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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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Selective relationships between HIF-2α and SERPINB3 up-regulationA,B. Analysis of SERPINB3, SERPINB4 and HIF-1α or HIF-2α transcripts by quantitative real-time PCR (Q-PCR) in HepG2 normoxic cells stably transfected in order to over-express either HIF-2α (H/2α, panel A) or HIF-1α (H/1α, panel B) (*p< 0.01 vs control values of transcripts in HepG2 cells transfected with empty vector, H/pCMV6).
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Figure 4: Selective relationships between HIF-2α and SERPINB3 up-regulationA,B. Analysis of SERPINB3, SERPINB4 and HIF-1α or HIF-2α transcripts by quantitative real-time PCR (Q-PCR) in HepG2 normoxic cells stably transfected in order to over-express either HIF-2α (H/2α, panel A) or HIF-1α (H/1α, panel B) (*p< 0.01 vs control values of transcripts in HepG2 cells transfected with empty vector, H/pCMV6).

Mentions: In order to confirm the selective cause-effect relationships between HIF-2α and SERPINB3 up-regulation we performed further experiments in HepG2 cells stably transfected in order to over-express either HIF-1α (H/1α) or HIF-2α (H/2α) (Figure 4A,B). As evaluated by Q-PCR only HepG2 cells genetically manipulated to over-express HIF-2α also showed up-regulation of SERPINB3 mRNA levels, but not of SERPINB4, confirming the selectivity of the relationships.


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)

Selective relationships between HIF-2α and SERPINB3 up-regulationA,B. Analysis of SERPINB3, SERPINB4 and HIF-1α or HIF-2α transcripts by quantitative real-time PCR (Q-PCR) in HepG2 normoxic cells stably transfected in order to over-express either HIF-2α (H/2α, panel A) or HIF-1α (H/1α, panel B) (*p< 0.01 vs control values of transcripts in HepG2 cells transfected with empty vector, H/pCMV6).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385846&req=5

Figure 4: Selective relationships between HIF-2α and SERPINB3 up-regulationA,B. Analysis of SERPINB3, SERPINB4 and HIF-1α or HIF-2α transcripts by quantitative real-time PCR (Q-PCR) in HepG2 normoxic cells stably transfected in order to over-express either HIF-2α (H/2α, panel A) or HIF-1α (H/1α, panel B) (*p< 0.01 vs control values of transcripts in HepG2 cells transfected with empty vector, H/pCMV6).
Mentions: In order to confirm the selective cause-effect relationships between HIF-2α and SERPINB3 up-regulation we performed further experiments in HepG2 cells stably transfected in order to over-express either HIF-1α (H/1α) or HIF-2α (H/2α) (Figure 4A,B). As evaluated by Q-PCR only HepG2 cells genetically manipulated to over-express HIF-2α also showed up-regulation of SERPINB3 mRNA levels, but not of SERPINB4, confirming the selectivity of the relationships.

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