PAX5 Controls Lineage Interconvert Between Neuroendocrine Carcinoma And Non-neuroendocrine Carcinoma

Objectives and Backgrounds: Tumor drug resistance is a huge problem faced by cancer treatment in clinic.Cell lineage conversion is one of the mechanisms of tumor drug resistance.Small-cell neuroendocrine carcinoma(SCNC)is a type of cancer that originates in all epithelial tissues and organs,has a high degree of malignancy,has a high degree of invasiveness and metastasis,and is accompanied by a neuroendocrine(NE)phenotype Malignant tumor.Combination chemotherapy of cisplatin and etoposide is the standard treatment for SCNC.Patients often respond very sensitively in the early stage of treatment,but they will soon develop resistance and relapse within a year and gain a higher metastatic capacity.After relapse,SCNC loses NE cell phenotype and switches from NE lineage to non-NE lineage.In addition,some adenocarcinomas in the clinic(including 14% of lung adenocarcinomas and 20%-40% of prostate adenocarcinomas)can be converted into NE cancers after receiving chemotherapy or targeted drugs and become resistant.It can be seen that the phenomenon of NE and non-NE lineage conversion of tumor cells after treatment resistance is widespread,but the epigenetic mechanism of its occurrence is still unclear.This article aims to find the key regulators of lineage conversion between NE and non-NE cancer cells,so as to provide a treatment plan for the treatment of SCNC and prevent the conversion of adenocarcinoma lineage to NE lineage.Methods: 1.Isolate fresh primary SCLC clinical tissue samples for 10×Genomics single cell transcription profiling(sc RNA-seq).Use inferred CNV and UMAP for grouping(mainly divided into two groups: NE-SCLC and non-NE-SCLC).Transcription factor binding motif enrichment analysis was used to enrich the active transcription factors in NE-SCLC(the nerve/lymphocyte-specific transcription factor PAX5 was found to be significantly activated in NE-SCLC).2.Immunohistochemical detection of PAX5 expression in lung cancer clinical samples;using non-small cell lung cancer cell line H1155 with neuroendocrine phenotype to knock out PAX5 using CRISPR/Cas9 to detect transcriptional profiles and cell biological behavior;and To explore the behavioral changes of cells and the sensitivity to SCLC standard treatment drugs through mouse subcutaneous tumor experiments.3.Overexpress PAX5 in lung adenocarcinoma cell line A549,and explore the behavioral changes of cells in vivo;generate Tet-op-h PAX5 knock-in mice,Tet-op-h EGFR-TD/CC10 rt TA hybridization was used to observe the lung tumor changes in h PAX5/h EGFR-TD/CC10 rt TA mice;the molecular mechanism of PAX5 affecting the biological changes of tumor cells was studied by microarray and Ch IP-Seq.4.Overexpress PAX5 in EGFR-mutated lung adenocarcinoma cell line HCC827,perform subcutaneous tumor implantation,and test whether PAX5 affects the sensitivity of tumor cells to EGFR-TKIs drug Erotinib.5.To explore the effect of the tumor microenvironment on PAX5 regulation of adenocarcinoma to neuroendocrine carcinoma.A549-PAX5 subcutaneous tumors and h PAX5/h EGFR-TD/CC10 rt TA mouse lung tumors and control groups were subjected to neovascular immunofluorescence double staining;Bevacizumab was used to conduct in vivo drug experiments on A549 subcutaneously implanted mice,and the effect of inhibiting angiogenesis on PAX5-induced adenocarcinoma to neuroendocrine cancer transformation was examined.6.In prostate cancer,explore the universal role of PAX5 in promoting the conversion of non-NE to NE lineage,and detect the expression of PAX5 in prostate cancer;after ectopic expression of PAX5,the biological behavior and expression profile of prostate adenocarcinoma cells are changed.Results: 1.PAX5 regulates lineage exchange between NE and non-NE cancer cells 1.1PAX5 is a key transcription factor for maintaining the SCLC neuroendocrine lineage(1)It is determined that the nerve/lymphocyte-specific transcription factor PAX5 is an active transcription factor in NE-SCLC cells.PAX5 is mainly expressed in lung cancer cell lines and tissues with neuroendocrine phenotype,and only 10% of lung adenocarcinomas express PAX5.