MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

Ovarian cancer (OC) cells survive in the peritoneal cavity in a complex microenvironment composed of diverse cell types. The interaction between tumor cells and non-malignant cells is crucial to the success of the metastatic process. Macrophages activate pro-metastatic signaling pathways in ovarian cancer cells (OCCs), induce tumor angiogenesis, and orchestrate a tumor suppressive immune response by releasing anti-inflammatory cytokines. Understanding the interaction between immune cells and tumor cells will enhance our ability to combat tumor growth and dissemination. When co-cultured with OCCs, macrophages induce projections consistent with tunneling nanotubes (TnTs) to form between OCCs. TnTs mediate transfer of material between cells, thus promoting invasiveness, angiogenesis, proliferation, and/or therapy resistance. Macrophage induction of OCC TnTs occurs through a soluble mediator as macrophage-conditioned media potently induced TnT formation in OCCs. Additionally, EGFR-induced TnT formation in OCCs through MAPK signaling may occur. In particular, inhibition of ERK and RSK prevented EGFR-induced TnTs. TnT formation in response to macrophage-conditioned media or EGFR signaling required MAPK signaling. Collectively, these studies suggest that inhibition of ERK/RSK activity may dampen macrophage-OCC communication and be a promising therapeutic strategy

ARID3B increases ovarian tumor burden and is associated with a cancer stem cell gene signature

Ovarian cancer is the most deadly gynecological malignancy since most patients have metastatic disease at the time of diagnosis. Therefore, identification of critical pathways that contribute to ovarian cancer progression is necessary to yield novel therapeutic targets. Recently we reported that the DNA binding protein ARID3B is overexpressed in human ovarian tumors. To determine if ARID3B has oncogenic functions in vivo, ovarian cancer cell lines stably expressing ARID3B were injected intraperitoneally into nude mice. Overexpression of ARID3B increased tumor burden and decreased survival. To assess how ARID3B contributes to the increased tumor growth in vivo, we identified ARID3B induced genes in tumor ascites cells. ARID3B induced expression of genes associated with metastasis and cancer stem cells (CD44, LGR5, PROM1 (CD133), and Notch2). Moreover, ARID3B increased the number of CD133+ (a cancer stem cell marker) cells compared to control cells. The increase in CD133+ cells resulting from ARID3B expression was accompanied by enhanced paclitaxel resistance. Our data demonstrate that ARID3B boosts production CD133+ cells and increases ovarian cancer progression in vivo

Enrichment for chemoresistant ovarian cancer stem cells from human cell lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse

Arid3b Is Critical for B Lymphocyte Development

Arid3a and Arid3b belong to a subfamily of ARID (AT-rich interaction domain) transcription factors. The Arid family is involved in regulating chromatin accessibility, proliferation, and differentiation. Arid3a and Arid3b are closely related and share a unique REKLES domain that mediates their homo- and hetero-multimerization. Arid3a was originally isolated as a B cell transcription factor binding to the AT rich matrix attachment regions (MARS) of the immunoglobulin heavy chain intronic enhancer. Deletion of Arid3a results in a highly penetrant embryonic lethality with severe defects in erythropoiesis and hematopoietic stem cells (HSCs). The few surviving Arid3a-/- (<1%) animals have decreased HSCs and early progenitors in the bone marrow, but all mature lineages are normally represented in the bone marrow and periphery except for B cells. Arid3b-/- animals die around E7.5 precluding examination of hematopoietic development. So it is unclear whether the phenotype of Arid3a loss on hematopoiesis is dependent or independent of Arid3b. In this study we circumvented this limitation by also examining hematopoiesis in mice with a conditional allele of Arid3b. Bone marrow lacking Arid3b shows decreased common lymphoid progenitors (CLPs) and downstream B cell populations while the T cell and myeloid lineages are unchanged, reminiscent of the adult hematopoietic defect in Arid3a mice. Unlike Arid3a-/- mice, HSC populations are unperturbed in Arid3b-/- mice. This study demonstrates that HSC development is independent of Arid3b, whereas B cell development requires both Arid3a and Arid3b transcription factors

