Suzanne M. Quartuccio

Early transformative changes in normal ovarian surface epithelium induced by oxidative stress require Akt upregulation, DNA damage and epithelial–stromal interaction

Ovarian cancer is the deadliest gynecological malignancy due to detection of cancer at a late stage when the disease has metastasized. One likely progenitor cell type of ovarian cancer is the ovarian surface epithelium (OSE), which proliferates rapidly in the presence of inflammatory cytokines and oxidative stress following ovulation. To determine whether oxidative stress induces DNA damage leading to spontaneous transformative changes in normal OSE, an immortalized mouse OSE cell line (MOSE cells) or normal mouse ovarian organoids were treated with hydrogen peroxide (H2O2) and loss of contact inhibition was assessed by soft agar assay. In response to H2O2, OSE cells grown in 3D exhibited growth in soft agar but MOSE cells grown on 2D plastic did not, indicating a critical role for epithelial-stromal interactions in neoplastic initiation. Loss of contact inhibition in response to H2O2 correlated with an increase in proliferation, DNA damage and upregulation of the oncogene Akt1. Use of a reactive oxygen species scavenger or Akt inhibitor blocked H2O2-induced proliferation and growth in soft agar. Although parental MOSE cells did not undergo transformation by H2O2, MOSE cells stably overexpressing constitutively active myristoylated Akt or knockdown of phosphatase and tensin homolog (PTEN) exhibited loss of contact inhibition and increased proliferation. This study indicates that normal OSE undergo transformative changes induced by oxidative stress and that this process requires Akt upregulation and activation. A 3D model that retains tissue architecture is critical for studying this process and may lead to development of new intervention strategies directed at early stages of ovarian cancer.

Alginate hydrogels for three-dimensional organ culture of ovaries and oviducts.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States(1). The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae(2,3). Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease. Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation(4). Alginate hydrogels can be used to support the growth of many types of tissues(5). Alginate is a linear polysaccharide composed of repeating units of β-D-mannuronic acid and α-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues(6,7). Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling(5). The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro. A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Mutant p53 expression in fallopian tube epithelium drives cell migration

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high‐grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the “p53 signature,” or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53‐mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro‐migratory genes in p53R273H MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53R273H with KRASG12V activation caused transformation of MOE into high‐grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53R273H in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the “p53 signature” thereby, improving survival rates.

Conditional Inactivation of p53 in Mouse Ovarian Surface Epithelium Does Not Alter MIS Driven Smad2-Dominant Negative Epithelium-Lined Inclusion Cysts or Teratomas

Epithelial ovarian cancer is the most lethal gynecological malignancy among US women. The etiology of this disease, although poorly understood, may involve the ovarian surface epithelium or the epithelium of the fallopian tube fimbriae as the progenitor cell. Disruptions in the transforming growth factor beta (TGFβ) pathway and p53 are frequently found in chemotherapy-resistant serous ovarian tumors. Transgenic mice expressing a dominant negative form of Smad2 (Smad2DN), a downstream transcription factor of the TGFβ signaling pathway, targeted to tissues of the reproductive tract were created on a FVB background. These mice developed epithelium-lined inclusion cysts, a potential precursor lesion to ovarian cancer, which morphologically resembled oviductal epithelium but exhibited protein expression more closely resembling the ovarian surface epithelium. An additional genetic “hit” of p53 deletion was predicted to result in ovarian tumors. Tissue specific deletion of p53 in the ovaries and oviducts alone was attempted through intrabursal or intraoviductal injection of Cre-recombinase expressing adenovirus (AdCreGFP) into p53 flox/flox mice. Ovarian bursal cysts were detected in some mice 6 months after intrabursal injection. No pathological abnormalities were detected in mice with intraoviductal injections, which may be related to decreased infectivity of the oviductal epithelium with adenovirus as compared to the ovarian surface epithelium. Bitransgenic mice, expressing both the Smad2DN transgene and p53 flox/flox, were then exposed to AdCreGFP in the bursa and oviductal lumen. These mice did not develop any additional phenotypes. Exposure to AdCreGFP is not an effective methodology for conditional deletion of floxed genes in oviductal epithelium and tissue specific promoters should be employed in future mouse models of the disease. In addition, a novel phenotype was observed in mice with high expression of the Smad2DN transgene as validated through qPCR analysis, characterized by teratoma-like lesions implicating Smad signaling in teratoma development.

