Tumor Heterogeneity In Recurrent Ovarian Cancer As Demonstrated By Polycomb Group Proteins Expression

BackgroundUnequivocally, ovarian cancer is the leading cause of death from gynecological malignancies, and recurrence is the major reason that the5-year survival for ovarian cancer remains around30%. Recent researches have put forward a challenge to the dogma that cancer is a homogeneous population of a malignantly transformed cell. A new concept has been suggested, that is tumor heterogeneity, referring to tumor evolution and adaptation during tumor progression resulting in different sub-clones of cells within a single tumor. Tumor heterogeneity makes the personalized medicine required for the treatment of cancer. Polycomb group (PcG) proteins not only are essential for the normal development of multicellular organisms, but also play an important role in tumorigenesis. Its close relation to tumor expansion, metastasis, recurrence, as well as chemoresistance provides great potential for PcG proteins to be the molecular biomarkers of tumor heterogeneity in ovarian cancer.Purposes1. To determine whether PcG proteins can be used as molecular biomarkers of tumor heterogeneity during the recurrence of ovarian serous cystadenocarcinoma.2. To verify the correlation between PcG proteins expression and the prognosis of patients with ovarian serous cystadenocarcinoma.3. To investigate whether the heterogeneous expression of PcG proteins could induce functional heterogeneity in epithelial ovarian cancer cells.4. To explore the potential mechanisms involved in the heterogeneous expression of PcG proteins, and search for poteinal targets for the treatment of recurrent ovarian cancer.Materials and Methods1. Tissue microarrays including matched primary sample, abdominal disseminated sample, lymphatic metastatic sample and recurrent sample from the same patient with ovarian serous cystadnocarcinoma were constructed. Corresponding clinical information of patients with ovarian serous cystadnocarcinoma was collected. Immunohistochemistry was used to examine PcG proteins expression in each of the four sites. For evaluation of the staining, a semi-quantitative scoring criterion was used, in which both staining intensity and positive areas were recorded. Proteins showing significant different expression levels among these four sites were picked out. Correlation analyses were performed between expression of proteins and clinicopathological features, and survival analyses were carried out to evaluate the impact of proteins expression on survival before recurrence and overall survival of patients with ovarian serous cystadenocarcinoma.2. Real-time PCR and Western Blot were used to detect the expression of PcG genes at both mRNA and protein levels in seven serous ovarian cancer cell lines, and cell models with high level and low level of PcG proteins expression were established. MTT assay and flow cytometry were adopted to detect the impact of PcG proteins expression on proliferation and cell cycle of serous ovarian cancer cells. Wound healing assay and Transwell insert assay were performed to detect the difference in migration and invasion ability. MTT assay was used to detect the effects of PcG proteins on the sensitivity to chemotherapeutics such as cisplatin and taxol. Colony formation assay was performed to observe the ability of independent survival.3. Fluorescence in situ hybridization was utilized to examine the amplification of PcG core genes, BMI1and EZH2, in matched primary tumors and recurrent lesions from a single patient with ovarian serous cystadennocarcinoma. miRNA chip was used to compare the difference in miRNA profile between primary and recurrent tumors. miRNAs showing significantly different expression level were verified by real-time PCR in enlarged samples, and their potential targets were predicted through bioinformatic softwares.Results1. PcG proteins expression presented with an increasing tendency during the dissemination, metastasis and recurrence of ovarian serous cystadenocarcinoma. PcG proteins could be used as molecular biomarkers of tumor heterogeneity in ovarian cancer. EZH2and BMI1expression in lymph node metastases, RING1and CBX2expression in abdominal dissemination, could be used as independent prognostic parameters for survival before recurrence. EZH2expression in lymph node metastases, BMI1and PHF1expression in recurrent lesions could be used as independent prognostic parameter for overall survival.2. Serous ovarian cancer cells with high expression of PcG proteins showed stronger ability of proliferation, migration, invasion, resistance to chemotherapy, and independent survival compared with their counterparts with low expression of PcG proteins.3. Amplification of BMI1and EZH2could be observed in the recurrent lesions compared with their matched primary tumors, in thirteen and sixteen patients, respectively, with intensive expression of proteins by immunohistochemical staining simultaneously. miRNA chip results indicated that there were8human miRNAs (hsa-miR-514b-5p, hsa-miR-4261, hsa-miR-298, hsa-miR-3147, hsa-miR-149*, hsa-miR-3621, hsa-miR-3676, hsa-miR-425*) showing significantly different expression levels between primary tumors and recurrent lesions. miR-298å'ŒmiR-4261might regulate the expression of EZH2through transcriptional factors ILF3and ZNF207.Conclusions1. PcG proteins could be used as molecular biomarkers of tumor heterogeneity in ovarian serous cystadenocarcinoma.2. Several PcG proteins could be used as independent prognostic parameters for the survival before recurrence and overall survival in patients with ovarian serous cystadenocarcinoma.3. Expression heteroneity of PcG proteins could induce functional heterogeneity of epithelial ovarian cancer cells.4. Elevation of PcG proteins expression in recurrent tumor lesions could be the results of both gene amplification and miRNA epigenetic regulation.5. Tumor heterogeneity necessitates personalized medicine in the treatment of recurrent ovarian cancer, and PcG genes, as well as miR-298and miR-4261have great potiental to be the specific targets.