Functional And Mechanistic Studies Of HMGA2in The Regulation Of Epithelial-Mesenchymal Transition In Ovarian Cancer

Epithelial ovarian cancer (EOC) is the leading cause of death among women with reproductive tract carcinomas in China. Because signs and symptoms of ovarian cancer are frequently subtle at its early stage,70%of patients are diagnosed at the advanced stage (stage Ⅲ or stage Ⅳ). EOC is characterized by high rate of metastases and resistance to chemotherapy and radiotherapy. At present, its exact cause remains unknown. Several factors are related to ovarian cancer, such as genetically determined susceptibility, hormones, alcohol, etc. Base on pathological and genetic features, ovarian cancers can be classified into Type Ⅰ and Type Ⅱ. Type I EOC (mucinous, clear cell, endometrioid, low-grade serous carcinoma) is characterized by mutations and dysfunction of K-RAS, B-RAF, PTEN. Type Ⅱ tumor (high-grade serous carcinoma) is very aggressive and characterized by mutations of TP53and BRCA1. Because of its high rate of mortality, the functional and mechanistic study on ovarian cancer is always a hot topic in the area of cancer research.HMGA2gene (High Mobility Group AT-Hook2), which is located in12q13-15, consists of five exons and encodes a17kd DNA binding protein. The first three exons encode for three AT-hook domains, which can bind to the minor groove of AT-rich stretches of DNA and introduce structural alterations in chromatin. Therefore, HMGA2protein was called an architectural transcription factor. HMGA2is involved in many biological pathways. For example, HMGA2can promote neural stem cell self-renewal in mice through indirectly repressing p16Ink4a and p19Arf expression. In pituitary adenomas, HMGA2can directly bind to the promoter of CCNB2gene and promote cell proliferation by up-regulating CCNB2expression at the level of transcription. Moreover, HMGA2is also involved in DNA repair processes. It can suppress nonhomologous end joining repair (NHEJ) by impairing the function of DNA-dependent protein kinase. It can also inhibit nucleotide excision repair (NER) and leads to genomic instability by directly inhibiting transcription of ERCC1. HMGA2is abundant in embryonic tissues where cells are proliferating rapidly and undifferentiated. However, it is undetectable in most normal adult tissues. HMGA2is an oncofetal protein and is highly expressed in many malignant cancers, such as lung carcinoma, pancreatic cancer, colorectal cancer, breast cancer, ovarian cancer, liposarcoma and osteosarcoma, indicating that it plays an important role in carcinogenesis and tumor progression.Epithelial-Mesenchymal Transition (EMT) is characterized by the loss of epithelial markers, gain of mesenchymal markers, loss of cell-cell and cell-matrix adhesion, remodeling of cytoskeleton and gain of motility. It is essential for embryonic development, tumor invasion and tumor metastasis. One of the most important steps for EMT is loss of E-cadherin function. Various signal pathways can trigger transition of epithelial cells into mesenchymal-like cells, such as TGF-β, Wnt, MAPK and AKT pathways. Accumulating evidences indicate that HMGA2is associated with EMT in many types of cancers. According to the study by Sylvie et al, in mouse mammary epithelial cells, HMGA2is involved in TGF-β/Smads pathway. Their data showed that HMGA2and Smads could bind to the proximal region of the Snail1promoter and directly activate its transcription. Because Snail1is the transcriptional repressor of E-cadherin, EMT will be elicited by the loss of E-cadherin. In human pancreatic cancer, it is involved in MEK pathway. HMGA2, activated by MEK, could directly bind to Snail1promoter. Subsequently, Snail1activation could cause cells to undergo EMT by repressing the expression of E-cadherin, and stimulating the expression of Vimentin and N-cadherin. In summary, functioning as a transcriptional factor, HMGA2plays an important role in EMT and tumor metastasis.As previously reported, we found that HMGA2was overexpressed in75%of serous tubal intraepithelial carcinoma (STIC) which was a pre-cancer lesion, and overexpressed in70%of high-grade papillary serous carcinoma (HG-PSC) which is invasive and aggressive. We utilized two immortalized ovarian