Devon A. Thompson

Up-regulation of Akt3 in Estrogen Receptor-deficient Breast Cancers and Androgen-independent Prostate Cancer Lines

We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20–60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.

GATA‐3 is expressed in association with estrogen receptor in breast cancer

To better understand the molecular basis for the hormone‐responsive phenotype in breast cancer, we have used a human cDNA array to compare patterns of gene expression between breast carcinoma cell lines discordant for estrogen receptor (ER) expression. These experiments indicated abundant expression of the transcription factor GATA‐3 in the ER‐positive cell lines MCF7 and T‐47D, with minimal or no expression in the ER‐negative cells lines MDA‐MB‐231 and HBL‐100. Northern blot analysis of a panel of human breast carcinoma cell lines demonstrated a correlation between ER and GATA‐3 expression. Studies of MCF7 cells grown in the absence or presence β‐estradiol indicated that GATA‐3 expression was not responsive to estradiol. Protein immunoprecipitation and gel shift analysis confirmed the presence of functional GATA‐3 protein in MCF7 but not in HBL‐100 nuclear extracts. A panel of 47 primary breast cancers was characterized for expression of ER and GATA‐3 using immunoperoxidase assay. In primary tumors, a statistically significant correlation between ER and GATA‐3 expression was established (p < 0.0001, χ2). Our results indicate that GATA‐3, in association with ER, is likely to regulate genes critical to the hormone‐responsive breast cancer phenotype. Int. J. Cancer (Pred. Oncol.) 84:122–128, 1999.

Structures of the Cancer Related Aurora-A, FAK and EphA2 Protein Kinases from Nanovolume Crystallography

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.

Moesin expression is associated with the estrogen receptor–negative breast cancer phenotype

BACKGROUND Estrogen receptor (ER)-positive breast carcinomas possess a less aggressive phenotype than ER-negative breast carcinomas. We hypothesize that a set of genes exists that is expressed only in ER-negative breast carcinomas, which account for the more malignant phenotypic characteristics of these tumors. METHODS We have used a new technique of polymerase chain reaction select suppression subtractive hybridization to identify genes that are expressed only in ER-negative carcinomas. RESULTS Seventy-one cDNA clones generated by suppression subtractive hybridization were screened by Northern blot analysis with RNA from ER-positive MCF7 and ER-negative MDA-MB-231 breast carcinoma cell lines. Fifteen clones were differentially expressed in MDA-MB-231 cells. Five of these 15 clones were consistently found to be associated with the ER-negative phenotype in a panel of eight breast carcinoma cell lines. Sequence analysis demonstrated that three of these clones were derived from vimentin and two clones from moesin. Western blot analysis with antihuman moesin antibody confirmed that moesin protein was overexpressed in ER-negative breast carcinoma cell lines but absent from ER-positive breast carcinomas. Moesin mRNA was examined in a panel of 29 primary breast carcinomas with semi-quantitative reverse transcriptase-polymerase chain reaction. Moesin expression was found to be decreased significantly in ER-positive compared with ER-negative tumors (P < .01). CONCLUSIONS Vimentin and moesin are differentially expressed in association with the ER-negative breast cancer phenotype. Moesin is a membrane/actin filament protein involved in dynamic restructuring of the cell surface and filopodia, a cell structure needed for cell adhesion and motility. Moesin may play a role in the invasiveness and pattern of metastasis characteristic of ER-negative breast cancers.