Three-dimensional modeling of the human fallopian tube fimbriae

OBJECTIVE Ovarian cancer is the most lethal gynecological malignancy that affects women. Recent data suggests that the disease may originate in the fallopian fimbriae; however, the anatomical origin of ovarian carcinogenesis remains unclear. This is largely driven by our lack of knowledge regarding the structure and function of normal fimbriae and the relative paucity of models that accurately recapitulate the in vivo fallopian tube. Therefore, a human three-dimensional (3D) culture system was developed to examine the role of the fallopian fimbriae in serous tumorigenesis. METHODS Alginate matrix was utilized to support human fallopian fimbriae ex vivo. Fimbriae were cultured with factors hypothesized to contribute to carcinogenesis, namely; H2O2 (1mM) a mimetic of oxidative stress, insulin (5μg/ml) to stimulate glycolysis, and estradiol (E2, 10nM) which peaks before ovulation. Cultures were evaluated for changes in proliferation and p53 expression, criteria utilized to identify potential precursor lesions. Further, secretory factors were assessed after treatment with E2 to identify if steroid signaling induces a pro-tumorigenic microenvironment. RESULTS 3D fimbriae cultures maintained normal tissue architecture up to 7days, retaining both epithelial subtypes. Treatment of cultures with H2O2 or insulin significantly induced proliferation. However, p53 stabilization was unaffected by any particular treatment, although it was induced by ex vivo culturing. Moreover, E2-alone treatment significantly induced its canonical target PR and expression of IL8, a factor linked to poor outcome. CONCLUSIONS 3D alginate cultures of human fallopian fimbriae provide an important microphysiological model, which can be further utilized to investigate serous tumorigenesis originating from the fallopian tube.

Mutation or Loss of p53 Differentially Modifies TGFβ Action in Ovarian Cancer

Ovarian cancer is the most lethal gynecological disease affecting women in the US. The Cancer Genome Atlas Network identified p53 mutations in 96% of high-grade serous ovarian carcinomas, demonstrating its critical role. Additionally, the Transforming Growth Factor Beta (TGFβ) pathway is dysfunctional in various malignancies, including ovarian cancer. This study investigated how expression of wild-type, mutant, or the absence of p53 alters ovarian cancer cell response to TGFβ signaling, as well as the response of the ovarian surface epithelium and the fallopian tube epithelium to TGFβ. Only ovarian cancer cells expressing wild-type p53 were growth inhibited by TGFβ, while ovarian cancer cells that were mutant or null p53 were not. TGFβ induced migration in p53 null SKOV3 cells, which was not observed in SKOV3 cells with stable expression of mutant p53 R273H. Knockdown of wild-type p53 in the OVCA 420 ovarian cancer cells enhanced cell migration in response to TGFβ. Increased protein expression of DKK1 and TMEPAI, two pro-invasive genes with enhanced expression in late stage metastatic ovarian cancer, was observed in p53 knockdown and null cells, while cells stably expressing mutant p53 demonstrated lower DKK1 and TMEPAI induction. Expression of mutant p53 or loss of p53 permit continued proliferation of ovarian cancer cell lines in the presence of TGFβ; however, cells expressing mutant p53 exhibit reduced migration and decreased protein levels of DKK1 and TMEPAI.

Abstract POSTER-TECH-1108: A novel spontaneously transformed model of early-stage serous cancer from murine oviductal epithelial cells

