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Hypoxia up-regulates SERPINB3 through HIF-2α in human liver cancer cells.

Cannito S, Turato C, Paternostro C, Biasiolo A, Colombatto S, Cambieri I, Quarta S, Novo E, Morello E, Villano G, Fasolato S, Musso T, David E, Tusa I, Rovida E, Autelli R, Smedile A, Cillo U, Pontisso P, Parola M - Oncotarget (2015)

Bottom Line: SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence.HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS.Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Biological Sciences, Unit of Experimental Medicine and Interuniversity Center for Liver Pathophysiology, University of Torino, Italy.

ABSTRACT
SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

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Hypoxia-dependent SERPINB3 (SB3) up-regulation involves HIF-2αPanel A. WB analysis of HIF-2α protein levels performed on total extracts of HepG2 cells (equal loading monitored by re-blotting membranes for β-actin) that were maintained in normoxic conditions (control, C) or exposed to hypoxia (HYP) for the indicated times. Panel B. WB analysis of HIF-2α expression in HepG2 cells not transfected, transfected with non-silencing control (NsC) or transfected with HIF-2α siRNA that were maintained in normoxic conditions (control, C) or exposed to hypoxia for 24 hrs. Panels C, D. Analysis of SERPINB3 transcripts by RT-PCR (panel C) or Q-PCR (panel D) in HepG2 cells treated as in (B) or as indicated. Panel E. ELISA analysis of intracellular SERPINB3 protein levels (ng/ml) in HepG2 cells treated as mentioned in (B-D). Data are expressed as means ± SEM of three independent experiments (* p<0.01 vs control values; # p<0.01 vs related treatment condition).
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Figure 3: Hypoxia-dependent SERPINB3 (SB3) up-regulation involves HIF-2αPanel A. WB analysis of HIF-2α protein levels performed on total extracts of HepG2 cells (equal loading monitored by re-blotting membranes for β-actin) that were maintained in normoxic conditions (control, C) or exposed to hypoxia (HYP) for the indicated times. Panel B. WB analysis of HIF-2α expression in HepG2 cells not transfected, transfected with non-silencing control (NsC) or transfected with HIF-2α siRNA that were maintained in normoxic conditions (control, C) or exposed to hypoxia for 24 hrs. Panels C, D. Analysis of SERPINB3 transcripts by RT-PCR (panel C) or Q-PCR (panel D) in HepG2 cells treated as in (B) or as indicated. Panel E. ELISA analysis of intracellular SERPINB3 protein levels (ng/ml) in HepG2 cells treated as mentioned in (B-D). Data are expressed as means ± SEM of three independent experiments (* p<0.01 vs control values; # p<0.01 vs related treatment condition).

Mentions: HIF-1α protein, a master regulator of hypoxia-induced responses, is increased in HepG2 cells exposed to hypoxia (Figure 2A) and consensus core HRE (hypoxia-responsive element) RCGTG sequences are present at the SERPINB3 promoter (Supplemental Figure 1) [32]. However, effective silencing of HIF-1α (evaluated as protein levels, Figure 2B) in HepG2 cells exposed to hypoxia by using a specific siRNA [10] did not prevent hypoxia-induced increase of SERPINB3 mRNA levels after 24 hrs of hypoxia (i.e., the time point with SERPINB3 peak transcription), as verified by RT-PCR (Figure 2C) and quantified by Q-PCR (Figure 2D). Since SERPINB3 promoter has, close to HRE sequences, one CGGA and four GGAC sequences (Supplemental Figure 1), which are ETS sites proposed to increase HIF-2α (not HIF-1α) binding affinity, [33,34] we then analyzed the possible involvement of HIF-2α. A time course analysis showed that hypoxia induced an early (30-60 min) and sustained (up to 24 hrs) increase of HIF-2α in HepG2 cells (Figure 3A). Moreover, following efficient silencing of HIF-2α with siRNA (Figure 3B), SERPINB3 transcripts, as verified by RT-PCR (Figure 3C) and quantified by Q-PCR (Figure 3D), as well as intracellular SERPINB3 protein levels (Figure 3E) were almost completely abolished in HepG2 cells exposed to hypoxia (24 hrs) as compared with related controls.


