763 (SB; 10 μmol/L), and rapamycin (Rap; 10 μmol/L) relative to DMSO control (Ctl) on hypoxia-induced HIF-1α expression. Nor, normoxia. Right, influence of shRNA-mediated depletion of FBW7 on the T315-induced suppression (1 and 2 μmol/L) of hypoxia-induced HIF-1α expression. Immunoblots and RT-PCR analyses are representative of three independent experiments.

Mentions: The role of ILK in regulating hypoxia-induced HIF-1α expression and EMT was further verified by using a proof-of-concept, small molecule ILK inhibitor, T315, in PC-3, LNCaP, and MCF-7 cells. The IC50 values of T315 in suppressing the viability of these three representative cell lines were: PC-3, 2 μmol/L; LNCaP, 2 μmol/L; MCF-7, 2.8 μmol/L [25]. As shown in Figure 3A, inhibition of ILK kinase activity by T315 dose-dependently suppressed hypoxia-induced increases in the expression of HIF-1α, ILK and YB-1, indicative of the ability of T315 to disrupt the HIF-1α-ILK regulatory loop. T315 also reversed hypoxia-induced changes in the EMT regulators/markers, Foxo3a, E-cadherin, vimentin, and/or Snail, in all three cell lines, restoring them to levels detected under normoxic conditions (Figure 3A). Reminiscent of the results observed after siRNA-mediated ILK knockdown, T315 suppressed the hypoxia-induced phosphorylation of Ser473-Akt and its downstream targets mTOR and Foxo3a in PC-3 and MCF-7 cells, but not in LNCaP cells. Accordingly, this differential effect of T315 on the Akt-Foxo3a signaling axis was manifested by differences in the cellular distribution of Foxo3a among these cell lines (Figure 3B). In PC-3 and MCF-7 cells, the suppression of hypoxia-induced Akt activation and Foxo3a phosphorylation by T315 (Figure 3A) was accompanied by the nuclear localization of Foxo3a (Figure 3B). In contrast, Foxo3a remained sequestered in the cytoplasm in T315-treated LNCaP cells (Figure 3B), reflecting the inability of T315 to inhibit Akt/Foxo3a phosphorylation. Nonetheless, T315 was able to downregulate GSK3β phosphorylation in all three cell lines (Figure 1A) as GSK3β represents a direct target of ILK [26].


A novel HIF-1α-integrin-linked kinase regulatory loop that facilitates hypoxia-induced HIF-1α expression and epithelial-mesenchymal transition in cancer cells.

Chou CC, Chuang HC, Salunke SB, Kulp SK, Chen CS - Oncotarget (2015)