surface epithelial cells (T29and T80) as our cell models to investigate the oncogenic properties of HMGA2in ovarian cancer,Firstly, we showed that HMGA2overexpression could induce cell transformation, render chemoresistance to cisplatin and paclitaxel, and promote cell migration and invasion in ovarian surface epithelial (OSE) cells. After T29and T80cell lines were stably transfected with HMGA2, we used soft agar assay, Matrigel invasion assay, MTS chemoresistance assay, and xenografts assay to characterize HMGA2's oncogenic roles in immortalized OSE cells. Soft agar assay was used to determine whether HMGA2overexpression could result in anchorage-independent proliferation. Matrigel invasion assay was used to determine HMGA2's role in cell migration and invasion. MTS chemoresistance assay was used to gauge whether HMGA2would increase the chemoresistance to two chemotherapy drugs, cisplatin and paclitaxel. Our results indicated that HMGA2overexpression could increase the oncogenic activities of OSE cells in vitro and in vivo.Secondly, we observed that repression of HMGA2expression could impede anchorage-independent cell growth, inhibit cell invasion, reduce cell proliferation and induce morphological transition from mesenchymal-like into epithelial-like cell type. We used let-7c, siRNA, shRNA to knockdown the expression of HMGA2in T29-HMGA2-with UTR cell lines, T29/T80-HMGA2-without UTR cells, SKOV3cells respectively. Then soft agar assay, Matrigel invasion assay and MTS proliferation assay were utilized to investigate oncogenic, invasive and proliferative ability of cell lines after HMGA2knockdown. And our results indicated that oncogenic activities could be reduced after down-regulation of HMGA2expression in ovarian cancer cells.Thirdly, we demonstrated that HMGA2could induce EMT by repressing Lumican expression at the level of transcription. We used gene expression profiling array and cancer-related microRNA array to screen for genes and microRNAs that are targeted by HMGA2. Lumican, which was most severely affected among the11down-regulated genes, was selected for further study. RT-PCR, Western Blot, siRNA transfection, and luciferase assay were used to test whether Lumican was regulated by HMGA2at the level of transcription. Subsequently, stable cell lines with Lumican overexpression and scramble control cells were established by the method of retrovirus infection. Then Matrigel invasion assay was carried out to test whether Lumican was associated with cell migration and invasion. Our results indicated that Lumican, an EMT-associated gene, could be repressed by HMGA2at the level of transcription.Finally, we showed that HMGA2could promote cell migration by inducing STC2expression at the level of transcription. Gene profile analysis also indicated an upregulation of STC2by HMGA2. Immunohistochemical analysis and RT-PCR were employed for measurement of HMGA2and STC2expression in HG-PSC tissues. Luciferase assay was used to determine whether STC2was regulated by HMGA2at transcriptional level. After that, stable knock-down cell lines (Caov-3-sh-STC2) and scramble control cells (Caov-3-pGPU6) were established. Wound healing assay and Transwell migration assay were conducted to examine whether STC2affected cell migration. Survival analysis was used to evaluate the prognostic value of STC2in patients of HG-PSC. Our results showed that HMGA2could promote cell migration by upregulating STC2. STC2could be a predictive factor for prognosis in HG-PSC.In summary, as an architectural transcription factor and an oncogene, HMGA2could induce epithelial-mesenchymal transition by regulating the expression of a series of genes including Lumican and STC2at the level of transcription in ovarian epithelial cells and ovarian cancer cells. PartⅠ HMGA2Overexpression Could Increase the Oncogenic Activities of OSE Cells In Vitro and In Vivo.In order to study HMGA2's oncogenic roles in immortalized ovarian surface epithelial cells, cell lines with stable HMGA2overexpression with (T29A2+) and without (T29A2-and T80A2-)3'untranslated region were established by the method of retrovirus infection. T29A2+cells were used to detect the role of let-7in ovarian cancer. Subsequently, soft agar colony formation assay, Matrigel invasion