Serous Endometrial Carcinoma In TP53 Knocking-out Mouse Model

Endometrial carcinoma is one of the most frequently diagnosed malignancies of the female genital tract. Based on histological appearance, clinical behavior and molecular differences, endometrial carcinomasare classified as type â…  and type â…¡ endometrial carcinomas. Type â…¡ endometrial carcinomas have not been overtly associated with hormonal risk factors; peak in the sixth decade of life, arise predominantly in the resting endometrium. These type â…¡ cancers are probably more intrinsically aggressive, present more frequently at a higher stage with a worse prognosis compared with type I cancers. Serous endometrial carcinoma are the primary group of endometrial carcinoma (60%-80%).In type â…¡ endometrial carcinomas, mutations of Tp53 are frequently involved at the earliest stage of development. In our previous study, we found clinicopathologic evidence that endometrial serous carcinoma might arise from endometrial glandular dysplasia (EmGD), which we believed as precursor lesions of AdCas. We also found that frequency of Tp53 gene mutations increased from p53 signature glands (42%) to EmGD (43%), to serous EIC (63% to 72%), and to ESC (96%). In addition, a significant number of identical Tp53 gene mutations are seen among lesions of p53 signature, EmGD, serous EIC, and ESC in samples obtained from the uteri with ESC. Therefore, that p53 gene mutation is a critical early step in endometrial serous carcinogenesis.This study focused endometrial carcinoma in mouse model which includes two parts as following:1. Establishment of a mouse model for type II endometrial carcinoma, whose Trp53 gene in the epithelia of the adult genitourinary system were knocked out.2. Molecular study of Trp53 gene knocked-out mouse model of serous endometrial carcinoma.Part â… :Establishment of a mouse model for type II endometrial carcinoma by specific Trp53 knocking-outOBJECTIVE:Tp53 mutation is the most striking genetic alteration in serous carcinoma. Approximately,90% of serous endometrial carcinoma present p53 (+). We observed that serous endometrial carcinoma and its early lesion, like EmGD, have Tp53 mutation. In addition, a highly concordant Tp53 mutation frequency was present among these lesions. Therefore, Tp53 mutated at the very early stage of serous endometrial carcinoma, and probably was the genetic etiology. This study is to knock out Trp53 gene specifically in endometrial epithelia of mice, to confirm if there were any lesions in endometrium after deletion of Trp53.MATERIALS AND METHODS:Trp53fl/fl and Ksp 1.3-Cre mice were involved for our study. Trp53fl/fl is a type of transgene mice with exons 2-10 of Trp53 gene flanked by loxp sites in this conditional targeted mutation. Ksp 1.3-Cre mice is a type of transgene mice that have a sequence of Cre recombinase knocked-in following Ksp 1.3 promoter. Ksp 1.3 promoter could encoding protein Cdh16, which is a member of the cadherin superfamily, genes encoding calcium-dependent, membrane-associated glycoproteins. We obtain a group of final genotype of (Trp53Δ/Δ), whose Trp53 gene are specifically knocked out in genitourinary system of Trp53Δ/Δ mice by crossing Trp53fl/fl and Ksp 1.3-Cre mice. To the uterus, Ksp 1.3-Cre recombinase activity gives rise to gene deletions in the endometrial epithelial cells of the lumen and glands. Besides, we take Trp53fl/fl (WT, Cohorts of littermate+/+; Trpfl/f) mice as the control group for our study. Generally, WT and Trp53Δ/Δ mice were analyzed every 2 weeks, starting from 10 weeks to 80 weeks (WT:Trp53Δ/Δ=1:2; WT, n=48; Trp53Δ/Δ, n=96). Each group of 10-30 weeks and 66-80 weeks (WT, n=l; Trp53Δ/Δ, n=2),32-64 weeks (WT, n=2; Trp53Δ/Δ, n=4). To determine lesions in Genito-urinary system of these mice, uterus, bilateral ovaries and fallopian tubes, kidney and bladder and any suspicious lesions from all the mice that involved in our study were entirely submitted for histopathologic