ERß-specific transcriptional profile in colon cancer
Valentina Martineti1, Massimiliano Mascherini4, Carmelo Mavilia1, Silvia Carbonell Sala1, Isabella Tognarini1, Chiara Azzari3, Federico Mattia Stefanini4, Francesco Tonelli2 & Maria Luisa Brandi1
Epidemiological data clearly evidence a protective role of estrogens against the development of colon cancer and ERβ has been identified as the predominant ER subtype in human colon. More recently it has been identified as a favourable prognostic marker in this disease, possibly explaining the protective effect of estrogens against colon cancer development. To understand the specific role and mechanism of action of ERβ in colon tumorigenesis we developed an in vitro engineered cell model through transfection and cloning of HCT8 human colon cancer cell line for stable over-expression of wild type human ERβ (HCT8β8), providing the first direct evidence that ERβ plays an important role in colon cancer as a regulator of cell proliferation through induction of G1-S phase transition arrest. To investigate the molecular events underlying growth arrest we analyzed specific ERβ-regulated genes by comparing expression profile of HCT8β8 cells versus its non-engineered counterpart using Agilents Human 1A Oligo Microarray (V2) chips harbouring over 22,000 human genes and ESTs. A list of 189 reproducibly ERβ-regulated targets, comprising 64 up-regulated and 125 down-regulated genes, emerged indicating that ERβ over-expression heavily affects different aspects of HCT8 cell function regarding both its intracellular metabolism and relationship with the extracellular milieu. According to their function, ERβ-modulated genes have been grouped into 16 categories, our interest for further validation (by quantitative real time RT-PCR and Western blotting) focused on cell cycle and mitosis genes category and this technique confirmed 50% of gene modulations. On the whole a trend to the slowing down of the cell cycle is demonstrated and one of the up-regulated genes is E4F transcription factor 1 (E4F1), which is already known to be an estrogen-modulated transcription factor. Two of their downstream targets are p21waf1 and cyclin E whose altered expression has already been documented in our cell model. We hypothesize that E4F1- p21WAF1-cyclin E is an ERβ specific pathway in colon cancer cells.