The Role Of Lymphocyte Restricted Transcription Factor SpiB In Lung Cancer

Objective:SpiB,a hematopoietic-specific of ETS family transcription factor,usually expresses in the development of lymphosytes,and plays an important role in the development and mature of B cell,the growth of T cells and the survival and development of plasmacytoid dendritic cell.SpiB^-/-mice display abnormal T-dependent antigenic responses,produce low levels of antigen-specific Ig G₁,Ig G_2aand Ig G_2b,and show a dramatic defect in germinal center formation and maintenance.Deletion of genes encoding PU.1 and Spi-B in B cells induces pre-B cell acute lymphoblastic leukemia.In solid tumors,immunohistochemical results of clinical samples show that SpiB protein expression levels are significantly increased in colon,liver,and stomach tumors,but the molecular mechanism of SpiB in solid tumors is still not clear.Here,we will study the role of ectopic expression of SpiB in lung cancer,and the connection between SpiB expression level with distal metastasis of lung cancer,which can determine whether SpiB become the target for diagnosis and therapy of patients with lung cancer.This study will help gain significant insight into the regulatory network associated with tumorigenesis and metastasis.Methods:1?Using RNA-seq to analyze the differential expression profiling between H209(SCLC)and A549(NSCLC),we verifed that SCLC expresses a series of trascripton factors normally restricted to lymphoid cells,including SpiB.Detecting SpiB expression level in lung cancer tissue through immunohistochemistry,we will dissent the connection between SpiB expression level and patients'prognosis.2?Using a subcutaneous injection model with C57BL/6,matrigel invasion assay,soft agar assay and matrigel 3D culture to study the effect of changing SpiB expression level on the biological behavior of cancer cells.3?Applying microarray to investigate the change of expression profile after overexpression of SpiB,and to identify the target genes of SpiB.4?Using 3C,Ch IP,luciferase assay to study the mechanism of SpiB regulating target genes.Results:1?Ectopic expressionof SpiB in lung cancerSpiB is ectopically expressed in lung cancer,and is negatively correlated with the prognosis of lung squamous cancer patients.2?SpiB promotes tumor metastasis and invasion through downregulating tight junction protein Claudin-2(encoded by CLDN2 gene)expression.Overexpression of SpiB significantly increases the lung metastasis of LLC1.Changing the SpiB expression level influences the cell-cell interactions.Microarray data shows that a series of genes related to tight junction are downregulated by SpiB,and metastasis-associated genes have been raised,such as MMP9.Overexpression of tight junction protein Claudin-2 can rescue the phenotype of SpiB overexpression in Matrigel 3D culture.Claudin-2 can also significantly inhibit LLC1 metastasis induced by SpiB in vivo.The results suggest that CLDN2 is the main target gene of SpiB.3?SpiB mediates the interaction between cis-regulatory element S1 and promoter of CLDN2 gene,silencing its transcription.Ch IP assay shows that several SpiB binding sites exist upstream of CLDN2promoter,and are also potential cis-regulatory element.3C technique,luciferase assay and EMSA reveal that cis-regulatory element S1 as a scilencer interacts with CLDN2promoter under SpiB overexpression,and SpiB directly binds to S1 sequence.Conclusion:SpiB,a hematopoietic-specific transcription factor,is ectopically expressed in lung cancers,binds to silencer S1 upstream of CLDN2 gene,mediates the interaction between S1 and the promoter,leading to CLDN2 transcription silenced,which disrupts the cell-cell interactions and results in tumor metastasis.This result further confirms that solid tumor cells can use the lymphocyte specific transcription factor to imitate the low adhesion of lymphocyte,achieving distal metastasis.This stuy links hematopoietic epigenetic regulatory network to solid tumor metastasis.