| Background:Aiolos, a zinc finger transcription factor of Ikaros gene family, is mainly expressed in lymphocytes and plays an important role in lymphocytes differentiation through activating or silencing distinct gene transcription. It has been reported that its dysregulation is related to hematopoietic malignancies. Recently, systematic bioinformatics analysis of expression levels of17,330human genes across9,783samples from175types of healthy and pathological tissues revealed upregulation of Aiolos in breast cancers, indicating that Aiolos is ectopically expressed in solid tumor cells. However, what are the consequences of Aiolos ectopic expression is completely unknown.Aim:The objective of our work is to study the function and regulation of Aiolos in human solid tumors derived from lung. Specifically, we will dissect the role of ectopic expression of Aiolos in cell adhesion related behavior and its mechanism, and the connection between Aiolos expression level with distal metastasis of lung cancer. Hopefully we may gain significant insight into the change of regulatory network associated with tumorigenesis and metastasis in solid tumors.Methods:1. Using immunohistochemistry and single cell RT-PCR to examine Aiolos expression in clinical lung cancer tissues.2. Using microarray to study the change of expression profile after transient expression of Aiolos.3. Using migration assay, matrigel invasion assay, adhesion assay, and MCF10A3D culture to study the change of biological behavior of cancer cells after overexpressing Aiolos.4. Study the role of Aiolos in cancer distal metastasis through tail vein injection of cancer cells into BALB/C nude mice.5. Using3C, ChIP, Luciferase assay to study the mechanism by which Aiolos regulates target gene transcription.Result:1. Aiolos is ectopically expressed in human lung cancers and its high expression level predicts survival disadvantage.Aiolos is not expressed in normal lung but ectopically expressed in lung cancer, especially in small cell lung cancer. Patients whose tumors had low Aiolos expression levels (33cases) had strikingly longer survival times than those whose tumors had high expression levels (25cases), with median survivals of41months (low Aiolos) vs.11months (high Aiolos).2. Aiolos ectopic expression results in global expression profile change. A number of genes related to focal adhesion and cell-ECM interactions are downregulated. One of downregulated genes is p66Shc who is previously demonstrated to induce anoikis in normal epithelial cells and function as a metastasis suppressor in vivo.3. Aiolos promotes anchorage independence by inhibiting p66Shc.In55human lung cancer samples, tumors frequently had significant levels of Aiolos, whereas surrounding lung parenchyma was negative. Conversely, normal lung showed diffuse p66Shc expression whereas tumors were frequently negative. When quantified, a highly significant negative correlation was found between Aiolos and p66Shc expression in these tumors.When Aiolos is over-expressed in HUVECs, A549and MCF10A cells, the cells show enhanced anoikis resistance. Re-expression of p66Shc rescues anoikis in these cells. These results indicate that Aiolos confers cancer cells anchorage independency through downregulation of p66Shc.In vivo, A549cells with or without Aiolos was introduced into nude mice through tail vein injection. After10weeks’ injection,6mice (100%) injected with Aiolos expressing A549cells died from lung cancer metastasis. However, only1out of6mouse injected with A549cells expressing empty vector died. This result suggest that Aiolos promotes cancer distal metastasis. 4. Aiolos associates with upstream of p66Shc gene, disrupts long range chromatin interactions and silences p66Shc transcription.Three cis-regulatory elements were found upstream of p66Shc promoter. E2exhibits strong enhancer function in lucifrase study. P66Shc expression requires long range chromatin interaction between E2and p66Shc promoter. In small cell lung cancer cells, however, the E2-p66Shc promoter physical interaction is disrupted.We detected occupancy of Aiolos in upstream of p66Shc promoter. Aiolos can inhibit function of E2in luciferase assay. Transient expression of Aiolos in A549cells disrupts the colocalizaiton of E2and p66shc promoter.Conclusion:Aiolos, a haematopoietic lineage restricted transcription factor, is frequently ectopically expressed in lung cancers and its expression reduces patient survival. Ectopic expression of Aiolos decreases E-cadherin mediated cell-cell junctions and integrin mediated cell-ECM adhesions and facilitates dissemination of carcinoma cells from epithelial sheet. More importantly, Aiolos expression permits anchorage-independent behavior in vitro and in vivo by directly silencing p66Shc, a detachment reporter that is frequently repressed in metastatic human lung cancer cells. These data link hematopoietic epigenetic gene control with changed cell adherence and anchorage independent behavior in metastatic cancer. |
PDF Full Text Request