Radiation-inducible PTEN Expression Radiosensitizes Hepatocellular Carcinoma Cells

Objective and Significance:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The worldwide estimated incidence of HCC is more than 620,000 per year. The overall male and female sex radio is about 4:1. In China, primary liver cancer is largely a problem in which 55% of the patients reside ranking second among all malignancies in incidence with about 130,000 patients dying of it. Among all malignant tumors, HCC is one of the lesions with low survival rate, high mortality rate and difficulty for therapy. So it is important to improve the therapeutic effects of HCC. Surgical resection is a preferred method for HCC, but feasible only in 5% to 20% of patients. Most patients are firstly diagnosed for HCC with mid- to later-stage and only 30% of them are suitable for operation. Other therapies for inoperable HCC patients such as TACE, PEI, RFA would be the treatment options. Similarly, these techniques are also not suitable for large tumors.Like surgical resection, radiotherapy is also a kind of local therapy. Recent technological and conceptual developments in the field of radiation therapy, such as image-guided radiation therapy (IGRT) have the potential to improve radiation treatment. However the role of radiotherapy for liver tumors had been limited by the tumor cells'relative radioresistance and the risk of radiation-induced liver disease (RILD). Therefore, the amount of the radiation that can be delivered is limited and dose escalation cannot be used to overcome the resistance to radiation. Hence it is important to investigate other strategies for sensitizing HCC to irradiation while decreasing adverse effect and toxic effect of adjacent normal liver.With the development of molecular biology, people realize that the gene changes may involve in the tumorigensis. Many researches for oncology have focused on gene therapy now, but no satisfying results have been achieved. At the same time, the developments of molecular biology also provide a theoretical support for radiotherapy. Combining gene therapy and radiotherapy protocols has the potential to overcome many of the limitations of adverse tumor biology on cancer treatment. Radiation-mediated gene activation, or "genetic radiotherapy", takes advantage of both the killing effect and the precise targeting potential of ionizing radiation to locally regulate transcription of genes encoding toxic or radiosensitizing proteins. X-irradiation activates the transcription of certain genes, including the early growth response gene Egr-1. These findings led us to the concept that promoters from these genes could be used to drive therapeutic transgenes introduced into irradiated tumor cells. In this strategy, designated genetic radiotherapy, radiation is combined with gene therapy, another local/regional modality, to spatially and temporally control transgene expression in the irradiated field. The choice of therapeutic genes is the key for genetic radiotherapy. So it is important to search new effective therapeutic genes.PTEN is the first tumor suppressor gene functioned with a dual-specificity lipid and protein phosphatase. PTEN is a multifunctional phosphatase with a dual-specificity lipid and protein phosphatase. PTEN with its lipid phosphatase activity can dephosphorylate phosphatidylinositol (3,4,5)-triphosphate (PIP-3) to phosphatidylinositol 4,5-bisphosphate (PIP2) which in turn could regulate Akt function and cellular growth. Consistent with its protein phosphatase activity PTEN may function to regulate cell migration/adhesion by decreasing in FAK phosphorylation and also physically interaction with this kinase. Importantly, hepatic deficiency of PTEN leads to the development of liver tumors. Pten +/- heterozygous mice exhibited neoplasms in multiple organs including liver. Frequent genetic alterations and loss of expression of the PTEN gene have been found in a variety of human cancers. In the HCC, PTEN expression is also downregulated mostly because of the promoter methylation and other epigenetic regulation. Although the enhancement of radiosensitivity by the expression of PTEN to HCC cells has not been previously studied, some reports demonstrated that PTEN gene transfer can sensitize cells to irradiation in breast cancer, prostate cancer, non-small cell lung cancer, colorectal cancer and malignant glioma. Considering frequent genetic alterations and loss of expression of the PTEN gene in HCC, we hypothesized that the loss of PTEN expression may effect the radiosensitivity in HCC and transfecting HCC cells with PTEN would sensitize them to the effects of radiation.Based on the theory of genetic radiotherapy, we combined an Egr-1 radio-responsive enhancer with tumor suppressor gene PTEN and constructed radiation-induced expression vectors to test the radiosensitive effect of PTEN in HCC and explore the mechanism on PTEN enhancement of radiosensitivity.At the same time, considering the lack of specificity in gene therapy especially in vivo studies when transfecting to the surrounding normal cells in a nonspecific manner, we also focused on the use of radiation-inducible promoters to activate gene expression. We used the Egr-1 promoter in our system to ensure that PTEN could be regulated by ionizing radiation in temporally, spatially and in a dose-dependent manner. At the same time we chose PTEN gene as target gene for HCC gene therapy. As we known, there have no studies about the enhancement of radiosensitivity by the expression of PTEN to HCC cells. Considering the important inhibition of PTEN on tumor cells, we think it is feasible to use PTEN for radiosensitization HCC. On the other hand, our study may also provide a theoretical support for the clinical application of genetic radiotherapy.Now the role of radiotherapy for liver tumors had been limited by the tumor-cell relatively radioresistance and the risk of radiation-induced liver disease (RILD). And the amount of the radiation that can be delivered is limited and dose escalation can not