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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

ILK inhibition by T315 reactivates Foxo3a gene expression under hypoxia by abolishing YB-1-mediated transcriptional repression in PC-3 cells(A) RT-PCR analyses of the effect of T315 on hypoxia-induced changes in YB-1 and Foxo3a expression. (B) Western blot analysis of the effect of siRNA-mediated knockdown (left) and ectopic expression (right) of YB-1 on expression of Foxo3a and E-cadherin. (C) Effect of ectopic expression of YB-1 on T315-induced upregulation of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells. Upper, Western blot; lower, RT-PCR. (D) Luciferase reporter assays of the effect of ectopically expressed YB-1 on Foxo3a promoter activity under normoxic and hypoxic conditions. Data are presented as means ± S.D. (n = 6). *p < 0.001, compared to the respective controls. (E) Upper, depiction of 4 regions (F1–4) in the Foxo3a gene promoter containing putative YB-1 binding elements (indicated by vertical bars). Lower left, ChIP analysis of selective YB-1 binding to different regions of the Foxo3a promoter in response to hypoxia. N, normoxia; H, hypoxia. Lower right, ChIP analysis of the effects of T315 (24 h) on hypoxia-induced YB-1 binding to the F3 region of the Foxo3a promoter.
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Figure 4: ILK inhibition by T315 reactivates Foxo3a gene expression under hypoxia by abolishing YB-1-mediated transcriptional repression in PC-3 cells(A) RT-PCR analyses of the effect of T315 on hypoxia-induced changes in YB-1 and Foxo3a expression. (B) Western blot analysis of the effect of siRNA-mediated knockdown (left) and ectopic expression (right) of YB-1 on expression of Foxo3a and E-cadherin. (C) Effect of ectopic expression of YB-1 on T315-induced upregulation of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells. Upper, Western blot; lower, RT-PCR. (D) Luciferase reporter assays of the effect of ectopically expressed YB-1 on Foxo3a promoter activity under normoxic and hypoxic conditions. Data are presented as means ± S.D. (n = 6). *p < 0.001, compared to the respective controls. (E) Upper, depiction of 4 regions (F1–4) in the Foxo3a gene promoter containing putative YB-1 binding elements (indicated by vertical bars). Lower left, ChIP analysis of selective YB-1 binding to different regions of the Foxo3a promoter in response to hypoxia. N, normoxia; H, hypoxia. Lower right, ChIP analysis of the effects of T315 (24 h) on hypoxia-induced YB-1 binding to the F3 region of the Foxo3a promoter.

Mentions: Consistent with our Western blot data (Figure 3A), RT-PCR analysis showed an inverse relationship between YB-1 and Foxo3a mRNA levels in response to T315 in hypoxia-exposed PC-3 cells (Figure 4A). The putative role of YB-1 as a transcriptional repressor of Foxo3a gene expression was supported by the abilities of siRNA-mediated knockdown and ectopic expression of YB-1 to increase and suppress, respectively, the expression of Foxo3a and its direct target E-cadherin (Figure 4B). Moreover, enforced expression of Flag-tagged YB-1 abolished the T315-induced restoration of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells, at both protein and mRNA levels (Figure 4C). The suppressive effect of YB-1 on Foxo3a expression was attributable to transcriptional repression, as enforced YB-1 expression reduced Foxo3a promoter activity under both normoxic and hypoxic conditions (Figure 4D).


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)

ILK inhibition by T315 reactivates Foxo3a gene expression under hypoxia by abolishing YB-1-mediated transcriptional repression in PC-3 cells(A) RT-PCR analyses of the effect of T315 on hypoxia-induced changes in YB-1 and Foxo3a expression. (B) Western blot analysis of the effect of siRNA-mediated knockdown (left) and ectopic expression (right) of YB-1 on expression of Foxo3a and E-cadherin. (C) Effect of ectopic expression of YB-1 on T315-induced upregulation of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells. Upper, Western blot; lower, RT-PCR. (D) Luciferase reporter assays of the effect of ectopically expressed YB-1 on Foxo3a promoter activity under normoxic and hypoxic conditions. Data are presented as means ± S.D. (n = 6). *p < 0.001, compared to the respective controls. (E) Upper, depiction of 4 regions (F1–4) in the Foxo3a gene promoter containing putative YB-1 binding elements (indicated by vertical bars). Lower left, ChIP analysis of selective YB-1 binding to different regions of the Foxo3a promoter in response to hypoxia. N, normoxia; H, hypoxia. Lower right, ChIP analysis of the effects of T315 (24 h) on hypoxia-induced YB-1 binding to the F3 region of the Foxo3a promoter.
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Figure 4: ILK inhibition by T315 reactivates Foxo3a gene expression under hypoxia by abolishing YB-1-mediated transcriptional repression in PC-3 cells(A) RT-PCR analyses of the effect of T315 on hypoxia-induced changes in YB-1 and Foxo3a expression. (B) Western blot analysis of the effect of siRNA-mediated knockdown (left) and ectopic expression (right) of YB-1 on expression of Foxo3a and E-cadherin. (C) Effect of ectopic expression of YB-1 on T315-induced upregulation of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells. Upper, Western blot; lower, RT-PCR. (D) Luciferase reporter assays of the effect of ectopically expressed YB-1 on Foxo3a promoter activity under normoxic and hypoxic conditions. Data are presented as means ± S.D. (n = 6). *p < 0.001, compared to the respective controls. (E) Upper, depiction of 4 regions (F1–4) in the Foxo3a gene promoter containing putative YB-1 binding elements (indicated by vertical bars). Lower left, ChIP analysis of selective YB-1 binding to different regions of the Foxo3a promoter in response to hypoxia. N, normoxia; H, hypoxia. Lower right, ChIP analysis of the effects of T315 (24 h) on hypoxia-induced YB-1 binding to the F3 region of the Foxo3a promoter.
Mentions: Consistent with our Western blot data (Figure 3A), RT-PCR analysis showed an inverse relationship between YB-1 and Foxo3a mRNA levels in response to T315 in hypoxia-exposed PC-3 cells (Figure 4A). The putative role of YB-1 as a transcriptional repressor of Foxo3a gene expression was supported by the abilities of siRNA-mediated knockdown and ectopic expression of YB-1 to increase and suppress, respectively, the expression of Foxo3a and its direct target E-cadherin (Figure 4B). Moreover, enforced expression of Flag-tagged YB-1 abolished the T315-induced restoration of Foxo3a and E-cadherin expression in hypoxia-treated PC-3 cells, at both protein and mRNA levels (Figure 4C). The suppressive effect of YB-1 on Foxo3a expression was attributable to transcriptional repression, as enforced YB-1 expression reduced Foxo3a promoter activity under both normoxic and hypoxic conditions (Figure 4D).

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