The Underlying Effects Of DNA Damage Repair Related Genes MDC1 And YAP1 In Ovarian Cancer

Ovarian cancer is the most lethal cancer of the gynecologic malignancies, largely due to the advanced stage at diagnosis in most patients and high rate of relapse. Even with decades of research, the overall survival rate does not improve a lot. Still, the underlying molecular mechanisms responsible for its onset and malignant behavior, remains unclear. As a result, effective potential candidates for early screening, as well as molecular targeting in the treatment, are in great need. We believe finding them is the key point to improve the prognosis of ovarian cancer.DNA is continuously damaged by genotoxic agents generated intracellularly or in the environment, such as radiation and chemicals. In normal cells, the integrity of the genome is ensured by the efficient DNA damage repair network. And cancer cells could arise through the accumulation of the genetic alterations. However, dysregulation of DNA repair related genes also affect the response of cancer cells to DNA damaging anti-cancer therapies. In other word, upregulation of DNA repair gene can cause resistance to chemotherapy.Based on the pathological and genetic data, ovarian carcinomas are classifies into 2 groups:Type Ⅰ and Type Ⅱ. Type Ⅰ tumors are not as aggressive as Type II, and usually present at early stage. While Type Ⅱ tumors, frequently present at an advanced stage, and tend to display poor prognosis. Previously, we selected ovarian carcinomas specimen with different types and performed proteomics analysis. The different expressed protein were analyzed and further validated. MDC1 and YAP1 were among them. In the present study, we performed related tumor biological assays to investigate the underlying effect of MDC1 and YAP1 on the progression of ovarian cancer. The discovery of the role of MDC1 and YAP1 in this field would definitely help us understand better of the molecular mechanism of ovarian cancer.PART ⅠThe role of DNA damage repair related gene MDC1 in ovarian cancerBACKGROUND AND OBJECTMDC1 plays important role in DNA damage repair network, which turns to be a hot field of tumor biology. On one side, many classical genes that participate in the DNA damage repair, such as p53 and BRCA1, are typical tumor suppressors. Their efficient response ensure the integrity of the genome. On the other, high expression of these genes also protect the cancer cells from chemotherapy drugs. Since DNA damage response is so much correlatedwith carcinogenesis and chemotherapy resistance, MDC1, as well as the other DNA damage repair genes, is drawing more and more attention in the field of cancer.Previous studies first advocated that MDC1 was a protein with oncogenic potential in both esophageal and cervical cancers. Afterward, MDC1 expression was observed to have a strong trend with tumor grade and chemotherapy treatment status in ovarian cancer. However, the role of MDC1 in ovarian cancer, especially its effect on metastasis, remains unknown.We investigate the role of MDC1 in the initiation and progression of ovarian cancer. And we believe MDC1 can be a potential candidate for tumor screening, as well as the molecular target in the treatment of ovarian cancer.METHODSTo investigate the role of MDC1 in ovarian cancer, we performed immunohistochmical staining assay to detect the expression of MDC1 in ovarian cancer patients’ tissues. The prognosis data was analyzed to detect the correlation between MDC1 expression level and it. By gene overexpression and knock out, we conduct in vitro and in vivo experiments to study MDC1 related cellular behavior and potential Epithelial-mesenchymal transition (EMT) mechanism.RESULTS1. High MDC1 expression correlates with poor prognosis of ovarian cancer patientsTo investigate the expression and clinical significance of MDC1 in human ovarian cancer, we performed immunohistochemistry to measure MDC1 expression in 130 paraffin-embedded ovarian cancer tissues. Patient characteristics of the population are summarized. High level expression of MDC1 was related to patients’ ages, while no statistical associations were observed between the expression of MDC1 and histological type, CA125 serum level or lymph node metastasis. Then, we analyzed the association between MDC1 level and patient overall