ARID3A and ARID3B induce stem promoting pathways in ovarian cancer cells

ARID3A and ARID3B are paralogs from the AT-Rich interactive Domain (ARID) family. ARID3A and ARID3B associate to regulate genes in B-cells and cancer. We were the first to demonstrate that ARID3B regulates stem cell genes and promotes the cancer stem cell phenotype. Importantly, different knockout phenotypes in mice and distinct patterns of expression in adult animals suggests that ARID3A and ARID3B may have unique functions. In addition, high levels of ARID3B but not ARID3A induce cell death. Our goal was to express ARID3A, ARID3B, or both genes at a moderate level (as can be observed in cancer) and then identify ARID3 regulated genes. We transduced ovarian cancer cells with ARID3A-GFP, ARID3B-RFP, or both. RNA-sequencing was conducted. ARID3A and ARID3B regulated nearly identical sets of genes. Few genes (<5%) were uniquely regulated by ARID3A or ARID3B. ARID3A/B induced genes involved in cancer and stem cell processes including: Twist, MYCN, MMP2, GLI2, TIMP3, and WNT5B. We found that ARID3A and ARID3B also induced expression of each other, providing evidence of the cooperativity. While ARID3A and ARID3B likely have unique functions in distinct contexts, they are largely capable of regulating the same stem cell genes in cancer cells. This study provides a comprehensive list of genes and pathways regulated by ARID3A and ARID3B in ovarian cancer cells

In vivo tumor growth of high-grade serous ovarian cancer cell lines

OBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community

Effect of oxygen on the expression of renin-angiotensin system components in a human trophoblast cell line

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin-angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development

Role of LKB1 in lung cancer development

Three phenotypically related genetic syndromes and their lesions (LKB1, PTEN, and TSC1/2) are identified as frequently altered in lung cancer. LKB1, a kinase inactivated in 30% of lung cancers, is discussed in this review. Loss of LKB1 regulation often coincident with KRAS activation allows for unchecked growth and the metabolic capacity to accommodate the proliferation

The hTERT Promoter Enhances the Antitumor Activity of an Oncolytic Adenovirus under a Hypoxic Microenvironment

Hypoxia is a microenvironmental factor that contributes to the invasion, progression and metastasis of tumor cells. Hypoxic tumor cells often show more resistance to conventional chemoradiotherapy than normoxic tumor cells, suggesting the requirement of novel antitumor therapies to efficiently eliminate the hypoxic tumor cells. We previously generated a tumor-specific replication-competent oncolytic adenovirus (OBP-301: Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives viral E1 expression. Since the promoter activity of the hTERT gene has been shown to be upregulated by hypoxia, we hypothesized that, under hypoxic conditions, the antitumor effect of OBP-301 with the hTERT promoter would be more efficient than that of the wild-type adenovirus 5 (Ad5). In this study, we investigated the antitumor effects of OBP-301 and Ad5 against human cancer cells under a normoxic (20% oxygen) or a hypoxic (1% oxygen) condition. Hypoxic condition induced nuclear accumulation of the hypoxia-inducible factor-1α and upregulation of hTERT promoter activity in human cancer cells. The cytopathic activity of OBP-301 was significantly higher than that of Ad5 under hypoxic condition. Consistent with their cytopathic activity, the replication of OBP-301 was significantly higher than that of Ad5 under the hypoxic condition. OBP-301-mediated E1A was expressed within hypoxic areas of human xenograft tumors in mice. These results suggest that the cytopathic activity of OBP-301 against hypoxic tumor cells is mediated through hypoxia-mediated activation of the hTERT promoter. Regulation of oncolytic adenoviruses by the hTERT promoter is a promising antitumor strategy, not only for induction of tumor-specific oncolysis, but also for efficient elimination of hypoxic tumor cells