Comprehensive Study Of HMGA2 In The Development Of Endometrial Serous Carcinoma

IntroductionEndometrial cancer, the most commonly diagnosed gynecologic malignancy, has the second highest mortality rate among gynecologic cancers. Endometrial cancer has been classified into two different types, based on its clinical, pathological and epidemiological observations. Type I endometrial cancer is endometrial endometrioid carcinoma, EEC, while the prototype of type II endometrial cancer is endometrial serous carcinoma, ESC. The rarer yet more clinically aggressive cancer, endometrial serous carcinoma (ESC) accounts for approximately 10-15% of all endometrial cancers but is responsible for a disproportionate 40% of the cancer related death. The high fatality rate that associated with ESC is probably, at least partly, because of the advanced stage at which a significant proportion of patients with endometrial serous carcinomas were diagnosised. Previous studies showed that the overall survival of corpus-confined, nonmyoinvasive endometrial serous carcinomas after a full staging is 83%-100%. Despite the significant advances about the carcinogenesis of endometrial endometrioid carcinomas, progresses about endometrial serous carcinoma carcinogenesis is significantly lacking. In brief, ESC arises mainly in the resting endometrium (RE), manifesting first as p53 signatures, then evolving to endometrial glandular dysplasia (EmGD), then serous endometrial intraepithelial carcinoma (EIC), and finally into fully developed malignancy, endometrial serous carcinoma (ESC).Therefore, the importance of researching endometrial serous carcinogenesis is highly needed. As the most important player in ESC development, more than 90% of endometrial serous carcinoma cases show TP53 mutation. The overexpression of P16 and Her2 has been discovered in more than 90% and 50% of endometrial serous carcinomas, while IMP3 overexpression has been found in about 94% of ESC cases. Another important player, loss of E-cadherin was found in more than 62% of endometrial serous carcinomas.Genetically Engineered Mouse Models (GEMMs) for endometrial serous carcinoma were built by conditional knockout Trp53 in genitourinary tract epithelium of female transgenic mice. This mice model successfully simulated the progression of human endometrial serous carcinoma and formed a series of precancer lesions, EmGD, and early stage lesions of ESC, EIC, which were samiliar with human endometrial serous carcinoma progression. This is a great advance of endometrial serous carcinoma animal model, which can be a very valuable research instrument for carcinogenesis of endometrial serous carcinoma.High-mobility group A2 (HMGA2), a member of the high-mobility group family, is a non-histone nuclear-binding oncofetal protein, which can modulate transcription by promoting conformational changes. HMGA2 is highly expressed in embryonic tissue and in many malignant neoplasms including the pancreas, thyroid, lung, and ovary, but rare in normal adult tissues. There is strong evidence from previous studies that expression of HMGA2 is associated with tumor aggressive behavior, which is probably due to its biologic function related to epithelial-mesenchymal transition and stem cell self-renewal ability.Previous studies have shown that the overexpression of HMGA2 was an important molecular event responsible for the carcinogenesis of ovarian serous carcinomas. A recent study found that HMGA2 was also overexpressed in advanced endometrial serous cancers by immunohistochemistry examing. However, whether HMGA2 plays an oncogenic function in the development of endometral serous career is still unclear. The goal of this study was to explore the role of HMGA2 in ESC and the specific mechanisms which includes the two following parts:1. Overexpression and oncogenic function of HMGA2 in endometrial serous carcinogenesis;2. HMGA2:a potential biomarker complement to P53 for detection of early-stage endometrial serous carcinoma.Part I. Overexpression and oncogenic function of HMGA2 in endometrial serous carcinogenesisOBJECTIVEHMGA2 is highly expressed in embryonic tissue and in many malignant neoplasms including the pancreas, thyroid, lung, and ovary, but rare in normal adult tissues. Previous studies demonstrated that expression of HMGA2 is associated with tumor aggressive behavior, which is probably due to its biologic function related to epithelial-mesenchymal transition. However, whether HMGA2 plays an oncogenic role in the development of ESC remains unclear. In this study, expression status of HMGA2 in ESC and EEC samples was evaluated. Its oncogenic function was tested by overexpression and interference of HMGA2 in endometrial cancer cell lines.METHODS59 Endometial serous carcinoma samples and 61 endometrial endometrioid carcinoma samples were collected to evaluate the expression status of HMGA2 by Immunological Histological Chemistry. Total RNA was extracted from endometrial cancer cells and baseline expression of HMGA2 was tested by real-time PCR. Given that HMGA2 was relatively low expression in SPEC-2 cells, but high is Ishikawa cells. SPEC-2 was selected to generate HMGA2 overexpressed cells by transfecting HMGA2 overexpression plasmid. Expression efficiency was validated by RT-PCR and western-blot. HMGA2 siRNA knockdown was performed in Ishikawa cells. MTT assay and clonogenic assay were performed to tested proliferation in vitro, while transwell assay were performed in vitro to tested invasion and migration. Subcutaneous tumor implantation and tailvein injections in nude mice were performed to tested proliferation, invasion and migration.RESULTSHMGA2 expression rate in full blown endometrial serous carcinoma was 66.1%(39 of 59), nearly 2/3. While the expression rate in full blown endometrial endometrioid carcinoma was lower, only 29.5%(18/61), less than 1/3. SPEC-2 was elected to be the cell line for HMGA2 overexpression and Ishikawa was elected to be the cell line for HMGA2 interfence based on their expression status. The results of MTT assay and clonogenic assay showed that overexpression of HMGA2 