assay, MTS chemoresistance assay and xenografts assay were used to examine the oncogenic activities of OSE cells, including transformation, invasion and chemoresistance.1. Establishment of HMGA2-overexpressing stable cell lines and scramble control cell lines:(1) Scramble control cell lines stably transfected by pBabe empty vector: T29-pB, T80-pB;(2) Cell lines stably transfected by pBabe-HMGA2-with UTR:T29A2+;(3) Cell lines stably transfected by pBabe-HMGA2-without UTR:T29A2-T80A2-.We observed a significant HMGA2induction in both of T29A2-and T29A2+cells. However, the level of HMGA2expression in T29A2-was much higher than that in T29A2+cells. It indicated that endogenous let-7s were biologically active. They repressed HMGA2in T29A2+cells, but not in T29A2-cells.2. HMGA2overexpression promotes cell anchorage-independent growth:(1) Numbers of colonies formed by T29A2-and T80A2-were higher than those byT29, T80, T29-pB, T80-pB cells;(2) Number of colonies formed by T29A2+was higher than those by T29,T80, T29-pB, T80-pB cells;(3) The number of colonies formed by T29A2+was half of that by T29A2-.3. HMGA2overexpression promotes cell migration and invasion.Matrigel invasion assay showed that T29A2-and T80A2-cells were more invasive and migratory than T29and T80cells.4. HMGA2overexpression increases chemoresistance to cisplatin and paclitaxel in OSE cells.5. HMGA2overexpression increases the oncogenic activities of OSE cells in vivo.(1) The rates of tumor formation in T29A2-and T80A2-cells were higher than those in T29and T80cells.(2) T29A2-and T80A2-tumors showed increased immunoreactivity for Vimentin and reduced immunoreactivity for E-cadherin.6. HMGA2overexpression impacts expression of genes involved in EMT.(1) Vimentin and N-cadherin were up-regulated in T29A2-and T80A2-cells.(2) Vimentin and N-cadherin were down-regulated in SKOV3-shHMGA2cells.(3) E-cadherin was down-regulated in T29A2-and T80A2-cells.(4) E-cadherin were up-regulated in SKOV3-shHMGA2cells.In this part, our results indicated that HMGA2overexpression could increase the oncogenic activities of OSE cells in vitro and in vivo.Part II Repression of HMGA2Could Reduce the Oncogenic Activities of OSE Cells in VitroIn this part, we used three different types of HMGA2-interfering molecules, let-7c, siRNA and shRNA, to test the oncogenic effect of HMGA2on different ovarian surface epithelial cells and ovarian cancer cells. Soft agar assay, matrigel invasion assay, MTS proliferation assay were utilized to test oncogenic, invasive, proliferative abilities of cell lines with repressed HMGA2.1. Generation of OSE cells and ovarian cancer cells with downregulated HMGA2:(1) Transient transfection of T29A2-and T80A2-cells by siRNA targeting HMGA2(2) Transient transfection of T29A2+cells by let-7c(3) Stable transfection of SKOV3cells by pGIPZ-sh-HMGA2: SKOV3-sh-HMGA2.2. HMGA2repression impedes anchorage-independent cell growth in OSE cells and ovarian cancer cells:(1) Numbers of colonies were significantly reduced when T29A2-and T80A2-cells were treated by HMGA2-siRNA.(2) Compared with control siRNA and anti-let-7, numbers of colonies were significantly reduced when T29A2+cells were treated by let-7c.3. HMGA2repression inhibits cell invasion in OSE cells and ovarian cancer cells: matrigel invasion assay showed that migration was significantly inhibited in T29A2-cells treated by HMGA2-siRNA, in T29A2+cells treated by let-7c and in SKOV3-sh-HMGA2cells.4. HMGA2repression inhibits cell proliferation in OSE cells and ovarian cancer cells: Cell proliferation was inhibited in T29A2-cells treated by HMGA2-siRNA, in T29A2+cells treated by let-7c and in SKOV3cells stably expressing sh-HMGA2.5. Cell morphology was changed from mesenchymal-like into epithelial-like type when SKOV3cells were stably transfected by sh-HMGA2.Taken together, these results indicated that repression of HMGA2expression could reduce the oncogenic activities of OSE cells in vitro.Part Ⅲ HMGA2Could Induce EMT by Repressing Lumican at the Level of TranscriptionTo gain insight into the mechanism by which HMGA2promotes tumorigenesis, gene expression array and cancer-related microRNA array were used to identify the genes and microRNAs targeted by HMGA2in T29/T29A2-and T80/T80A2-cells.1. A group of microRNAs are dysregulated by HMGA2overexpression.