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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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“In vivo” evidence for HIF-2α and SERPINB3 (SB3) correlation in human HCCPanels A-C. IHC analysis on serial sections of human HCC from cirrhotic patients carrying HCC showing different patterns of expression of SERPINB3 and HIF-2α: pattern 1, negative for either SERPINB3 or HIF-2α (panel A); pattern 2, weakly positive for SERPINB3 and positive, mainly cytoplasmic, staining for HIF-2α (panel B); pattern 3, intense positive stain for both SERPINB3 and HIF-2α, the latter showing mainly nuclear staining (panel C). Original magnification as indicated. Panel D. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in HCC specimens obtained from a cohort of 67 patients. Statistical analysis was performed using Spearman correlation. Panel E. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in relation to low expression (SERPINB3 low; N=52) or high expression (SERPINB3 high; N=15) of SERPINB3 mRNA. The y axis represents the relative mRNA amounts of the normalised gene. Central bars represent mean and external bars represent s.e.m. The analysis was performed with Mann-Whitney test.
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Figure 7: “In vivo” evidence for HIF-2α and SERPINB3 (SB3) correlation in human HCCPanels A-C. IHC analysis on serial sections of human HCC from cirrhotic patients carrying HCC showing different patterns of expression of SERPINB3 and HIF-2α: pattern 1, negative for either SERPINB3 or HIF-2α (panel A); pattern 2, weakly positive for SERPINB3 and positive, mainly cytoplasmic, staining for HIF-2α (panel B); pattern 3, intense positive stain for both SERPINB3 and HIF-2α, the latter showing mainly nuclear staining (panel C). Original magnification as indicated. Panel D. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in HCC specimens obtained from a cohort of 67 patients. Statistical analysis was performed using Spearman correlation. Panel E. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in relation to low expression (SERPINB3 low; N=52) or high expression (SERPINB3 high; N=15) of SERPINB3 mRNA. The y axis represents the relative mRNA amounts of the normalised gene. Central bars represent mean and external bars represent s.e.m. The analysis was performed with Mann-Whitney test.

Mentions: In a first series of experiments, designed to investigate a possible relationship between HIF-2α and SERPINB3 expression directly on HCC tissue, we performed immuno-histochemistry (IHC) on paraffin-included human liver specimens (n=18). These analysis offered an overall morphological scenario in which human samples were characterized by either i) negativity for SERPINB3 and negativity or low/moderately positivity for HIF-2α (group 1: 9 patients out of 18, Figure 7A), ii) low/moderate SERPINB3 positivity associated with more intense staining for HIF-2α (group 2: 5 patients out of 18, Figure 7B), iii) intense positive staining for both SERPINB3 and for nuclear HIF-2α (group 3: 4 out of 18 patients, Figure 7C). Along these lines, in a recent human study [30] some of us reported that high levels of SERPINB3 transcripts were detectable in 15 out of 67 human HCC specimens (approx. 22%) and that high expression levels of SERPINB3 were significantly associated with early tumor recurrence. In order to further verify the significance of the correlation between HIF-2α and SERPINB3 we performed Q-PCR analysis of both HIF-2α and SERPINB3 transcripts in human HCC specimens obtained from the same cohort of 67 well characterized patients recently described [30]. Statistical analysis extended to all available specimens revealed the existence of a positive correlation (Spearman r coefficient, p < 0.01) between HIF-2α and SERPINB3 transcript levels (Figure 7D), supporting the notion that also in vivo HIF-2α may play a relevant role in increasing SERPINB3 expression. In agreement with these findings, the highest levels of HIF-2α transcription were found in the sub-class of patients with high (> median value) SERPINB3 expression (Figure 7E) and previously described to carry the higher rate of early tumor recurrence [30].


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)

“In vivo” evidence for HIF-2α and SERPINB3 (SB3) correlation in human HCCPanels A-C. IHC analysis on serial sections of human HCC from cirrhotic patients carrying HCC showing different patterns of expression of SERPINB3 and HIF-2α: pattern 1, negative for either SERPINB3 or HIF-2α (panel A); pattern 2, weakly positive for SERPINB3 and positive, mainly cytoplasmic, staining for HIF-2α (panel B); pattern 3, intense positive stain for both SERPINB3 and HIF-2α, the latter showing mainly nuclear staining (panel C). Original magnification as indicated. Panel D. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in HCC specimens obtained from a cohort of 67 patients. Statistical analysis was performed using Spearman correlation. Panel E. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in relation to low expression (SERPINB3 low; N=52) or high expression (SERPINB3 high; N=15) of SERPINB3 mRNA. The y axis represents the relative mRNA amounts of the normalised gene. Central bars represent mean and external bars represent s.e.m. The analysis was performed with Mann-Whitney test.
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Related In: Results  -  Collection

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Figure 7: “In vivo” evidence for HIF-2α and SERPINB3 (SB3) correlation in human HCCPanels A-C. IHC analysis on serial sections of human HCC from cirrhotic patients carrying HCC showing different patterns of expression of SERPINB3 and HIF-2α: pattern 1, negative for either SERPINB3 or HIF-2α (panel A); pattern 2, weakly positive for SERPINB3 and positive, mainly cytoplasmic, staining for HIF-2α (panel B); pattern 3, intense positive stain for both SERPINB3 and HIF-2α, the latter showing mainly nuclear staining (panel C). Original magnification as indicated. Panel D. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in HCC specimens obtained from a cohort of 67 patients. Statistical analysis was performed using Spearman correlation. Panel E. SERPINB3 and HIF-2α mRNA were analysed by Q-PCR in relation to low expression (SERPINB3 low; N=52) or high expression (SERPINB3 high; N=15) of SERPINB3 mRNA. The y axis represents the relative mRNA amounts of the normalised gene. Central bars represent mean and external bars represent s.e.m. The analysis was performed with Mann-Whitney test.
Mentions: In a first series of experiments, designed to investigate a possible relationship between HIF-2α and SERPINB3 expression directly on HCC tissue, we performed immuno-histochemistry (IHC) on paraffin-included human liver specimens (n=18). These analysis offered an overall morphological scenario in which human samples were characterized by either i) negativity for SERPINB3 and negativity or low/moderately positivity for HIF-2α (group 1: 9 patients out of 18, Figure 7A), ii) low/moderate SERPINB3 positivity associated with more intense staining for HIF-2α (group 2: 5 patients out of 18, Figure 7B), iii) intense positive staining for both SERPINB3 and for nuclear HIF-2α (group 3: 4 out of 18 patients, Figure 7C). Along these lines, in a recent human study [30] some of us reported that high levels of SERPINB3 transcripts were detectable in 15 out of 67 human HCC specimens (approx. 22%) and that high expression levels of SERPINB3 were significantly associated with early tumor recurrence. In order to further verify the significance of the correlation between HIF-2α and SERPINB3 we performed Q-PCR analysis of both HIF-2α and SERPINB3 transcripts in human HCC specimens obtained from the same cohort of 67 well characterized patients recently described [30]. Statistical analysis extended to all available specimens revealed the existence of a positive correlation (Spearman r coefficient, p < 0.01) between HIF-2α and SERPINB3 transcript levels (Figure 7D), supporting the notion that also in vivo HIF-2α may play a relevant role in increasing SERPINB3 expression. In agreement with these findings, the highest levels of HIF-2α transcription were found in the sub-class of patients with high (> median value) SERPINB3 expression (Figure 7E) and previously described to carry the higher rate of early tumor recurrence [30].

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