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Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

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HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients.A. HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D.Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients (C,D left panels) and nonresponding patients (C,D, right panels). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples (n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p < 0.05. Fibrosis scores were determined by histopathology and using the Fibroscan method [22]. The underlying data for panels in this figure can be found in S1 Data.
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pbio.1002421.g001: HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients.A. HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D.Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients (C,D left panels) and nonresponding patients (C,D, right panels). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples (n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p < 0.05. Fibrosis scores were determined by histopathology and using the Fibroscan method [22]. The underlying data for panels in this figure can be found in S1 Data.

Mentions: HCV, along with several other liver conditions, is known to trigger hepatic inflammation. To establish a connection between the expression of Netrin-1 (Uniprot Acc. # O95631) and viral infection of the liver, we first measured the level of Netrin-1 mRNA (GenBank Acc. # NM_004822) in 418 liver biopsies, taken either from virus-free patients (165 samples), from HCV-infected patients (223 samples), or from HBV-infected patients (30 samples) (S1 Table). The latter were included as a positive control for chronic viral infection of the liver, and tissue biopsies revealed an 11-fold increase in the level of Netrin-1 mRNA compared to uninfected controls (Fig 1A). Interestingly, the HCV-infected samples displayed a further 2-fold increase in Netrin-1 transcripts versus HBV+ samples, totaling a 23-fold increase in Netrin-1 mRNA levels compared to the uninfected controls. Moreover, a positive correlation was found between the levels of Netrin-1 mRNA and HCV RNA in those liver biopsies (Fig 1B). Similarly, HCV RNA and Netrin-1 mRNA levels were measured in patients before and after first-time treatment with interferon and ribavirin, two antiviral compounds, in biopsies obtained from 18 HCV+/HBV- patients. Of these, 16 showed a partial treatment response (i.e., presented a decrease in viral load; Fig 1C, left panel), accompanied in all but one with a clear decrease in Netrin-1 mRNA levels (Fig 1D, left panel). The two patients who failed to respond to treatment (Fig 1C, right panel) showed stable or increased levels in Netrin-1 mRNA, which paralleled their stable or increased HCV RNA load (Fig 1D, right panel). These data support the HCV-dependent status of Netrin-1 upregulation in HCV-positive patients.


Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

Plissonnier ML, Lahlali T, Michelet M, Lebossé F, Cottarel J, Beer M, Neveu G, Durantel D, Bartosch B, Accardi R, Clément S, Paradisi A, Devouassoux-Shisheboran M, Einav S, Mehlen P, Zoulim F, Parent R - PLoS Biol. (2016)

HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients.A. HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D.Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients (C,D left panels) and nonresponding patients (C,D, right panels). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples (n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p < 0.05. Fibrosis scores were determined by histopathology and using the Fibroscan method [22]. The underlying data for panels in this figure can be found in S1 Data.
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Related In: Results  -  Collection

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Show All Figures
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pbio.1002421.g001: HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients.A. HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D.Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients (C,D left panels) and nonresponding patients (C,D, right panels). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples (n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p < 0.05. Fibrosis scores were determined by histopathology and using the Fibroscan method [22]. The underlying data for panels in this figure can be found in S1 Data.
Mentions: HCV, along with several other liver conditions, is known to trigger hepatic inflammation. To establish a connection between the expression of Netrin-1 (Uniprot Acc. # O95631) and viral infection of the liver, we first measured the level of Netrin-1 mRNA (GenBank Acc. # NM_004822) in 418 liver biopsies, taken either from virus-free patients (165 samples), from HCV-infected patients (223 samples), or from HBV-infected patients (30 samples) (S1 Table). The latter were included as a positive control for chronic viral infection of the liver, and tissue biopsies revealed an 11-fold increase in the level of Netrin-1 mRNA compared to uninfected controls (Fig 1A). Interestingly, the HCV-infected samples displayed a further 2-fold increase in Netrin-1 transcripts versus HBV+ samples, totaling a 23-fold increase in Netrin-1 mRNA levels compared to the uninfected controls. Moreover, a positive correlation was found between the levels of Netrin-1 mRNA and HCV RNA in those liver biopsies (Fig 1B). Similarly, HCV RNA and Netrin-1 mRNA levels were measured in patients before and after first-time treatment with interferon and ribavirin, two antiviral compounds, in biopsies obtained from 18 HCV+/HBV- patients. Of these, 16 showed a partial treatment response (i.e., presented a decrease in viral load; Fig 1C, left panel), accompanied in all but one with a clear decrease in Netrin-1 mRNA levels (Fig 1D, left panel). The two patients who failed to respond to treatment (Fig 1C, right panel) showed stable or increased levels in Netrin-1 mRNA, which paralleled their stable or increased HCV RNA load (Fig 1D, right panel). These data support the HCV-dependent status of Netrin-1 upregulation in HCV-positive patients.

Bottom Line: Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues.Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression.Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells.

View Article: PubMed Central - PubMed

Affiliation: Pathogenesis of Hepatitis B and C - Equipe labellisée LabEx DEVweCAN, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, F-69003 Lyon, France, Université de Lyon, F-69003 Lyon, Université Lyon 1, ISPB, Lyon, F-69622, France, CNRS UMR5286, F-69083 Lyon, France, Centre Léon Bérard, F-69008 Lyon, France.

ABSTRACT
Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

Show MeSH
Related in: MedlinePlus