Hypoxia up-regulates SERPINB3 through HIF-2α in human liver cancer cells.

Cannito S, Turato C, Paternostro C, Biasiolo A, Colombatto S, Cambieri I, Quarta S, Novo E, Morello E, Villano G, Fasolato S, Musso T, David E, Tusa I, Rovida E, Autelli R, Smedile A, Cillo U, Pontisso P, Parola M - Oncotarget (2015)

Hypoxia-dependent SERPINB3 (SB3) up-regulation involves HIF-2αPanel A. WB analysis of HIF-2α protein levels performed on total extracts of HepG2 cells (equal loading monitored by re-blotting membranes for β-actin) that were maintained in normoxic conditions (control, C) or exposed to hypoxia (HYP) for the indicated times. Panel B. WB analysis of HIF-2α expression in HepG2 cells not transfected, transfected with non-silencing control (NsC) or transfected with HIF-2α siRNA that were maintained in normoxic conditions (control, C) or exposed to hypoxia for 24 hrs. Panels C, D. Analysis of SERPINB3 transcripts by RT-PCR (panel C) or Q-PCR (panel D) in HepG2 cells treated as in (B) or as indicated. Panel E. ELISA analysis of intracellular SERPINB3 protein levels (ng/ml) in HepG2 cells treated as mentioned in (B-D). Data are expressed as means ± SEM of three independent experiments (* p<0.01 vs control values; # p<0.01 vs related treatment condition).
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Figure 3: Hypoxia-dependent SERPINB3 (SB3) up-regulation involves HIF-2αPanel A. WB analysis of HIF-2α protein levels performed on total extracts of HepG2 cells (equal loading monitored by re-blotting membranes for β-actin) that were maintained in normoxic conditions (control, C) or exposed to hypoxia (HYP) for the indicated times. Panel B. WB analysis of HIF-2α expression in HepG2 cells not transfected, transfected with non-silencing control (NsC) or transfected with HIF-2α siRNA that were maintained in normoxic conditions (control, C) or exposed to hypoxia for 24 hrs. Panels C, D. Analysis of SERPINB3 transcripts by RT-PCR (panel C) or Q-PCR (panel D) in HepG2 cells treated as in (B) or as indicated. Panel E. ELISA analysis of intracellular SERPINB3 protein levels (ng/ml) in HepG2 cells treated as mentioned in (B-D). Data are expressed as means ± SEM of three independent experiments (* p<0.01 vs control values; # p<0.01 vs related treatment condition).
Mentions: HIF-1α protein, a master regulator of hypoxia-induced responses, is increased in HepG2 cells exposed to hypoxia (Figure 2A) and consensus core HRE (hypoxia-responsive element) RCGTG sequences are present at the SERPINB3 promoter (Supplemental Figure 1) [32]. However, effective silencing of HIF-1α (evaluated as protein levels, Figure 2B) in HepG2 cells exposed to hypoxia by using a specific siRNA [10] did not prevent hypoxia-induced increase of SERPINB3 mRNA levels after 24 hrs of hypoxia (i.e., the time point with SERPINB3 peak transcription), as verified by RT-PCR (Figure 2C) and quantified by Q-PCR (Figure 2D). Since SERPINB3 promoter has, close to HRE sequences, one CGGA and four GGAC sequences (Supplemental Figure 1), which are ETS sites proposed to increase HIF-2α (not HIF-1α) binding affinity, [33,34] we then analyzed the possible involvement of HIF-2α. A time course analysis showed that hypoxia induced an early (30-60 min) and sustained (up to 24 hrs) increase of HIF-2α in HepG2 cells (Figure 3A). Moreover, following efficient silencing of HIF-2α with siRNA (Figure 3B), SERPINB3 transcripts, as verified by RT-PCR (Figure 3C) and quantified by Q-PCR (Figure 3D), as well as intracellular SERPINB3 protein levels (Figure 3E) were almost completely abolished in HepG2 cells exposed to hypoxia (24 hrs) as compared with related controls.

Bottom Line: SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence.HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS.Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical and Biological Sciences, Unit of Experimental Medicine and Interuniversity Center for Liver Pathophysiology, University of Torino, Italy.

ABSTRACT
SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells. Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively. Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

Show MeSH
Related in: MedlinePlus