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy. Many studies have implicated the ovarian surface epithelia as the origin of HGSC origin. Newer studies suggest other sources of HGSC, such as the distal fallopian tube (oviduct in mice). Current models have to use multiple genetic manipulations of either isolated human fallopian tube secretory cells (FTSECs) or in transgenic mice to form HGSC-like tumors. While these models are vital for identifying and understanding the key players in HGSC, they do not provide information regarding the step-wise development of HGSC. This information is critical for earlier diagnosis and treatments, since most women develop HGSC spontaneously (85%-90% of cases). Thus, development of a spontaneous transformation model offers the potential to interrogate, in a step-wise manner, premalignant changes that initially drive HGSC progression. A murine oviductal epithelial (MOE) cell line was developed from oviducts of CD-1 mice that were validated using known markers: oviductal glycoprotein 1, Pax8, and acetylated tubulin. The low passage cells, designated MOELOW, ( As compared to MOELOW cells, MOEHIGH cells were significantly more proliferative, resistant to damage by bulky DNA adducts, displayed increased clonogenicity, and anchorage-independent colony formation. RNA-Seq analysis elicited 7,425 differentially expressed transcripts. Database comparisons uncovered several well-known pathways altered in HGSC and a few unique pathways altered upon MOE transformation. Consistent with the results from The Cancer Genome Atlas (TCGA) both the Rb and FoxM1 pathways were altered with increased expression of cyclin E1, c-Myc, PCNA; and reduced expression of p16INK4A and hyperphosphorylation of Rb. Remarkably, the MOEHIGH cells do not contain a mutation in Trp53, the HGSC hallmark expressed in 96% of all cases, but rather an alternatively spliced transcript. This p53 transcript, termed p53AS, has both a frameshift and truncation within the negative-regulation domain at the C-terminus. This region loses the lysine residues for MDM2-mediated degradation thereby stabilizing the protein. When wild-type p53 is transiently transfected back into MOEHIGH cells, the cells still cannot induce p21 expression, functioning similar to a p53 DNA-binding mutation. Nude mice injected subcutaneously with MOEHIGH cells all formed tumors but MOELOW mice did not. MOEHIGH forms densely packed and necrotic tumors, stained for Ki67, Pax8, and OVGP1. Intraperitoneal injections of MOEHIGH cells did not metastasize in any mice. This spontaneous transformation model allows for the evaluation of the stepwise progression of HGSC and since tumors are not metastatic, this will be one of the oviductal-derived first early stage serous carcinoma cell lines. Citation Format: Michael P. Endsley, Georgette Moyle-Heyrman, Suzanne M. Quartuccio, Daniel D. Lantvit, Joanna E. Burdette. A novel spontaneously transformed model of early-stage serous cancer from murine oviductal epithelial cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1108.

Abstract B7: Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression

Ovarian cancer is the most lethal gynecological malignancy in women. This is primarily due to patients presenting in late stage disease when the cancer has already metastasized. Contributing to this delayed diagnosis is uncertainty regarding the origin of ovarian cancer and what causes initial transformation. It has recently been hypothesized that the distal epithelia of the fallopian tube is the origin of transformation leading to ‘ovarian’ cancer, particularly the serous histotype. A potential precursor lesion to serous cancer, known as the ‘p53 signature’, has been identified in the fallopian tube. However, little is known about the normal human fallopian tube and what factor(s) might lead this potential precursor lesion to develop into serous ovarian cancer. In order to examine the role of the fallopian tube in the development of serous cancer, as well as potential initiation factors that might be involved in early disease, a novel three-dimensional (3D) culture method was utilized. This 3D culture supports growth within the normal tissue architecture and retains epithelia in contact with its stromal environment using alginate hydrogels. This system was utilized for examination of normal human fallopian tissue collected from women undergoing salpingectomy for both benign and malignant conditions. Donated tissues were treated with factors hypothesized to have a role in the onset of serous cancer for 2 and 7 days in culture. Insulin (5 ug/ml) was used to stimulate glycolysis, hydrogen peroxide (H2O2, 1 mM) was used as an oxidative stress mimetic, and the dominant female steroid hormones estrogen (10 nM) and progesterone (100 nM) were utilized to investigate hormone controlled cell growth. Resulting cultured tissues retained normal fallopian morphology as demonstrated by immunohistochemistry for cytokeratin-8 and E-cadherin. Additionally, both tubal epithelial sub-types (secretory and ciliated) were retained in culture (denoted by PAX8 and acetylated-tubulin staining, respectively). Fimbriae cultures were further analyzed for changes in proliferation, DNA damage, and importantly, induction of p53 expression, which is thought to be linked to the onset of serous cancer. Initial findings reveal ex vivo inducible p53 expression can be acquired. These hotspots of p53 expression are secretory cell enriched similarly to those seen in vivo, but are not always expressed concomitantly with γH2AX, a marker of DNA damage. Additionally, it was noted that oxidative stress (H2O2) and insulin significantly increased the rate of proliferation as compared to vehicle control. Conversely, the effect of the steroid hormones on proliferation was less notable, with progesterone treatment consistent with possible promotion of fallopian epithelial cell death. Characterization of biological function within early putative preneoplastic lesions is integral to ovarian cancer research. Without identifying the tissue of origin, current research may be focusing on genetic or protein changes that are different only because they are compared to the wrong normal tissue of origin. Further, targeted therapies might fail due to identification of targets that are not in the correct progenitor cell population. In order to evaluate the p53 signature as a potential precursor lesion to serous cancer, we must understand what induces p53 expression in the fallopian tube and why. Citation Format: Sharon L. Eddie, Suzanne M. Quartuccio, Jessica A. Shepherd, Rajul Kothari, Joanna E. Burdette. Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B7.