Mary E. McAsey

Endoglin expression as a measure of microvessel density in cervical cancer.

Objective To evaluate endoglin, a membrane protein and member of the transforming growth factor β-1 receptor complex, as an endothelial marker of angiogenesis in cervical cancer tissues. Methods Tumor tissue was collected from 31 surgically treated stage IB nonbulky (under 5 cm) cervical cancer subjects, and samples were fixed in formalin and embedded in paraffin. Endoglin was stained on 5-μm slide sections by the DAKO Catalyzed Signal Amplification method (DAKO Corporation, Carpinteria, CA). Factor VIII was stained by standard immunohistochemistry. Positively stained microvessels were counted in “hot spots” at 200× magnification. Clinical data were correlated with vessel counts by Spearman correlation. Mean differences in counts were tested using paired t tests. Results This staining method for endoglin identified significantly more vessels than the factor VIII method (mean 92 ± 45 versus 33 ± 16, P < .001). Endoglin and factor VIII counts correlated significantly with deep stromal invasion (Spearman rho 0.466 and 0.522, respectively, P < .05); however, only endoglin counts correlated significantly with lymph node metastases (rho = .495, P < .01). Conclusion Endoglin is stimulated in tumor angiogenesis and might be relatively more specific than commonly used endothelial markers. The endoglin system was more sensitive for staining capillaries in neoplastic cervical tissue, better predicted lymph node metastases, and should be widely applicable for the study of other tumors.

WNT7A regulates tumor growth and progression in ovarian cancer through the WNT/β-catenin pathway.

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway. Mol Cancer Res; 10(3); 469–82. ©2012 AACR.

Estradiol regulation of astroglia and apolipoprotein E: an important role in neuronal regeneration.

The effects of ovarian hormone on neuronal growth and function are well known. However, equally important, but often neglected, are ovarian hormone effects on glia. Our in vivo and in vitro studies show that estradiol modifies both neuronal growth and glial activity and these effects are tightly linked. Estradiol stimulates neurite growth and the release of the glial apolipoprotein E (apoE) in culture studies. Estradiol-stimulated neurite growth in these cultures requires apoE. Estradiol replacement in ovariectomized mice transiently increases the expression of apoE, the low density lipoprotein receptor related protein (LRP) and synaptophysin throughout the brain. Continuous estradiol replacement over two months loses effect on apoE, LRP, and synaptophysin and suppresses reactive gliosis. Estrous cycle variation of glial activation (GFAP) and apoE are not identical. We propose that estradiol (and other ovarian hormones) functions as a zeitgeber to co-ordinate neuronal-glial interactions. Co-ordination assures temporally appropriate excitatory and inhibitory interactions between glia and neurons. With aging and the loss of ovarian cyclicity, some of this co-ordination must be diminished. These observations present significant clinical implications. Approaches to hormone therapy (HT), for diminishing the risk of chronic neurological diseases, need to consider the temporal nature of ovarian hormones in brain repair and plasticity. Moreover, approaches must consider apoE genotype. The neuroprotective effects of HT in numerous chronic age-related diseases may represent effective co-ordination of repair processes rather than direct disease-specific actions. Moreover, the role of glial-derived proteins in neuroprotection should not be ignored.

Inhibin‐ A and Pro‐αC Are Elevated in Preeclamptic Pregnancy and Correlate with Human Chorionic Gonadotropin

PROBLEM: Serum concentrations of the heterodimeric glycoprotein inhibin‐A (α‐βA) and its α‐subunit increase during pregnancy. The placenta is the predominant source of inhibin during pregnancy, and a paracrine role in the trophoblast has been suggested. Elevated serum concentrations of inhibin α‐subunit as well as the glycoprotein human chorionic gonadotropin (hCG) have been described in preeclamptic pregnancy. The objectives of this investigation were to compare serum concentrations of inhibin‐A and inhibin pro‐αC in preeclamptic and normotensive pregnancy, and to examine the relationship of hCG and inhibin‐A in those pregnancies.

Time course of response to estradiol replacement in ovariectomized mice: brain apolipoprotein E and synaptophysin transiently increase and glial fibrillary acidic protein is suppressed.

