Mechanism Of Selenium - Binding Protein 1 (SBP1) On Invasion And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common and aggressive human malignancies worldwide, and the third most common cause of cancer-related mortality. Although major improvements have been achieved in early diagnosis, surgical treatment and liver transplantation, only30%～40%HCC patients were suitable for surgery when diagnosis. Even with curative surgical treatment,60%～70%of the patients would suffer from recurrence and metastasis in the next five years. The5-year overall survival of patients with HCC is still about30%. Therefore, it is urgent to explore the mechanism of metastasis and recurrence of HCC, and find effective way to improve the prognosis of patients with HCC.Selenium is an essential trace element with cancer-preventing activities that have been shown in many epidemiologic studies. The cellular biochemistry of selenium is a complex system that involves the expression of a wide range of selenium-containing proteins. Of these proteins, selenium-binding protein1(SBP1) was found to be the most possible mediator of selenium’s anticancer functions. The previous studies have established a solid connection between SBP1and cancer. Decreased SBP1was found in a vast number of human cancers and was closely related with poor prognosis. However, the molecular mechanism underlying the tumor suppressive functions of SBP1remains unclear.Glutathione peroxidase (GPX1) is also an important selenium-containing protein, which can scavenge organic hydroperoxides and protect cells from reactive oxygen species (ROS). On the other hand, GPX1was also reported to protect cancer cells from oxidative stress and anticancer agents. Previous studies have already showed that these2distinct selenium-containing proteins (GPX1and SBP1) can form a physical association that facilitates their interactions. But their possible roles in cancer development are still unknown. SBP1is also known to have a hypoxia response element in its promoter region and to be a target gene of hypoxia-inducible factor-la (HIF-la), which is a fundamental mediator of cellular adaptation to microenvironmental stress, especially oxidative stress and plays a complex role in cancer biology. The relationship between GPX1and oxidative stress and the multifunctional role of HIF-la in cancer biology may be associated with the anti-tumor activity of SBP1, and requires our further investigation.In this study, we compared the expression of SBP1in HCC cell lines with different metastatic potentials, explored the role of SBP1in cancer cell migration, invasion, proliferation and apoptosis, and investigated the possible mechanism of its anti-tumor activity. At last, nineteen fresh tumor tissues and323parafin-embedded samples were used to validate our in vitro findings and to detect the prognostic significance of SBP1, respectively. Part OneSBP1expression in HCC cell lines and its anti-tumor effectsThe aim of this part is to investigate the expression of SBP1in normal liver cells and HCC cell lines, and further validate SBP1’s anti-tumor effect using molecular and cell biology assays.We chose normal hepatic cell line L-02and HCC cell lines Hep3B, HepG2, Huh7, SMMC7721, PLC/PRF/5, MHCC97-L, MHCC97-H, HCCLM3and HCCLM6using Western blot to validate protein expression of SBP1. siRNA were used to knock out the expression of SBP1in SMMC7721and further molecular cell biology assays were taken in SBP1-silenced and control groups to investigate the role of SBP1in cancer biology.As a result, SBP1was highly expressed in normal liver cells while barely detected in HCC cell lines with the exception of SMMC7721. Inhibition of SBP1effectively increased cell motility, promoted cell proliferation and inhibited apoptosis only under hydrogen peroxide treatment.Taken together, these results showed that SBP1was highly expressed in normal liver cells while greatly decreased in most of the HCC cell lines. Inhibition of SBP1could help cancer cells maintain their proliferation ability and escape apoptosis under hydrogen peroxide treatment. These findings suggested that the anti-tumor activity of SBP1might be related with the regulation of reactive oxygen species (ROS). Part TwoPossible mechanisms underlying SBP1’s anti-tumor activity and the relationship among SBP1, GPX1and HIF-laThe aim of this part is to investigate the possible mechanisms of SBP1inhibiting proliferation and inducing apoptosis under hydrogen peroxide treatment, and further illustrate the relationship among SBP1, GPX1and HIF-la.First, we examined the expression level of SBP1, GPX1and HIF-la in SBP1-silenced and control groups under different treatment; GPX1activities in different groups were also measured. The interaction of SBP1and GPX1under different treatment was studied by Immunofluorescence.Results showed that the expression of HIF-la and SBP1in the control group