Screening The Liver Metastasis Functional Genes Of Serous Ovarian Cancers By Combining Laser Capture Microdissection With CDNA Microarray And Further Investigating Molecular Mechanism Of Selected Targets

Background and ObjectiveOvarian cancer is one of the most threatening malignant tumors in females due to the frequent occurrence of metastasis that precedes diagnosis. The morbidity is in the second place and the mortality is in the first one. Clinical characteristics of ovarian carcinoma include no particular symptoms in the early stage, delitescent pathogenetic condition and difficult finding. 70%-80% patients belong to clinical advanced stage (stageâ…¢ or stage â…£) when finally diagnosed. When the volume of primary lesion is not large at all (clinical early stage), it is capable of generating general dissemination metastasis in pelvis cavity as well as abdominal cavity, and frequently accompanied with ascites. Ovarian serous papillary cystadenocarcinomas posses the characteristics of the frequent occurrence of metastasis, especially liver metastasis that precedes diagnosis. Abdominopelvic cavity dissemination and lymphatic metastasis of post-peritoneal membrane are the major diffusion and metastasis pathways of ovarian cancer, and also are the most important and frequent reasons for unpleasant therapeutic efficacy in cancer patients. Therefore, investigating the invasion and metastasis mechanisms of ovarian serous papillary cystadenocarcinoma and searching more effective therapeutic targets become more and more enthusiastic in molecular oncologic research fields. In order to elevate the availability of molecular targeted therapy in ovarian cancer, it is important to analyze ovarian malignant phenotype gene correctly and select the best candidate medicine targets. It had been reported that the functional factors such as growth factor bFGF, cytokines (TNF-a and IL-8), VEGF, cell adhesive molecules (CD44H, E-cadherin, Integrin avb3 and a5b1), laminin receptor, ECM proteolysis erzymes(u-PA/u-PAR, MMPs), CXCL12/CXCR4 and p53 participated the recurrence and implantation metastasis to abdominal cavity of ovarian cancer. In the recent years experimental investigations were reported that some molecules above resisted recurrence and metastasis of ovarian cancer. Morever, overall therapeutic effects were still unsatisfactory. There might be three causes: 1. the effective factors for controlling tumor recurrence and metastasis determined by cultured tumor cell lines in vitro did not truly reflect the practical conditions of complicated microenvironment of tumor in vivo; 2. the research materials obtained through traditional tissue purification methods were applied and the informations obtained could not literally reflect the interactions between metastatic cancer cells and proximal cells or extracellular matrix; 3. tumor metastasis involved in multiple genes and the genetic background of metastatic cancer cells were different, so the process of metastasis appeared were not accomplished at the same time. Laser capture microdissection (LCM) is a novel technique for tissue purification that can be generally applied in genomics and proteomics fields. LCM can in situ obtain pure targeted cell subgroup or even a single cell quickly and precisely under the microscope, thus the problem of tissue heterogeneity in molecular analysis can be tackled successfully. Because LCM can provide pure targeted cells literally reflecting the actual conditions in vivo, the molecular events appeared in physiological and pathological process were actually researched. cDNA microarray is another novel technique that can simultaneously analyze gene expressions of thousands of genes, even full genome. According to the basis above, LCM was used to in situ harvested the homogeneous ovarian cancer cells of primary sites from frozen ovarian serous cystadenocarcinoma membrane sections and its matched cancer cells from frozen liver metastases tissue sections. The small amount of total RNA was extracted using the RNAqueous micro kit and the purified RNA was linearly amplified in vitro. The 25-mer oligonucleotide microarrays including 19 thousand genes with high through input and efficiency was primarily applied to screen the functional gene clusters differentially expressed by analyzing the gene expression profiles between microdissected tumor cells from primary sites and liver metastases of ovarian cancer. The most interesting findings are that metastasis inhibitor CD9 and regulatory factor of vesicle trafficking Rab25 were over-expressed in liver metastases, thus CD9 and Rab25 were selected to identify their mRNA expressions in ten fresh clinic specimens from matched-pairs of metastatic serous ovarian carcinomas and four matched-pairs of tumor cell lines with high and low metastasis potentials for elevating the correlation between CD9 and Rab25 expression and tumor metastasis. Because the chief