The GGLEAM Study: Understanding Glaucoma in the Ohio Amish.

Glaucoma leads to millions of cases of visual impairment and blindness around the world. Its susceptibility is shaped by both environmental and genetic risk factors. Although over 120 risk loci have been identified for glaucoma, a large portion of its heritability is still unexplained. Here we describe the foundation of the Genetics of GLaucoma Evaluation in the AMish (GGLEAM) study to investigate the genetic architecture of glaucoma in the Ohio Amish, which exhibits lower genetic and environmental heterogeneity compared to the general population. To date, we have enrolled 81 Amish individuals in our study from Holmes County, Ohio. As a part of our enrollment process, 62 GGLEAM study participants (42 glaucoma-affected and 20 unaffected individuals) received comprehensive eye examinations and glaucoma evaluations. Using the data from the Anabaptist Genealogy Database, we found that 80 of the GGLEAM study participants were related to one another through a large, multigenerational pedigree containing 1586 people. We plan to integrate the health and kinship data obtained for the GGLEAM study to interrogate glaucoma genetics and pathophysiology in this unique population.

Altered ErbB receptor signaling and gene expression in cisplatin-resistant ovarian cancer.

The majority of ovarian cancer patients are treated with platinum-based chemotherapy, but the emergence of resistance to such chemotherapy severely limits its overall effectiveness. We have shown that development of resistance to this treatment can modify cell signaling responses in a model system wherein cisplatin treatment has altered cell responsiveness to ligands of the erbB receptor family. A cisplatin-resistant ovarian carcinoma cell line PE01CDDP was derived from the parent PE01 line by exposure to increasing concentrations of cisplatin, eventually obtaining a 20-fold level of resistance. Whereas PE01 cells were growth stimulated by the erbB receptor-activating ligands, such as transforming growth factor-alpha (TGFalpha), NRG1alpha, and NRG1beta, the PE01CDDP line was growth inhibited by TGFalpha and NRG1beta but unaffected by NRG1alpha. TGFalpha increased apoptosis in PE01CDDP cells but decreased apoptosis in PE01 cells. Differences in extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling were also found, which may be implicated in the altered cell response to ligands. Microarray analysis revealed 51 genes whose mRNA increased by at least 2-fold in PE01CDDP cells relative to PE01 (including FRA1, ETV4, MCM2, AXL, MT3, TRAP1, and FANCG), whereas 36 genes (including IGFBP3, TRAM1, and KRT4 and KRT19) decreased by a similar amount. Differential display reverse transcriptase-PCR identified altered mRNA expression for TCP1, SLP1, proliferating cell nuclear antigen, and ZXDA. Small interfering RNA inhibition of FRA1, TCP1, and MCM2 expression was associated with reduced growth and FRA1 inhibition with enhanced cisplatin sensitivity. Altered expression of these genes by cytotoxic exposure may provide survival advantages to cells including deregulation of signaling pathways, which may be critical in the development of drug resistance.

Neuregulin expression, function, and signaling in human ovarian cancer cells.

PURPOSE: To investigate the expression and function of neuregulin (NRG) isoforms in ovarian cancer cell lines and tumor samples. EXPERIMENTAL DESIGN: Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry and mRNA by RT-PCR. erbB receptor levels and downstream signaling proteins were measured by Western blot analysis. RESULTS: Expression of NRG-1alpha and NRG-1beta proteins were detected by immunohistochemistry in 46 of 53 (87%) and 41 of 53 (77%) ovarian carcinomas, respectively. Serous carcinomas express higher levels of NRG-1alpha than endometrioid carcinomas (P = 0.017). NRG mRNA was detected by RT-PCR in 20 of 24 (83%) of ovarian carcinomas and eight of nine (89%) ovarian cancer cell lines. NRG-1alpha stimulated the growth of 5 of 14 cell lines whereas NRG-1beta stimulated 7 of 14 cell lines. The magnitude of NRG growth response was significantly associated with erbB2 expression levels. NRG-1alpha and -1beta (1 nM) growth-stimulated cell lines PE01 and PE06 demonstrated increased tyrosine phosphorylation of erbB2 and elevated tyrosine phosphorylation of ERK1 and ERK2. In contrast, the SKOV-3 cell line, the growth of which was unaffected, did not show these downstream responses. An anti-erbB3 receptor antibody (clone H3.105.5) blocked NRG-1beta growth changes and signaling in these cell lines. Conversely, the anti-erbB4 antibody (clone H4.72.8) enhanced NRG-beta1 growth stimulation. Herceptin also inhibited growth. CONCLUSIONS: With NRG expression in the majority of ovarian carcinomas and cell lines, there is the potential for autocrine regulation of cell growth. Interfering with ligand-receptor interactions by receptor blocking antibodies suggests erbB3 is primarily involved in NRG-1beta-induced proliferation, with erbB4 having a more complex role.

Raf-1 is the predominant Raf isoform that mediates growth factor-stimulated growth in ovarian cancer cells.

There is currently much interest in the role of the Raf family in cancer, particularly since mutated B-Raf has been shown to be oncogenic in certain disease types. In this study we have explored the expression, signaling and function of the three known Raf isoforms (Raf-1, A-Raf and B-Raf) in patients with ovarian cancer. While increased expression of Raf-1 was associated with poor survival, increased expression of B-Raf was associated with improved survival. Using a panel of ovarian cancer cell lines, all three isoforms were shown to be involved in growth factor initiated signaling. Antisense inhibition of function in ovarian cancer cell lines indicated that both Raf-1 and A-Raf, but not B-Raf, were linked to cell proliferation. Raf-1 (but not A-Raf or B-Raf) was also associated with reduced apoptosis. While individual Raf reduction by isoform-targeted antisense oligonucleotides (ODNs) produced growth inhibition in some cell lines, similar use of the MEK inhibitor UO126 produced growth inhibition in all cell lines tested. These data suggest that Raf-1 is the predominant Raf isoform responsible for regulating cellular growth in ovarian cancer cells and may be particularly important in high grade serous ovarian cancers.

Role of TGF alpha stimulation of the ERK, PI3 kinase and PLC gamma pathways in ovarian cancer growth and migration.

The Epidermal Growth Factor Receptor (EGFR) and its structural relative erbB2 are frequently over-expressed in ovarian cancers and both are strongly associated with poor patient survival. To investigate the relative roles of these receptors in the regulation of cell growth and migration, a panel of ovarian carcinoma cell lines were stimulated with TGF alpha and NRG1beta. TGF alpha had a much greater influence on cell migration than NRG1beta where growth effects were equivalent. The extent of TGF alpha-stimulated migration on collagen in these assays could be associated with erbB2 expression levels. In addition, TGF alpha was found to stimulate activation of the ERK, PI3 kinase and PLC gamma pathways. Direct blockade of the TGF alpha-interacting receptor EGFR inhibited both cell growth and migration, as well as downstream signaling induced by the growth factor. Specific blockade of the downstream proteins MEK and PI3 kinase significantly affected TGF alpha-induced mitogenesis in the cell lines tested but had less impact upon migration. Conversely, inhibition of the PLC gamma pathway had little effect on cell growth but significantly decreased TGF alpha-driven migration. These results corroborate the likely importance of migration as well as growth in erbB receptor over-expressing ovarian cancers and directly implicate the roles of ERK and PI3 kinase in growth control, and PLC gamma in the regulation of migration in this disease.