Hepatitis C virus NS5A drives a PTEN‐PI3K/Akt feedback loop to support cell survival

Decreased levels of phosphatase and tensin homologue (PTEN) are associated with hepatocellular carcinoma (HCC) pathogenesis and poor prognosis in hepatitis C virus (HCV)‐infected HCC patients. The molecular processes governing the reduction in PTEN and outcome of PTEN dysfunction in hepatocytes are poorly understood.


Abstract
Background & Aims: Decreased levels of phosphatase and tensin homologue (PTEN) are associated with hepatocellular carcinoma (HCC) pathogenesis and poor prognosis in hepatitis C virus (HCV)-infected HCC patients. The molecular processes governing the reduction in PTEN and outcome of PTEN dysfunction in hepatocytes are poorly understood. Methods: The levels of proteins and mRNA were assessed by real time PCR and immunoblot. PTEN promoter activity was measured by reporter assay. Signalling pathways were perturbed using siRNAs or pharmacological inhibitors. Results: Here, we report that HCV down-regulates PTEN expression at the transcriptional level by decreasing its promoter activity, mRNA transcription, and protein levels. We further identify NS5A protein as a key determinant of PTEN reduction among HCV proteins. NS5A-mediated down-regulation of PTEN occurs through a cooperation of reactive oxygen species (ROS)-dependent Nuclear Factor-kappa B (NF-jB) and ROS-independent phosphoinositol-3-kinase (PI3K) pathways. Moreover, NS5A protects cells against apoptosis. In addition, we found that down-regulation of PTEN relieves its inhibitory effect on PI3K-Akt pathway and triggers cumulative activation of Akt. This PTEN-PI3K/Akt feedback network mediates the suppression of cell apoptosis caused by NS5A. Conclusions: These data demonstrate that HCV NS5A down-regulates PTEN expression through a cooperation of ROS-dependent and -independent pathways that subsequently drives a PTEN-PI3K/Akt feedback loop to support cell survival. Our findings provide new insights suggesting that NS5A contributes to HCV-related hepatocarcinogenesis.
Chronic infection with hepatitis C virus (HCV) is a major contributor to the high and rising incidence of hepatocellular carcinoma (HCC) worldwide (1). Phosphatase and tensin homologue (PTEN), a tumour suppressor gene located at human chromosome 10q23, is involved in many cellular processes, such as tumourigenesis, viral replication, and glucose and lipid metabolism in the liver (2,3). Previous reports have found that PTEN is down-regulated in HCV-positive cirrhotic HCC patients (4). PTEN expression has been further described as a prognostic factor for the survival of HCV-positive cirrhotic HCC patients (5). Although several studies have reported that PTEN dysfuntion caused by HCV Jc1 or core protein of HCV 3a leads to hepatic steatosis and virus secretion (6,7), the possibility of a direct effect of HCV on PTEN expression in HCV JFH1 or full-length HCV 1b replicon cells have not been investigated.
Phosphatase and tensin homologue is positively and negatively regulated by multiple mechanisms (8). Mitogen-activated protein kinase kinase-4 (MAPK4) has been shown to inhibit PTEN transcription by activating nuclear factor-kappa B (NF-jB) (9). It has also been suggested that reactive oxygen species (ROS) inactivate PTEN, which subsequently leads to the insulin-mediated activation of protein kinase Akt (10). In contrast, active p38 mitogen-activated protein kinase (p38 MAPK) has been suggested to up-regulate PTEN gene expression (11). P53 can also up-regulate PTEN by binding to its promoter (12). The outcomes of PTEN dysfunction are still unclear. Importantly, a decrease in PTEN contributes to the occurrence of HCC through activation of the PI3K/PTEN/Akt pathway (13). PTEN induces apoptosis and cell cycle arrest through PI3K/Akt-dependent andindependent pathways and inhibits cellular proliferation and invasion (14,15). Accumulating experimental evidence suggests that HCV NS5A utilizes multiple mechanisms to inhibit both extrinsic and intrinsic apoptotic stimuli (10-12, 16, 17). We previously reported that the NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates NF-jB (14). NF-jB has been further found to mediate a negative feedback mechanism to suppress TNF-a-induced apoptosis (18).
In this study, we investigated the regulatory mechanism and biological significance of PTEN expression in response to NS5A-induced cell survival in hepatoma cells. NF-jB and PI3K are activated by NS5A through ROS-dependent and -independent mechanisms, which in turn contribute to PTEN reduction. PTEN reduction acts as a feedback regulator of PI3K/Akt and consequently increases Akt activation. Subsequently, PTEN reduction and Akt activation cooperatively support cell survival.

