Mechanism Of POU5F1in Invasion And Metastasis Of Lung Cancer And Preliminary Study On Immune Escape Of Glioma Stem Cells

Lung cancer is a serious threat to human health. According to the statistics of theWHO, lung cancer patients will account for19%of cancer deaths in2025. In our country,the number of patients with lung cancer approaches to one million per year. Conventionaltherapy of lung cancer is composed of a surgical resection in combation with radiotherapyand chemotherapy, but the effect is not ideal.There are several reasons attributed to this phenomenon. On one hand, The5-yearsurvival rate of patients with lung cancer is less than15%as a result of high morbidity andmortality. There is no effctive way to deal with early distant metastasis which happens inthe early stage of tumorigenesis. On the other hand, the cancer stem cells (CSCs, alsonamed as cancer-initiating cells, CICs or cancer stem-like cells, CSLCs) are found invarious malignancies. Hypothesis about CSCs, the great contribution to cancer research,holds that tumor is drived by small cell populations that are multipotent with the ability ofself-renewal, and can replicate the characteristics of cancer which they are derived,including tumor type and morphology. The most important feature of CSCs is tumorigenicin vivo. Recent results have showed that CSCs play an important role in tumor initiation,progression, migration, invasion, and recurrence. Exploration of the biologicalcharacteristics of cancer stem-like cells (CSLCs) will open new avenues in lung cancerdiagnosis and therapy.In order to uncover the mechanism of POU transcription factor family POU5F1intumor invasion and metastasis, we choose lung cancer as research model. First of all, weisolated and identified cancer stem-like cells (Lung Adenocarcinoma Cancer Stem-likeCells, LACSLCs) from A549cell line. We futher compared the invasive property betweenLACSLCs and ordinary tumor cells. Secondly, we performed knockdown on POU5F1genein LACSLCs to study its role on cell migration and invasion. Then, we detected the expression level of matrix metalloproteinase2(MMP2)-a well-known molecule relatedwith tumor invasion and metastasis. Finally, we studied the relationship between POU5F1,MMP2and the patients’ prognoses.Metastasis of tumor cells to distant organ or tissue is mainly through the blood streamor lymphatic channels. During this process, tumor cells will be recognized and killed by theimmune system. However, a large number of studies have found that tumor cells can evadethe monitoring mechanism of immune system. Two aspects are involved in such process:(1)change of the tumor cells, such as "antigen coverage" or closed on cell surface, antigendownregulation or deletion, immune response down-regulated or absent expression ofmolecules, costimulatory molecule expression inhibited, cell death signaling pathwaydefects; and (2) suppression of the immune system function, such as reduced infiltration ofimmune cells, inhibition of immune cell function，induced apoptosis of immune cells, andinduced regulatory T cells (Treg).Researches on the correlationship between cancer stem cells and immune system isstill in the early stage. Dendritic cells (DCs) are the strongest professional antigen-presenting cells. The interaction of DCs with CSCs has not yet been fully addressed.Therefore, the other part of the study is investigation of the role of DCs on the mostcommon primary central nervous system tumor-malignant glioma. Firstly, we isolated andidentified CSCs from mouse glioma cell line GL261, human glioma cell line U87andhuman primary glioma cells. Secondly, mouse dendritic cell lines DC2.4and humandendritic cells were isolated, cultured and identified. Thirdly, supernatant from GCSLCs/adherent tumor cells (Adhered cells, ACs) was used to treat DCs. Finally, we explored thepossible mechanism underlying the process that GCSLCs/ACs suppressed DCs’differentiation.Contents and major findings：1. The tumorsphere from A549cell line has more invasive ability;(1) Isolation and identification of GSCs from A549cell line.○1The tumorsphere from A549cell line displayed higher ability for tumor colonyformation than adhered counterparts.○2The tumorsphere expressed abundant CD133, Sox2, ALDH1and POU5F1, and lower levels of differentiation markers CK8and CK18. In contrast, adhered cells containedlittle CD133, Sox2, ALDH1and POU5F1, and high levels of CK8and CK18.○3As few as1×104tumorsphere cells were able to initiate tumor formation. Incontrast,50-fold higher number of adhered cells was needed to form tumor in NOD/SCIDmice.(2) The tumorsphere cells displayed highly migratory and invasive abilities.The tumorsphere cells migrated into the scratched area more rapidly than A549monolayer cells. Results showed that A549LACSLCs displayed higher invasive capacitythan A549monolayer cells from an in vitro Matrigel invasion assay.2. POU5F1and MMP2are aberrantly high expressed in LACSLCs.Immunostaining and western bloting analyses showed that POU5F1and MMP2wereaberrantly high expressed in A549LACSLCs.3. Knockdown of POU5F1disrupts cell invasion, migration and tumorigenesispotentials of LACSLCs.(1) LACSLCs containing sh-POU5F1migrated much slower than control cells.(2) A549LACSLCs with POU5F1knockdown resulted in significantly decreasednumber of invading cells through the Matrigel to the lower chamber compared withsh-control-infected A549LACSLCs.(3) The tumors formed by A549LACSLCs with POU5F1knockdown were muchsmaller than control cells.4. POU5F1knockdown represses expression and activity of MMP2.(1) The protein level of MMP2was markedly reduced in A549LACSLCs containingsh-POU5F1.(2) POU5F1can bind to MMP2promoter, suggesting that POU5F1may be responsiblefor transcriptional activation of MMP2.(3) Knockdown of POU5F1significantly reduces luciferase activity of reporterconstruct containing MMP2promoter.5. Glioma stem cells inhibit maturation of dendritic cells by paracrine cytokines.We used supernatant from GCSLCs to induce DCs, and found that paracrine cytokines(VEGF-A, IL-10) secreted by GCSLCs can suppress differentiation and maturation of DCs. The experiment data and main conclusions are listed as follows:1. POU5F1directly regulates transcription of matrix metalloproteinase2(MMP2).2. The pathological detection of the double-positive expressions for POU5F1andMMP2will be useful as diagnostic and prognostic biomarkers in LAC to advanceanti-metastasis therapy.3. Glioma stem cells inhibit maturation of dendritic cells by paracrine suppressivecytokines VEGF-A, IL-10.