Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression.

The late stages of human breast cancer development are poorly understood complex processes associated with the expression of genes by cancers that promote specific tumorigenic activities, such as angiogenesis. Here, we describe the identification of periostin as a mesenchyme-specific gene whose acquired expression by human breast cancers leads to a significant enhancement in tumor progression and angiogenesis. Undetectable in normal human breast tissues, periostin was found to be overexpressed by the vast majority of human primary breast cancers examined. Tumor cell lines engineered to overexpress periostin showed a phenotype of accelerated growth and angiogenesis as xenografts in immunocompromised animals. The underlying mechanism of periostin-mediated induction of angiogenesis was found to derive in part from the up-regulation of the vascular endothelial growth factor receptor Flk-1/KDR by endothelial cells through an integrin alpha(v)beta(3)-focal adhesion kinase-mediated signaling pathway. These findings demonstrate the presence of a novel mechanism by which tumor angiogenesis is acquired with the expression of a mesenchyme-specific gene as a crucial step in late stages of tumorigenesis.

High expression of tumor necrosis factor-related apoptosis-inducing ligand is associated with favorable ovarian cancer survival.

PURPOSE: The molecular determinants of survival in ovarian cancer are poorly understood. Using expression microarrays, we recently found that high expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene is associated with prolonged survival in advanced ovarian cancer. TRAIL has also been shown to synergize with chemotherapeutic agents to induce apoptosis in ovarian cancer cell lines. We therefore sought to confirm the association between TRAIL expression and survival in a larger group of women with ovarian cancer. EXPERIMENTAL DESIGN: TRAIL expression was measured using quantitative real-time PCR in 120 epithelial ovarian cancers (11 stage I/II, 109 stage III/IV) and 8 normal ovarian surface epithelial samples. RESULTS: Ovarian cancers demonstrated 10-fold higher mean TRAIL expression than normal ovarian epithelial samples (P < 0.001). Among ovarian cancers, high TRAIL expression was associated with prolonged survival and was 2.2-fold higher in cancers from patients who lived more than 5 years compared with patients who died within 1 year (P = 0.03). CONCLUSIONS: TRAIL expression is higher in ovarian cancers relative to normal ovarian epithelium. High TRAIL expression is associated with favorable ovarian cancer survival, which may be attributable to increased chemosensitivity of cancers that express the most TRAIL. The use of TRAIL to enhance sensitivity of ovarian cancers to therapy represents an appealing molecular therapeutic strategy worthy of further investigation.

Prediction and uncertainty in the analysis of gene expression profiles.

We have developed a complete statistical model for the analysis of tumor specific gene expression profiles. The approach provides investigators with a global overview on large scale gene expression data, indicating aspects of the data that relate to tumor phenotype, but also summarizing the uncertainties inherent in classification of tumor types. We demonstrate the use of this method in the context of a gene expression profiling study of 27 human breast cancers. The study is aimed at defining molecular characteristics of tumors that reflect estrogen receptor tatus. In addition to good predictive performance with respect to pure classification of the expression profiles, the model also uncovers conflicts in the data with respect to the classification of some of the tumors, highlighting them as critical cases for which additional investigations are appropriate.