Construction of PTEN promoter reporter plasmid and luciferase assay
The PTEN promoter reporter plasmid was cloned by PCR from human genomic DNA using the following oligonucleotides: sense 5 0 -GATGAGCTCGAGGAGTG GCACCAGTTTG-3 0 and antisense 5 0 -GAGAAGCTTGC TGCTCAGTGT AGAGGGAA-3 0 . The sequences were designed based on the published sequence of PTEN in the GenBank databases (accession number AF067844). The resultant 1098-bp fragment was inserted into the SacI and HindIII sites of the pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA), which contained the human PTEN gene sequences spanning the region between -1467 and -370-bp. Cells were cotransfected with PTEN promoter construct (PTEN-Luc) expressing fireflylu ciferase and construct pRL-TK

Key Points
• NF-jB and PI3K were activated by HCV NS5A through ROS-dependent and -independent manners, respectively, which in turn contributes to PTEN reduction.
• PTEN reduction reacted as a feedback regulator of PI3K/Akt and increased Akt activation.
• PTEN-PI3K/Akt feedback network mediates the suppression of cell apoptosis caused by NS5A.
• Our data demonstrated the regulatory mechanism and biological significance of PTEN expression in response to HCV NS5A-induced cell survival in human hepatoma cells.

ROS measurements
Cells were seeded at a density of 10 4 cells/well (100 ll of DMEM with 10% FBS) in 96-well white plates for 14 h. The cells were washed with phosphate-buffered saline (PBS) and then incubated with 10 lM carboxyl derivative of fluorescein (carboxy-H2DCFDA, Invitrogen, Carlsbad, CA, USA) for 1 h according to the manufacturer's protocol. Then the treated cells were incubated in 10% FBS for 5 min. ROS were assessed by measuring the fluorescence at an excitation of 492 nm and an emission of 525 nm.

Caspase 3/7 activity assay
The relative caspase 3 and 7 activity was measured using Caspase-Glo â 3/7 assay system (Promega, Madison, WI, USA) according to the manufacturer's protocol. Cells were seeded at a density of 10 4 cells/well (100 ll ofDMEM with 10% FBS) in 96-well white plates for 48 h. 100 ll of Caspase-Glo â 3/7 reagent was then added to each well. The plates were mixed for 30 s on an orbital shaker and incubated at room temperature in the dark for 30 min. The luminescence was measured using a FLX800 micro-plate reader (BioTek, Winooski, VT, USA).

Data analysis
Data analysis was performed using a 2-tailed Student's t-test with pooled variance. The data are expressed as the mean ± SD of at least four sample replicates, unless stated otherwise. In the figures, '*' denotes P < 0.05.

