Mechanism Investigation And Therapeutical Strategies Exploration Of Endocrine-Related Urogenital Tumors

Endocrine-related urogenital tumors mainly include neuroendocrine tumors and reproductive endocrine tumors.This article explored the pathogenesis and potential treatment strategies of endocrine-related urogenital tumors from these two categories.In the first part of this thesis,we studied the neuroendocrine tumors.Bladder neuroendocrine tumor is a tumor mainly derived from urothelial pluripotent stem cells,of which the bladder small cell carcinoma has the highest malignancy.The incidence rate of bladder neuroendocrine tumor is about 1% of all malignant bladder tumors,.Due to the lower incidence and higher malignancy,researches on bladder small cell cancer are very limited,and its genomic characteristics,pathogenesis,and treatments have not been fully studied.In this study,we collected 12 clinical samples of bladder small cell carcinoma and did the whole genome,whole exome and transcriptome sequencing.By analyzing the DNA sequencing results,we found that the mutation spectra of bladder neuroendocrine tumor is very similar to urothelial carcinoma,suggesting that they have the same origin.Through analysis of mutation characteristics,we found that bladder neuroendocrine tumors are closely related with age factors and APOBEC.At the same time,we also found that the tumor formation was significantly associated with aristolochic acid in one sample,which has been proved to cause urothelial cancer.Gene mutations and copy number variations affect genes in three major signaling pathways in cells,including the P53 / RB1 pathway,the RTK pathway,and the epigenetic regulatory pathway.Transcriptome sequencing analysis revealed that gene expression profiles of tumor tissues were significantly different from normal tissues.At the same time,we also found some new abnormal gene fusions,such as PVT-ERBB2,which can significantly upregulate the expression of HER2 receptor after the fusion occurs.Further mechanism exploration,we found that simultaneous knocking out of TP53 and RB1 in urothelial cancer cells induce them transformation into cancer cells with neuroendocrine characteristics through lineage switching,resulting in reduced sensitivity to targeted drugs.In this study,we first identified the gene mutation spectra and landscape of neuroendocrine bladder cancer through multiple second-generation sequencing methods,clarified the molecular characteristics and pathogenic factors of neuroendocrine bladder cancer,and provided basis for the development of novel therapeutic strategies.In the second part of this thesis,we investigated reproductive endocrine tumors.Ovarian cancer,as a type of reproductive endocrine tumor,has the highest lethality rate among gynecological tumors.Over the past few decades,there have been few breakthroughs in the standard treatments of ovarian cancer,leading to high mortality rates.Therefore,exploration the pathogenesis and development new therapeutic strategies are particularly important for the treatment of ovarian cancer.Due to the genome characteristics of ovarian cancer is low-frequency mutations,drugs targeting tumor-specific driver mutation is not applicable.Large sample omics analysis of The Cancer Genome Atlas(TCGA)shows that almost all High-grade serous ovarian cancer(the main pathological type of ovarian cancer)has large-scale chromosome copy number variation,which is its main pathogenic molecular mechanism.The change of gene copy number will not lead to abnormal sequence and function of the encoded protein,but to promote and maintain the malignant development of tumors through the regulation of gene and protein expression levels.Thus,our study aims to investigate the roles of gene replication and transcription in ovarian cancer,and try to explore the underlying therapies.We adopted the CRISPR-Cas9 gene knockout strategy to screen the 23 members of the 3 ? transcription termination complex core in ovarian cancer cell lines,and found that CPSF family genes are particularly important for ovarian cancer cell proliferation.Knockout of WDR33,CPSF1,CPSF2,and CPSF3 in the CPSF family significantly inhibited ovarian cancer cell proliferation in in vivo and in vitro experiments.Further analysis of the relationship between the members of the CPSF complex suggested that WDR33 played a key role in maintaining the stability of the entire complex.Taking advantage of photobleaching technique,we found that WDR33 formed phase separation to promote the assembly and function of CPSF complexes.Through RNAseq analysis,we found that knockout of WDR33,CPSF1,CPSF2,and CPSF3 obviously decreased the expression of functional m RNA related with chromatin assembly,leading to abnormal chromatin assembly and cell death.At the same time,RNA could not complete the cleavage at the termination site after knock-out of WDR33,CPSF1,CPSF2,and CPSF3,which was unable to detach from the DNA and formed an R-loop,resulting in the increased cellular DNA damage.Due to DNA damage repair depends on pathways such as PARP,combination of PARP inhibitors in WDR33,CPSF1,CPSF2,and CPSF3 knockout cells further promoted tumor cell death.The above results revealed that the CPSF family of transcription termination complexes plays a key role in the development of ovarian cancer.Targeting WDR33,CPSF1,CPSF2,and CPSF3 can significantly inhibited the proliferation of ovarian cancer cells,and in combination with PARP inhibitors induced a synergistic lethal effect.Our study first clarified the roles of transcription termination complexes in ovarian cancer and indicated synergistic lethal effect with PARP inhibitors,which suggested to be a potential therapeutic target.