survivals and found that high MDC1 expression was significantly associated with poor overall survival (P=0.0052).2. Generation of stable ovarian cancer cells with MDC1 overexpression or knockdownIn order to conduct in vitro and in vivo experiments to study the role of MDC1 in ovarian cancer, we established stable cell lines with MDC1 overexpression or knockdown. We first measured baseline expression level of MDC1 in six ovarian cancer cell lines by Western Blot. MDC1 was highly expressed in most of these cell lines (HEY, HO8910PM, OVCAR3 and A2780), and was relatively low expression in HO8910 cells and SKOV3 cell lines. So we chose HO8910 to generate MDC1 overexpressed cells (MDC1-OVE-HO8910), HO8910PM and OVCAR3 to generate knocked down stable cell lines (shMDC1-HO8910PM and shMDCl-OVCAR3). Western Blot was performed to prove the successful generation of these three cell lines.3. MDC1 promotes migratory and invasive behaviors in ovarian cancer cellsTo test whether MDC1 promotes tumor cell migration and invasion in vitro, the migration and invasion were then measured by transwell assay in our three cell lines with MDC1 altered expression. When compared with control cells, the MDC1-OVE-HO8910 line was highly invasive, while shMDC1-HO8910PM and shMDCl-OVCAR3 exhibited reduced migration and invasion. These data imply that MDC1 promotes migration and invasion of ovarian cancer cell lines in vitro.4. MDC1 induces epithelial-mesenchymal transition (EMT) through modulation EMT markersWe observed morphology changes in MDC1 knockdown cells compared to control cells. MDC1-OVE-HO8910 cells exhibited relatively fibroblastic morphology while shMDCl-HO8910PM cells exhibited epithelial morphology compared to their control cells.To further confirm, we measured the expression level of EMT markers using Western Blot. The results showed that E-cadherin increased, accompanied by down-regulation of N-cadherin and Vimentin in shMDC1-OVCAR3. While in MDC1 overexpression cell line MDC1-OVE-HO8910, vimentin was increased and there were no obvious changes of E-cadherin and N-cadherin. We further measured expression of of N-cadherin by immunofluorescence assay and found immunofluorescent signal was reduced in MDCl knockdown cells compared to control cells. All these results support that MDC1 promotes metastasis by inducing EMT in ovarian cancer.5. Knockdown of MDC1 inhibits tumor metastasis of HO8910PM cells in vivoTo evaluate the effect of MDCl on ovarian cancer metastasis in vivo, NC-HO8910PM and shMDCl-HO8910PM cells were injected into nude mice through the tail veins. As expected, the lungs from mice injected with shMDCl-HO8910PM cells (n=6) developed less metastatic foci than the control group (n=6). This result gives a support that MDCl promotes metastasis of ovarian cancer.6. MDC1 has no effect on cell cycle and apoptosisAfter MDC1 siRNA treated, cancer cells showed no change in cell cycle detected by Flow Cytometry (FCM), which meant MDCl was not involved in cell cycle regulation of ovarian cancer cells. Ovarian cancer cells also did not show increased apoptosis cell numbers detected by Flow Cytometry (FCM) after MDC1 siRNA treated, which meant MDC1 was not involved in apoptosis of ovarian cancer cells.7. MDC1 is not responsible for CDDP and ADR drug sensitivityAfter MDC1 was interfered by siRNA, CDDP and ADR treated ovarian cancer cells did not decrease in their viabilities in MTT assay.CONCLUSIONS1.High MDC1 expression correlates with poor prognosis of ovarian cancer.2. MDC1 promotes ovarian cancer metastasis by inducing EMT.3. MDC1 has no effect on cell cycle and apoptosis, as well as CDDP and ADR drug sensitivity.PART ⅡThe role of DNA damage repair related gene YAP1 in ovarian cancerBACKGROUND AND OBJECTHippo signaling pathway plays important roles in controlling organ sizes. To achieve this, the signaling pathway is tightly involved in cellular apoptosis and proliferation regulation. YAP1, as a pivotal downstream transcription co-activator in the Hippo pathway, also effects in the same field. YAP1 is wildly expressed in organs and tissues, while its functions domains of YAP1 are conservative. Many studies reported high expression of YAP1 in malignancies, and YAP1 overexpression promotes tumor progression. YAP1 also behaves as a DNA damage repair related gene. As transcription