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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 and HIF-1α form a regulatory feedback loop in regulating hypoxia-induced EMT(A) Effects of doxycycline (Dox)-responsive shRNA-mediated ILK knockdown on the expression and/or phosphorylation levels of ILK, HIF-1α, Akt, YB-1, Foxo3a, and EMT effectors after 48-h exposure to normoxic or hypoxic condition. Left, Western blot; right, RT-PCR. (B) Effects of siRNA-mediated knockdown of ILK on the expression/phosphorylation levels of the same markers as in (A) in LNCaP and MCF-7 cells. (C) Effects of ectopic expression of GFP-ILK on the expression/phosphorylation levels of these markers in PC-3 cells. Immunoblots are representative of three independent experiments.
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Figure 2: Evidence that ILK and HIF-1α form a regulatory feedback loop in regulating hypoxia-induced EMT(A) Effects of doxycycline (Dox)-responsive shRNA-mediated ILK knockdown on the expression and/or phosphorylation levels of ILK, HIF-1α, Akt, YB-1, Foxo3a, and EMT effectors after 48-h exposure to normoxic or hypoxic condition. Left, Western blot; right, RT-PCR. (B) Effects of siRNA-mediated knockdown of ILK on the expression/phosphorylation levels of the same markers as in (A) in LNCaP and MCF-7 cells. (C) Effects of ectopic expression of GFP-ILK on the expression/phosphorylation levels of these markers in PC-3 cells. Immunoblots are representative of three independent experiments.

Mentions: Pursuant to these findings, we demonstrated that ILK, in turn, could regulate HIF-1α expression, thereby forming a positive feedback loop in maintaining HIF-1α expression, and thus EMT, under hypoxic conditions. Using a stable clone of PC-3 cells that overexpress ILK shRNA under Tet-on control (PC-3TRE-shILK), we showed that doxycycline-induced knockdown of ILK, as verified by parallel reduction of YB-1 expression, suppressed HIF-1α protein expression under normoxic conditions, and abrogated the hypoxia-induced upregulation of HIF-1α (Figure 2A, left). This ILK knockdown-induced suppression of HIF-1α expression occurred at the posttranscriptional level as the abundance of HIF-1α mRNA remained unchanged in response to hypoxia and/or doxycycline treatment (right). Equally important, knockdown of ILK also blocked the effects of hypoxia on Ser-473-Akt phosphorylation, as well as the protein expression of various EMT regulators/markers in PC-3 cells (Figure 2A). Similar effects were also noted in LNCaP and MCF-7 cells, with the exception of an effect on Akt phosphorylation in LNCaP cells which was unaffected (Figure 2B). Mechanistically, this discrepancy is in-line with our earlier finding that Ser473-Akt phosphorylation is regulated in a cell line-specific manner by ILK and mTORC2 in PTEN-deficient PC-3 and LNCaP cells, respectively [25]. Conversely, enforced expression of constitutively active ILK in PC-3 cells increased HIF-1α expression, accompanied by parallel changes in Akt phosphorylation and expression of EMT-associated regulators/markers (Figure 2C).


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 and HIF-1α form a regulatory feedback loop in regulating hypoxia-induced EMT(A) Effects of doxycycline (Dox)-responsive shRNA-mediated ILK knockdown on the expression and/or phosphorylation levels of ILK, HIF-1α, Akt, YB-1, Foxo3a, and EMT effectors after 48-h exposure to normoxic or hypoxic condition. Left, Western blot; right, RT-PCR. (B) Effects of siRNA-mediated knockdown of ILK on the expression/phosphorylation levels of the same markers as in (A) in LNCaP and MCF-7 cells. (C) Effects of ectopic expression of GFP-ILK on the expression/phosphorylation levels of these markers in PC-3 cells. Immunoblots are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480751&req=5

Figure 2: Evidence that ILK and HIF-1α form a regulatory feedback loop in regulating hypoxia-induced EMT(A) Effects of doxycycline (Dox)-responsive shRNA-mediated ILK knockdown on the expression and/or phosphorylation levels of ILK, HIF-1α, Akt, YB-1, Foxo3a, and EMT effectors after 48-h exposure to normoxic or hypoxic condition. Left, Western blot; right, RT-PCR. (B) Effects of siRNA-mediated knockdown of ILK on the expression/phosphorylation levels of the same markers as in (A) in LNCaP and MCF-7 cells. (C) Effects of ectopic expression of GFP-ILK on the expression/phosphorylation levels of these markers in PC-3 cells. Immunoblots are representative of three independent experiments.
Mentions: Pursuant to these findings, we demonstrated that ILK, in turn, could regulate HIF-1α expression, thereby forming a positive feedback loop in maintaining HIF-1α expression, and thus EMT, under hypoxic conditions. Using a stable clone of PC-3 cells that overexpress ILK shRNA under Tet-on control (PC-3TRE-shILK), we showed that doxycycline-induced knockdown of ILK, as verified by parallel reduction of YB-1 expression, suppressed HIF-1α protein expression under normoxic conditions, and abrogated the hypoxia-induced upregulation of HIF-1α (Figure 2A, left). This ILK knockdown-induced suppression of HIF-1α expression occurred at the posttranscriptional level as the abundance of HIF-1α mRNA remained unchanged in response to hypoxia and/or doxycycline treatment (right). Equally important, knockdown of ILK also blocked the effects of hypoxia on Ser-473-Akt phosphorylation, as well as the protein expression of various EMT regulators/markers in PC-3 cells (Figure 2A). Similar effects were also noted in LNCaP and MCF-7 cells, with the exception of an effect on Akt phosphorylation in LNCaP cells which was unaffected (Figure 2B). Mechanistically, this discrepancy is in-line with our earlier finding that Ser473-Akt phosphorylation is regulated in a cell line-specific manner by ILK and mTORC2 in PTEN-deficient PC-3 and LNCaP cells, respectively [25]. Conversely, enforced expression of constitutively active ILK in PC-3 cells increased HIF-1α expression, accompanied by parallel changes in Akt phosphorylation and expression of EMT-associated regulators/markers (Figure 2C).

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