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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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Hypoxic conditions up-regulate SERPINB3 (SB3) expressionPanels A,B. Time-dependent analysis of SERPINB3 transcripts by quantitative real-time PCR (Q-PCR) in HepG2 (panel A) or Huh7 (panel B) cells not exposed (control, C) or exposed to hypoxic conditions (HYP) for the indicated times (*p< 0.05 vs control values of SB3; **p< 0.01 vs control values of SB3). Panels C,D. Time-dependent analysis of SERPINB3 protein levels (ng/ml) by ELISA in cellular extracts (white columns) or culture medium (black columns) of HepG2 (panel C) or Huh7 (panel D) cells not exposed (control, C) or exposed to hypoxia (HYP) for the indicated times. Data are expressed as means ± SEM of three independent experiments (*p< 0.01 vs control values of SB3 in cellular extracts; #p< 0.01 vs control values of SB3 release in culture medium).
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Figure 1: Hypoxic conditions up-regulate SERPINB3 (SB3) expressionPanels A,B. Time-dependent analysis of SERPINB3 transcripts by quantitative real-time PCR (Q-PCR) in HepG2 (panel A) or Huh7 (panel B) cells not exposed (control, C) or exposed to hypoxic conditions (HYP) for the indicated times (*p< 0.05 vs control values of SB3; **p< 0.01 vs control values of SB3). Panels C,D. Time-dependent analysis of SERPINB3 protein levels (ng/ml) by ELISA in cellular extracts (white columns) or culture medium (black columns) of HepG2 (panel C) or Huh7 (panel D) cells not exposed (control, C) or exposed to hypoxia (HYP) for the indicated times. Data are expressed as means ± SEM of three independent experiments (*p< 0.01 vs control values of SB3 in cellular extracts; #p< 0.01 vs control values of SB3 release in culture medium).

Mentions: In order to identify a possible link between hypoxia and SERPINB3 expression we performed immuno-histochemistry analyses on serial sections obtained from a series of human HCC specimens (n=18) developed in cirrhotic livers (HCV etiology, G1 and G2 grading) and positive for SERPINB3. This preliminary analysis, showed that SERPINB3 (SB3) immuno-positivity was detectable in the same cells and areas positive for either HIF-1α and VEGF, supporting the hypothesis of a correlation between hypoxia and SERPINB3 expression (Supplemental Figure 2A,B). We therefore started to investigate the link between hypoxia and SERPINB3 expression by performing a first series of experiments in which two human liver cancer cell lines (HepG2 and Huh7 cells) were initially exposed to moderate hypoxia (3% O2) for up to 96 hrs. Under these experimental conditions HepG2 and Huh7 cells showed no significant signs of cell death, either necrotic or apoptotic, as already previously reported [10]. Exposure to hypoxia (3% O2) resulted in a significant up-regulation of SERPINB3 mRNA transcription, as quantified by Q-PCR (Figure 1A,B), that was apparently earlier in HepG2 cells (6 hr) than in Huh7. In HepG2, hypoxia-induced SERPINB3 transcription was maximal at 24 hrs and declined at later time points (Figure 1A). In preliminary experiments exposure of HepG2 to more severe conditions of hypoxia (0.1% O2) still resulted in early up-regulation of SERPINB3 transcripts (1 and 3 hrs, Supplemental Figure 3) but also in subsequent induction of significant cell death (data not shown). Whether the synthesis of the protein is concerned, as determined by ELISA, exposure of both HepG2 and Huh7 cells to hypoxia (3% O2) similarly induced (Figure 1C,D): a) a marked increase in intracellular SERPINB3 protein levels, that became detectable after 24 hrs of incubation, lasted until 72 hrs to decline at 96 hrs time point; b) a marked and delayed release of SERPINB3 in the extracellular medium that became detectable after 72 hrs (HepG2 cells) or 48 hrs (Huh7 cells). Up-regulation of SERPINB3 mRNA levels and protein synthesis as well as the delayed release of SERPINB3 protein in the extracellular medium was also detected in HT-29 human colon cancer cells exposed to identical conditions of hypoxia (Supplemental Figure 4A,B), suggesting that hypoxia-dependent up-regulation of SERPINB3 may be not limited to hepatic cancer cells. On the other hand, further experiments showed that hypoxia-induced up-regulation of SERPINB3 is a selective response since no significant change in transcript levels for SERPINB4 (i.e., a serine-protease inhibitor known to share a high degree of homology with SERPINB3 [21,23-26]) was detected in either HepG2 or HuH7 cells exposed to hypoxic conditions (Suppl. Figure 5A,B).