solve radiation resistance. Our study will give a potential option for HCC radiotherapy and provide a theoretical support for the clinical application of genetic radiotherapy.Methods:Radiation-induced expression vectors of wild-type PTEN (pEgr-PTEN) or mutant PTEN (loss of lipid phosphatase activity) (pEgr-PTEN-G129E) were constructed respectively. Then we evaluated the responses of Egr-1 promoter to radiation treatment by luciferase report assay. After stably transfected, the expressions of PTEN in difference groups of SMMC-7721 cells were detected by Western blot analysis 24 hours after irradiation. To investigate if PTEN could inhibit cells growth, we carried out a MTT assay using SMMC-7721 cells that had been stably transfected with pEgr-PTEN, pEgr-PTEN-G129E or pEgr-C1 (mock) and treated them with 8 Gy irradiation. The radiosensitivity of cells in pEgr-PTEN, pEgr-PTEN-G129E or pEgr-C1 was assessed by clonogenic survival assays and we used GraphPad Prism 5 software to fit cell survival curve in accordance with standard linear-quadratic (LQ) model and one-hit multi-target model and then get the radiation biology parameters. We next determined the effect of PTEN on cell-cycle phases in SMMC-7721 cells by using Fluorescence Activating Cell Sorter (FACS) analysis. To quantitate apoptosis, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP biotin nick end labeling (TUNEL) assay was done. Immunofluorescence was used to detect gamma-histone H2A (γ-H2AX) foci formation and then to track the repair of DSB produced by radiation at several times following irradiation. To explore the mechanism on PTEN enhancement of radiosensitivity we also detected the expression of Akt and its phosphorylated form (pAkt) by Western blot analysis. We also treated cells with LY294002, a specific inhibitor of PI3K/Akt pathway. The cell viability was assessed by MTT assay and the radiosensitivity was assessed by clonogenic survival assays, cell survival curve was fitted in accordance with standard linear-quadratic (LQ) model and one-hit multi-target model using GraphPad Prism 5 software after treating with LY294002. Similarly, the effect of LY294002 on cell-cycle phases in SMMC-7721 cells was determined by using Fluorescence Activating Cell Sorter (FACS) analysis and the apoptosis/rate was quantitated by TUNEL assay. Immunofluorescence was done to detect gamma-histone H2A (y-H2AX) foci formation to detect the repair of DSB at several times following irradiation.Results:1. We constructed the radiation-induced expression vectors of wild-type PTEN (pEgr-PTEN) or mutant PTEN (loss of lipid phosphatase activity) (pEgr-PTEN-G129E) respectively.2. By luciferase activity based promoter assays, we demonstrated that the Egr-1 promoter was induced by irradiation, and the response was dose-dependent. Peak responses were observed in SMMC-7721 cells at 24 h after 8 Gy irradiation.3. Little expression of PTEN was detected with no irradiation, while the response to irradiation was observed where PTEN was expressed higher than that of the non-irradiated cells in the pEgr-PTEN and pEgr-PTEN-G129E groups and that of the pEGFP-PTEN and pEGFP-PTEN-G129E groups after 8 Gy irradiation.4. By MTT assay we found that PTEN expression caused the decreases in the cell viability and proliferation decreased after irradiation.5. The survival fraction of 2 Gy (SF2), DO and Dq in pEgr-PTEN group were lower than that in the pEgr-PTEN-G129E and pEgr-C1 groups, while there was a higher value for both a andα/βin pEgr-PTEN group. Transfection of pEgr-PTEN increased the sensitivity of SMMC-7721 cells to irradiation, whereas that of pEgr-PTEN-G129E and pEgr-C1 induced no changes.6. A significant G2/M arrest at 24 h after irradiation of cells with pEgr-PTEN was found, while cells in the pEgr-PTEN-G129E or pEgr-C1 groups displayed minimal levels of G2/M arrest when compared with the non-irradiated groups.7. We found that the TUNEL positive cells in pEgr-PTEN group were increased at 24 h after irradiation and found a greater number of apoptotic cells at 48h after irradiation.8. During investigations of the DNA repair processes after irradiation in our study, we found that although all the cells incurred an equivalent number of initial DSB, the repair of irradiation-induced DSB was retarded in wild-type PTEN cells, but not in PTEN G129E mutant cells or the mock cells.9. When we detected the expression of phosphorylated Akt, we found that transfecting cells with wild-type PTEN decreased pAkt but not with the PTEN G129E mutant and mock transfections, indicating that PTEN negatively controled the phosphoinositide 3-kinase/PIP3/Akt signaling pathway.10. We treated cells with LY294002, a specific inhibitor of the PI3K/Akt pathway, before irradiation with 8 Gy. By MTT assays, we observed an obvious decrease in absorbance. Correspondingly, the radio sensitivity of cells was enhanced by LY294002 after assessment by clonogenic survival assays. Similarly, treatment of cells with LY294002 and irradiation induced G2/M arrest, and the TUNEL assay showed that the number of apoptotic cells treated with LY294002 only was higher than the other non-irradiated groups which increased after irradiation. The retarding of DSB repair was also seen with LY294002 and irradiation treatments demonstrating that radiosensitivity correlates with an active PTEN-PI3K-Akt pathway.Conlusions:1. Our present study shows that the presence of wild-type PTEN enhances the radiosensitization effects in the HCC line SMMC-7721.2. Restoring PTEN function is correlated with G2/M arrest, increase of apoptosis and the retardation of the repair of radiation-induced DSB.3. PTEN enhanced the radiosensitization effects specifically by its lipid phosphatase activity mediated through the PTEN-PI3K-Akt signaling pathway.4. These findings suggest that strategies designed to restore the expression of PTEN combining with radiation-inducible technique may be promising therapies by sensitizing HCC cells to irradiation and provide a theoretical support for the clinical application of genetic radiotherapy.