Chromatin immunoprecipitation (ChIP) methodology and readouts

The identification of direct nuclear hormone receptor gene targets provides clues to their contribution to both development and cancer progression. Until recently, the identification of such direct target genes has relied on a combination of expression analysis and in silico searches for consensus binding motifs in gene promoters. Consensus binding motifs for transcription factors are often defined using in vitro DNA binding strategies. Such in vitro strategies fail to account for the many factors that contribute significantly to target selection by transcription factors in cells beyond the recognition of these short consensus DNA sequences. These factors include DNA methylation, chromatin structure, posttranslational modifications of transcription factors, and the cooperative recruitment of transcription factor complexes. Chromatin immunoprecipitation (ChIP) provides a means of isolating transcription factor complexes in the context of endogenous chromatin, allowing the identification of direct transcription factor targets. ChIP can be combined with site-specific PCR for candidate binding sites or alternatively with cloning, genomic microarrays or more recently direct high throughput sequencing to identify novel genomic targets. The application of ChIP-based approaches has redefined consensus binding motifs for transcription factors and provided important insights into transcription factor biology.

Terminal and progenitor lineage-survival oncogenes as cancer markers

Tumour classification has traditionally focused on differentiation and cellular morphology, and latterly on the application of genomic approaches. By combining chromatin immunoprecipitation with expression array, it has been possible to identify direct gene targets for transcription factors for nuclear hormone receptors. At the same time, there have been great strides in deriving stem and progenitor cells from tissues. It is therefore timely to propose that pairing the isolation of these cell subpopulations from tissues and tumours with these genomics approaches will reveal conserved gene targets for transcription factors. By focusing on transcription factors (lineage-survival oncogenes) with roles in both organogenesis and tumourigenesis at multiple organ sites, we suggest that this comparative genomics approach will enable developmental biology to be used more fully in relation to understanding tumour progression and will reveal new cancer markers. We focus here on neurogenesis and neuroendocrine differentiation in tumours.

A role for neurotensin in bicalutamide resistant prostate cancer cells

BACKGROUND: Anti-androgens are administered as a principal treatment for prostate cancer. Aggressive hormone refractory disease is characterized in some cases by the development of a neuroendocrine phenotype. However little attention has been paid to resistance pathways selected for by long-term treatment with non-steroidal anti-androgens.

METHODS: Using a resistant sub-line, LNCaP-Bic, we performed a comparative gene expression profiling using cDNA microarrays and target validation by qRT-PCR. Targets were then explored using cell proliferation, cell cycle analysis and in vitro invasion assays using siRNA technology.

RESULTS: Neurotensin/Neuromedin N (NTS) was upregulated in the LNCaP-Bic line at both the transcript and protein level. The resistant line was found to have an increased proliferation rate, more rapid cell cycle progression and increased invasiveness through Matrigel. Each phenotypic difference could be reduced using siRNA knockdown of NT.

CONCLUSION: Increased expression of NT in bicalutamide resistant prostate cancer cells induces cell proliferation and invasion suggesting that this peptide may contribute to the development of bicalutamide resistant prostate cancer.