(2)The expression profile of KO-PAX5-H1155 cells is consistent with the expression profile of non-NE-SCLC in SCLC sc RNA-seq,and a transition to non-NE cell lineage occurs;the growth mode of KO-PAX5-H1155 cells in vitro culture is from suspension growth Converted to adherent growth;in vivo experimental results of subcutaneous tumors showed that the cell transfer ability after KO-PAX5 was enhanced(renal metastasis occurred),and the pathological morphology of in situ tumor cells changed to non-NE cell morphology and did not express NE markers;for SCNC Of standard chemotherapy drugs have decreased sensitivity.1.2.PAX5 is the driving gene for the conversion of lung adenocarcinoma to SCLC lineage(1)In vivo experiments of A549-PAX5 obviously resulted in cell migration to the lung,the pathological morphology changed from adenocarcinoma to SCLC,and the expression of NE markers increased;h PAX5/h EGFR-TD/CC10 rt TA mouse lung pathology and NE markers The change of expression is consistent with that of A549-PAX5 subcutaneous tumor.HCC827 subcutaneously implanted tumors.After being treated with Erotinib,the HCC827-PAX5 group was resistant to Erotinib.2.PAX5 directly regulates the expression of neuroendocrine pathway related genes GESA analyzed the results of A549-PAX5 microarray and found that PAX5 up-regulates genes of neuroendocrine pathways;PAX5 can be combined with the promoter regions of these neuroendocrine genes for direct regulation.The SCLC cell line grows in suspension under in vitro culture conditions.A549 overexpresses PAX5 and cannot induce cell suspension growth,but it can induce lung adenocarcinoma to SCLC transformation in vivo,suggesting that depend on the interaction between tumor cells and tumor microenvironment.effect.3.PAX5 promotes angiogenesis in the tumor microenvironment to achieve non-NE and NE lineage conversion H&E staining revealed that there were increased blood vessels in the lungs of A549-PAX5 subcutaneous tumors and h PAX5/h EGFR-TD/CC10 rt TA mice,which were confirmed by CD105 and ?-SMA immunofluorescence double staining.After Bevacizumab treatment of A549-PAX5 tumor-bearing mice inhibited the formation of new blood vessels,it significantly reduced tumor lung metastasis and inhibited tumor transformation from adenocarcinoma to SCLC.Overexpression of PAX5 up-regulates the expression of angiogenic factors such as VEGFA and TNF-?.4.Transcription factor PAX5 promotes the conversion of prostate adenocarcinoma to SCPC lineage PAX5 is highly expressed in SCPC cell lines and tissues;PAX5 is sporadicly low in 10% of prostate adenocarcinoma(ADPC)tissue samples.Ectopic expression of PAX5 in ADPC cell line can promote the transformation of prostate adenocarcinoma lineage to NE lineage.In situ tumors of RM-1-PAX5 cells have increased intravascular tumors,and the use of angiogenesis inhibitors can effectively prevent in-situ tumors from lung metastasis and APDC to SCPC pathological morphology.Conclusions: We found that the transcription factor PAX5,which is only expressed in the nervous system and lymphocytes under physiological conditions,is ectopically activated in epithelial cell carcinoma and is a decisive factor for the maintenance of the SCNC lineage.The absence of PAX5 causes SCNC to lose neuroendocrine properties and become resistant,PAX5 ectopic activation in adenocarcinoma will promote the transformation of adenocarcinoma to SCNC and drug resistance.These findings have found a molecular mechanism for the phenomenon of lineage conversion between NE and non-NE cancer cells in the clinic;because PAX5 is a recognized transcription factor that determines the differentiation of B lymphocyte lineages,our findings are also related to lymphocyte development and SCNC differentiation provides a specific molecular association and provides a mechanistic explanation for the histological and clinical similarities(including metastatic ability and rich vascularity)between SCNC and lymphoma.Inhibition of PAX5-promoted angiogenesis can prevent PAX5-induced non-NE tumor cells from switching to the NE lineage.Loss of PAX5 expression in SCNC can promote SCNC resistance to standard cisplatin and etoposide chemotherapy drugs.These studies provide a reference treatment for SCNC treatment and prevention of PAX5 expressing adenocarcinoma to SCNC lineage transformation.