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Q6, a novel hypoxia-targeted drug, regulates hypoxia-inducible factor signaling via an autophagy-dependent mechanism in hepatocellular carcinoma.

Liu XW, Cai TY, Zhu H, Cao J, Su Y, Hu YZ, He QJ, Yang B - Autophagy (2013)

Bottom Line: Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy.These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC.Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

View Article: PubMed Central - PubMed

Affiliation: Zhejiang Province Key Laboratory of Anti-Cancer Drug Research; Institute of Pharmacology and Toxicology; College of Pharmaceutical Sciences; Zhejiang University; Hangzhou, China.

ABSTRACT
Tumor hypoxia underlies treatment failure and yields more aggressive and metastatic cancer phenotypes. Although therapeutically targeting these hypoxic environments has been proposed for many years, to date no approaches have shown the therapeutic value to gain regulatory approval. Here, we demonstrated that a novel hypoxia-activated prodrug, Q6, exhibits potent antiproliferative efficacy under hypoxic conditions and induces caspase-dependent apoptosis in 2 hepatocellular carcinoma (HCC) cell lines, with no obvious toxicity being detected in 2 normal liver cell lines. Treatment with Q6 markedly downregulated HIF1A [hypoxia inducible factor 1, α subunit (basic helix-loop-helix transcription factor)] expression and transcription of the downstream target gene, VEGFA (vascular endothelial growth factor A). This dual hypoxia-targeted modulation mechanism leads to high potency in suppressing tumor growth and vascularization in 2 in vivo models. Intriguingly, it is the autophagy-dependent degradation pathway that plays a crucial role in Q6-induced attenuation of HIF1A expression, rather than the proteasome-dependent pathway, which is normally regarded as the predominant mechanism underlying posttranslational regulation of HIF1A. Inhibition of autophagy, either by short interfering RNA (siRNA) or by chemical inhibitors, blocked Q6-induced HIF1A degradation. Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy. Additionally, silencing of SQSTM1 inhibited Q6-induced HIF1A degradation. These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC. Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

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Figure 5. SQSTM1 regulates degradation of HIF1A. (A) HepG2 and Bel-7402 cells were exposed to Q6 (0, 5 μM) in the presence/absence of bafilomycin A1 (BafA1; 0.2 μM) for 6 h in hypoxia and then HIF1A and SQSTM1 protein levels were analyzed by confocal microscopy. Scale bar, 20 μm. (B) HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 5 μM) or Q6 (0, 5 μM) + bafilomycin A1 (0.2 μM) for 6 h and then assayed for protein expression levels as indicated by immunoprecipitation or western blotting. 10% of lysate used for IP was loaded for WCL. (C) After transfection with SQSTM1 siRNA for 36 h, HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control.
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Figure 5: Figure 5. SQSTM1 regulates degradation of HIF1A. (A) HepG2 and Bel-7402 cells were exposed to Q6 (0, 5 μM) in the presence/absence of bafilomycin A1 (BafA1; 0.2 μM) for 6 h in hypoxia and then HIF1A and SQSTM1 protein levels were analyzed by confocal microscopy. Scale bar, 20 μm. (B) HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 5 μM) or Q6 (0, 5 μM) + bafilomycin A1 (0.2 μM) for 6 h and then assayed for protein expression levels as indicated by immunoprecipitation or western blotting. 10% of lysate used for IP was loaded for WCL. (C) After transfection with SQSTM1 siRNA for 36 h, HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control.

Mentions: Accumulating studies have demonstrated that autophagy is an important mechanism to prevent the accumulation of polyubiquitinated protein aggregates, and recent studies have revealed that SQSTM1 recognizes polyubiquitinated protein aggregates and then binds to LC3B on autophagosomal membranes, thereby delivering the aggregates for degradation.22 Therefore, we hypothesized that SQSTM1 could recognize the polyubiquitinated protein HIF1A. The first supporting evidence was provided by immunofluorescence analysis, where a significant increase in levels of HIF1A was found in the cytoplasm after Q6 treatment, and thus the colocalization between HIF1A and SQSTM1 was enhanced (Fig. 5A). The coimmunoprecipitation analysis using SQSTM1 and HIF1A antibodies further evaluated that under basal conditions there is no association between endogenous SQSTM1 and HIF1A, and this link is significantly increased after Q6 treatment. Particularly, cotreatment Q6 with bafilomycin A1, causes HIF1A and SQSTM1 to accumulate in autophagosomes and lysosomes, thus the more evident HIF1A band was observed. (Fig. 5B).


Q6, a novel hypoxia-targeted drug, regulates hypoxia-inducible factor signaling via an autophagy-dependent mechanism in hepatocellular carcinoma.