co-activators, YAP family assist DNA damage repair, and may be related with clinical chemotherapy resistance.The study would be much helpful in revealing the molecular mechanism of tumor growth. In this way, we may find a new therapeutic target for ovarian cancer clinics.METHODSWe performed immunohistochmical staining to detect the expression of YAP 1 in ovarian cancer patients’ tissues. The prognosis data was analyzed to explain the correlation between YAP1 expression level and the prognosis. We established stable cell lines with YAP1 overexpression or knockdown and conducted related in vitro and in vivo experiments to study the proliferation behavior. We also detect the CDDP sensitivity with MTT assay in our stable ovarian cancer cell lines. To scan the downstream target gene of YAP1, we performed RT-PCR to detect the related gene expression level of stable cell lines with YAP1 overexpression or konckdown.RESULTS1. High nuclear YAP1 expression correlates with poor prognosis of ovarian cancer patientsTo investigate the expression and clinical significance of YAP 1 in human ovarian cancer, we performed immunohistochemical staining to measure YAP1 expression in 76 paraffin-embedded ovarian cancer tissues. We found expression both in the cytoplasm and the nucleus. Patients with high nuclear YAP1 level tend to have poor prognosis. Then, we analyzed the association between nuclear YAP1 level and patient overall survivals and found that high nuclear YAP1 expression was significantly associated with poor overall survival (P=0.0163). However, cytoplasmic YAP1 expression was not associated with the overall survival (P=0.7143). When taking both the nuclear YAP1 and cytoplasmic YAP1 both into consideration, the YAP1 expression was signigicantly associated with a poor prognosis (P=0.0001).2. High YAP1 level in ovarian cancerWe used IHC and Western Blot to detect YAP1 expression in ovarian cancer tissue compared to normal fallopian tube tissue. YAP1 expression level was high in ovarian cancer tissue. We Western Blot to detect YAP1 expression in ovarian cancer cell lines compared to normal fallopian tube cell line (FTE187). YAP1 expresssion level was high in ovarian cancer cell lines and low in FTE187.3. Generation of stable ovarian cancer cells with YAP1 overexpression or knockdownIn order to conduct in vitro and in vivo experiments to study the role of YAP1 in ovarian cancer, we established stable cell lines with YAP1 overexpression or knockdown. We first measured baseline expression level of YAP 1 in ovarian cancer cell lines by Western Blot. And we chose A2780 and SKOV3 to generate YAP1 overexpressed cells YAP1-OVE-A2780 and YAP1-OVE-SKOV3, HO8910 and HEY to generate knocked down stable cell lines (shYAPl-HO8910 and shYAP1-HEY). Western Blot was performed to prove the successful generation of these four cell lines. 4. YAP1 promotes proliferation in ovarian cancer cellsTo test whether YAP1 promotes proliferation, MTT assays were performed in ourfour cell lines. When compared with control cells, the YAP1-OVE-A2780 and YAP1-OVE-SKOV3 line showed high proliferation rate, while shYAP1-HO8910 and shYAP1-HEY exhibited reduced rate. Clone forming assays were also performed to prove that. These data imply that YAP1 promotes proliferation of ovarian cancer cell lines. 5. YAP1 reduced sensitivity of ovarian cancer cells to CDDPWe evaluated the effect of CDDP on ovarian cancer cells and discovered lowsensitivity in YAP1 overexpression ovarian cancer cells. We further performed Western Blot to detect related cell signaling pathway, only to find the role of YAP 1 in reduced CDDP sensitivity may be related to up-regulation of pJNK and down-regulation of pATF2.6. YAP1 downstream target gene scanTo scan the downstream target gene of YAP1, we performed RT-PCR to detect the related gene expression level of stable cell lines with YAP1 overexpression or konckdown. We found ATF2, RAD51AP1 and MYCL1 expression was in consistent with YAP1 expression, which meant they might be the downstream target gene of YAP1.CONCLUSIONS1. YAP1 expression is high in ovarian cancer tissues, and high nuclear YAP1 level correlates with poor prognosis. YAP1 promotes ovarian cancer proliferation. YAP1 confers to CDDP low sensitivity in ovarian cancer.2. ATF2, RAD51AP1 and MYCL1 might be the downstream target genes of YAP1.