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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 Snail expression through multiple mechanisms in hypoxic conditions(A) Luciferase reporter assays of the effects of (left) hypoxia and ectopic expression of HIF-1α and ILK on Snail promoter activity, and (right), the ectopic expression of HIF-1α and ILK on T315-induced suppression of Snail promoter activity in PC-3 cells. Data are presented as means ± S.D. (n = 6). (B) Effect of T315 on the expression/phosphorylation levels of Snail and GSK3β in hypoxia-treated PC-3 cells. Upper, Western blot; lower, immunoprecipitation. (C & D) Effect of ILK inhibition on cellular distribution of ectopically expressed GFP-tagged Snail under hypoxic conditions. (C) Immunocytochemical analysis of T315-treated PC-3 and doxycycline (Dox)-treated PC-3TRE-ILKi cells. Scale bar, 50 μm. (D) Western blot analysis of cytoplasmic and nuclear fractions from T315-treated PC-3 cells. Histone H3 and α-tubulin were used as internal markers for the nucleus and cytoplasm, respectively. (E) Western blot analysis of the protective effect of ectopic Myc-ΔF-β-TrCP expression on T315-mediated degradation of Snail in hypoxia-treated PC-3 cells. Immunoblots are representative of three independent experiments.
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Figure 5: Evidence that ILK regulates Snail expression through multiple mechanisms in hypoxic conditions(A) Luciferase reporter assays of the effects of (left) hypoxia and ectopic expression of HIF-1α and ILK on Snail promoter activity, and (right), the ectopic expression of HIF-1α and ILK on T315-induced suppression of Snail promoter activity in PC-3 cells. Data are presented as means ± S.D. (n = 6). (B) Effect of T315 on the expression/phosphorylation levels of Snail and GSK3β in hypoxia-treated PC-3 cells. Upper, Western blot; lower, immunoprecipitation. (C & D) Effect of ILK inhibition on cellular distribution of ectopically expressed GFP-tagged Snail under hypoxic conditions. (C) Immunocytochemical analysis of T315-treated PC-3 and doxycycline (Dox)-treated PC-3TRE-ILKi cells. Scale bar, 50 μm. (D) Western blot analysis of cytoplasmic and nuclear fractions from T315-treated PC-3 cells. Histone H3 and α-tubulin were used as internal markers for the nucleus and cytoplasm, respectively. (E) Western blot analysis of the protective effect of ectopic Myc-ΔF-β-TrCP expression on T315-mediated degradation of Snail in hypoxia-treated PC-3 cells. Immunoblots are representative of three independent experiments.

Mentions: In addition to Foxo3a upregulation, the ability of T315 to suppress the EMT master regulator Snail and its target Zeb1 is noteworthy. The overexpression of ILK was reported to increase gene transcription of Snail in human colon carcinoma cells [36]. Here, we obtained evidence that T315 regulates the expression and cellular fate of Snail in hypoxia-treated cells through several distinct mechanisms. First, recent reports indicate that Snail expression is regulated by HIF-1α [37, 38] and YB-1 [39]. Accordingly, ectopic expression of HIF-1α or ILK in PC-3 cells mimicked the stimulatory effect of hypoxia on Snail promoter activity (Figure 5A, left) and partially protected cells against the suppressive effect of T315 on this activity (right). Second, GSK3β has been shown to phosphorylate Snail, leading to its cytoplasmic sequestration and degradation [40]. Consistent with this finding, T315-mediated suppression of hypoxia-induced Snail expression was associated with the dephosphorylating activation of GSK3β, and the consequent increase in Snail phosphorylation (Figure 5B). Immunofluorescence analysis indicated that inhibition of ILK, by either T315 or siRNA-mediated repression, resulted in the nuclear to cytoplasmic translocation of green fluorescent protein (GFP)-tagged Snail (Figure 5C). This finding was corroborated by Western blot analysis of the cytosolic versus nuclear distribution of GFP-Snail using anti-GFP antibodies (Figure 5D). Finally, as β-TrCP is the E3 ligase responsible for GSK3β-facilitated Snail degradation [40], enforced expression of ΔF-β-TrCP, an F-box-deleted, dominant-negative mutant form of β-TrCP [41], protected hypoxia-exposed PC-3 cells against T315-induced Snail downregulation (Figure 5D).