could be increased by hydrogen peroxide treatment, while the same treatment could not increase the expression of HIF-la in SBP1-silenced group. The expression of GPX1was not associated with either SBP1or HIF-la; however, compared to the control group, the GPX1activities were significantly increased in SBP1-silenced group (P<0.001). Immunofluorescence results indicated that SBP1and GPX1could form nuclear bodies and co-localized under oxidative stress.As a conclusion, ROS could elevate the expression of HIF-la, and simultaneously increase the expression of the HIF-1α targeted gene SBP1. However, the silencing of SBP1expression by siRNA could also counter inhibit the expression of HIF-la. On the other hand, although the expression of GPX1was not associated with SBP1, SBP1could greatly inhibit the activity of GPX1through molecular interactions. SBP1might exert its anti-tumor activity through the modulation of the tumor redox microenvironment. Part ThreeSBP1expression in HCC tissues and corresponding clinical significanceThe aim is to explore the expression of SBP1in HCC tissues, further validate the inhibition of GPX1activity by SBP1in vivo, and analyze the relationship between SBP1expression and clinical pathology, invasiveness and prognosis of HCC.Patients with HCC (n=342) who underwent surgical treatment at the Zhongshan Hospital were enrolled in this study. SBP1expressions and GPX1activities in19freshly isolated HCC samples were validated and analyzed. To analyze the relationship between SBP1expression and clinical pathology, invasiveness and prognosis of HCC, we used TMA technology and immunochemistry to dectect SBP1expression in a group of323HCC patients, and then made correlation analysis between SBP1with other clinical-pathological characters and survival rates. We also compared SBP1expression in predicting the reccurence of HCC after curative resections, and evaluated the significance of SBP1as a new predictive marker for the prognosis of HCC.As a result, samples with low expression of SBP1had relatively high GPX1activities while samples with high expression of SBP1had limited GPX1activities. Overall, samples with vascular invasion had a lower SBP1expression and relatively higher GPX1activity. Based on TMA results, negative SBP1expression in tumor tissues was significantly correlated with patient age (P=0.045), AFP (P<0.001), tumor size (P=0.005), tumor number (P=0.019), tumor encapsulation (P=0.034), vascular invasion (P<0.001) and recurrence (P<0.001). Both univariate and multivariate analyses showed that SBP1expression in tumor cells was an independent risk factor for both overall survival (P<0.001) and recurrence (P<0.001). Based on Kaplan-Meier survival curves, SBP1was an independent risk factor for overall survival and disease recurrence, patients with lower SBP1expression experienced shorter overall survival periods and higher rates of disease recurrence (P<0.001). Further analyses indicated that the predictive power of SBP1was more significant for patients beyond the Milan criteria than for patients within the Milan criteria. Taken together, Decreased expression of SBP1could promote tumor invasiveness by increasing GPX1activity and diminishing HIF-la expression in HCC;SBP1could be a novel biomarker for predicting prognosis and guiding personalized therapeutic strategies, especially in patients with advanced HCC. Conclusions1.SBP1was highly expressed in normal liver cells while greatly decreased in most of the HCC cell lines.2. Inhibition of SBP1could help cancer cells maintain their proliferation ability and escape apoptosis under oxidative stress.3. SBP1could greatly inhibit the activity of GPX1through molecular interactions. SBP1might exert its anti-tumor activity through the modulation of the tumor redox microenvironment.4. SBP1could be a novel biomarker for predicting prognosis and guiding personalized therapeutic strategies, especially in patients with advanced HCC.Novelty1.Our findings indicate that SBP1may act as a pro-oxidant rather than anti-oxidant through the interaction with GPX1and HIF-1α in tumor biology.2. To our knowledge, this is the first report to demonstrate the prognostic value of SBPl in large cohorts of patients with HCC. SBP1could be a novel biomarker for guiding personalized therapeutic strategies, especially in patients with advanced HCC.Potential Application1.SBP1could be a novel biomarker for predicting prognosis and guiding personalized therapeutic strategies, especially in patients with advanced HCC.2. Determination of SBP1expression is especially useful for personalized therapeutic strategies and decisions regarding individuals beyond Milan criteria who could benefit from more aggressive treatment, such as chemotherapy or liver transplantation.3. The use of anti-oxidants such as glutathione in HCC patients especially patients with advanced-stage cancer should be completed with caution.