PTTG1 Levels Are Predictive of Saracatinib Sensitivity in Ovarian Cancer Cell Lines.

Src kinase is recognized as a key target for molecular cancer therapy. However, methods to efficiently select patients responsive to Src inhibitors are lacking. We explored the sensitivity of ovarian cancer cell lines to the Src kinase inhibitor saracatinib to identify predictive markers of drug sensitivity using gene microarrays. Pituitary tumor transforming gene 1 (PTTG1) was selected as a potential biomarker as mRNA levels were correlated with saracatinib resistance, as well as higher PTTG1 protein expression. PTTG1 expression was correlated with proliferation, cell division, and mitosis in ovarian cancer tissues data sets. In sensitive cell lines, saracatinib treatment decreased PTTG1 and fibroblast growth factor 2 (FGF2) protein levels. Downregulating PTTG1 by siRNAs increased saracatinib sensitivity in two resistant cell lines. Our results indicate PTTG1 may be a valuable biomarker in ovarian cancer to predict sensitivity to saracatinib, and could form the basis of a targeted prospective saracatinib trial for ovarian cancer.

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.

Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.

A tumor cell line, HCC1937, was established from a primary breast carcinoma from a 24-year-old patient with a germ-line BRCA1 mutation. A corresponding B-lymphoblastoid cell line was established from the patient's peripheral blood lymphocytes. BRCA1 analysis revealed that the tumor cell line is homozygous for the BRCA1 5382insC mutation, whereas the patient's lymphocyte DNA is heterozygous for the same mutation, as are at least two other family members' lymphocyte DNA. The tumor cell line is marked by multiple additional genetic changes including a high degree of aneuploidy, an acquired mutation of TP53 with wild-type allele loss, an acquired homozygous deletion of the PTEN gene, and loss of heterozygosity at multiple loci known to be involved in the pathogenesis of breast cancer. Comparison of the primary tumor with the cell line revealed the same BRCA1 mutation and an identical pattern of allele loss at multiple loci, indicating that the cell line had maintained many of the properties of the original tumor. This breast tumor-derived cell line may provide a useful model system for the study of familial breast cancer pathogenesis and for elucidating BRCA1 function and localization.

Mutations in the BRCA1-associated RING domain (BARD1) gene in primary breast, ovarian and uterine cancers.

Germline alterations of BRCA1 result in susceptibility to breast and ovarian cancer. The protein encoded by BRCA1 interacts in vivo with the BRCA1-associated RING domain (BARD1) protein. Accordingly, BARD1 is likely to be a critical factor in BRCA1-mediated tumor suppression and may also serve as a target for tumorigenic lesions in some human cancers. We have now determined the genomic structure of BARD1 and performed a mutational analysis of 58 ovarian tumors, 50 breast tumors and 60 uterine tumors. Seven polymorphisms were detected within the 2.34 kb coding sequence of BARD1 . Somatically acquired missense mutations were observed in one breast carcinoma and one endometrial tumor; in at least one of these cases, tumor formation was accompanied by loss of the wild-type BARD1 allele, following the paradigm for known tumor suppressor genes. In addition, a germline alteration of BARD1 was identified in a clear cell ovarian tumor (Gln564His); again, loss of the wild-type BARD1 allele was observed in the malignant cells of this patient. The Gln564His patient was also diagnosed with two other primary cancers: a synchronous lobular breast carcinoma and a stage IA clear cell endometrioid cancer confined to an endometrial polyp 6 years earlier. These findings suggest an occasional role for BARD1 mutations in the development of sporadic and hereditary tumors.

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.

BRCA1 and BRCA2 mRNA levels are coordinately elevated in human breast cancer cells in response to estrogen.

The steady state levels of BRCA1 and BRCA2 mRNAs were shown to be coordinately elevated by the steroid hormone estrogen but not progesterone in the human breast cancer cell lines BT-483 and MCF-7. Two different antiestrogens, trans 4'-hydroxytamoxifen and ICI 182,780, blocked the elevation of BRCA1 and BRCA2 mRNA levels, confirming that the effect was mediated through the estrogen receptor. In BT-483 cells, BRCA1 and BRCA2 mRNA levels were both elevated 18 to 24 h after estrogen stimulation, suggesting that the effect of estrogen was indirect. Cycloheximide blocked the estrogen effect implying that estrogen induces synthesis of an unidentified estrogen-responsive protein(s) that then result in the elevation of BRCA1 and BRCA2 mRNAs.