HCV down-regulates PTEN expression in cultured human hepatoma cells
To analyse PTEN expression in the presence of HCV in human hepatoma cell lines, we used Huh7-2-3 cells, which were Huh7-derived cells stably harbouring the full-length HCV RNA (genotype 1b). The presence of the HCV 1b replicon led to the reduction in PTEN protein levels detected by Western blotting in Huh7-2-3 cells (Fig. 1A). PTEN mRNA detected by quantitative PCR was down-regulated in Huh7-2-3 cells by 62% (P < 0.05) compared with parental Huh7 cells (Fig. 1B). Furthermore, a PTEN promoter reporter vector was constructed and a luciferase assay was conducted. We found that PTEN promoter activity in Huh7-2-3 cells was significantly lower than that in parental Huh7 cells (down to 38% of parental Huh7 cells, P < 0.05; Fig. 1C). These data suggested that PTEN expression is down-regulated by HCV in human liver cells.
To extend our findings to more than one HCV genotype, PTEN expression was assessed in the human hepatoma cell line Huh7.5.1, in the presence or absence of the HCV genotype 2a infectious clone, JFH1. Western blotting showed a significant decrease in PTEN protein in the presence of infectious clone JFH1 compared with uninfected Huh7.5.1 cells (Fig. 1D). mRNA transcription and PTEN promoter activity were down-regulated by 35% (P < 0.05; Fig. 1B) and 40% (P < 0.05; Fig. 1C) in JFH1-infected cells compared with uninfected Huh7.5.1 cells respectively. Taken together, these data demonstrated that HCV represses PTEN expression at the transcriptional level by decreasing its promoter activity, mRNA transcription and protein levels in vitro.

Identification of NS5A as a key determinant for PTEN reduction among HCV proteins
It has been shown that HCV core and NS5A repress expression of some host proteins (21,22). NS3/4A is a protease that cleaves several host proteins. To evaluate the effect of core, NS3/4A or NS5A on PTEN expression, cells were harvested from Huh7 cells, Huh7-2-3 cells and Huh7 cells following transient transfection with expression constructs for HCV core, NS3/4A or NS5A, all derived from genotype 1b HCV. PTEN mRNA transcription was decreased by 57% in Huh7-2-3 cells, and approximately 42% in Huh7 cells transfected with NS5A (P < 0.05 in both cases; Fig. 2A). HCV core exhibited less inhibitory effect on PTEN mRNA levels than did NS5A (À14% vs À42%), while no significant effect on PTEN mRNA levels was observed following NS3/4A transfection. PTEN protein levels were significantly decreased in Huh7 cells transfected with NS5A compared to Vector control (Fig. 2B). Huh7 cells transfected with core showed slight reductions in PTEN protein expression, while no change in PTEN protein expression was found following NS3/4A transfection (Fig. 2B). PTEN promoter activity was also significantly suppressed by NS5A. (Fig. 2C).
To investigate further whether the NS5A-mediated down-regulation of PTEN was consistent in other human hepatoma cell lines, we assessed PTEN expression in HepG2 cells transfected with NS5A protein at different doses. We found that NS5A at the dose of 2.0 lg downregulated PTEN mRNA transcripts more significantly in HepG2 cells than that in Huh7 cells, decreasing by 73% compared to the corresponding cells transfected with vector alone (P < 0.001; Fig. 2D). NS5A protein exhibited a similar inhibitory effect on PTEN protein in HepG2 cells as that observed with PTEN mRNA expression (Fig. 2D). These data collectively indicate that NS5A protein efficiently represses PTEN expression.

