Differential expression of ETS family members in prostate cancer tissues and androgen-sensitive and insensitive prostate cancer cell lines.

The ETS family of transcription factors plays important roles in both normal and neoplastic cells for different biological processes such as proliferation, differentiation, development, transformation, apoptosis, migration, invasion and angiogenesis. The 27 ETS factors are probably a part of complex regulatory networks including interactions among family members. In human prostate cancer, rearrangements have been found in several genes of the ETS family resulting in chimeric oncoproteins. In a previous study we found that the ETS family prototype, Ets-1 affects biological properties of PC3 prostate cancer cells. In a first effort to understand the cooperative interactions between different ETS factors in prostate cancer, in the present study we examined the expression pattern of all 27 ETS members using quantitative RT-PCR (qRT-PCR) in the androgen-sensitive VCaP and LNCaP, and the androgen-insensitive PC3 and DU-145 prostate cancer cell lines as well as in human prostate cancer tissue samples. We further investigated whether the ETS family prototype, Ets-1, regulates other ETS family members by examining the effect of Ets-1 blockade in PC3 cells on their expression. We found an expression specificity of various ETS family members in the prostate cancer cell lines which might reflect their different biological properties. In human prostate samples only 3 among the 27 ETS family members (Ehf, Elk-4 and Ets-2) showed significant expression differences between normal and cancerous prostate glands. We finally demonstrate that the family prototype, Ets-1, regulates the family members Elf-1, Elf-2, Elk-1, Etv-5 and Spi-1 in PC3 prostate cancer cells. Chimeric oncoproteins containing ETS family members arising due to frequent translocations in prostate cancer are probably part of a regulatory network involving other ETS family members as well.

Comprehensive gene expression microarray analysis of Ets-1 blockade in PC3 prostate cancer cells and correlations with prostate cancer tissues: Insights into genes involved in the metastatic cascade.

Ets-1 is the prototype of the ETS family of transcription factors and is suggested to play an important role in the malignant progression of prostatic carcinomas. Therefore, in this study we investigated the effect of blocking Ets-1 in PC3 prostate cancer cells on genes involved in the metastatic cascade, and correlated these findings with prostate cancer tissues. Two stable PC3 cell cultures were established by transfection with either an Ets-1 inverse antisense expression vector or a mock control vector. The effect of blocking Ets-1 on genes involved in the metastatic cascade was assessed by a comprehensive gene expression microarray analysis of Ets-1 inverse and mock control cells. Correlating the sets of genes found in the PC3 microarray data with prostate cancer tissues was performed by verifying the genes in a comprehensive gene expression microarray analysis of RNA extracted from laser microdissected normal prostate glands and from carcinoma glands taken from prostate cancer patients. Western blot analysis confirmed the presence of Ets-1 in mock cells and its absence in Ets-1 inverse cells. In the Ets-1 blockade microarray, many differentially expressed genes were found; however, only genes with a greater than 10-fold up- or down-regulation between the Ets-1 blockade and mock control were considered significant. The genes were placed into four groups that play a role in the so-called metastatic cascade based on their known functions in proliferation, apoptosis, migration and angiogenesis. The genes found in the Ets-1 blockade microarray analysis were verified for their presence in the microarray analysis of prostate cancer tissues. Genes found in the microarray analysis of prostate cancer tissues with an >2-fold change and a p-value <0.01 were considered significant. We identified sets of genes that are involved in the metastatic cascade and are known to be implicated in prostate cancer to show changes in the expression patterns due to the Ets-1 blockade in PC3 cells. Correlating these sets of genes with the findings in prostate cancer tissues, we identified 16 genes that are up- or down-regulated in healthy compared to tumor prostate glands. Further investigation revealed that 4 out of the 16 genes have been reported to be regulated by members of the ETS family. These findings provide in vitro and in vivo evidence for the importance of Ets-1 in the development and progression of prostate cancer.

Ets-1 is implicated in the regulation of androgen co-regulator FHL2 and reveals specificity for migration, but not invasion, of PC3 prostate cancer cells.

Different members of the Ets-family of transcription factors are involved in TMPRSS-2-Ets translocations frequently found in human prostate cancers. We previously reported that Ets-1, which is the prototype of Ets-family members, promotes both migration and invasion of melanoma, Hela and glioma cells. Here, we examined whether Ets-1 has a similar effect upon migration and invasion of PC3 prostate cancer cells, and whether it is implicated in the regulation of the androgen co-regulator four and a half LIM only protein-2 (FHL2). Two stable PC3 cell cultures were established by transfection with either an Ets-1 inverse antisense expression vector or a mock control vector. Western blot analysis confirmed presence of Ets-1 in mock and absence in Ets-1 inverse cells. Microarray and qRT-PCR revealed an up-regulation of FHL2 in Ets-1 blocked cells, compared to mock. To examine the effects of Ets-1 upon cell migration, a wound assay was performed, and demonstrated that wounds were completely colonized by mock compared to Ets-1 blocked cells after 55 h. Evaluation of the effect upon invasion was examined using the Boyden chamber, which revealed no significant difference between mock and Ets-1 blocked cells. In conclusion, our study demonstrated for the first time that Ets-1 is implicated in the regulation of the androgen co-regulator FHL2, and reveals specificity of action for migration, but not invasion of PC3 prostate cancer cells.

Regulation of protein tyrosine kinases in tumour cells by the transcription factor Ets-1.

Tyrosine phosphorylation is one of the key covalent modifications that occurs in multicellular organisms as a result of intercellular communication. The family of tyrosine kinases (PTKs) are responsible for part of the cellular phosphorylation and are involved in a broad variety of cellular functions including differentiation, proliferation, migration, invasion, angiogenesis and survival under physiological as well as pathological conditions. Aberration in PTK signalling occurs in inflammatory diseases and diabetes, and aberrant expression can lead to benign proliferative conditions as well as to various forms of cancer. Indeed, more than 70% of the known oncogenes and proto-oncogenes involved in cancer code for PTKs. Therefore, these enzymes are now used as targets in the treatment of different tumours. Ets-1 is a transcription factor expressed in a number of human malignancies with demonstrated roles within both neoplastic cells and tumour stroma. These roles include stimulation of tumour cell proliferation and invasion as well as tumour angiogenesis. Database searches have revealed that ETS binding sites are present in several promoters of PTK-encoding genes. We investigated the role of Ets-1 in transcriptional regulation of a panel of 89 PTKs in epithelial HeLa tumour cells. In this study, HeLa cells stably overexpressing and underexpressing Ets-1 were used for real-time PCR analysis of all known human PTKs. The results suggest that Ets-1 is an essential transcription factor that cannot be substituted by other members of the ETS family. Transcription of most PTKs was found to be increased by Ets-1. In contrast Ets-1 seems to act as a transcriptional repressor of other PTKs. The data presented here underscore the importance of Ets-1 in tumour development and progression.