FOXA1 repression is associated with loss of BRCA1 and increased promoter methylation and chromatin silencing in breast cancer.

FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.

pSURF-2, a modified BAC vector for selective YAC cloning and functional analysis.

A modified bacterial artificial chromosome (BAC) vector, pSURF-2, adapted for the selective subcloning of yeast artificial chromosome (YAC) sequences was constructed. DH10B-U, a pyrF derivative of the highly transformable E. coli strain DH10B was also constructed and used for the detection of Ura+ recombinants carrying DNA linked to YAC right arms. The vector's properties were illustrated in two main ways. (i) An intact 25-kb YAC containing a mouse tyrosinase minigene was cloned into pSURF-2. Appropriately spliced tyrosinase RNA was detected by reverse transcription (RT)-PCR in extracts of cells transiently lipofected with the cloned YAC. (ii) Cells expressing human cystic fibrosis transmembrane conductance regulator (CFTR) from an integrated pSURF-2 recombinant containing a cDNA expression cassette were selected using the hygromycin-resistance (HyTK) marker of the vector and characterized by RT-PCR and immunoprecipitation. The unique I-SceI site and HyTK marker of pSURF-2 are designed to facilitate subsequent functional studies of cloned DNA.

Evaluation in vitro and in vivo of cationic liposome-expression construct complexes for cystic fibrosis gene therapy.

We have tested the cationic liposome N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl-ammoniummethylsul phate, (DOTAP), for gene delivery in vitro and in vivo with a view to clinical use in gene therapy for cystic fibrosis. Delivery of lacZ cDNA-DOTAP complexes via aerosol showed promoter-dependent differences in the pattern and longevity of expression. Repeated administration was well tolerated. The potential for the transfer of foreign genes into reproductive tissue was investigated by intravenous injection of DNA-DOTAP into female mice. Foreign DNA was undetectable in the ovaries by Southern blot analysis at 1 and 7 days after injection. Our results suggest that DOTAP merits testing in cystic fibrosis patients for delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the respiratory tract and that substitution of the cytomegalovirus (CMV) promoter for the simian virus (SV) promoter may improve on the transitory response reported previously.

Insertion of natural intron 6a-6b into a human cDNA-derived gene therapy vector for cystic fibrosis improves plasmid stability and permits facile RNA/DNA discrimination.

BACKGROUND: The gene therapy vector pCMV-CFTR containing human CFTR cDNA shows high segregational instability during growth in Escherichia coli. METHODS: By host strain screening and optimization of fermentation, satisfactory levels of pCMV-CFTR production were achieved. However, the vector was also vulnerable to structural instability manifested by the appearance during fermentation of a more stable mutant form in which the bacterial insertion sequence IS1 had transposed into exon 7 of plasmidborne CFTR. The instability of pCMV-CFTR is attributable to transcription from an upstream cryptic promoter leading to the production of CFTR peptide fragments known to be toxic when expressed in E. coli. To address this, we inserted the 1.1 kb natural human 6a-6b intron into pCMV-CFTR. RESULTS: The new vector pCMV-CFTR-int6ab is more stable in E. coli than either pCMV-CFTR or the IS1 mutant, grows to high cell density giving higher DNA yields and expresses CFTR appropriately in transfected cells. Thus, the intron has a stabilizing effect comparable to the IS1 insertion yet retains full functionality for gene therapy. We describe a PCR assay using primers directed to sequences flanking the intron that allows differentiation between DNA and mature mRNA. The T936C mutation present only in vector DNA has also been exploited to allow transgene CFTR to be distinguished and its dose-dependent expression to be detected in human cellular backgrounds. CONCLUSIONS: Instability of a plasmid vector for gene therapy has been minimized by rational modification. The introduction of an intron for this purpose offers the additional advantage of providing a discriminatory RT-PCR assay.

Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR-DOTAP cationic liposome complex. A single dose of 400 micrograms pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA-liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR-DOTAP aimed at treating CF lung disease.

Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP.

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.