Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways

Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.published_or_final_versio

Cancer Genomics Identifies Regulatory Gene Networks Associated with the Transition from Dysplasia to Advanced Lung Adenocarcinomas Induced by c-Raf-1

Background: Lung cancer is a leading cause of cancer morbidity. To improve an understanding of molecular causes of disease a transgenic mouse model was investigated where targeted expression of the serine threonine kinase c-Raf to respiratory epithelium induced initialy dysplasia and subsequently adenocarcinomas. This enables dissection of genetic events associated with precancerous and cancerous lesions. Methodology/Principal Findings: By laser microdissection cancer cell populations were harvested and subjected to whole genome expression analyses. Overall 473 and 541 genes were significantly regulated, when cancer versus transgenic and non-transgenic cells were compared, giving rise to three distinct and one common regulatory gene network. At advanced stages of tumor growth predominately repression of gene expression was observed, but genes previously shown to be upregulated in dysplasia were also up-regulated in solid tumors. Regulation of developmental programs as well as epithelial mesenchymal and mesenchymal endothelial transition was a hall mark of adenocarcinomas. Additionaly, genes coding for cell adhesion, i.e. the integrins and the tight and gap junction proteins were repressed, whereas ligands for receptor tyrosine kinase such as epi- and amphiregulin were up-regulated. Notably, Vegfr- 2 and its ligand Vegfd, as well as Notch and Wnt signalling cascades were regulated as were glycosylases that influence cellular recognition. Other regulated signalling molecules included guanine exchange factors that play a role in an activation of the MAP kinases while several tumor suppressors i.e. Mcc, Hey1, Fat3, Armcx1 and Reck were significantly repressed. Finally, probable molecular switches forcing dysplastic cells into malignantly transformed cells could be identified. Conclusions/Significance: This study provides insight into molecular pertubations allowing dysplasia to progress further to adenocarcinoma induced by exaggerted c-Raf kinase activity

Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness via targeting GRB7

The human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. We and others have reported that GRB7 is frequently upregulated and associated with the progression of human cancers. Clinicopathological analysis has shown that the overexpressed GRB7 is correlated with high grade and metastatic ovarian cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. In this study, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. We found that the protein level of GRB7 was much higher than its mRNA levels in a subset of ovarian cancer cell lines, indicating aberrant post-transcriptional alterations in GRB7. Using an in silico study, we identified miR-193a-3p and its isoform, miR-193b-3p, by specifically targeting the conserved site (GGCCAGT) at position 332–338 in the 3’ UTR (length: 387) of the human GRB7 gene. QPCR and western blot analyses revealed that only the expression levels of miR-193a-3p but not miR-193b-3p were inversely correlated in ovarian cancer cell lines accompanied with high levels of GRB7 protein levels. Further biochemical analyses using luciferase reporter assays in combination with mutational analyses identified miR-193a-3p specifically targeting GRB7 3’UTR, confirming miR-193a-3p, instead of its isoform miR-193b-3p, has a more important role in GRB7 post-transcriptional regulation in ovarian cancer cells. Constitutive expression or knockdown of miR-193a-3p could significantly alter GRB7 expression, as well as oncogenic capacities of ovarian cancer cells such as cell growth, cell migration and cell invasion. Importantly, we observed that there was a significant stepwise decrease of miR-193a-3p expression from low to high grade ovarian cancers, and was inversely correlated with GRB7 expressions. Intriguingly, treatment with the DNA methyl transferase inhibitor (5’-Aza-dc) could restore the expression of miR-193a-3p, suggesting the epigenetic inhibition of miR-193a-3p is clinically relevance. Importantly, such epigenetic alteration may be the clue leading to overexpression of GRB7 in ovarian cancer cells. Taken together, our findings suggest that epigenetically silencing of miR-193a-3p upregulates GRB7 and contribute to ovarian cancer aggressiveness in tumor progression

Targeting lipid metabolism with a metabolic inhibitor cocktail eradicates peritoneal metastases of ovarian cancer