(1)18microRNAs are up-regulated by at least a2-fold change, including let-7a, miR-126, let-7c and miR-193b. (2)4microRNAs are down-regulated by at least a2-fold change, including miR-29b, miR-18a, miR-15a, and miR-22.2. MiR-29b expression is negatively correlated with HMGA2expression in HG-PSC and normal ovarian tissues.(1) HMGA2is up-regulated in HG-PSC, but down-regulated in normal ovarian tissues.(2) MiR-29b is down-regulated in HG-PSC, but up-regulated in normal ovarian tissues.(3) MiR-29b expression is inversely correlated with HMGA2expression in HG-PSC and ovarian tissues by RT-PCR assay.3. A group of genes are dysregulated by HMGA2overexpression.(1)11genes are down-regulated by at least a2-fold change, including Lumican and WNT2.(2)25genes are up-regulated by at least a2-fold change, including STC2and CA9.4. Lumican, which is significantly downregulated in T29A2-and T80A2-cells, was further evaluated for its role in EMT.(1) Lumican expression is significantly higher in293T-shHMGA2and SKOV3-shHMGA2cells than in293T-pGIPZ and SKOV3-pGIPZ cells.(2) Lumican expression is inversely correlated with HMGA2expression in six ovarian surface epithelial cells and ovarian cancer cells, including T29, T29A2-, T29H, HEY, SKOV3, and OVCAR3cells.(3) Luciferase assay shows that Lumican could be transcriptionally down-regulated by HMGA2, and the region from+1to-800of Lumican promoter contained regulatory sequences by HMGA2.(4) Stable cell lines with Lumican overexpression were established by the method of retrovirus infection:HEY-Lumican and scramble control cells HEY-pBabe.(5) The number of migrating cells in HEY-Lumican cells was significantly lower than that in HEY-pBabe cells.(6) Compared with normal ovarian surface and fallopian tube epithelia, Lumican expression is down-regulated in HG-PSC tissues by immunohistochemical analysis.In this part, our results indicated that HMGA2could induce EMT by repressing Lumican expression at the level of transcription.Part IV HMGA2Could Promote Cell Migration by Upregulating STC2Expression at the Level of TranscriptionSTC2was upregulated in HMGA2overexpressing cells. In order to investigate the relationship between HMGA2and STC2, and to determine the oncogenic roles of STC2in ovarian cancer, STC2was chosen for further validation by RT-PCR, Western Blot, siRNA transfection, and luciferase assay. Subsequently, stable cell lines with STC2knockdown were established. Survival analysis was used to evaluate the prognostic value of STC2in patients of HG-PSC.1. STC2expression was positively correlated with HMGA2expression in HG-PSC tissues.2. Compared with that in T29and T80cells, STC2expression was significantly increased in T29A2-and T80A2-cells.3. STC2was up-regulated by HMGA2at the level of transcription.(1) Compared with control siRNA, HMGA2and STC2expression were reduced when SKOV3cells were treated by HMGA2-siRNA.(2) Compared with control siRNA and HMGA2-siRNA, HMGA2and STC2expression were significantly increased when SKOV3cells were treated by anti-let-7.4. Luciferase assay showed that STC2could be transcriptionally up-regulated by HMGA2and the region from-647bp to-290bp of STC2promoter contained regulatory sequences by HMGA2.5. Stable cell lines with STC2knockdown (Caov-3-sh-STC2) and scramble control cell lines (Caov-3-pGPU6) were established. 6. Cell morphology was changed from mesenchymal-like into epithelial-like type when Caov-3cells were stably transfected by sh-STC2.7. Compared with control Caov-3-pGPU6cells, Caov-3-sh-STC2cells showed evident delays in wound healing.8. Migrating ability of Caov-3-sh-STC2cells was significantly lower than that of Caov-3and Caov-3-pGPU6cells.9. Stable knock-down of STC2decreases the oncogenic activities of Caov-3cells in vivo.(1) The rate of tumor formation in Caov-3-sh-STC2cells was higher than those in Caov-3and Caov-3-pGPU6cells.(2) Caov-3-sh-STC2cells shows significant regression of tumor growth and reduction in tumor weight.10. Survival analysis showed that high STC2expression was positively correlated with poor outcome of patients with HG-PSC.In this part, our results indicated that HMGA2could promote cell migration by upregulating STC2expression at the level of transcription.