Inhibition of Radiation-Induced Lung Adenocarcinoma Cell Metastasis by Adenovirus of PIAS 3 Overexpression Driven by Radiation-Inducible Promoter ( Ad-pig 3 RRP-PIAS 3 )

Radiotherapy is one of important approaches for pulmonary adenocarcinoma. However, many studies have shown that radiation can also enhance the ability of tumor cells metastasis, although the lung adenocarcinoma could be killed. The increased metastasis induced by radiation is associated with the activation of STAT3 in lung adenocarcinoma cells. Based on the importance role of STAT3 in cell proliferation and survival, we can construct an adenovirus vector of PIAS3 overexpress driven by radiation-induced promoter to inhibit the activation of STAT3 specifically. In this way, when STAT3 was activated by radiation, the expression of PIAS3 will be increased at the same time; this lead to the inhibition of invasion and metastasis caused by STAT3 in lung adenocarcinoma cells. These researches are expected to develop a novel target and method for radiotherapy and molecular therapy of lung adenocarcinoma.


STAT3-Key Target of Radiation-Induced Metastasis in Lung Adenocarcinoma
In the current study, it was indicated that radiation can promote the metastasis of pancreatic cancer cells [1] [2], glioma cells [3] [4], hepatoma carcinoma cells [5], breast cancer cells [6], melanoma cells [7], ductal carcinoma cells [8] and lung adenocarcinoma cells [9], although the tumor cells can be killed.Radiation was found to activate the phosphorylation of STAT3 which resulted in the increase of invasion of A549 cells [9].By using special inhibitor to block radiation-induced STAT3 activation, the increase of invasion and migration of A549 cell induced by radiation can be reduced significantly [10].All above comes down to one point: STAT3 plays a key role in radiation-induced invasion and metastasis and is a potential target of therapy in lung adenocarcinoma.

PIAS3-Specific STAT3 Inhibitor Protein
PIAS3 (protein inhibitor of activated STAT3) is one of PIAS (including PIAS1, PIAS2, PIAS3 and PIAS4) family members, which can inhibit STAT3 specifically [11].As an endogenous inhibitor of STAT3, PISA3 is expressed at high level in many normal human tissues and cells, while shows a very low level or even no expression in the tumor tissues and cells.Dabric [12] found that protein expression with PIAS3 is in a low level which affected survival upon mesothelioma patients and indicated PIAS3 as having potential for development into a novel therapeutic target.When the expression of PISA3 has been silenced, the activity of STAT3 increased continuously, which resulted in the significant increasing of growth and proliferation of tumor.Our group study showed that the expression of PISA3 is in a low level, and is not affected by γ-ray radiation.As the radiation-induced activation of STAT3 plays an important role on invasion and metastasis of A549 cells, it can be inferred that PIAS3 also played a prominent role in the invasion and metastasis of lung adenocarcinoma cells via STAT3.

PIG3-Radiation Sensitive Gene
PIG3, one of PIG family members regulated by p53 protein, participated in oxidative stress of cells and cell apoptosis pathway mediated by radiation.Our study group has drawn a conclusion that the expressions of PIG3 in mRNA and protein level were enhanced 15 to 20 times by radiation in a dose-dependent manner from 0 to 10 Gy.Other group also discovered that the PIG3 gene expression was enhanced by radiation in AHH-1 and HPBL cells [13].These results demonstrated that the promoter region of PIG3 gene contains a radiation-sensitive region, which could activate the gene expression of downstream when the cells were irradiated.It indicated that we can turn-on or turn-off the gene expression by controlling the radiation at any time.

Adenovirus Drug-An Effective Gene Therapy Means
Adenovirus has no genetic toxicity to the human body because adenovirus DNA does not integrate into the host cell genome.Adenovirus drugs are wildly considered to be safe and effective, with the launching of gendicine, which is the first commercial adenovirus gene therapy product and have been used in clinical for many years.Yu M. [14] reported that Recombinant adenovirus-p53 is effective for pulmonary metastasis in hepatocellular carcinoma.Jinluan Li [15] reported that Recombinant adenovirus-p53 (Gendicine) enhances radiosensitivity of a pancreatic carcinoma cell line.The future of adenovirus drugs is attractive and vast.

Discussion
Based on the fact that PISA3 can inhibit the activation of STAT3 specifically, we can utilize recombinant adenovirus vector which can be administrated in clinical to transfer the radiation-sensitive PISA3 gene into cancer cells.Under these circumstances, when STAT3 was activated by radiation, the expression of PIAS3 would be increased at the same time.So while radiation is utilized to kill tumor cells, meanwhile, the invasion and metastasis induced by radiation will be prevented effectively in lung adenocarcinoma cells.The vector will have no function until the cells transfected with it are exposed to radiation.Therefore, if the normal tissue cells, which will not be irradiated during radiotherapy, intake the vector, the PIAS3 expression should not be initiated.This eliminates the potential effect of vector on normal tissue cells.On the basis of these findings, we intend to identify the radiosensitive region of PIG3 promoter, and subsequently clone this region into the adenovirus expression vector of PIAS3, in order to construct a radiosensitive adenovirus vector encoding PIAS3 (Ad-pig3RRP-PIAS3).
Based on the function of PIAS3 on inhibiting cell proliferation [16] [17] and the response of STAT3 to radiation [18] [19], we will further investigate the inhibiting effect of Ad-pig3RRP-PIAS3 on metastasis which is induced by radiation in lung adenocarcinoma cells and the correlation mechanism.From a new perspective, a novel target and treatment means which combined molecular target therapy with radiotherapy will be explored in lung adenocarcinoma.