condition of determining the best candidate pharmaceutical targets for anti-metastasis of ovarian cancer is the efficacy of molecular targets, their functions of CD9 and Rab25 genes were further investigated. Based on the prevalence of altered CD9 and Rab25 expression and function in different metastases of ovarian cancers, it was of interest to consider the therapeutic potential of controlling CD9 and Rab25 function or their regulated pathways through pharmacologic or genetic modulation.MethodsThe tumor cells in frozen sections were fixed and stained by rapid hematoxylin and eosinophilic staining. Homogeneous ovarian cancer cells of primary sites were obtained from frozen ovarian serous cystadenocarcinoma membrane sections and its matched cancer cells from frozen liver metastases tissue sections by using LCM. The small amount of total RNA was extracted using the RNAqueous micro kit and was linearly amplified in vitro. The 25-mer oligonucleotide microarrays including 19 thousand genes were then hybridized against the cDNA probe mixture with high through input and efficiency and the fluorescent signals were scanned. The obtained data were analyzed using Feature Extraction software. CD9 and Rab25 mRNA expression of ten fresh clinic specimens from matched-pairs of metastatic serous ovarian carcinoma and four matched-pairs of tumor cell lines with high and low metastasis potentials were detected by fluoresce quantitative real time PCR (qPCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of MRP-1/CD9 and E-cadherin were detected by immunohistochemistry Envision^TMtwo steps method and semiquantitative analysis in 32 cases of advanced serous ovarian carcinoma complicated with different pelvic cavity, abdominal cavity, vessels (blood vascular and lymphatic included) metastasis and 14 cases of normal ovarian tissues; the clinical pathological factors were also analysed. CD9 and Rab25 full-length eukaryotic expression vectors were constructed using the technique of gene engineering; the ovarian carcinoma and breast carcinoma cell clones stable expressing CD9 and Rab25 were screened by G418. After transfection, the changes of cell proliferation and motility were detected by CFSE-flow cytometry and monolayer wound healing assay, respectively. The changes of AKT, P-AKT,E-cadherin 6-catenin and Bcl-X/L expression in A2780/Rab25 and A2780/Rab25B cell clones were detected by RT-PCR and western blotting. The capabilities of cell growth, cell motility, in vitro invasion and cell adhesion in A2780/Rab25 and A2780/Rab25B cell clones were performed by MTT assay, monolayer wound healing assay, Transwell migration assay and cell adhesion assay, respectively. Laser co focal microscopy imaging system was used to find the effect of high expression of Rab25 and Rab25B on the cytoskeletons of A2780 cell clones. Transmission electron microscope was used to watch the changes of ultramicrostructure in A2780/Rab25 and A2780/Rab25B cell clones. The effects of over-expression of Rab25 and Rab25B on metastasis phenotypes of ovarian cancer were surveyed by athymic mouse animal model of ovarian cancer.Results1. LCM combined with RNA linear amplification in vitro can successfully be applied to obtain pure, sufficient and intact RNA for the further research of gene expression, which is a feasible technique line for procuring high-quailty RNA.2. 458 functional genes differentially expressed were obtained by comparing gene expression profiles of the primary sites of serous ovarian cancer with those of the liver metastases. The most interesting findings are that metastasis inhibitor CD9 and the important mediator of vesicle transport Rab25 were over-expressed in liver metastases of ovarian cancer.3. mRNA expression of CD9 and Rab25 increased (at least 7-fold) in different metastases of abdominal cavity compared with the primary sites of ovarian carcinomas; and their mRNA levels were significantly higher in ovarian carcinoma cell line A2780 than in Skov-3. In contrast, CD9 mRNA expression was notably down-regulated in tumor cell lines with high metastasis potentials such as MDA-MB-231, PC-3M-1E8 and PG-BE1 compared with lowly metastatic cancer cell lines MCF-7, PC-3M-2B4 and PG-LH7; The expression levels of Rab25 mRNA were obviously increased in PC-3M-1E8 and PG-BE1, but decreased in MDA-MB-231. For the fist time we found a novel variant of Rab25 gene, named as Rab25B, and then constructed CD9, Rab25, Rab25B full-length eukaryotic expression vector successfully, and obtained ovarian carcinoma and breast carcinoma cell clones which stably expressed CD9, Rab25 and Rab25B. These results indicated that abnormal-expression of CD9 and Rab25 gene closely correlated with neoplasm metastasis. Morever their over-expression promoted cancer metastasis in ovrian cancers.4. This study found the tumor cells present in primary sites, different metastases and vessels of ovarian cancers in a special clump