HCV down-regulates PTEN expression through ROS-NF-jB and PI3K pathways
We previously reported that NS5A induces ROS production (14) and enhanced PI3K kinase activity (17,21,23,24). Recent studies have reported that ROS are possible upstream signalling molecules for p38 MAPK, NF-jB and ERK (25). To analyse which pathways are involved in PTEN reduction caused by NS5A, stable NS5A-or Vector-Huh7 cells were incubated with selective inhibitors for the major signalling pathways, including oxidative stress, p38 MAPKs, ERK1/2, PI3K-Akt, and NF-jB. First, we monitored PTEN expression and PTEN promoter activity in stable NS5A-Huh7 cells. We found that the ROS inhibitor DPI, NF-jB activation inhibitor QNZ, and PI3K inhibitor LY294002 each partially rescued PTEN mRNA (P < 0.05, Fig. 3A), protein levels ( Fig. 3A) and PTEN promoter reporter activity (Fig. 3B) in NS5A-Huh7 cells. Interestingly, the Akt inhibitor Akt IV had no effect on NS5A-mediated PTEN reduction ( Fig. 3A and 3B), suggesting that Akt was not involved in PTEN regulation; at least in our model, Akt does not appear to lie upstream of PTEN. Similarly, the ERK1/2 inhibitor PD98059 and p38 MAPK inhibitor SB203580 also had no effect on PTEN reduction ( Fig. 3A and 3B). These data demonstrated that ROS, NF-jB, and PI3K kinases are involved in NS5A-induced PTEN reduction.
Next, we examined ROS production and the interactions of ROS generation with these kinases. NS5A indeed induced ROS production by 1.35 ± 0.13 fold compared with the Vector control (P < 0.05, Fig. 3C). DPI completely abrogated NS5A-induced ROS production. In contrast, QNZ, LY294002, Akt IV, PD98059 and SB203580 had no effect on NS5A-induced ROS production (Fig. 3C), indicating that NF-jB, PI3K, Akt, ERK1/ 2 and p38 MAPK were not involved in NS5A-induced ROS enhancement. Furthermore, Western blotting showed that NS5A induced the phosphorylation of NF-jB and Akt. DPI blocked phosphorylation of NF-jB to levels comparable to those seen with its specific inhibitor QNZ (Fig. 3D), summarized in Fig. 2C, confirming that ROS lies upstream of NF-jB and that NS5A-induced activation of NF-jB was ROS-dependent. Moreover, LY294002 and Akt IV completely blocked Akt phosphorylation. DPI moderately blocked Akt phosphorylation compared with LY294002 or Akt IV (Fig. 3D), combined with Fig. 2C, suggesting that NS5A induced PI3K-Akt activation independent of ROS.
Taken together, these data suggest that HCV NS5A activates NF-jB and PI3K pathways through ROSdependent and -independent mechanisms respectively. These data also indicate that ROS-NF-jB and PI3K pathways mediated NS5A-induced down-regulation of PTEN expression.

NF-jB and PI3K pathways cooperatively down-regulate PTEN expression
To further estimate the specific effect of PI3K and NF-jB pathways on PTEN expression, we performed siRNAs to knock-down these kinases. We found that the down-regulation of PTEN mRNA transcription was partially rescued by PI3K (up to 84%) and NF-jB siR-NAs (up to 75%) compared with the negative control siRNAs (39%, P < 0.05; Fig. 4A) in NS5A-Huh7 cells. In addition, the combination of NF-jB and PI3K siR-NAs cooperatively rescued PTEN mRNA transcription to a higher level than that seen with individual NF-jB or PI3K siRNAs (up to 92-94%, P < 0.05; Fig. 3A). Western blotting confirmed that the expression levels of PI3K and NF-jB protein were knocked down by PI3K and NF-jB siRNAs respectively (Fig. 4B). In addition, PI3K and NF-jB siRNAs also partially rescued PTEN protein levels compared with the negative control siR-NAs in NS5A-Huh7 cells, which confirmed our previous data (Fig. 4B). Taken together, these results demonstrate further that HCV NS5A-induced activation of NF-jB and PI3K cooperatively down-regulated PTEN expression.

NS5A-induced down-regulation of PTEN expression leads to cumulative activation of Akt
We originally found that PI3K negatively mediates NS5A-induced down-regulation of PTEN expression and that Akt was not involved in PTEN reduction. Pre-vious reports have shown that PTEN was identified as a negative regulator of the PI3K-Akt signalling pathway (26).Therefore, we speculated that PTEN reduction leads to the cumulative activation of Akt. Western blotting showed that PTEN expression was specifically knocked down by PTEN siRNAs. The phosphorylation levels of Akt were increased by PTEN siRNAs compared with the negative control siRNAs in NS5A-Huh7 cells, whereas PTEN overexpression abolished NS5A-induced phosphorylation of Akt to a similar extent as that caused by PI3K siRNAs (Fig. 4C), These data indicated that PTEN is indeed antagonistic to PI3K-induced activation of Akt. Indeed, PTEN reduction relieved its inhibition effect on PI3K-Akt pathway, which in turn led to the cumulative activation of Akt. These results also suggested that PTEN-PI3K/Akt acted as part of a positive feedback loop.

