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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

Suppressive effects of ILK inhibition on hypoxia-induced aggressive phenotype in PC-3 cells(A) Effects of T315 on cell viability (left), migratory activity (left center), and invasiveness (right center) after 12 h of treatment. Data are presented as mean ± S.D. (n = 6). Right, trend analysis of the rates of decrease in the viability, migration, and invasion of the T315-treated PC-3 cells. Individual data points from each treatment group in each assay are shown. (B) Left, photomicrographs of PC-3 colonies after growth in BME under normoxic or hypoxic conditions in the presence of T315 or DMSO control (Ctl). Scale bar, 100 μm. Right, Western blot analysis of the expression of HIF-1α, YB-1, Foxo3a, and EMT effectors in PC-3 cells grown in monolayers under normoxic conditions (Norm) versus that of the invasive colonies described in the left panel. (C) Effects of T315 versus DMSO control (Ctl) on the F-actin cytoskeleton in PC-3 cells after 24 h of treatment. Scale bar, 50 μm.
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Figure 6: Suppressive effects of ILK inhibition on hypoxia-induced aggressive phenotype in PC-3 cells(A) Effects of T315 on cell viability (left), migratory activity (left center), and invasiveness (right center) after 12 h of treatment. Data are presented as mean ± S.D. (n = 6). Right, trend analysis of the rates of decrease in the viability, migration, and invasion of the T315-treated PC-3 cells. Individual data points from each treatment group in each assay are shown. (B) Left, photomicrographs of PC-3 colonies after growth in BME under normoxic or hypoxic conditions in the presence of T315 or DMSO control (Ctl). Scale bar, 100 μm. Right, Western blot analysis of the expression of HIF-1α, YB-1, Foxo3a, and EMT effectors in PC-3 cells grown in monolayers under normoxic conditions (Norm) versus that of the invasive colonies described in the left panel. (C) Effects of T315 versus DMSO control (Ctl) on the F-actin cytoskeleton in PC-3 cells after 24 h of treatment. Scale bar, 50 μm.

Mentions: T315 inhibited PC-3 cell migration and invasion in vitro in a dose-dependent manner under both normoxic and hypoxic conditions (Figure 6A, center two panels). Although treatment with T315 under the same experimental conditions also caused a dose-dependent reduction in cell viability (left panel), the inhibition of cell motility and invasion was not attributable to cell death as the concentration-dependent rates of decrease in invasion and migration were significantly greater than that in viability (right panel). In addition, the suppressive effect of T315 on the metastatic potential of PC-3 cells was interrogated using the three-dimensional (3-D) colony formation assay, which is frequently used to assess metastatic capacity [42]. PC-3 cell colonies that formed under normoxic conditions were characterized by round spheroid morphology with few protrusions (Figure 6B, left). Colonies generated under hypoxic conditions exhibited stellate projections that bridged multiple colonies, indicating an invasive phenotype, which was blocked by T315, as indicated by the loss of stellate morphology. Western blot analysis of cell lysates collected from these colonies confirmed the ability of T315 to suppress hypoxia-induced changes in EMT-associated markers, restoring their expression to levels comparable to, or even lower than, those observed under normoxic conditions (Figure 6B, right). In addition, staining with FITC-conjugated phalloidin revealed the induction of pseudopod formation in hypoxia-exposed cells, which was blocked by T315 (Figure 6C). Together, these findings suggest that ILK inhibition by T315 suppressed the metastatic phenotype of PC-3 cells.


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)

Suppressive effects of ILK inhibition on hypoxia-induced aggressive phenotype in PC-3 cells(A) Effects of T315 on cell viability (left), migratory activity (left center), and invasiveness (right center) after 12 h of treatment. Data are presented as mean ± S.D. (n = 6). Right, trend analysis of the rates of decrease in the viability, migration, and invasion of the T315-treated PC-3 cells. Individual data points from each treatment group in each assay are shown. (B) Left, photomicrographs of PC-3 colonies after growth in BME under normoxic or hypoxic conditions in the presence of T315 or DMSO control (Ctl). Scale bar, 100 μm. Right, Western blot analysis of the expression of HIF-1α, YB-1, Foxo3a, and EMT effectors in PC-3 cells grown in monolayers under normoxic conditions (Norm) versus that of the invasive colonies described in the left panel. (C) Effects of T315 versus DMSO control (Ctl) on the F-actin cytoskeleton in PC-3 cells after 24 h of treatment. Scale bar, 50 μm.
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Figure 6: Suppressive effects of ILK inhibition on hypoxia-induced aggressive phenotype in PC-3 cells(A) Effects of T315 on cell viability (left), migratory activity (left center), and invasiveness (right center) after 12 h of treatment. Data are presented as mean ± S.D. (n = 6). Right, trend analysis of the rates of decrease in the viability, migration, and invasion of the T315-treated PC-3 cells. Individual data points from each treatment group in each assay are shown. (B) Left, photomicrographs of PC-3 colonies after growth in BME under normoxic or hypoxic conditions in the presence of T315 or DMSO control (Ctl). Scale bar, 100 μm. Right, Western blot analysis of the expression of HIF-1α, YB-1, Foxo3a, and EMT effectors in PC-3 cells grown in monolayers under normoxic conditions (Norm) versus that of the invasive colonies described in the left panel. (C) Effects of T315 versus DMSO control (Ctl) on the F-actin cytoskeleton in PC-3 cells after 24 h of treatment. Scale bar, 50 μm.
Mentions: T315 inhibited PC-3 cell migration and invasion in vitro in a dose-dependent manner under both normoxic and hypoxic conditions (Figure 6A, center two panels). Although treatment with T315 under the same experimental conditions also caused a dose-dependent reduction in cell viability (left panel), the inhibition of cell motility and invasion was not attributable to cell death as the concentration-dependent rates of decrease in invasion and migration were significantly greater than that in viability (right panel). In addition, the suppressive effect of T315 on the metastatic potential of PC-3 cells was interrogated using the three-dimensional (3-D) colony formation assay, which is frequently used to assess metastatic capacity [42]. PC-3 cell colonies that formed under normoxic conditions were characterized by round spheroid morphology with few protrusions (Figure 6B, left). Colonies generated under hypoxic conditions exhibited stellate projections that bridged multiple colonies, indicating an invasive phenotype, which was blocked by T315, as indicated by the loss of stellate morphology. Western blot analysis of cell lysates collected from these colonies confirmed the ability of T315 to suppress hypoxia-induced changes in EMT-associated markers, restoring their expression to levels comparable to, or even lower than, those observed under normoxic conditions (Figure 6B, right). In addition, staining with FITC-conjugated phalloidin revealed the induction of pseudopod formation in hypoxia-exposed cells, which was blocked by T315 (Figure 6C). Together, these findings suggest that ILK inhibition by T315 suppressed the metastatic phenotype of PC-3 cells.

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