Overexpression of BLM promotes DNA damage and increased sensitivity to platinum salts in triple-negative breast and serous ovarian cancers.

Background: Platinum-based therapy is an effective treatment for a subset of triple-negative breast cancer and ovarian cancer patients. In order to increase response rate and decrease unnecessary use, robust biomarkers that predict response to therapy are needed. Patients and methods: We performed an integrated genomic approach combining differential analysis of gene expression and DNA copy number in sensitive compared with resistant triple-negative breast cancers in two independent neoadjuvant cisplatin-treated cohorts. Functional relevance of significant hits was investigated in vitro by overexpression, knockdown and targeted inhibitor treatment. Results: We identified two genes, the Bloom helicase (BLM) and Fanconi anemia complementation group I (FANCI), that have both increased DNA copy number and gene expression in the platinum-sensitive cases. Increased level of expression of these two genes was also associated with platinum but not with taxane response in ovarian cancer. As a functional validation, we found that overexpression of BLM promotes DNA damage and induces sensitivity to cisplatin but has no effect on paclitaxel sensitivity. Conclusions: A biomarker based on the expression levels of the BLM and FANCI genes is a potential predictor of platinum sensitivity in triple-negative breast cancer and ovarian cancer.

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.

This corrects the article DOI: 10.1038/onc.2016.243.

Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut.

BACKGROUND: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. METHODS: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. RESULTS: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. CONCLUSIONS: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data.

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.

Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

An autoregulatory function of Dfos during Drosophila endoderm induction.

The endoderm of Drosophila is patterned during embryogenesis by an inductive cascade emanating from the adhering mesoderm. An immediate-early endodermal target gene of this induction is Dfos whose expression is upregulated in the middle midgut by Dpp signalling. Previous evidence based on a dominant-negative Dfos construct indicated that Dfos may cooperate with Dpp signalling to induce the HOX gene labial, the ultimate target gene of the inductive cascade. Here, we examine kayak mutants that lack Dfos to establish that Dfos is indeed required for labial induction. We provide evidence that Dfos acts through a CRE-like sequence, previously identified to be a target for signalling by Dpp and by the Epidermal growth factor receptor (Egfr) in the embryonic midgut. We show that Dfos expression is stimulated by Egfr signalling. Finally, we find that Dfos function is required for its own upregulation. Thus, endoderm induction is based on at least four tiers of positive autoregulatory feedback loops.

LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator.

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Functional intertwining of Dpp and EGFR signaling during Drosophila endoderm induction.

Endoderm induction in Drosophila is mediated by the extracellular signals Decapentaplegic (Dpp) and Wingless (Wg). We discovered a secondary signal with a permissive role in this process, namely Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor (EGFR) and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and EGFR signaling show that these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. We discuss the potential significance of why an inductive process might use a secondary signal whose function is intertwined with that of the primary signal.

Antagonism between EGFR and Wingless signalling in the larval cuticle of Drosophila.

Signalling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, we show that EGFR signalling specifies the anterior denticles in each segment of the larval abdomen. We provide evidence that these denticles derive from a segmental zone of embryonic cells in which EGFR signalling activity is maximal. Within each segment, there is a competition between the denticle fate specified by EGFR signalling and the naked cuticle fate specified by Wingless signalling. The final pattern of the denticle belts is the product of this antagonism between the two signalling pathways. Finally, we show that the segmental zones of high EGFR signalling activity depend on bithorax gene function and that they account for the main difference in shape between abdominal and thoracic denticle belts.