HCV NS5A protects cells against apoptosis
Accumulating experimental evidences suggest that NS5A suppresses apoptosis (25,27). A recent report has shown that HCV JFH1 induces apoptosis (28). These results seem to be inconsistent with the inhibitory effect exerted by NS5A on cell apoptosis. Thus, we decided to determine the effect of NS5A on cell apoptosis and investigate this discrepancy between NS5A and JFH1 on cell apoptosis. The JFH1-infected cells used in this experiment were analysed at 4 and 25 days postinfection (dpi). We employed staurosporine to induce apoptosis. We used caspase-3/7 activity, cleaved caspase-3 and PARP protein levels, and Annexin V as apoptotic markers for this study (29). We found that Caspase 3/7 activity in NS5A-Huh7 cells was significantly lower by 57% (P < 0.05) than that in Vector-Huh7 cells (Fig. 5A). Consistently, Huh7-2-3 cells exhibited a similar reduction by 32% (P < 0.05) in Caspase 3/7 activity as that observed in NS5A-Huh7 cells (Fig. 5A). Likewise, the apoptosis effect was reduced in 7701-NS5A cells than 7701 cells (Fig. 5B). Interestingly, we extended the infection days and found that JFH1 cells dpi 25 suppressed Caspase 3/7 activity by 45% compared with the uninfected Huh7.5.1 cells (Fig. 5A), suggesting that chronic JFH1 cells exert a similar inhibitory effect on cell apoptosis as that observed in NS5A-Huh7 cells. Western blotting confirmed that cleaved caspase-3 and PARP were much lower in NS5A-Huh7 cells and JFH1 dpi25 than that in Huh7 cells (Fig. 5A), indicating that NS5A inhibited cell apoptosis and exhibited the consistent effect with chronic HCV infection in hepatoma cells. Annexin V-FITC experiments confirmed that cell apoptosis was reduced in JFH1-infected cells than in Huh7.5.1 cells (Fig. 5B). Taken together, these data demonstrate that HCV NS5A prevented cell apoptosis.

PTEN-PI3K/Akt positive feedback loop driven by HCV NS5A triggers cells survival
Next, we explored whether NS5A protects against cell apoptosis attributed to a PTEN-PI3K/Akt positive feedback loop. We evaluated apoptotic markers in the presence of PTEN overexpression, Akt inhibition and the combination of PTEN overexpression and Akt inhibition. We found that PTEN overexpression increased caspase-3/7 activity in Vector-Huh7 and NS5A-Huh7 cells compared with their corresponding control cells by 2.55-and 1.98-fold respectively (both P < 0.05, Fig. 5C), suggesting that PTEN reduction contributed to cell survival and that PTEN overexpression conversely led to cell apoptosis.
The Akt IV inhibitor also increased caspase-3/7 activity in Vector-Huh7 and NS5A-Huh7 cells by 2.89-and 2.31-fold respectively (both p < 0.05, Fig. 5C). The combination of PTEN overexpression and Akt IV inhibitor additionally increased caspase 3/7 activity compared with individual PTEN overexpression or Akt IV inhibitor in Vector-Huh7 and NS5A-Huh7 cells by 3.79-and 3.58-fold respectively (both P < 0.05, Fig. 5C). Either PTEN overexpression or Akt IV inhibitor increased cleaved caspase-3 and PARP protein expression in Vector-Huh7 and NS5A-Huh7 cells compared to their corresponding controls (Fig. 5C), demonstrating that PTEN reduction and Akt activation cooperatively mediated cell survival in NS5A-Huh7 cells.