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 Snail expression through multiple mechanisms in hypoxic conditions(A) Luciferase reporter assays of the effects of (left) hypoxia and ectopic expression of HIF-1α and ILK on Snail promoter activity, and (right), the ectopic expression of HIF-1α and ILK on T315-induced suppression of Snail promoter activity in PC-3 cells. Data are presented as means ± S.D. (n = 6). (B) Effect of T315 on the expression/phosphorylation levels of Snail and GSK3β in hypoxia-treated PC-3 cells. Upper, Western blot; lower, immunoprecipitation. (C & D) Effect of ILK inhibition on cellular distribution of ectopically expressed GFP-tagged Snail under hypoxic conditions. (C) Immunocytochemical analysis of T315-treated PC-3 and doxycycline (Dox)-treated PC-3TRE-ILKi cells. Scale bar, 50 μm. (D) Western blot analysis of cytoplasmic and nuclear fractions from T315-treated PC-3 cells. Histone H3 and α-tubulin were used as internal markers for the nucleus and cytoplasm, respectively. (E) Western blot analysis of the protective effect of ectopic Myc-ΔF-β-TrCP expression on T315-mediated degradation of Snail in hypoxia-treated PC-3 cells. Immunoblots are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Evidence that ILK regulates Snail expression through multiple mechanisms in hypoxic conditions(A) Luciferase reporter assays of the effects of (left) hypoxia and ectopic expression of HIF-1α and ILK on Snail promoter activity, and (right), the ectopic expression of HIF-1α and ILK on T315-induced suppression of Snail promoter activity in PC-3 cells. Data are presented as means ± S.D. (n = 6). (B) Effect of T315 on the expression/phosphorylation levels of Snail and GSK3β in hypoxia-treated PC-3 cells. Upper, Western blot; lower, immunoprecipitation. (C & D) Effect of ILK inhibition on cellular distribution of ectopically expressed GFP-tagged Snail under hypoxic conditions. (C) Immunocytochemical analysis of T315-treated PC-3 and doxycycline (Dox)-treated PC-3TRE-ILKi cells. Scale bar, 50 μm. (D) Western blot analysis of cytoplasmic and nuclear fractions from T315-treated PC-3 cells. Histone H3 and α-tubulin were used as internal markers for the nucleus and cytoplasm, respectively. (E) Western blot analysis of the protective effect of ectopic Myc-ΔF-β-TrCP expression on T315-mediated degradation of Snail in hypoxia-treated PC-3 cells. Immunoblots are representative of three independent experiments.
Mentions: In addition to Foxo3a upregulation, the ability of T315 to suppress the EMT master regulator Snail and its target Zeb1 is noteworthy. The overexpression of ILK was reported to increase gene transcription of Snail in human colon carcinoma cells [36]. Here, we obtained evidence that T315 regulates the expression and cellular fate of Snail in hypoxia-treated cells through several distinct mechanisms. First, recent reports indicate that Snail expression is regulated by HIF-1α [37, 38] and YB-1 [39]. Accordingly, ectopic expression of HIF-1α or ILK in PC-3 cells mimicked the stimulatory effect of hypoxia on Snail promoter activity (Figure 5A, left) and partially protected cells against the suppressive effect of T315 on this activity (right). Second, GSK3β has been shown to phosphorylate Snail, leading to its cytoplasmic sequestration and degradation [40]. Consistent with this finding, T315-mediated suppression of hypoxia-induced Snail expression was associated with the dephosphorylating activation of GSK3β, and the consequent increase in Snail phosphorylation (Figure 5B). Immunofluorescence analysis indicated that inhibition of ILK, by either T315 or siRNA-mediated repression, resulted in the nuclear to cytoplasmic translocation of green fluorescent protein (GFP)-tagged Snail (Figure 5C). This finding was corroborated by Western blot analysis of the cytosolic versus nuclear distribution of GFP-Snail using anti-GFP antibodies (Figure 5D). Finally, as β-TrCP is the E3 ligase responsible for GSK3β-facilitated Snail degradation [40], enforced expression of ΔF-β-TrCP, an F-box-deleted, dominant-negative mutant form of β-TrCP [41], protected hypoxia-exposed PC-3 cells against T315-induced Snail downregulation (Figure 5D).

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