The current study examined the effect of long-term estradiol replacement in ovariectomized mice. Estradiol-17beta (E2) pellets or vehicle pellets were implanted at the time of ovariectomy (OVX) in young adult female mice. Five mice from each group were sacrificed at 5, 14, 28 and 49 days after OVX and pellet replacement. Western blotting of homogenates from somatosensory cortex, hippocampus, olfactory bulb and cerebellum was performed to obtain concentrations of glial fibrillary acidic protein (GFAP), apolipoprotein E (apoE) and synaptophysin (SYN). At 5 days after OVX, GFAP levels were not affected by E2 replacement. In contrast to GFAP, synaptophysin and apoE concentrations were significantly elevated by 15% and 25%, respectively, in the E2-replaced group compared to the vehicle-replaced group at 5 days but by 14 days concentrations were equivalent. Late in the time course of this study, at 49 days, GFAP concentrations were higher in the E2-deprived mice but did not increase in the E2-replaced group. Immunocytochemistry for GFAP confirmed this observation. Of note was that these effects occurred in all four brain regions measured. These observations suggest that estradiol is able to suppress reactive gliosis. In addition, E2 replacement in OVX mice is associated with transiently higher levels of apoE and synaptophysin.

Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17β estradiol

Studies have suggested that 17beta estradiol (E2) can modify apolipoprotein E (apoE) expression. The current study determined if apoE protein varied in different regions of the mouse brain as a function of the estrous cycle and if E2 could increase apoE protein expression. In this study apoE concentration was lowest on estrus in the hippocampus, cingulate cortex and frontal cortex. In contrast, apoE concentration was highest on estrus in the olfactory bulb and cerebellum. There were no differences in the striatal apoE expression throughout the estrous cycle. Exogenous E2 significantly raised tissue levels of apoE in the olfactory bulb and cerebellum at 5 days after treatment. There was a slight, but nonsignificant increase in cortical expression of apoE and no change in striatum. Immunocytochemical localization studies found estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in cortical neurons and glia. In the cerebellum and olfactory bulb, ERbeta was seen primarily in glia. ERalpha was not observed in the cerebellum and was rare in the olfactory bulb. Neither ERalpha nor ERbeta was seen in the striatum. Our data show regional differences in the production of apoE throughout the estrous cycle. In addition, exogenous E2 has regionally specific effects on apoE expression. Regional variability in apoE production appears to vary as a function of the estrogen receptor subtype.

Apolipoprotein E may be a critical factor in hormone therapy neuroprotection.

In this review we examine the evidence for ovarian hormone neuroprotection in chronic neurological diseases, including stroke. We propose that neuroprotection may involve the ability of estrogens to modulate apolipoprotein E (apoE) and its receptor, the low density lipoprotein receptor related protein (LRP). Results from numerous studies have demonstrated that (1) nerve regeneration is severely delayed in apoE-gene knockout (KO) mice as compared to wild-type (WT) littermates; (2) 17beta estradiol replacement in ovariectomized mice resulted in a significant increase in levels of apoE and LRP, in the olfactory bulb (OB) and other brain areas; (3) estradiol treatment increased both apoE and neurite outgrowth in cortical and olfactory neuronal cultures; and (4) estradiol treatment had no effect on neurite outgrowth in cultures deprived of apoE or in the presence of apoE4. In essence these studies suggest that apoE is a critical intermediary for the beneficial effects of 17beta estradiol on nerve repair, which can lead to functional reorganization (plasticity). Future studies of HT should evaluate the effects of apoE genotype and production estradiol on neuroprotection.

Estradiol replacement increases the low-density lipoprotein receptor related protein (LRP) in the mouse brain

Numerous epidemiology studies have shown protective effects of hormone therapy (HT) on chronic neurological diseases. We have proposed that some of the neuroprotective effects of estrogen are mediated by apolipoprotein E (apoE). Polymorphisms of receptors for apoE modify the risk for dementia. To our knowledge, no reports exist showing CNS effects of estrogen replacement on members of the low-density lipoprotein receptor family. The current study focused on the effect of estradiol-17beta (E2) replacement on protein expression of two members of the receptor family, the low-density lipoprotein receptor (LDL-r) and low-density lipoprotein receptor related protein (LRP) in ovariectomized mice. Five days of E2 replacement significantly increased LRP expression in the hippocampus, olfactory bulb and neocortex but not in cerebellum. In contrast, E2 treatment decreased LDL-r protein expression in olfactory bulb. HT modification of both apoE and LRP could have wide-spread effects on cellular function given LRPs manifold signaling functions.

