Matthew Dean

Factors affecting mitochondrial DNA quality from museum preserved Drosophila simulans

In this study we investigate how traditional killing methods and storage regimes affected mitochondrial DNA quality in Drosophila simulans. Here we define quality with three criteria: (1) size of extracted DNA, (2) extraction yield, and (3) ability to amplify from four target regions. Killing methods had a significant effect on extraction yield, but not on PCR success. Highest DNA yields were extracted from specimens exposed to cyanide, while the lowest were from specimens killed in 70% ethanol. Specimens stored for two years contained badly sheared DNA, which translated into a significant decrease in PCR success compared to freshly assayed specimens. The most dramatic decrease in PCR success occurred in the 1822 bp and 1332 bp amplicons, compared to the 959 bp and 291 bp fragments. Naphthalene did not affect any aspect of DNA quality; time of storage affected PCR success regardless of naphthalene environment. This study serves to further refine our understanding of DNA degradation.

PAX2 function, regulation and targeting in fallopian tube-derived high-grade serous ovarian cancer

The fallopian tube epithelium (FTE) is one of the progenitor populations for high-grade serous ovarian cancer (HGSC). Loss of PAX2 is the earliest known molecular aberration in the FTE occurring in serous carcinogenesis followed by a mutation in p53. Pathological studies report consistent loss of PAX2 in benign lesions as well as serous tumors. In the current study, the combined loss of PAX2 and expression of the R273H p53 mutant protein in murine oviductal epithelial (MOE) cells enhanced proliferation and growth in soft agar in vitro but was insufficient to drive tumorigenesis in vivo. A serially passaged model was generated to investigate the role of aging, but was also insufficient to drive tumorigenesis. These models recapitulate early benign lesions and suggest that a latency period exists between loss of PAX2, p53 mutation and tumor formation. Stathmin and fut8 were identified as downstream targets regulated by loss of PAX2 and mutation of p53 in MOE cells. Re-expression of PAX2 in PAX2-null human HGSC cells reduced cell survival via apoptosis. Phosphatase and tensin homolog (PTEN)shRNA negatively regulated PAX2 expression and stable re-expression of PAX2 in MOE:PTENshRNA cells significantly reduced proliferation and peritoneal tumor formation in athymic nude mice. PAX2 was determined to be a direct transcriptional target that was activated by wild-type p53, whereas mutant p53 inhibited PAX2 transcription in MOE cells. A small molecule screen using the proximal PAX2 promoter driving luciferase identified four small molecules that were able to enhance PAX2 mRNA expression in MOE cells. PAX2 re-expression in HGSC cells and PTEN-deficient oviductal tumors may have the potential to induce apoptosis. In summary, mutant p53 and PTEN loss negatively regulated PAX2 and PAX2 re-expression in HGSC cells induced cell death.

Activin A Stimulates Migration of the Fallopian Tube Epithelium, an Origin of High-Grade Serous Ovarian Cancer, through Non-Canonical Signaling.

Factors that stimulate the migration of fallopian tube epithelial (FTE)-derived high-grade serous ovarian cancer (HGSOC) to the ovary are poorly elucidated. This study characterized the effect of the ovarian hormone, activin A, on normal FTE and HGSOC. Activin A and TGFβ1 induced an epithelial-to-mesenchymal transition in murine oviductal epithelial (MOE) cells, but only activin A increased migration. The migratory effect of activin A was independent of Smad2/3 and required phospho-AKT, phospho-ERK, and Rac1. Exogenous activin A stimulated migration of the HGSOC cell line OVCAR3 through a similar mechanism. Activin A signaling inhibitors, SB431542 and follistatin, reduced migration in OVCAR4 cells, which expressed activin A subunits (encoded by INHBA). Murine superovulation increased phospho-Smad2/3 immunostaining in the FTE. In Oncomine, transcripts for the activin A receptors (ACVR1B and ACVR2A) were higher in serous tumors relative to the normal ovary, while inhibitors of activin A signaling (INHA and TGFB3) were lower. High expression of both INHBA and ACVR2A, but not TGFβ receptors or co-receptors, was associated with shorter disease-free survival in serous cancer patients. These results suggest activin A stimulates migration of FTE-derived tumors to the ovary.