Liu XW, Cai TY, Zhu H, Cao J, Su Y, Hu YZ, He QJ, Yang B - Autophagy (2013)

Figure 5. SQSTM1 regulates degradation of HIF1A. (A) HepG2 and Bel-7402 cells were exposed to Q6 (0, 5 μM) in the presence/absence of bafilomycin A1 (BafA1; 0.2 μM) for 6 h in hypoxia and then HIF1A and SQSTM1 protein levels were analyzed by confocal microscopy. Scale bar, 20 μm. (B) HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 5 μM) or Q6 (0, 5 μM) + bafilomycin A1 (0.2 μM) for 6 h and then assayed for protein expression levels as indicated by immunoprecipitation or western blotting. 10% of lysate used for IP was loaded for WCL. (C) After transfection with SQSTM1 siRNA for 36 h, HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Figure 5. SQSTM1 regulates degradation of HIF1A. (A) HepG2 and Bel-7402 cells were exposed to Q6 (0, 5 μM) in the presence/absence of bafilomycin A1 (BafA1; 0.2 μM) for 6 h in hypoxia and then HIF1A and SQSTM1 protein levels were analyzed by confocal microscopy. Scale bar, 20 μm. (B) HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 5 μM) or Q6 (0, 5 μM) + bafilomycin A1 (0.2 μM) for 6 h and then assayed for protein expression levels as indicated by immunoprecipitation or western blotting. 10% of lysate used for IP was loaded for WCL. (C) After transfection with SQSTM1 siRNA for 36 h, HepG2 (left) and Bel-7402 (right) cells were treated with Q6 (0, 2.5, 5 μM) for 6 h, after which HIF1A protein levels were measured by western blot analysis. ACTB was measured as the loading control.
Mentions: Accumulating studies have demonstrated that autophagy is an important mechanism to prevent the accumulation of polyubiquitinated protein aggregates, and recent studies have revealed that SQSTM1 recognizes polyubiquitinated protein aggregates and then binds to LC3B on autophagosomal membranes, thereby delivering the aggregates for degradation.22 Therefore, we hypothesized that SQSTM1 could recognize the polyubiquitinated protein HIF1A. The first supporting evidence was provided by immunofluorescence analysis, where a significant increase in levels of HIF1A was found in the cytoplasm after Q6 treatment, and thus the colocalization between HIF1A and SQSTM1 was enhanced (Fig. 5A). The coimmunoprecipitation analysis using SQSTM1 and HIF1A antibodies further evaluated that under basal conditions there is no association between endogenous SQSTM1 and HIF1A, and this link is significantly increased after Q6 treatment. Particularly, cotreatment Q6 with bafilomycin A1, causes HIF1A and SQSTM1 to accumulate in autophagosomes and lysosomes, thus the more evident HIF1A band was observed. (Fig. 5B).

Bottom Line: Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy.These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC.Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

View Article: PubMed Central - PubMed

Affiliation: Zhejiang Province Key Laboratory of Anti-Cancer Drug Research; Institute of Pharmacology and Toxicology; College of Pharmaceutical Sciences; Zhejiang University; Hangzhou, China.

ABSTRACT
Tumor hypoxia underlies treatment failure and yields more aggressive and metastatic cancer phenotypes. Although therapeutically targeting these hypoxic environments has been proposed for many years, to date no approaches have shown the therapeutic value to gain regulatory approval. Here, we demonstrated that a novel hypoxia-activated prodrug, Q6, exhibits potent antiproliferative efficacy under hypoxic conditions and induces caspase-dependent apoptosis in 2 hepatocellular carcinoma (HCC) cell lines, with no obvious toxicity being detected in 2 normal liver cell lines. Treatment with Q6 markedly downregulated HIF1A [hypoxia inducible factor 1, α subunit (basic helix-loop-helix transcription factor)] expression and transcription of the downstream target gene, VEGFA (vascular endothelial growth factor A). This dual hypoxia-targeted modulation mechanism leads to high potency in suppressing tumor growth and vascularization in 2 in vivo models. Intriguingly, it is the autophagy-dependent degradation pathway that plays a crucial role in Q6-induced attenuation of HIF1A expression, rather than the proteasome-dependent pathway, which is normally regarded as the predominant mechanism underlying posttranslational regulation of HIF1A. Inhibition of autophagy, either by short interfering RNA (siRNA) or by chemical inhibitors, blocked Q6-induced HIF1A degradation. Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy. Additionally, silencing of SQSTM1 inhibited Q6-induced HIF1A degradation. These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC. Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

Show MeSH
Related in: MedlinePlus