Monique A. Spillman

Identification of a RING protein that can interact in vivo with the BRCA1 gene product

The hereditary breast and ovarian cancer gene, BRCA1, encodes a large polypeptide that contains the cysteine–rich RING motif, a zinc–binding domain found in a variety of regulatory proteins. Here we describe a novel protein that interacts in vivo with the N–terminal region of BRCA1. This BRCA1–associated RING domain (BARD1) protein contains an N–terminal RING motif, three tandem ankyrin repeats, and a C–terminal sequence with significant homology to the phylogenetically conserved BRCT domains that lie near the C terminus of BRCA1. The BARD1/BRCA1 interaction is disrupted by BRCA1 missense mutations that segregate with breast cancer susceptibility, indicating that BARD1 may be involved in mediating tumour suppression by BRCA1.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

OBJECTIVES Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Frequent IGF2/H19 Domain Epigenetic Alterations and Elevated IGF2 Expression in Epithelial Ovarian Cancer

Overexpression of the imprinted insulin-like growth factor-II (IGF2) is a prominent characteristic of gynecologic malignancies. The purpose of this study was to determine whether IGF2 loss of imprinting (LOI), aberrant H19 expression, and/or epigenetic deregulation of the IGF2/H19 imprinted domain contributes to elevated IGF2 expression in serous epithelial ovarian tumors. IGF2 LOI was observed in 5 of 23 informative serous epithelial ovarian cancers, but this did not correlate with elevated expression of IGF2 H19 RNA expression levels were also found not to correlate with IGF2 transcript levels. However, we identified positive correlations between elevated IGF2 expression and hypermethylation of CCCTC transcription factor binding sites 1 and 6 at the H19 proximal imprint center (P = 0.05 and 0.02, respectively). Hypermethylation of CCCTC transcription factor sites 1 and 6 was observed more frequently in cancer DNA compared with lymphocyte DNA obtained from women without malignancy (P < 0.0001 for both sites 1 and 6). Ovarian cancers were also more likely to exhibit maternal allele-specific hypomethylation upstream of the imprinted IGF2 promoters when compared with normal lymphocyte DNA (P = 0.004). This is the same region shown previously to be hypomethylated in colon cancers with IGF2 LOI, but this was not associated with LOI in ovarian cancers. Elevated IGF2 expression is a frequent event in serous ovarian cancer and this occurs in the absence of IGF2 LOI. These data indicate that the epigenetic changes observed in these cancers at the imprint center may contribute to IGF2 overexpression in a novel mechanistic manner. (Mol Cancer Res 2006;4(4):283–92)

Type-Specific Cell Line Models for Type-Specific Ovarian Cancer Research

Background Ovarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies. Methods We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis. Results Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements. Conclusions: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.

Restoration of miR-200c to Ovarian Cancer Reduces Tumor Burden and Increases Sensitivity to Paclitaxel

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III β-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose. Mol Cancer Ther; 11(12); 2556–65. ©2012 AACR.

Tissue-Specific Pathways for Estrogen Regulation of Ovarian Cancer Growth and Metastasis

Menopausal estrogen (E2) replacement therapy increases the risk of estrogen receptor (ER)-positive epithelial ovarian cancers (EOC). Whether E2 is tumorigenic or promotes expansion of undiagnosed preexisting disease is unknown. To determine E2 effects on tumor promotion, we developed an intraperitoneal mouse xenograft model using ZsGreen fluorescent ER(-) 2008 and ER(+) PEO4 human EOC cells. Tumor growth was quantified by in vivo fluorescent imaging. In ER(+) tumors, E2 significantly increased size, induced progesterone receptors, and promoted lymph node metastasis, confirming that ERs are functional and foster aggressiveness. Laser-captured human EOC cells from ER(-) and ER(+) xenografted tumors were profiled for expression of E2-regulated genes. Three classes of E2-regulated EOC genes were defined, but <10% were shared with E2-regulated breast cancer genes. Because breast cancer selective ER modulators (SERM) are therapeutically ineffective in EOC, we suggest that our EOC-specific E2-regulated genes can assist pharmacologic discovery of ovarian-targeted SERM.

A phase II trial of brivanib in recurrent or persistent endometrial cancer: An NRG Oncology/Gynecologic Oncology Group Study

PURPOSE Brivanib, an oral, multi-targeted tyrosine kinase inhibitor with activity against vascular endothelial growth factor (VEGF) and fibroblast growth factor receptor (FGFR) was investigated as a single agent in a phase II trial to assess the activity and tolerability in recurrent or persistent endometrial cancer (EMC). PATIENTS AND METHODS Eligible patients had persistent or recurrent EMC after receiving one to two prior cytotoxic regimens, measurable disease, and performance status of ≤2. Treatment consisted of brivanib 800 mg orally every day until disease progression or prohibitive toxicity. Primary endpoints were progression-free survival (PFS) at six months and objective tumor response. Expression of multiple angiogenic proteins and FGFR2 mutation status was assessed. RESULTS Forty-five patients were enrolled. Forty-three patients were eligible and evaluable. Median age was 64 years. Twenty-four patients (55.8%) received prior radiation. Median number of cycles was two (range 1-24). No GI perforations but one rectal fistula were seen. Nine patients had grade 3 hypertension, with one experiencing grade 4 confusion. Eight patients (18.6%; 90% CI 9.6%-31.7%) had responses (one CR and seven PRs), and 13 patients (30.2%; 90% CI 18.9%-43.9%) were PFS at six months. Median PFS and overall survival (OS) were 3.3 and 10.7 months, respectively. When modeled jointly, VEGF and angiopoietin-2 expression may diametrically predict PFS. Estrogen receptor-α (ER) expression was positively correlated with OS. CONCLUSION Brivanib is reasonably well tolerated and worthy of further investigation based on PFS at six months in recurrent or persistent EMC.

Gab2 regulates the migratory behaviors and E-cadherin expression via activation of the PI3K pathway in ovarian cancer cells

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the advanced stage when the cancer cells have already migrated and invaded into other tissues and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is essential to the design of effective therapy. In this study, we investigated the function of scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration and invasion, and downregulates E-cadherin expression in ovarian cancer cells with low-Gab2 expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By expressing Gab2 wild-type and Gab2 mutants that are defective in activation of the PI3K and Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes characteristics of EMT in ovarian cancer cells.

Clinical report - Maternal-fetal intervention and fetal care centers

The past 2 decades have yielded profound advances in the fields of prenatal diagnosis and fetal intervention. Although fetal interventions are driven by a beneficence-based motivation to improve fetal and neonatal outcomes, advancement in fetal therapies raises ethical issues surrounding maternal autonomy and decision-making, concepts of innovation versus research, and organizational aspects within institutions in the development of fetal care centers. To safeguard the interests of both the pregnant woman and the fetus, the American College of Obstetricians and Gynecologists and the American Academy of Pediatrics make recommendations regarding informed consent, the role of research subject advocates and other independent advocates, the availability of support services, the multidisciplinary nature of fetal intervention teams, the oversight of centers, and the need to accumulate maternal and fetal outcome data.

Risk-reducing salpingectomy as preventative strategy for pelvic serous cancer.

Abstract The systemic failure to detect early-stage ovarian cancer may be attributed to a significant amount of pelvic serous cancers arising from the fallopian tube rather than the ovarian surface epithelium. This article reviews the possibility of applying risk-reducing salpingectomy as a new paradigm for the prevention of pelvic serous cancer in both high- and low-risk women.