Modification of oestrogen signalling during the acquisition of hormone resistance in breast cancer cells

The oestrogen receptor (ER) mediates both normal and malignant breast development. The ER can exert its ligand-dependent gene regulatory functions directly by association with target genes and subsequent dynamic formation of a transcription complex through its oestrogen response element. The complex receptorligand signalling network has received much interest as a potential therapeutic target. Endocrine agents, such as tamoxifen, have been developed to reverse oestrogen (E2) stimulated gene transcription and tumour growth, however progression to endocrine resistance provides a major obstacle in breast cancer treatment. This study investigates changes in endocrine response and ER transcription activation during the acquisition of endocrine resistance.Breast cancer cell lines were selected that encompassed the range of oestrogen and anti-oestrogen sensitivities from the E^-dependent and tamoxifen-sensitive MCF7 cell line through the less sensitive LCC-1 and LCC-2 lines to the insensitive LCC-9 and independently derived LY2 line. All lines had been derived from MCF-7 cells. These models represent different endocrine phenotypes and were designed to reflect sequential changes in the clinical progression from hormone sensitive to hormone insensitive and antioestrogen resistant. MDA-MB-231 breast cancer cells were used as ERα negative controls. Growth assays confirmed these phenotypes and proliferative behaviour in response to E₂ and tamoxifen.To help evaluate the role of the ERα in the development of endocrine resistance, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis was carried out to assess transcriptional and translational receptor status for each phenotype. All resistant lines possessed greater levels of ERα mRNA than wild-type MCF-7 cells. E₂ downregulated ERα mRNA and protein. Results suggest ERa functionality in these cell lines. Analysis of mRNA levels of several ER target genes (pS2, progesterone receptor (PR), cathepsin D and MYC) indicated informative differences between lines. pS2 was highly expressed in resistant lines as was PR in most lines compared to MCF-7 cells. E2 and tamoxifen produced attenuated or no effects in pS2 expression compared to the marked induction produced by E2 in MCF-7 cells. Minor changes were also observed in the expression levels of various coactivators (SRC-1, SRC-2, SRC-3) and corepressors (NCoR, SMRT and RIP140) investigated by qRT-PCR and Western blot.A subset of three cell lines (MCF-7, LCC-1 and LCC-9) was used to examine transcription complex assembly at the pS2 promoter in response to E2 utilizing chromatin immunoprecipitation. This identified a dynamic cycle of increasing H4 acetylation (indicative of active transcription) and ERα as well as cofactor recruitment upon E₂ addition in MCF-7 cells. A distinctive H4 acetylation pattern was revealed for LCC-1 and LCC-9 cells. While ERα recruitment was similar to MCF-7 cells, particularly strong SRC-1 and SRC-3 recruitment was detected in LCC-1 but most markedly in LCC-9 cells, implying altered pS2 transcription complex assembly.The data suggest that a functional oestrogen receptor may remain in these endocrine resistant models. Cell proliferation and E₂ target gene expression in LCC-1 cells proved to be oestrogen and antioestrogen independent but responsive in the presence of the ligand whereas LCC-9 cells manifest complete endocrine resistance. In addition, changes have been detected in the assembly of a gene transcription complex at the pS2 promoter. Taken together, this is evidence for modified ER mediated transcription activity. These results may help to identify potential mechanisms of endocrine resistance

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)

International audienceTrichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production