PTEN loss in the fallopian tube induces hyperplasia and ovarian tumor formation

The signaling events involved in the onset of ovarian cancer from the fallopian tube epithelium (FTE) are crucial for early detection and treatment of the disease, but they remain poorly defined. Conditional homozygous knockout of PTEN mediated by PAX8-cre recombinase was sufficient to drive endometrioid and serous borderline ovarian carcinoma, providing the first model of FTE-derived borderline tumors. In addition, heterozygous PTEN deletion in the FTE resulted in hyperplasia, providing a model to study early events of human ovarian pathogenesis. To uncover the mechanism underlying the invasion of cancerous oviductal cells to the ovary, PTEN-deficient murine oviductal cells were developed and tagged with green fluorescent protein. Loss of PTEN increased cell migration, invasion, and upregulated WNT4, a key regulator of Müllerian duct development during embryogenesis. Further investigation revealed that WNT4 was required for increased migration and colonization of the ovary by PTEN-deficient oviductal cells in a β-catenin independent manner. Human tumor microarrays and ovarian cancer cells lines confirmed WNT4 expression in cancer and its role in migration. Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis.

Phytosteroids beyond estrogens: Regulators of reproductive and endocrine function in natural products

Foods and botanical supplements can interfere with the endocrine system through the presence of phytosteroids - chemicals that interact with steroids receptors. Phytoestrogens are well studied, but compounds such as kaempferol, apigenin, genistein, ginsenoside Rf, and glycyrrhetinic acid have been shown to interact with non-estrogen nuclear receptors. These compounds can have agonist, antagonist, or mixed agonist/antagonist activity depending on compound, receptor, cell line or tissue, and concentration. Some phytosteroids have also been shown to inhibit steroid metabolizing enzymes, resulting in biological effects through altered endogenous steroid concentrations. An interesting example, compound A (4-[1-chloro-2-(methylamino)ethyl]phenyl acetate hydrochloride (1:1)) is a promising selective glucocorticoid receptor modulator (SGRM) based on a phytosteroid isolated from Salsola tuberculatiformis Botschantzev. Given that

Activation of the IGF1 receptor stimulates glycogen synthesis by mink uterine epithelial cells

6.9 billion of herbal supplements are sold each year, is clear that further identification and characterization of phytosteroids is needed to ensure the safe and effective use of botanical supplements.

Irilone from Red Clover (Trifolium pratense) Potentiates Progesterone Signaling

Glycogen synthesis by mink uterine epithelial cells is stimulated by estradiol (E2) during estrus, although the mechanism/s through which the steroid promotes glycogen accumulation are unknown. Our aim was to determine if insulin is required for E2 induced glycogen synthesis by an immortalized mink uterine epithelial cell line (GMMe). We show that the cells expressed the genes for glycogen metabolizing enzymes (hexokinase 1, glucose‐6‐phosphatase 3, glycogen synthase 1, and glycogen phosphorylase‐muscle), receptors for insulin, insulin‐like growth factor 1 and E2 (Esr1). Interestingly, treatment of cells with E2 alone failed to stimulate glycogen production, whereas supraphysiological concentrations of insulin (50 μg/ml) only, significantly increased glycogen content. Moreover, insulin + E2 increased glycogen content when compared to insulin alone (p < 0.05), an affect that was blocked when cells were treated with the pure E2 receptor antagonist ICI 182,780. Glycogen synthesis in response to insulin was significantly inhibited when cells were pre‐treated with picropodophyllotoxin, an IGF1R antagonist. Treatment of cells with LY294002, a phosphatidylinositol 3‐kinase (PI3K) antagonist, blocked insulins effects on glycogen production whereas treatment with U0126, an inhibitor of mitogen activated kinase‐kinase (MEK1/2) was without effect. These findings suggest to us that the affects of E2 on glycogen synthesis by GMMe cells is mediated through Esr1 and increased responsiveness of the cells to insulin. Because picropodophylotoxin blocked the effects of insulin on glycogen production, and both insulin and IGF1 act through PI3K, it is possible that IGF1 plays a role in glycogen production by these cells.