Session 4: Organismal MetabolismOvarian cancer is an intra-abdominal tumor in which the presence of ascites enables metastatic dissemination, and correlates with poor prognosis. Malignant ascites provide a key reservoir of growth factors and nutrients to augment the aggressiveness of ovarian cancer. However, the significance of metabolic alterations in ovarian cancer cells in the ascites microenvironment remains unclear. Here, we report that ovarian cancer cells exhibited increased aggressiveness in ascites microenvironment via reprogramming of lipid metabolism. Comparative proteomic analyses revealed that lipid metabolism is highly active in ovarian cancer cells when cultured in omental conditioned medium (OCM) that mimics the ascites microenvironment. Under these conditions, knockdown of Glut1, Glut3 or Glut4, or treatment with a glycolysis inhibitor (STF31) did not alter cell growth. By contrast, depletion of acetyl-CoA carboxylase-1 or -2 (ACC1/ACC2) or fatty acid synthase (FASN), or treatment with the FASN inhibitor orlistat remarkably attenuated ATP production and cell growth, indicating a metabolic shift from aerobic glycolysis to β-oxidation in ovarian cancer cells. This shift was evidenced by increases in lipogenesis and fatty acid β-oxidation to generate ATP for supporting oncogenic capacities of ovarian cancer cells. However, the activity of AMP-activated protein kinase (AMPK), a crucial energy sensor, was initially induced to trigger fatty acid oxidation but was then gradually reduced due to the feedback effect of high ATP production. The reduced AMPK activity led to significant activation of mTOR and TAK1/NF-κB signaling, which in turn, enhanced the growth of ovarian cancer cells. Notably, combined treatment of AMPK activators, TAK1 inhibitor and FASN inhibitor could significantly impair OCM-mediated oncogenic augmentation of ovarian cancer cells in vitro and in vivo. Collectively, our findings suggest that the ascites microenvironment compels metabolic reprogramming in ovarian cancer cells to support their aggressiveness, while targeting lipid metabolism and TAK1/NF-κB signaling axis could impede ovarian cancer peritoneal metastases

The roles of AMPK and TAK1/NF-kB signaling cascade in governing cancer cell aggressiveness in omental microenvironemnt

This journal suppl. is the Proceedings Book of EACR-24INTRODUCTION: Ovarian cancer is a common and the most lethal gynecological malignancy among women. The high mortality rate of this disease is due to poor prognosis and most patients present at an advanced stage. Metastatic dissemination is a common occurrence of advanced-stage ovarian cancer and usually causes critical problem in clinical management. The omentum is an adipose tissue and ovarian cancer cells preferentially metastasize to this organ. However, the underlying molecular mechanisms for the dynamic interplay of omentum and ovarian cancer cells remain unknown. MATERIAL AND METHOD: An omental conditioned Medium (OCM) was established by collecting the surgical resected omental tissues from ovarian cancer patients and by incubating with DMEM at 38o C for 24 hours. The accumulation of lipid in ovarian cancer cells was examined by florescent staining and cellular ATP level was quantified after co-treatment with OCM. XTT cell proliferation, Transwell cell migration and invasion were performed to assess the cell growth, cell migration/invasion capacities of ovarian cancer cells in OCM. RESULT: Ovarian cancer cells co-cultured with OCM exhibited an increase in cell growth, cell migration and invasion through activation of TAK1 and NF-kB signaling activity. Intriguingly, an increase of Acetyl-CoA Carboxylase activity (phospho-ACC) was also observed, indicating the tumor cells synthesized ATP cellular energy by fatty acid oxidation for the rapid cell growth. Indeed, ovarian cancer cells cultured in OCM showed increased lipid droplets accumulation in cytosol and ATP content. However, AMPK acts as an upstream of ACC, only slight elevated initially and progressively reduced. The reduced AMPK activity led to an increase of TAK1 and NFkB signaling activities in a time dependent manner upon treatment of OCM. In contrast, co-treatment of metformin, a known AMPK activator, or knockdown of AMPKa1/2, or blocking TAK1/NFkB signaling cascade using TAK1 specific inhibitor, (5Z)-7-Oxozeaenol, could remarkably inhibit OCM-mediated cell growth, cell migration and invasion of ovarian cancer cells. CONCLUSION: These data suggest that the free fatty acid in OCM provide energy for ovarian cancer cells to support their cell growth, cell migration/invasion

Targeting lipid metabolism eradicates peritoneal metastases of ovarian cancer

Ovarian cancer is one of the deadliest women malignancies in the world. The chemoresistance accompanied with metastasis lead to high mortality of this disease. Different from other solid tumors, the advanced ovarian cancers prefer transcoelomic route in metastasis. Clinical observation indicates ovarian cancer patients with intraperitoneal metastases are closely association with poor prognosis. Emerging evidence has indicated that ascites fluids which acts as a reservoir providing plenty of growth factors, chemokines and importantly, free fatty acids etc. However, the significance of ascites microenvironment in governing ovarian cancer metastatic progression remain unclear. Herein, we report that the ascites microenvironment alters metabolism that in turn, enhances ovarian cancer cell aggressiveness. By mimicking the ascites microenvironment, we established omental conditioned medium (OCM). Ovarian cancer cells co-cultured in OCM showed enhanced ovarian cancer oncogenic capacities as observed in ascites. By RNA-Seq and proteomics analyses, we found the activities of lipid metabolism were elevated in ovarian cancer cells cultured in OCM. Depletion of Glut-1/-3/-4 by shRNAi approach or treatment of STF31, a glycolysis inhibitor, showed no or slight effect on ATP production and cell growth of ovarian cancer cells. In contrast, knockdown of Acetyl-CoA Carboxylase- orCCor treatment of FASN inhibitor, Orlistat, significantly inhibited the ATP production and cell growth, suggesting ovarian cancer cells undergone a metabolic shift from aerobic glycolysis to oxidation when co-cultured in fatty acid enriched OCM. On the other hand, AMP-activated protein kinase (AMPK) is a key energy sensor and affects oncogenic capacities of many cancer cells. We found that its activity was induced initially for triggering fatty acid oxidation in ATP production. But the AMPK activity was gradually reduced due to the negative feedback loop of high ATP content. The lowered AMPK activity led to mTOR and TAK1/NF-B signaling activation that result in the increased oncogenic capacities of ovarian cancer cells. Notably, inhibition of the above signaling pathways by a combined cocktails of AMPK activator, TAK1 and FASN inhibitors could remarkably impair OCM-mediated oncogenic properties of ovarian cancer cells in vitro and in vivo. Taken together, these findings suggest that the ascites microenvironment induces metabolic reprogramming for ovarian cancer cells in production of energy supporting their aggressiveness, while targeting the lipid metabolism and associated signalings could impede peritoneal metastases of ovarian cancer

The AMPK/TAK1/NF-kB signaling axis is indispensable for modulating ovarian cancer cell metabolism and peritoneal metastases

Ovarian cancer is an intra-abdominal tumor and metastatic dissemination is usually confined in the peritoneal cavity accompanied with the presence of ascites. Malignant ascites provides a key source of soluble growth factors, chemokines and fatty acids determining the morbidity and mortality of ovarian cancer patients. However, the underlying molecular mechanisms for the dynamic interplay of the ascites microenvironment and ovarian cancer cells in terms of cell metabolism and altered oncogenic signalings in sustaining metastatic progression remains unknown. Here we report that the ovarian cancer cells in ascites microenvironment exhibit lipid metabolism for energy production for metastatic progression by modulating of the AMPK activities in lipogenesis and oncogenic signalings; AMPK/mTOR and TAK1/NFkB signaling axis. Omental metastasis is one of the preferential routes in peritoneal metastases of ovarian cancers and the omentum is usually removed by omentectomy. By mimicking the ascites microenvironement, an omental explant culture system (OCM) was established and ovarian cancer cells demonstrated a significant increase in cell growth, cell migration and invasion when co-cultured in this system. Biochemical analyses proved that ovarian cancer cells displayed an increased rate of lipid synthesis and ATP content when cultured in OCM. This was supported by the metabolic reprogramming from the use of aerobic glycolysis to lipid metabolism via AMPK/ACC signaling mediated lipogenesis. Intriguingly, the activated AMPK activity is quickly reduced by the high ATP production in comparison of the ACC activity in ovarian cancer cells. The reduced AMPK activity was accompanied with not only enhanced mTOR activity but also increased TAK1/NFkB signaling in ovarian cancer cells upon treatment of OCM for a longer time. Noticeably, co-treatment of TAK1 activator, PGE2, nor TAK1 specific inhibitor, (5Z)-7-Oxozeaenol (5ZO) and knockdown of TAK1, could not alter the AMPK activity. In contrast, knockdown of AMPKa1 or activating AMPK by co-treatment of metformin could significantly alter TAK1/NFkB signaling, suggesting that AMPK acts as the upstream effector of TAK1/ NFkB signaling in ovarian cancer cells. Therefore, knockdown of AMPKa1 could enhance, while co-treatment of metformin or TAK1 inhibitor 5ZO could inhibit OCM-mediated cell growth, cell migration and invasion, ex vivo omental metastasis and in vivo dissemination of ovarian cancer cells. Taken together, our findings suggest that the metastatic ovarian cancer cells utilize fatty acids as energy fuel through AMPK mediated ACC-lipogenesis, mTOR and TAK1/NF-kB signaling axis in ovarian cancer development and metastasis

GRO-α and IL-8 enhance ovarian cancer metastatic potential via the CXCR2-mediated TAK1/NFκB signaling cascade

Intraperitoneal metastasis is a common occurrence and is usually involved in the poor prognosis of ovarian cancer. Its specific metastatic pattern implies that certain indispensable microenvironmental factors secreted in the peritoneal cavity can direct metastatic ovarian cancer cells to permissive niches for secondary lesion formation. However, the underlying molecular mechanisms are ill defined. Our previous report showed that omentum conditioned medium (OCM), mimicking the omental tumor microenvironment, promotes the oncogenic properties of ovarian cancer cells. Herein, we report that GRO-α and IL-8 are predominately upregulated in OCM derived from either normal or cancerous omenta and are associated with increased ovarian cancer aggressiveness. Both chemokines can activate TAK1/NFκB signaling via the CXCR2 receptor. Intriguingly, TAK1/NFκB signaling activity was higher in metastatic ovarian cancer cells; this higher activity makes them more susceptible to OCM-induced tumor aggressiveness. Treatment of ovarian cancer cells with GRO-α and IL-8 neutralizing antibodies or depletion of CXCR2 by shRNA gene knockdown, CRISPR/Cas9 gene knockout, or CXCR2 inhibitor SB225002 treatment significantly attenuated TAK1/NFκB signaling and decreased in vitro and in vivo oncogenic and metastatic potential, suggesting CXCR2 plays a key role in the GRO-α and IL-8-governed metastatic spreading of ovarian cancer cells in the intraperitoneal cavity. Taken together, our study highlights the significance of GRO-α and IL-8 as the key chemokines in the peritoneal tumor microenvironment and suggests the utility of targeting their receptor CXCR2 as a potential target-based therapy for peritoneal metastases of ovarian cancer

Retrospective analysis of survival in 81 women with early epithelial ovarian carcinoma who had not undergone systematic lymphadenectomy

Objective: This study aims to investigate whether postoperative adjuvant chemotherapy had any influence on the survival rate of those women with early ovarian carcinoma who did not receive pelvic lymphadenectomy and secondary surgery. Methods: This work conducted a retrospective review on all apparently stage I epithelial ovarian carcinoma patients who had not undergone lymphadenectomy in Queen Mary hospital, Hong Kong, within a ten-year period, from January of 1992 to December of 2002. Results: This work collected the clinical data from 81 patients. Twenty-three patients were apparently at stage IA, 1 patient at stage IB, and 57 patients at stage IC. The mean age was 47 years old (21-81 years) and the median follow-up period was 110 months (50-179 months). Totally 64 (79.0%) patients received adjuvant chemotherapy. The 5-year overall survival rate was 91.4% and 4-year progression free survival rate was 85.2%, which were similar to the survival rates described in the literature for patients who had undergone systematic lymphadenectomy. Conclusion: With provision of appropriate adjuvant chemotherapy, women with apparent stage I disease who have not undergone pelvic lymphadenectomy may achieve similar long-term survival rate to those who have undergone systematic lymphadectomy.link_to_subscribed_fulltex

The significance of proliferating cell nuclear antigen in human trophoblastic disease: An immunohistochemical study

The expression of proliferating cell nuclear antigen (PCNA) in human trophoblastic disease was assessed immunohistochemically in tissue from 29 spontaneous abortions, 33 partial moles, 40 complete moles and 23 choriocarcinomas using the monoclonal antibody PC10. PCNA immunoreactivity occurred predominantly in the cytotrophoblasts in each of the four types of tissue. Quantitative analysis showed that the choriocarcinoma group gave a statistically significant higher PCNA index than the other three. There was no significant difference between the groups of spontaneous abortion, partial or complete mole. Sixteen of the 73 patients with partial and complete moles developed persistent gestational trophoblastic disease and there was no significant difference between the patients requiring chemotherapy and those who did not. We conclude that choriocarcinoma has a significantly higher PCNA proliferative index whilst hydatidiform moles cannot be distinguished from abortions by such analysis. The PCNA index does not appear to be useful in predicting the progression of molar pregnancies to persistent trophoblastic diseases.link_to_subscribed_fulltex