Discussion
Hepatitis C Virus infection can lead to liver fibrosis, cirrhosis and HCC through multiple mechanisms. Dysregulated PTEN expression likely plays an important role in the occurrence of HCV-positive cirrhotic HCC. However, direct evidence of PTEN dysfunction in HCVmediated liver diseases is lacking. This study therefore examined the effect of HCV on PTEN expression. Our study reveals that HCV inhibits PTEN expression by decreasing its promoter activity, mRNA transcription and protein expression in hepatocytes. Furthermore, we identified that NS5A plays the critical role in HCV-mediated down-regulation of PTEN.
Previous reports have shown that NF-jB decreases PTEN expression through binding to the PTEN promoter (30). We have identified that NS5A protein induces oxidative stress and activates NF-jB (14). It has been shown that PTEN activity is down-regulated by ROS and NF-jB (9). Suppression of PTEN expression by NF-jB has been further reported to prevent apoptosis (31). Our study provides new evidence that NS5A induces ROS generation and subsequently activates downstream NF-jB. The ROS-NF-jB pathway in turn negatively regulates PTEN expression.
Our study demonstrates that NS5A-induced PI3K-Akt activation is ROS-independent. We further show that only PI3K activation is involved in negative regulation of PTEN expression. Although the mechanism by which PI3K negatively regulates PTEN remains to be determined, a previous study has shown that the PI3K catalytic subunit p110 inactivates PTEN through a pathway involving RhoA and ROCK (8). We also show that PTEN is antagonistic to PI3K-induced activation of Akt. One possibility is that PTEN hydrolyses the 3 0 -phosphate on PIP3 to generate PIP2, thereby directly antagonizing the function of PI3K (32).
Apoptosis plays a key role in the host defence against viral infection and tumourigenesis. Apoptosis of liver cells also plays a significant role in the pathogenesis of HCV. Once a chronic infection becomes established, HCV can utilize various strategies to support cell survival, which in turn is beneficial for viral survival (21).

Liver International (2015)
So we extended the JFH1 infection duration to establish a chronic infection model and found that chronic JFH1 infection indeed repressed cell apoptosis, which is con-sistent with the effect of NS5A protein on cell apoptosis. Here, we provided new evidences to prove our previous speculation. Several reports have suggested that NS5A inhibits apoptosis through multiple mechanisms, including activation of the PI3K-Akt survival pathway (27,33). In contrast, PTEN induces apoptosis (34). Our results revealed that NS5A prevented cell apoptosis and confirmed the anti-apoptotic role of NS5A. Moreover, we demonstrated that NS5A specifically protected cells against apoptosis via the PTEN-PI3K/Akt feedback loop.
We propose a unique model in which NS5A inhibits the expression of the tumour suppressor gene PTEN through the cooperative regulation of ROS-dependent NF-jB and ROS-independent PI3K (Fig. 5D). Using inhibitors for several signalling pathways and siRNAs, our study is the first systematic investigation of the mechanisms by which NS5A directly inhibits the expression of PTEN through distinct ROS-dependent and ROS-independent manners in vitro. NS5A induces ROS generation which in turn stimulates NF-jB phosphorylation. In contrast, NS5A activates the PI3K-Akt pathway independent of ROS. Activated NF-jB and PI3K cooperatively down-regulate PTEN expression. Reduced PTEN acts as a positive feedback regulator to the PI3K-Akt pathway, leading to cumulative activation of Akt. As a result, PTEN-PI3K/Akt positive feedback loop protects cells against apoptosis. Our data provide new evidence for a direct mechanism by which NS5A drives a PTEN-PI3K/Akt feedback loop to support cell survival. These data also provide new insights into the mechanisms by which NS5A could modulate HCV-related HCC by protesting cells against apoptosis.