Neocortical and Hippocampal Glial Fibrillary Acidic Protein Immunoreactivity Shows Region-Specific Variation during the Mouse Estrous Cycle

Ovarian hormones modulate both neuronal and glial activation during the estrous cycle. These effects are particularly well characterized in the hypothalamus. Ovarian hormones also affect brain regions not directly related to reproductive function. In this study we used glial fibrillary acidic protein (GFAP) immunocytochemistry to quantify astroglial cells and process density in both the neocortex and hippocampus during the estrous cycle. Our data show that the density of GFAP immunoreactive processes in the hippocampus peaks on proestrus although cell density does not change. In contrast, both GFAP immunoreactive cell and process densities are elevated on diestrus and proestrus in the supragranular layer of the somatosensory cortex and reach a nadir on estrus and metestrus. This activation pattern is not apparent in the motor or cingulate cortex. Neocortical GFAP immunoreactivity appears to follow the distribution of estrogen receptor-α-like immunoreactivity. Our data show that ovarian hormones have regionally specific effects on glial activation within the neocortex. Characterizing glial activation by ovarian hormones is important since astroglia are the source of numerous trophic factors and play an important, although often unrecognized, role in neuronal metabolism and function.

Abstract 3904: Potential role of WNT7A in ovarian cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Most ovarian cancers are of epithelial origin, arising from a single layer of simple epithelial cells that covers the surface of the ovary. Epithelial ovarian cancer (EOC) remains the most common cause of death from a gynecological malignancy. The high mortality related to EOC is thought to be because of the advanced stage of the disease at presentation. Therefore, the early stage of detection and diagnosis as well as staging of ovarian cancer ultimately lead patients to the chance for a complete cure and long-term survival. The WNT system is known to play an important role in the regulation of developmental events where they govern cellular proliferation and differentiation. While WNT signaling plays a key role in the embryonic development of the ovary as well as normal follicular development and ovarian function, abnormal activation the WNT/CTNNB1 signaling pathway has been associated with ovarian tumorigenesis. It is likely that the WNT signaling pathway is involved in ovarian cancer development via multiple, diverse mechanisms such as gene mutations and changes in pathway components such as extracellular inhibitors and intranuclear transcription cofactors. However, the ligand partners associated with WNT signaling have not been examined in ovarian cancers. WNT7A is exclusively expressed in the luminal epithelium in reproductive tracts, whereas role of WNT7A in the normal ovary has not been reported, because WNT7A is not detected in the normal ovarian surface epithelium. Therefore, the objective is to study the functional role of WNT7A in ovarian cancer, especially to investigate WNT7A regulation of known metastatic inducing factors. First, we found that WNT7A was widely expressed in highly metastatic epithelial ovarian cancer cell lines (SKOV3.ip1). WNT7A was very low or almost undetectable in the ovarian biopsies taken from normal tissues, moderately expressed in the benign, but exclusively increased in the malignant ovarian tumors. Further, PLAU, MMP7, MMP9 and CDH1 were highly expressed in the same malignancy cases. To determine whether WNT7A activates the CTNNB1-TCF/LEF signal transduction pathway in ovarian tumors, SKOV3 cells, which do not express WNT7A, were transfected with the TOP-FLASH reporter, then treated with WNT7A conditioned media. The activity of the TOP-FLASH reporter in the SKOV3 cells was significantly stimulated by WNT7A conditioned media. WNT7A siRNA treatment reduced WNT7A mRNA to 27% (P<0.05). Down-regulation of WNT7A led to significant reduction in the mRNA levels of MMP7 (64%), MMP9 (47%) and CDH1 (72%) in the SKOV3.ip1 cells. While immunoreactive CTNNB1 was observed in the cytoplasmic and membrane of the SKOV3.ip1 and SKOV3 cells, nuclear CTNNB1 was only limited in the SKOV3.ip1 cells, but not in the SKOV3 cells. Further, WNT7A increased directional cell migration and attachment. Therefore, these results suggest that WNT7A is active as a secreted protein and a critical regulator of ovarian tumorigenesis as a metastatic factor of EOC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3904.