significantly promoted proliferation. Transwell assay showed that ability of migration and invasion were increased in endometrial cancer cells after HMGA2 overexpression. Overexpression of HMGA2 promotes tumor growth and metastasis was further proved in vivo by subcutaneous tumor-burdened experiment and the tail vein injection of pulmonary metastases model. After HMGA2 transfected, tumor volume of SPEC-2 cells increased obviously, metastases significantly increased, and there were visible metastases formed to the naked eye.CONCLUSIONHMGA2 is overexpressed in ESC. Functional characterization of HMGA2 revealed that the gene has both tumor growth promotion and metastasis in ESC. In addition, HMGA2 induced epithelial-mesenchymal transition (EMT) through modulation vimentin and p-catenin.Part II. HMGA2:a potential biomarker complement to P53 for detection of early-stage endometrial serous carcinoma.OBJECTIVEOne of the most important players in ESC development, up to 90% of ESC cases showed p53 mutation. It’s the most important event in ESC carcinogenesis. Genetically Engineered Mouse Models (GEMMs) for ESC were built by conditional knockout Trp53 in genitourinary tract epithelium of female transgenic mice. This mice model successfully simulated the progression of human ESC and formed a series of precancer lesions, EmGD, and early stage lesions of ESC, EIC, which were samiliar with human ESC progression. This is a great advance of ESC animal model, which can be a very valuable research instrument for carcinogenesis of ESC. In this study, Trp53 conditional knockout mice model and IHC of normal endometrium, endometrial glandular dysplasia, EmGD, endometrial intraepithelial carcinoma, EIC and ESC samples was evaluated to test the expression status of HMGA2 in ESC progression. Further researching was needed to elevate the valve for HMGA2 in detection of early-stage ESC complement with P53.METHODSTrp53fl/fl mice and Kspl.3-Cre mice were matched to generate+/+; Trp 53fl/f (Wild Type) and Kspl.3-Cre/+; Trp 53fl/f mice. Exons 2-10 of Trp53 gene, the mouse homologue of human TP53, were flanked by loxP in this conditional targeted knockout. Exons 2-10 of Trp53 gene were flanked by loxP in this conditional targeted knockout. Trp53 was conditional knockout in epithelia of the adult genitourinary system. The female mice were euthanized gradually at the age of 20-80 weeks and FFPE uteri were submitted to immunohistochemical analysis. Morphological changes and HMGA2 expression were evaluated in the transgenie mice model. For human sample, normal endometrium, EmGD and EIC samples were collocted to test the expression status of HMGA2 and verify the result from mice model. P53 and HMGA2 expression were conjoint analysised to elvate the function of HMGA2 as a biomarker for early detection of ESCs.RESULTSThe mice with endometrium specific deletion of Trp53 gene initially exhibited precursor lesions of type Ⅱ endometrial carcinomas in humans and later developed carcinomas representing all type Ⅱ subtypes. HMGA2 expression was examined by IHC staining in mice tissue and it was found HMGA2 first appeared expression in 28 week-old mice. The femal mice at 40-60 weeks showed HMGA2 positive was 57.1% (16 of 28), while the femal mice at 60-80 weeks showed HMGA2 positive was 76.9% (10 of 13). HMGA2 expression was also found in EmGD, EIC and endometrial serous carcinomas. These results support the result that a gain of HMGA2 expression is a very early event in endometrial serous carcinomas progression. HMGA2 expression was barely detected in benign endometrium samples (2 of 28). However, HMGA2 expression increased significantly from precancerous lesion endometrial glandular dysplasia (7 of 17,41.2%), to serous endometrial intraepithelial carcinoma (5 of 8, 62.5%) and to full blown endometrial serous carcinoma (39 of 59,66.1%).22 normal endometrium,13 EmGDs,7 EICs and 41 full blown ESC samples were elvated by IHC for HMGA2 and P53. HMGA2 could improve the detection rate of early-stage endometrial serous carcinoma complement to P53 in EmGD and EIC samples.CONCLUSIONThese results support the result that HMGA2 overexpression started from very early stage of endometrial serous carcinoma. HMGA2 could work as a potential biomarker complement to P53 for detection of early-stage endometrial serous carcinoma.Collectively, this study presented analysis of expression status of HMGA2 in ESC, proved that HMGA2 is an early event of ESC. The creative points of the study are reflected in the following aspects:1. HMGA2 is overexpressed in ESCs.2. This study conducted comprehensive in vitro, in vivo and tissue samples experiments to explore that HMGA2 has both tumor growth promotion and metastasis promotion in ESC.3. HMGA2 overexpression was identified as an early event in the process of ESC development, which means it can works as one of the early diagnose biomarker in clinical practice.4. HMGA2 promote matastasis of cancer cells in ESC is implemented by improving Epithelial-Mesenchymal TransitionThe defects and the future directions of our job and study include:1. This study explored that HMGA2 has both tumor growth promotion and metastasis promotion in ESC. But because of the constraints of patients’information, whether HMGA2 has any correlation with poor prognosis was unclear, more research is needed.2. In this paper, the function of HMGA2 in ESC drug sensitivity change still needs further researching.3. This thesis found that HMGA2was overexpressed in some advanced EECs, too, while the positive rate was significantly lower than ESC, but HMGA2 anomalies in the development progression of EEC and HMGA2 expressed in other types of EC situation still needs further research.4. This paper has shown that HMGA2 was an early diagnostic marker combined with P53 to detect early stage ESC. But the number of samples we elvated was still not enough, further expand of experiment range was needed.5. Previous researching has shown that HMGA2 expression could be negative post-transcriptional regulation by microRNA let-7. Whether let-7 can be a targeted therapy drug of ESC needs further research.