form. The special phenomenon in this study was called as "conglomerate infiltration and metastasis of tumor cells or collective movement of tumor cells". In different metastases of advanced serous cystadenocarcinoma, CD9 expression mainly localized to cytoplasm and its levels significantly up-regulated compared with the primary sites as well as correlated well to tumor grade, tumor clinical stage and prognosis. These results indicated that CD9 may involve in ovarian cancer invasion and metastasis through the special cluster movement form of tumor cells mediated by E-cadherin.5. After transferring CD9 gene into human ovarian carcinoma cell lines SKOV-3, CD9 increased the abilities of cell proliferation and motility of SKOV-3 cells, and these functions might be regulated by activating PI4K/AKT intracellular signaling pathway.6. Forced Rab25 expression made AKT phosphorylated at Ser473 site and regulated the recycling of internalized E-cadherin. Because of changing the function of E-cadherin/catenin complex retaining cell polarity and tissue structure, Rab25 over-expression contributed to aggressive progression of human ovarian cancer cell lines A2780. By contrast, over-expression of Rab25B reversed the malignant phenotypes of A2780 cell lines.7. Animal in vivo experiments indicated that forced Rab25 expression improved tumor growth of ovarian cancer cells and contributed to implantation metastasis of abdominopelvic cavity and distant metastasis to mediastinum thoracis. By contrast, stable Rab25B expression reversed the metastasis phenotypes of A2780 cell lines.ConclusionLaser capture microdissection was used to in situ harvested the homogeneous ovarian cancer cells of primary sites from frozen ovarian serous cystadenocarcinoma membrane sections and its matched cancer cells from frozen liver metastases tissue sections. The small amount of total RNA was extracted using the RNAqueous micro kit and the purified RNA was linearly amplified in vitro. The 25-mer oligonucleotide microarrays including 19 thousand genes with high through input and efficiency was applied to analyze the gene expression profiles of microdissected tumor cells from liver metastases and primarily screened the functional genes differentially expreesed of ovarian cancer metastasis to liver. Metastasis inhibitor CD9 and regulatory factor of vesicle trafficking Rab25 over-expressed in liver metastases were selected to further investigate.Identification of CD9 and Rab25 mRNA expression in ten fresh clinic specimens from matched-pairs of metastatic serous ovarian carcinoma and four matched-pairs of tumor cell lines with high and low metastasis potentials showed that abnormal-expression of CD9 and Rab25 gene closely correlated with neoplasm metastasis, moreover, in ovarian cancers CD9 and Rab25 over-expression promoted cancer metastasis. The first findings during identification were as follows: one was that the new variant of Rab25 gene, named as Rab25B in this study, produced the loss of second exon 196 base pair determining the function of Rab25 gene, the other was that CD9 expression not lost but significantly re-overexpressed in liver metastases compared with the primary sites as well as correlated well to tumor grade, tumor clinical stage and prognosis and then its expression mode was the same as that of E-cadherin.To further investigate the molecular mechanism of CD9, Rab25 and Rab25B in ovarian cancer invasion and metastasis, the present studies showed that forced CD9 expression enhanced the capabilities of cell growth and motility of Skov-3 cells through up-regulating the PI4K/AKT signaling; Over-expression of Rab25 gene made AKT phosphorylated at Ser473 site and regulated the recycling of internalized E-cadherin. Because of changing the function of E-cadherin/catenin complex retaining cell polarity and tissue structure, Rab25 over-expression contributed to aggressive progression of human ovarian cancer cell lines A2780. In contrast, over-expression of Rab25B reversed metastasis phenotypes of A2780 cell lines. Animal in vivo experiments indicated that forced Rab25 expression improved tumor growth of ovarian cancer cells and contributed to implantation metastasis of abdominopelvic cavity and distant metastasis to mediastinum thoracis. By contrast, stable Rab25B expression reversed the metastasis phenotypes of A2780 cell lines.In summary, CD9 may involve in ovarian cancer metastasis in the special collective movement form mediated by E-cadherin. The role of Rab25 gene in ovarian cancer metastasis was contradictory to Rab25B and the second exon of Rab25 gene was the important function domain playing a great role in facilitating tumor metastasis. Thus CD9 and Rab25 were novel and effective target molecules for therapeutic invention of invasion and metastasis, especially, the second exon of Rab25 gene was the more precise function domain for anti-invasion and anti-metastasis of ovarian cancers.