Evidence that ILK regulates HIF-1α expression through different mechanisms in PC-3 and MCF-7 cells versus LNCaP cells(A) Upper, Western blot analysis of the effects of T315 on hypoxia-induced changes in the expression/phosphorylation levels of HIF-1α, ILK, YB-1, GSK3β, and markers of EMT and Akt signaling. Lower, RT-PCR analysis of the corresponding effects on hypoxia-induced changes in the mRNA levels of HIF-1α and EMT markers. (B) Immunocytochemical analysis of the effect of T315 (1 μmol/L; 24 h) versus DMSO control on the cellular distribution of ectopically expressed GFP-Foxo3a under hypoxic conditions. Scale bar, 50 μm. (C) Left, effects of T315 (T; 2 μmol/L), SB-216763 (SB; 10 μmol/L), and rapamycin (Rap; 10 μmol/L) relative to DMSO control (Ctl) on hypoxia-induced HIF-1α expression. Nor, normoxia. Right, influence of shRNA-mediated depletion of FBW7 on the T315-induced suppression (1 and 2 μmol/L) of hypoxia-induced HIF-1α expression. Immunoblots and RT-PCR analyses are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 3: Evidence that ILK regulates HIF-1α expression through different mechanisms in PC-3 and MCF-7 cells versus LNCaP cells(A) Upper, Western blot analysis of the effects of T315 on hypoxia-induced changes in the expression/phosphorylation levels of HIF-1α, ILK, YB-1, GSK3β, and markers of EMT and Akt signaling. Lower, RT-PCR analysis of the corresponding effects on hypoxia-induced changes in the mRNA levels of HIF-1α and EMT markers. (B) Immunocytochemical analysis of the effect of T315 (1 μmol/L; 24 h) versus DMSO control on the cellular distribution of ectopically expressed GFP-Foxo3a under hypoxic conditions. Scale bar, 50 μm. (C) Left, effects of T315 (T; 2 μmol/L), SB-216763 (SB; 10 μmol/L), and rapamycin (Rap; 10 μmol/L) relative to DMSO control (Ctl) on hypoxia-induced HIF-1α expression. Nor, normoxia. Right, influence of shRNA-mediated depletion of FBW7 on the T315-induced suppression (1 and 2 μmol/L) of hypoxia-induced HIF-1α expression. Immunoblots and RT-PCR analyses are representative of three independent experiments.
Mentions: The role of ILK in regulating hypoxia-induced HIF-1α expression and EMT was further verified by using a proof-of-concept, small molecule ILK inhibitor, T315, in PC-3, LNCaP, and MCF-7 cells. The IC50 values of T315 in suppressing the viability of these three representative cell lines were: PC-3, 2 μmol/L; LNCaP, 2 μmol/L; MCF-7, 2.8 μmol/L [25]. As shown in Figure 3A, inhibition of ILK kinase activity by T315 dose-dependently suppressed hypoxia-induced increases in the expression of HIF-1α, ILK and YB-1, indicative of the ability of T315 to disrupt the HIF-1α-ILK regulatory loop. T315 also reversed hypoxia-induced changes in the EMT regulators/markers, Foxo3a, E-cadherin, vimentin, and/or Snail, in all three cell lines, restoring them to levels detected under normoxic conditions (Figure 3A). Reminiscent of the results observed after siRNA-mediated ILK knockdown, T315 suppressed the hypoxia-induced phosphorylation of Ser473-Akt and its downstream targets mTOR and Foxo3a in PC-3 and MCF-7 cells, but not in LNCaP cells. Accordingly, this differential effect of T315 on the Akt-Foxo3a signaling axis was manifested by differences in the cellular distribution of Foxo3a among these cell lines (Figure 3B). In PC-3 and MCF-7 cells, the suppression of hypoxia-induced Akt activation and Foxo3a phosphorylation by T315 (Figure 3A) was accompanied by the nuclear localization of Foxo3a (Figure 3B). In contrast, Foxo3a remained sequestered in the cytoplasm in T315-treated LNCaP cells (Figure 3B), reflecting the inability of T315 to inhibit Akt/Foxo3a phosphorylation. Nonetheless, T315 was able to downregulate GSK3β phosphorylation in all three cell lines (Figure 1A) as GSK3β represents a direct target of ILK [26].

Bottom Line: We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3β phosphorylation.In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1.Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

ABSTRACT
Here, we described a novel regulatory feedback loop in which hypoxia induces integrin-linked kinase (ILK) expression through a HIF-1α-dependent mechanism and ILK, in turn, stimulates HIF-1α expression through cell type- and cell context-dependent pathways. HIF-1α increased ILK via transcriptional activation. ILK increased HIF-1α levels by promoting mTOR-mediated translation in PC-3 and MCF-7 cells, and by blocking GSK3β-mediated degradation in LNCaP cells, consistent with the cell line-/cellular context-specific functions of ILK as a Ser473-Akt kinase. We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3β phosphorylation. Also, ILK can de-repress HIF-1α signaling through the YB-1-mediated inhibition of Foxo3a expression. In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1. Thus, the ILK-HIF-1α regulatory loop could underlie the maintenance of high HIF-1α expression levels and the promotion of EMT under hypoxic conditions. Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.

No MeSH data available.


Related in: MedlinePlus