examination. A blinded evaluation of the immunostained slides were performed without clinical data. Using proteinase K to dissolve mouse tail to extract DNA for genotyping PCR.RESULTS:1. We successfully established a mouse model for endometrial serous carcinoma. While obvious serous tumor (which had a part of undifferentiation carcinoma) were observed in female Trp53Δ/Δ mice of 80 weeks of age, ESC showed starting from 54 weeks of age.2. The whole process of "REâ†'EmGD â†'EICâ†' ESC" showed completely during the progress of tumor. EmGD were observed in female Trp53Δ/Δ mice from 12 weeks, the earliest, to older cohorts. EIC were observed in female Trp53Δ/Δ mice from 32 weeks, the earliest, to 80 weeks, the oldest we had in our study. ESC started from 54 weeks of age. These lesions could appeared simultaneously in different part of one uterus.3. No lesions were found in ovaries, fallopian tubes, kidney and bladder until at least 80 weeks of age.4. No lesions were found in ovaries, fallopian tubes, kidney or bladder of these Trp53fl/fl transgenetic mice until at least 80 weeks of age.CONCLUSION:Trp53 gene is a critical factor in serous endometrial carcinomas. Specific Trp53Δ/Δ mice could generate serous endometrial carcinoma. Moreover, in the process of serous endometrial carcinoma, EmGD and EIC could be observed.Part II:Molecular study of Trp53 gene knocked-out mouse model of serous endometrial carcinomaOBJECTIVE:Type II endometrial carcinoma, especially serous endometrial cancer, frequently exhibit mutations in the TP53 (90%) and HER2/neu genes (16%-80%), loss of ER (< 30%) and PR (< 5%), and high expression of p16 and IMP3. This study is to find out the knocking-out site of Trp53 in uterus, fallopian tube and ovary. Using IHC to learn the molecular characteristic to confirm the accuracy of this mouse model. In addition, other changed molecular and pathway in this serous endometrial carcinoma model are studied by microarray analysis.MATERIALS AND METHODS:Use immunohistochemistry to stain Cdh16 in order to confirm the deletion site of Trp53. Distinctive markers (ER, PR, Vimentin, IMP3) were stained by immunohistochemistry to confirm the histopathologic classification.Do microarray analysis with four uterine segments and four fallopian tube and ovaries to determine molecular changes.RESULTS:1. Immunohistochemistryof Cdhl6 showed that the deletion of Trp53 is in the glandular epithelia of uteri and the epithelia of fallopian tube.2. Immunohistochemistry results of the tumor present ER (20%-40%), PR(<5%), vimentin(-), IMP3 (+)which belongs to human serous endometrial carcinoma. In Trp53Δ/Δmice, EmGD, EIC showed high expression of IMP3, which is the same as ESC. Howere, Trp53fl/fl mice did not.3. Microarray analysisMicorarray analysis show 738 genes significantly changed in in the uteri of Trp53Δ/Δmice, of which 210 genes up-regulation,528 genes down-regulation.Trp53 was down-regulation(pTrp53=0.006, Fold change= 3.7, FDR= 0.005) Microarray of fallopian tube and ovaries showed that, there is no statistic change in Trp53 and any related protein between Trp53Δ/Δ mice and Trp53fl/fl mice (p> 0.05).4. Gene Ontology (GO) analysis of different genes.We did GO analysis of different genes. It showed that in Biological process,203 items were up-regulation and 180 items were down-regulation; in Molecular function,70 items were up-regulation and 77 items were down-regulation; in Cellular component,38 items were up-regulation and 18 items were down-regulation.5. Pathway analysis of different genesWe did pathway analysis of different genes. It showed that in the uterus of Trp53Δ/Δ mice, there were 63 pathways significantly changed, of which 22 pathways were up-regulation and 41 pathways were down-regulation.CONCLUSION:In this study,Trp53 knock-out site is endometrium. Molecular study proved accurity of this mouse model we established for serous endometrial carcinoma. We also molecularly confirmed EmGD is the precancer lesion for ESC.