Identification of an intronic regulatory element in the human protein C (PROC) gene

Regulatory DNA elements responsible for human protein C (PROC) gene expression have previously been identified in the upstream promoter region and first (untranslated) exon of the gene. Here we show that an additional sequence element located more than 500 bp downstream of the core promoter within intron 1 further enhances PROC promoter-driven reporter gene expression in human hepatoma cells. In common with core promoter constructs used in previous studies, the activity of this 3'-extended regulatory region is diminished by a naturally occurring promoter mutation. However, in contrast to constructs lacking intronic sequence, the promoter/intron regulatory region is repressed rather than activated by the transcription factor HNF-1. Using both conventional alignment procedures and complexity analysis to study the human and canine PROC sequences, we identified two conserved intronic regions, which were tested for their involvement in gene regulation. High-level gene expression from the intron-coupled promoter was dependent upon the integrity of a 142 bp sequence element, a duplicate copy of which is located in an upstream region of the PROC gene that possesses enhancer activity. These findings emphasise the potential importance of intragenic sequences for gene regulation and serve to illustrate that the results of PROC promoter/reporter gene experiments are critically dependent upon the sequence context. The identification of such intragenic elements is relevant to the analysis of human genetic disease since it will facilitate the detection and functional evaluation of regulatory mutations and polymorphisms

Inactivating <i>NF1</i> Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

Purpose Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Experimental design Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.Results We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro . Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant.Conclusions Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations