Growth Of Epithelial Ovarian Cancer Research Articles

Current insights into the metastasis of epithelial ovarian cancer - hopes and hurdles.

Ovarian cancer is the most lethal gynecologic cancer and the fifth leading cause of cancer-related mortality in women worldwide. Despite various attempts to improve the diagnosis and therapy of ovarian cancer patients, the survival rate for these patients is still dismal, mainly because most of them are diagnosed at a late stage. Up to 90% of ovarian cancers arise from neoplastic transformation of ovarian surface epithelial cells, and are usually referred to as epithelial ovarian cancer (EOC). Unlike most human cancers, which are disseminated through blood-borne metastatic routes, EOC has traditionally been thought to be disseminated through direct migration of ovarian tumor cells to the peritoneal cavity and omentum via peritoneal fluid. It has recently been shown, however, that EOC can also be disseminated through blood-borne metastatic routes, challenging previous thoughts about ovarian cancer metastasis. Here, we review our current understanding of the most updated cellular and molecular mechanisms underlying EOC metastasis and discuss in more detail two main metastatic routes of EOC, i.e., transcoelomic metastasis and hematogenous metastasis. The emerging concept of blood-borne EOC metastasis has led to exploration of the significance of circulating tumor cells (CTCs) as novel and non-invasive prognostic markers in this daunting cancer. We also evaluate the role of tumor stroma, including cancer associated fibroblasts (CAFs), tumor associated macrophages (TAMs), endothelial cells, adipocytes, dendritic cells and extracellular matrix (ECM) components in EOC growth and metastasis. Lastly, we discuss therapeutic approaches for targeting EOC. Unraveling the mechanisms underlying EOC metastasis will open up avenues to the design of new therapeutic options. For instance, understanding the molecular mechanisms involved in the hematogenous metastasis of EOC, the biology of CTCs, and the detailed mechanisms through which EOC cells take advantage of stromal cells may help to find new opportunities for targeting EOC metastasis.

Use of Transabdominal Ultrasound for the detection of intra-peritoneal tumor engraftment and growth in mouse xenografts of epithelial ovarian cancer.

To evaluate intraperitoneal (IP) tumor engraftment, metastasis and growth in a pre-clinical murine epithelial ovarian cancer (EOC) model using both transabdominal ultrasound (TAUS) and bioluminescence in vivo imaging system (IVIS). Ten female C57Bl/6J mice at six weeks of age were included in this study. Five mice underwent IP injection of 5x106 ID8-luc cells (+ D- luciferin) and the remaining five mice underwent IP injection of ID8-VEGF cells. Monitoring of tumor growth and ascites was performed weekly starting at seven days post-injection until study endpoint. ID8-luc mice were monitored using both TAUS and IVIS, and ID8-VEGF mice underwent TAUS monitoring only. Individual tumor implant dimension and total tumor volume were calculated. Average luminescent intensity was calculated and reported per mouse abdomen. Tumor detection was confirmed by gross evaluation and histopathology. All data are presented as mean +/- standard deviation. Overall, tumors were successfully detected in all ten mice using TAUS and IVIS, and tumor detection correlated with terminal endpoint histology/ H&E staining. For TAUS, the smallest confirmed tumor measurements were at seven days post-injection with mean long axis of 2.23mm and mean tumor volume of 4.17mm3. However, IVIS imaging was able to detect tumor growth at 14 days post-injection. Ascites formation was detected in mice at 21 days post-injection. TAUS is highly discriminatory for monitoring EOC in pre-clinical murine model, allowing for detection of tumor dimension as small as 2 mm and as early as seven days post-injection compared to IVIS. In addition, TAUS provides relevant information for ascites development and detection of multiple small metastatic tumor implants. TAUS provides an accurate and reliable method to detect and monitor IP EOC growth in mouse xenografts.

LncRNA ABHD11-AS1, regulated by the EGFR pathway, contributes to the ovarian cancer tumorigenesis by epigenetically suppressing TIMP2.

ObjectiveEpithelial ovarian cancer (EOC) is a common gynecologic malignancy characterized by extensive peritoneal metastasis and high mortality rate. ABHD11 Antisense RNA1 (ABHD11‐AS1) has recently been identified as a regulator of growth and metastasis in multiple tumors, including EOC. However, the biological function and potential mechanism of ABHD11‐AS1 in EOC remains poorly understood.MethodsImmunohistochemistry, western blot, and qRT‐PCR analysis were used to determine the expression pattern of ABHD11‐AS1 and epidermal growth factor receptor (EGFR) in both EOC tissues and cell lines, respectively. Colony formation, transwell and wound healing assays were performed to evaluate the roles of EGFR and ABHD11‐AS1 on the capacity of cell proliferation, migration, and invasion. Western blot analysis was performed to measure the regulation of EGFR pathway on STAT3. Moreover, chromatin immunoprecipitation was employed to demonstrate the interaction between ABHD11‐AS1 and STAT3. RNA immunoprecipitation was subjected to prove the direct binding between ABHD11‐AS1 and EZH2. Immunofluorescence staining was performed to measure the expression and localization of TIMP2. EOC mouse model was conducted for validating the role of ABHD11‐AS1 in vivo.ResultsEGFR and ABHD11‐AS1 were highly expressed in EOC tissues and cell lines. Knockdown of EGFR or ABHD11‐AS1 inhibited cell growth, migration, and invasion of EOC cells. Expression of ABHD11‐AS1 was regulated by the activation of EGFR signaling pathway, mediated by STAT3. Besides, ABHD11‐AS1 was shown to silence TIMP2 by binding to chromatin‐modifying enzyme EZH2. Furthermore, inhibition of EGFR pathway or ABHD11‐AS1 repressed the tumor growth of EOC.ConclusionWe defined the regulatory relationship between the EGFR signaling pathway, ABHD11‐AS1, EZH2, and TIMP2 suggesting that ABHD11‐AS1 may act as an oncogene and a potential target for antitumor therapies in ovarian cancer.

LINC00968 accelerates the progression of epithelial ovarian cancer via mediating the cell cycle progression.

The aim of this study was to clarify the potential role of LINC00968 in the progression of epithelial ovarian cancer (EOC) and the underlying mechanism. The relative expression level of LINC00968 in EOC tissues (n=40) and normal ovarian tissues (n=40) was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). LINC00968 expression in human-derived ovarian cancer cell lines was examined by qRT-PCR as well. After transfection of LINC00968 small-interfering RNA (siRNA) in ovarian cancer cells, cell cycle progression and cell proliferation were evaluated through flow cytometry, Cell Counting Kit-8 (CCK-8) and colony formation assay, respectively. Tumor xenograft was conducted in nude mice to elucidate the function of LINC00968 in EOC tumorigenesis in vivo. Furthermore, the relative expression levels of cell cycle factors and protein kinase B/extracellular-signal-regulated kinase (AKT/ERK) in ovarian cancer cells influenced by LINC00968 were detected by Western blot. LINC00968 was significantly up-regulated in EOC tissues when compared with normal control tissues. Meanwhile, LINC00968 expression was positively correlated with the prognosis of EOC. Transfection of LINC00968 siRNA in HEY and HO8910 cells markedly attenuated proliferative ability and arrested cell cycle in the G1 phase. Knockdown of LINC00968 remarkably suppressed tumor growth of EOC in nude mice. The silence of LINC00968 significantly downregulated Cyclin D, Cyclin E and CDK4, whereas upregulated p16 and p21. In addition, AKT and ERK pathways were inhibited by knockdown of LINC00968 in ovarian cancer cells. LINC00968 expression is markedly upregulated in EOC. Meanwhile, it arrests the cell cycle in the G1 phase by inhibiting the ERK and AKT pathways, thus accelerating EOC progression.

The contribution of adipocyte reservoirs towards ovarian cancer growth in time and site-specific manner

Objective: Free fatty acids derived from omentum adipocytes have been shown to meet the energy demands of rapidly proliferating epithelial ovarian cancer (EOC) cells. We have previously shown that EOC progresses aggressively in high-fat diet (HFD)-fed obese mice, highlighting the importance of overall adipocyte reserves in promoting EOC progression. Since major adipose reservoirs exist at multiple body sites (omental, retroperitoneal, subcutaneous, visceral, and gonadal), the aim of the study is to determine the contribution of individual adipocyte reserves in promoting EOC growth in time and site-specific manner.

Myeloid derived suppressor cells promote ovarian cancer progression in a murine model of ovarian cancer

Abstract Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous population of immature myeloid cells that create an immunosuppressive environment by inhibiting T-cell function. MDSCs also promote angiogenesis, tumor invasion, and metastasis. Increased MDSCs in epithelial ovarian cancer (EOC) are been associated with poor prognosis. The aim of our study was to investigate if MDSCs can influence EOC progression and if their depletion would improve immune response and restrict EOC growth. The intraperitoneal ID8 syngeneic mouse EOC model in B6 mice was used. ID8 tumor bearing mice exhibited significantly higher levels of MDSCs (CD11b+Gr1+) in bone marrow, blood, ascites and tumor compared to non-tumor control groups, including both sub-populations; granulocytic (CD11b+Ly6G+Ly6Clo) and monocytic (CD11b+Ly6G−Ly6Chi). Depleting MDSCs either by anti-Gr1 (Ly6G/Ly6C mAb) monoclonal antibody or gemcitabine treatment significantly slowed EOC progression, as reflected by decreased ascites and reduced tumor burden at the peritoneum, bowel, liver, spleen, and kidney. This was accompanied with an increased number of IFNγ-producing CD4 and CD8 T-cells. Adoptive transfer of MDSCs (Gr1hiLy6G+) enhanced ID8 tumor progression, further supporting their pathogenic role in EOC. Moreover, depletion of MDSCs enhanced the effects of anti-PD-1 immunotherapy in the ID8 EOC mouse model. Overall, we report here that depleting MDSCs improves the antitumor immune response, restricts EOC progression, and enhances tumor responsiveness to immunotherapy. Thus, targeting MDSCs is a potential therapeutic modality in EOC that may be beneficial in combination with other immunotherapies.

Natural antisense transcript of hypoxia-inducible factor 1 regulates hypoxic cell apoptosis in epithelial ovarian cancer.

PurposeHypoxia is a key stress that triggers apoptosis in various tumors, including epithelial ovarian cancer (EOC). Previous researches identified a hypoxia-upregulated lncRNA named “a natural antisense transcript of hypoxia-inducible factor 1 (aHIF)” in some tumors. However, the contribution of aHIF to EOC remains unclear. Here, we aimed to investigate the expression, function, and underlying mechanisms of aHIF in EOC progression under hypoxia.Materials and methodsExpression levels of aHIF in EOC tissues were tested. In vitro and in vivo assays were conducted to explore the function and mechanism of aHIF in hypoxia-induced EOC progression.ResultsaHIF levels were increased in EOC tissues and were upregulated by hypoxia in EOC cells. Functional data revealed that aHIF knockdown accelerated cell apoptosis under hypoxia and inhibited EOC tumorigenesis and tumor growth in vivo. Additionally, aHIF overexpression inhibited cell apoptosis and enhanced cell proliferation under hypoxia in EOC. Mechanistically, the dysregulation of certain key mitochondrial apoptosis pathway-related genes, including Bcl-2, Bax, Caspase-7, and Caspase-9, may partially explain aHIF-regulated EOC apoptosis and growth under hypoxia.ConclusionThese data provide the first convincing evidence that aHIF may inhibit EOC apoptosis and thereby promote tumor growth through activation of the mitochondrial apoptosis pathway under hypoxia. Our findings help clarify the role of lncRNA in hypoxia-induced EOC progression.

Combination of triapine, olaparib, and cediranib suppresses progression of BRCA-wild type and PARP inhibitor-resistant epithelial ovarian cancer

PARP inhibitors target BRCA mutations and defective homologous recombination repair (HRR) for the treatment of epithelial ovarian cancer (EOC). However, the treatment of HRR-proficient EOC with PARP inhibitors remains challenging. The objective of this study was to determine whether the combination of triapine (ribonucleotide reductase inhibitor), cediranib (vascular endothelial growth factor receptor tyrosine kinase inhibitor), and the PARP inhibitor olaparib synergized against BRCA wild-type and HRR-proficient EOC in xenograft mouse models. In addition, the mechanisms by which cediranib augmented the efficacy of triapine and olaparib were investigated. BRCA-wild type and PARP inhibitor-resistant EOC cell lines were implanted subcutaneously (s.c.) into nude mice or injected intraperitoneally (i.p.) into SCID-Beige mice. Mice were then treated i.p. with olaparib, cediranib, triapine, various double and triple combinations. The volume of s.c tumor in nude mice and the abdominal circumference of SCID-Beige mice were measured to evaluate the effectiveness of the treatment to delay tumor growth and prolong the survival time of mice. In both xenograft mouse models, the combination of triapine, olaparib and cediranib resulted in marked suppression of BRCA-wild type EOC growth and significant prolongation of the survival time of mice, with efficacy greater than any double combinations and single drugs. Furthermore, we identified that cediranib abrogated pro-survival and anti-apoptotic AKT signaling, thereby enhancing the efficacy of triapine and olaparib against BRCA-wild type EOC cells. Taken together, our results demonstrate a proof-of-principle approach and the combination regiment holds promise in treating BRCA-wild type and PARP inhibitor-resistant EOC.

Clinical associations between ASCT2 and p‑mTOR in the pathogenesis and prognosis of epithelial ovarian cancer

Alanine serine cysteine‑preferring transporter2 (ASCT2; also known as SLC1A5) is an important glutamine transporter, and it serves a crucial role in tumor growth and progression. ASCT2 is highly expressed in numerous types of cancer, but the pathological significance of its expression in epithelial ovarian cancer (EOC) remains unclear. The mechanistic target of rapamycin (mTOR) level is hyperelevated in a number of tumor types, including ovarian cancer. The aim of the present study was to elucidate the prognostic role of ASCT2 and phosphorylated (p)‑mTOR in EOC. The levels of ASCT2 and p‑mTOR/mTOR were detected in normal ovarian tissues, benign ovarian tumors, borderline ovarian tumors and EOC tissues by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot assays. The protein levels of ASCT2 and p‑mTOR in EOC patients were then detected by immunohistochemistry (IHC). Furthermore, EOC tumor sections were stained for Ki‑67 and cluster of differentiation34 (CD34) to assess proliferation and microvessel density by IHC. The results of RT‑qPCR and western blot analysis demonstrated that ASCT2 and p‑mTOR protein levels were significantly higher in EOC tissues compared with those in other groups. IHC analysis of 104 EOC tissues suggested that ASCT2 expression was associated with clinicopathological parameters, including International Federation of Gynecology and Obstetrics stage, pathological grade, serum cancer antigen 125 level, Ki‑67 status and CD34 status. Kaplan‑Meier survival curve analysis indicated that high expression of ASCT2 and p‑mTOR were important factors predicting a poor prognosis for patients with EOC. The expression levels of ASCT2 and p‑mTOR in EOC were positively correlated (r=0.385, P<0.001). This positive correlation between ASCT2 and p‑mTOR indicates that they have a synergistic effect on the growth and development of early EOC. The combined detection of ASCT2 and p‑mTOR may serve as a potential marker to inform diagnosis, postoperative follow‑up requirements and targeted therapy options for patients with early‑stage EOC, but not for terminal‑stage patients.

Upregulation of IGF1 by tumor-associated macrophages promotes the proliferation and migration of epithelial ovarian cancer cells.

Ovarian cancer (OC), of which epithelial ovarian cancer (EOC) is the most common, is the deadliest gynecological tumor because of the difficulties in detection at early stages, and metastasis and chemoresistance at advanced stages. Tumor-associated macrophages (TAMs) differentiate through alternative pathways and play important roles in tumor growth and metastasis. However, the underlying mechanism remains unclear. Here, we established a mouse TAM model using bone marrow monocytes and conditioned medium (CM) of TAMs to culture ID8 mouse EOC cells. The results showed that TAMCM accelerated the proliferation and migration of ID8 cells. In a previous study, gene chip analysis showed that human TAMs expressed significantly higher levels of insulin-like growth factor‑1 (IGF1) than undifferentiated M0 myeloid cells. In the present study, we observed that the IGF1 level was higher in human EOC specimens than that in benign ovarian tumor specimens, and further analysis showed that a higher level of IGF1 was related to more advanced clinical stage and liver metastasis. Therefore, we hypothesized that TAMs may accelerate the proliferation and migration of EOC cells by upregulating IGF1. As expected, increased IGF1 expression at both the mRNA and protein levels was observed in ID8 cells cultured with TAMCM, whereas blockade of the IGF1 pathway in ID8 cells with an IGF1 neutralizing antibody effectively reversed the promotion of proliferation and migration. Finally, we inhibited the phosphorylation of insulin-like growth factor‑1 receptor (IGF1R) and its downstream molecules Akt and Erk with the IGF1R inhibitor linsitinib, and observed that the treatment effectively suppressed the proliferation and migration of ID8 cells exposed to TAMCM. Thus, we demonstrated that TAMs may promote the growth and metastasis of EOC via the activation of the IGF1 pathway; thus, targeting the IGF1 pathway may be promising for EOC therapy.

TMED2 promotes epithelial ovarian cancer growth.

TMED2 is involved in morphogenesis of the mouse embryo and placenta. We found that expression of TMED2 was higher in epithelial ovarian cancer tissues than normal ovarian tissues. Silencing TMED2 decreased cell proliferation, migration, and invasion. Ectopic expression of TMED2 increased cell proliferation, migration and invasion. Silencing TMED2 inhibited ovarian cancer growth in mice. Silencing TMED2 inhibited IGF2/IGF1R/PI3K/Akt pathway. In agreement, ectopically expressed TMED2 activated IGF2/IGF1R/PI3K/Akt pathway. Mechanistic study revealed that TMED2 directly binds to AKT2, thereby facilitating its phosphorylation. We also found that TMED2 increased IGF1R expression by competing for miR-30a. Thus, TMED2 is oncogenic and a potential target for epithelial ovarian cancer therapy.

Elevated Th22 cells and related cytokines in patients with epithelial ovarian cancer.

This study is conducted to investigate the involvement of T-helper (Th) cells and regulatory T cells in epithelial ovarian cancer (EOC).The percentages of Th22, Th17, Th1, and regulatory T cells in the peripheral blood of EOC patients, benign ovarian epithelial neoplasm (BOEN) patients, and healthy control (HC) were examined by flow cytometry. Enzyme-linked immunosorbent assay was used to determine serum levels of interleukin (IL)-22, IL-17, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α).Th22 and Th17 were significantly increased in EOC patients. The plasma concentrations of IL-22 and TNF-α were significantly elevated in EOC patients compared with BOEN patients and HC. In EOC patients, there was an increased trend of Th22, IL-22, and TNF-α in stage III-IV patients compared with stage I-II patients. A positive correlation was seen among Th22, Th17, and Th1 cells in EOC patients. Similarly, positive correlations were detected between Th22 cells and IL-22 or TNF-α and between Th1 cells and interferon-γ (IFN-γ) in EOC patients. Besides, no significant difference was found in Th1 cells and regulatory T cells among EOC and BOEN patients and HC.There is a higher circulating frequency of Th22, Th17 cells, IL-22, and TNF-α concentration in EOC patients, which may conjointly participate in the pathogenesis and growth of EOC.

ElncRNA1, a long non-coding RNA that is transcriptionally induced by oestrogen, promotes epithelial ovarian cancer cell proliferation.

Previously, the novel oestrogen (E2)-upregulated lncRNA TC0101441, was identified by us, via microarray analysis. However, the detailed mechanism by which E2 upregulates TC0101441 and the role of TC0101441 in epithelial ovarian cancer (EOC) progression have not been elucidated. In the present study, we further analysed TC0101441, which we designated oestrogen-induced long non-coding RNA-1 (ElncRNA1). We showed that E2 transcriptionally upregulates ElncRNA1 through the oestrogen receptor α (ERα)-oestrogen response element (ERE) pathway using RNA stability assays, bioinformatics-based searches for ERE binding sites, chromatin immunoprecipitation (ChIP) assays and dual luciferase reporter assays. Clinically, ElncRNA1 levels are significantly higher in EOC tissues than in normal ovarian surface epithelium. Invitro and invivo loss-of-function assays revealed that ElncRNA1 promotes EOC cell proliferation. This pro-proliferation effect of ElncRNA1 was partially mediated by the regulation of CDK4, CDK6 and cyclinD1. These findings provide the first evidence that E2 upregulates ElncRNA1 at the transcriptional level through the ERα-ERE pathway and that this novel E2-upregulated lncRNA has an oncogenic role in EOC growth. The placement of ElncRNA1 in the E2-ERα-ERE signalling pathway may provide greater insight into the effects of oestrogen on EOC progression from the perspective of lncRNA.

MicroRNA‑139 suppressed tumor cell proliferation, migration and invasion by directly targeting HDGF in epithelial ovarian cancer.

The current study investigated the expression and functional roles of microRNA‑139 (miR‑139) on human epithelial ovarian cancer (EOC). Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to measure miR‑139 expression in EOC tissues and cell lines. The effects of miR‑139 on EOC cell proliferation, migration and invasion were assessed using MTT, cell migration and invasion assays, respectively. Subsequently, the molecular mechanism underlying the tumor suppressive roles of miR‑139 on EOC was determined by bioinformatics analysis, RT‑qPCR, western blotting and the luciferase reporter assay. According to the results, it was identified that miR‑139 was significantly downregulated in EOC tissues and cell lines. In addition, restoration of miR‑139 suppressed tumor cell proliferation, migration and invasion in EOC. Furthermore, hepatoma‑derived growth factor (HDGF) was identified as a target of miR‑139 in EOC. Upregulation of HDGF could rescue the inhibitory effects exerted by miR‑139 overexpression on EOC cell proliferation, migration and invasion. Collectively, the results indicated that miR‑139 was downregulated in EOC, and acted as a tumor suppressor by directly targeting HDGF. To the best of our knowledge, this was the first study to identify that miR‑139 contributes to the growth and metastasis of EOC.

Guidance of Signaling Activations by Cadherins and Integrins in Epithelial Ovarian Cancer Cells.

Epithelial ovarian cancer (EOC) is the deadliest tumor among gynecological cancer in the industrialized countries. The EOC incidence and mortality have remained unchanged over the last 30 years, despite the progress in diagnosis and treatment. In order to develop novel and more effective therapeutic approaches, the molecular mechanisms involved in EOC progression have been thoroughly investigated in the last few decades. At the late stage, peritoneal metastases originate from the attachment of small clusters of cancer cells that shed from the primary site and carried by the ascites adhere to the abdominal peritoneum or omentum. This behavior suggests that cell–cell or cell–matrix adhesion mechanisms regulate EOC growth and dissemination. Complex downstream signalings, which might be influenced by functional cross-talk between adhesion molecules and co-expressed and activated signaling proteins, can affect the proliferation/survival and the migration/invasion of EOC cells. This review aimed to define the impact of the mechanisms of cell–cell, through cadherins, and cell–extracellular matrix adhesion, through integrins, on the signaling cascades induced by membrane receptors and cytoplasmic proteins known to have a role in the proliferation, migration and invasion of EOC cells. Finally, some novel approaches using peptidomimetic ligands to cadherin and integrins are summarized.

Ludartin treatment exhibits promising inhibitory effect on the epithelial ovarian cancer growth and metastasis

&lt;p class="Abstract"&gt;In the present study, the effect of ludartin on OVCAR5 ovarian carcinoma cells was investigated. MTT cell assay was used for the analysis of cell viability and wound healing assay for migration potential. Results revealed that ludartin treatment at 10 and 30 µM doses reduced the cell viability from 94 to 38%. The invasive potential was decreased from 62 to 24% with increase in ludartin concentration from 10 to 30 µM. Western blot analysis showed that ludartin treatment reduced the expression of p-FAK in OVCAR5 cells significantly. Ludartin treatment for 48 hours also caused a significant decrease in the expression of MMP-2 and MMP-9 in OVCAR5 cells at a concentration of 30 µM. The metastatic regions were absent in the rats treated with 30 µM/kg doses of ludartin daily for 15 days. Thus, ludartin treatment inhibits the proliferation, invasion and migration of ovarian cancer cells through down-regulation of pFAK and MMPs, therefore can be used for the treatment of ovarian cancer.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Video Clip:&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;a href="https://youtube.com/v/GMpEHXdGSeI"&gt;Cell proliferation assay:&lt;/a&gt; 2 min 45 sec&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

MiR-494 Inhibits Epithelial Ovarian Cancer Growth by Targeting c-Myc.

BackgroundEpithelial ovarian cancer (EOC) is the most lethal malignant gynecological cancer. MicroRNAs (miRNAs) play important roles in the pathogenesis of ovarian cancer. The role of miR-494 in EOC has not been fully investigated.Material/methodsMiR-494 levels in ovarian cancer tissues and cells were tested by qRT-PCR. Cells were transfected with miR-494 mimics or miR-494 ASO by Lipofectamine. Bioinformatics algorithms from TargetScanHuman were used to predict the target genes of miR-494. The c-Myc protein level was assayed by Western blot. The interaction between miR-494 and c-Myc was confirmed by dual luciferase assays.ResultsMiR-494 showed low levels in EOC tissues and cells. Overexpression of miR-494 inhibited cell growth and migration of EOC cells and vice versa. c-Myc is the targeted gene of miR-494.ConclusionsMiR-494 has an anti-tumor role in EOC via c-Myc.

Abstract A42: TWIST1 as a driver of accelerated tumor growth and drug resistance: Potential novel pathways and therapeutics.

Abstract Introduction: Epithelial ovarian cancer (EOC) is particularly deadly due to its fast rate of growth, dissemination, and chemoresistant recurrences. Novel therapies are urgently needed to limit metastasis and restore platinum sensitivity. We have investigated the role of TWIST1, a basic helix-loop-helix transcription factor, in these processes. TWIST1 is a well-documented regulator of metastasis from solid tumors, which has been linked to cancer stem cell phenotypes, drug resistance, and poor prognosis in several tumor types. Here we present in vitro and in vivo data supporting TWIST1's role in EOC growth and drug resistance, and propose a therapeutic modality to target TWIST1. Methodology: In vitro and in vivo studies: We created two cell lines, derived from the EOC line OVCAR8, that differ only in their TWIST1 expression. The cells were stably transfected with a G418-selectible vector containing either TWIST1 or an shRNA targeting TWIST1, under the control of the CMV viral promoter. Cells from each line were injected IP (intraperitoneally) into NSG mice, allowed to grow for 7 weeks, and tumor burden was evaluated at necropsy. In addition, RNA from each of these OVCAR8 cell lines (TWIST1 overexpressing versus knockdown) was isolated and used for RNA-seq analysis. Raw data was processed using the Galaxy online platform, and differentially expressed genes were further analyzed using Ingenuity Pathway Analysis. To determine the effect of TWIST1 on drug resistance, OVCAR8 cells with and without TWIST1 were treated with varying doses of cisplatin, and survival was quantified using colorimetric cytotoxicity assays. Therapeutic Application: Since we and others have shown that TWIST1's transactivation/protein binding (WR) domain is required for TWIST1 function, we produced a fusion protein comprised of GFP and the WR domain, and used CoIP to evaluate its ability to competitively inhibit TWIST1 binding in HEK 293 cells. Results: TWIST1 knockdown resulted in reduced tumor size and overall tumor burden in an NSG mouse model of EOC. Specifically, all mice receiving TWIST1-overexpressing cells developed primary ovarian tumors which were scored +4 on H&amp;E by a veterinary pathologist, whereas only 50% of animals with TWIST1 knockdown developed ovarian tumors, and only 1 of 4 mice had a tumor that scored +4. In addition, fewer metastases were present in mice receiving TWIST knockdown cells, and no mice with TWIST1 knockdown tumors produced ascites. RNA-seq analysis of the TWIST overexpressing versus knockdown cell lines revealed marked differences in genes and pathways responsible for cell migration and cell survival, including upregulation of genes implicated in metastasis, DNA repair, and resistance to apoptosis in TWIST1 overexpressing cells. TWIST1 overexpressing cells also showed less cell death following cisplatin treatment. Finally, in our in vitro inhibitor tests, a GFP-WR domain fusion protein successfully reduced TWIST1 binding to its partner protein NFkB-p65 in HEK 293 cells. Conclusions: The importance of TWIST1 for tumor growth has been demonstrated for multiple tumor types – including ovarian cancer, as presented here. Our RNA-seq analysis reveals a complex network of TWIST1-mediated factors implicated in cell movement, survival, and resistance to DNA damaging agents, and we are working to determine the most important of these pathways in EOC. Most importantly, we have shown that TWIST1 expression in EOC cells leads to greater tumor burden in a mouse model, and by knocking down TWIST1, we were able to prevent peritoneal metastases and reduce tumor size. Therefore, we believe that TWIST1 is an attractive therapeutic target in EOC, owing to its roles at the crossroads of metastasis, drug resistance, and tumor growth. Citation Format: Cai M. Roberts, Michelle A. Tran, James Finlay, Sophia Allaf Shahin, Joana Loeza, Leslie Cisneros, Emily Ye, Thanh Dellinger, Carlotta A. Glackin. TWIST1 as a driver of accelerated tumor growth and drug resistance: Potential novel pathways and therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A42.

PSMB4 expression associates with epithelial ovarian cancer growth and poor prognosis.

In this study, we investigated the expression and role of PSMB4 in human epithelial ovarian cancer(EOC). Western blot was used to evaluate the expression of PSMB4 in EOC tissues, and immunohistochemical analysis was performed on 115 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between PSMB4 and clinicopathological parameters. Starvation and re-feeding assay was used to assess cell cycle. CCK-8 assay and plate colony formation assay showed the influence of PSMB4 on proliferation of EOC cells. The expression of PSMB4 in EOC tissues was higher than normal ovary tissues and was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of PSMB4 was related to poor prognosis of EOC patients. Starvation and re-feeding assay suggested that PSMB4 played a critical role in EOC cell proliferation. CCK-8 assay and plate colony formation assay showed that EOC cells treated with PSMB4-siRNA reduced cell proliferation of EOC cells. Additionally, PSMB4 knockdown decreased NF-κB activity. PSMB4 also regulated the expression of NF-κB mediated proteins, including cyclin D1, and cyclin E which involved in cell proliferation. Our findings implied that PSMB4 is involved in the progression of EOC and could serve as potential therapeutical target of EOC. These data suggested that PSMB4 may promote cell proliferation via the NF-κB-target gene in EOC.

High expression of CDC6 is associated with accelerated cell proliferation and poor prognosis of epithelial ovarian cancer

Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with the oncogenic activities in human cancers, but the biological function and clinical significance of CDC6 in EOC remain unclear. The aim of the present study is to examine the effect of CDC6 on epithelial ovarian cancer (EOC) cells proliferation. We found that CDC6 protein level was up-regulated in EOC tissues compared with the normal ovary tissues. CDC6 expression correlated significantly with FIGO stage (p<0.001), differentiation grade (p=0.002), ascites (p<0.001), malignant tumor cells in ascites (p=0.004), and lymph node status (p<0.001). In vitro, after the release of ovarian cancer cell line (HO8910) from serum starvation, the expression of CDC6, cyclinD1, and PCNA was up-regulated, whereas p16 expression was down-regulated. Furthermore, down-regulation of CDC6 in HO8910 cells decreased cell proliferation and colony formation. HO8910 cells transfected with sh CDC6#1 underwent G1 phase cell cycle arrest. Collectively, this study provides a novel regulatory signaling pathway of CDC6-regulated EOC growth and a new potential therapeutic target for EOC patients.