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)

Hypoxic conditions up-regulate SERPINB3 (SB3) expressionPanels A,B. Time-dependent analysis of SERPINB3 transcripts by quantitative real-time PCR (Q-PCR) in HepG2 (panel A) or Huh7 (panel B) cells not exposed (control, C) or exposed to hypoxic conditions (HYP) for the indicated times (*p< 0.05 vs control values of SB3; **p< 0.01 vs control values of SB3). Panels C,D. Time-dependent analysis of SERPINB3 protein levels (ng/ml) by ELISA in cellular extracts (white columns) or culture medium (black columns) of HepG2 (panel C) or Huh7 (panel D) cells not exposed (control, C) or exposed to hypoxia (HYP) for the indicated times. Data are expressed as means ± SEM of three independent experiments (*p< 0.01 vs control values of SB3 in cellular extracts; #p< 0.01 vs control values of SB3 release in culture medium).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Hypoxic conditions up-regulate SERPINB3 (SB3) expressionPanels A,B. Time-dependent analysis of SERPINB3 transcripts by quantitative real-time PCR (Q-PCR) in HepG2 (panel A) or Huh7 (panel B) cells not exposed (control, C) or exposed to hypoxic conditions (HYP) for the indicated times (*p< 0.05 vs control values of SB3; **p< 0.01 vs control values of SB3). Panels C,D. Time-dependent analysis of SERPINB3 protein levels (ng/ml) by ELISA in cellular extracts (white columns) or culture medium (black columns) of HepG2 (panel C) or Huh7 (panel D) cells not exposed (control, C) or exposed to hypoxia (HYP) for the indicated times. Data are expressed as means ± SEM of three independent experiments (*p< 0.01 vs control values of SB3 in cellular extracts; #p< 0.01 vs control values of SB3 release in culture medium).
Mentions: In order to identify a possible link between hypoxia and SERPINB3 expression we performed immuno-histochemistry analyses on serial sections obtained from a series of human HCC specimens (n=18) developed in cirrhotic livers (HCV etiology, G1 and G2 grading) and positive for SERPINB3. This preliminary analysis, showed that SERPINB3 (SB3) immuno-positivity was detectable in the same cells and areas positive for either HIF-1α and VEGF, supporting the hypothesis of a correlation between hypoxia and SERPINB3 expression (Supplemental Figure 2A,B). We therefore started to investigate the link between hypoxia and SERPINB3 expression by performing a first series of experiments in which two human liver cancer cell lines (HepG2 and Huh7 cells) were initially exposed to moderate hypoxia (3% O2) for up to 96 hrs. Under these experimental conditions HepG2 and Huh7 cells showed no significant signs of cell death, either necrotic or apoptotic, as already previously reported [10]. Exposure to hypoxia (3% O2) resulted in a significant up-regulation of SERPINB3 mRNA transcription, as quantified by Q-PCR (Figure 1A,B), that was apparently earlier in HepG2 cells (6 hr) than in Huh7. In HepG2, hypoxia-induced SERPINB3 transcription was maximal at 24 hrs and declined at later time points (Figure 1A). In preliminary experiments exposure of HepG2 to more severe conditions of hypoxia (0.1% O2) still resulted in early up-regulation of SERPINB3 transcripts (1 and 3 hrs, Supplemental Figure 3) but also in subsequent induction of significant cell death (data not shown). Whether the synthesis of the protein is concerned, as determined by ELISA, exposure of both HepG2 and Huh7 cells to hypoxia (3% O2) similarly induced (Figure 1C,D): a) a marked increase in intracellular SERPINB3 protein levels, that became detectable after 24 hrs of incubation, lasted until 72 hrs to decline at 96 hrs time point; b) a marked and delayed release of SERPINB3 in the extracellular medium that became detectable after 72 hrs (HepG2 cells) or 48 hrs (Huh7 cells). Up-regulation of SERPINB3 mRNA levels and protein synthesis as well as the delayed release of SERPINB3 protein in the extracellular medium was also detected in HT-29 human colon cancer cells exposed to identical conditions of hypoxia (Supplemental Figure 4A,B), suggesting that hypoxia-dependent up-regulation of SERPINB3 may be not limited to hepatic cancer cells. On the other hand, further experiments showed that hypoxia-induced up-regulation of SERPINB3 is a selective response since no significant change in transcript levels for SERPINB4 (i.e., a serine-protease inhibitor known to share a high degree of homology with SERPINB3 [21,23-26]) was detected in either HepG2 or HuH7 cells exposed to hypoxic conditions (Suppl. Figure 5A,B).

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