Prolactin signaling drives tumorigenesis in human high grade serous ovarian cancer cells and in a spontaneous fallopian tube derived model

The use of botanical dietary supplements is becoming increasingly popular for the alleviation of hormonal-based conditions such as hot flashes, premenstrual syndrome, and fertility. Estrogen and progesterone receptors (ER and PR) play an essential role in these processes. However, despite the fact that many therapies used to alleviate gynecological conditions act through PR-mediated mechanisms, few studies have investigated or identified any herbal natural product components that act on this receptor. In the current study, we used a progesterone response element (PRE)-luciferase (Luc) reporter assay to identify four phytoprogestins present in a standardized red clover ( Trifolium pratense) extract. We found that the component irilone (1) potentiated the effect of progesterone in both endometrial and ovarian cancer cell lines. In these cancers, progesterone action is generally associated with positive outcomes; thus the potentiating effect of 1 may provide entirely new strategies for enhancing progesterone signaling as a means of mitigating conditions such as fibroids and endometriosis. Formononetin (3) and biochanin A (4) exhibited mixed agonist activity, while prunetin (2) acted only as an antagonist. Collectively, these results suggest that the effects of red clover extract repeatedly observed in cultured cells and the inverse correlation between risk of various cancers and flavonoid intake may be due, in part, to altered progesterone signaling.

Imaging Mass Spectrometry Reveals Crosstalk between the Fallopian Tube and the Ovary that Drives Primary Metastasis of Ovarian Cancer

The pathways responsible for tumorigenesis of high grade serous ovarian cancer (HGSOC) from the fallopian tube epithelium (FTE) are still poorly understood. A human prolactin (PRL) like gene, Prl2c2 was amplified >100 fold in a spontaneous model of FTE-derived ovarian cancer (MOEhigh - murine oviductal epithelium high passage). Prl2c2 stable knockdown in MOEhigh cells demonstrated a significant reduction in cell proliferation, 2-dimensional foci, anchorage independent growth, and blocked tumor formation. The overall survival of ovarian cancer patients from transcriptome analysis of 1868 samples was lower when abundant PRL and prolactin receptors (PRL-R) were expressed. A HGSOC cell line (OVCAR3) and a tumorigenic human FTE cell line (FT33-Tag-Myc) were treated with recombinant PRL and a significant increase in cellular proliferation was detected. A CRISPR/Cas9 mediated PRL-R deletion in OVCAR3 and FT33-Tag-Myc cells demonstrated significant reduction in cell proliferation and eliminated tumor growth using the OVCAR3 model. PRL was found to phosphorylate STAT5, m-TOR and ERK in ovarian cancer cells. This study identified Prl2c2 as a driver of tumorigenesis in a spontaneous model and confirmed that prolactin signaling supports tumorigenesis in high grade serous ovarian cancer.

Genome-wide transcriptional regulation of estrogen receptor targets in fallopian tube cells and the role of selective estrogen receptor modulators

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women. New evidence suggests that HGSOC arises in the fallopian tube and then colonizes the ovary before spreading into the peritoneal space. Therefore, due to the proximity of this metastasis, an experimental design was optimized using imaging mass spectrometry to capture the spatial composition of small molecules uniquely expressed when fallopian-tube-derived tumor cells were grown in the microenvironment of the ovary as a model of primary metastasis. The observed mass-to-charge ratios (m/z’s) that were induced specifically in coculture represent small molecules that may contribute to the metastasis of HGSOC selectively to the ovary. Human fallopian tube epithelial HGSOC and tumorigenic murine oviductal epithelial cells, but not normal cell types, repeatedly induced a signal from the ovary at m/z 170. This signal was identified as norepinephrine, which was confirmed to stimulate invasion of ovarian cancer cells lacking wild-type p53. These molecules may reveal pathways that contribute to metastasis and biological targets for therapeutic intervention to block ovarian metastasis of fallopian-tube-derived HGSOC. The developed mass spectrometry method can be adapted to other mammalian-based model systems for investigation of untargeted metabolomics that facilitate metastasis.