Solid tumours are complex structures in which the interdependent relationship between tumour and endothelial cells modulates tumour development and metastasis dissemination. The tumour microenvironment plays an important role in this cell interplay, and changes in its features have a major impact on tumour growth as well as on anticancer therapy responsiveness. Different studies have shown irregular blood flow in tumours, which is responsible for hypoxia and reoxygenation phases, also called intermittent hypoxia. Intermittent hypoxia induces transient changes, the impact of which has been underestimated for a long time. Recent in vitro and in vivo studies have shown that intermittent hypoxia could positively modulate tumour development, inducing tumour growth, angiogenic processes, chemoresistance, and radioresistance. In this article, we review the effects of intermittent hypoxia on tumour and endothelial cells as well as its impacts on tumour development.

Motivation: Because of its low cost, amplicon sequencing, also known as ultra-deep targeted sequencing, is now becoming widely used in oncology for detection of actionable mutations, i.e. mutations influencing cell sensitivity to targeted therapies. Amplicon sequencing is based on the polymerase chain reaction amplification of the regions of interest, a process that considerably distorts the information on copy numbers initially present in the tumor DNA. Therefore, additional experiments such as single nucleotide polymorphism (SNP) or comparative genomic hybridization (CGH) arrays often complement amplicon sequencing in clinics to identify copy number status of genes whose amplification or deletion has direct consequences on the efficacy of a particular cancer treatment. So far, there has been no proven method to extract the information on gene copy number aberrations based solely on amplicon sequencing.Results: Here we present ONCOCNV, a method that includes a multifactor normalization and annotation technique enabling the detection of large copy number changes from amplicon sequencing data. We validated our approach on high and low amplicon density datasets and demonstrated that ONCOCNV can achieve a precision comparable with that of array CGH techniques in detecting copy number aberrations. Thus, ONCOCNV applied on amplicon sequencing data would make the use of additional array CGH or SNP array experiments unnecessary.Availability and implementation: http://oncocnv.curie.fr/Contact: valentina.boeva@curie.frSupplementary information: Supplementary data are available at Bioinformatics online.

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The presence of hypoxia in tumor and its role in promoting angiogenesis are well-established. Recently, in addition to chronic hypoxia, cycling or intermittent hypoxia has also been demonstrated. However, its role in inducing new blood vessel formation is less clear. This work is aimed to investigate whether intermittent hypoxia can induce a pro-angiogenic phenotype in endothelial cells, in vitro. We studied changes in the expression of genes involved in inflammation and angiogenesis under intermittent and chronic hypoxia. We evidenced genes specifically expressed under intermittent hypoxia, suggesting different cell responses induced by intermittent versus chronic hypoxia. An increase in the expression of pro-angiogenic and pro-inflammatory genes under intermittent hypoxia, translating a pro-angiogenic effect of intermittent hypoxia was detected. In parallel, we investigated the activity of three transcription factors known to be activated either under hypoxia or by reoxygenation: HIF-1, Nrf2, and NF-kappaB. HIF-1alpha stabilization and an increase in HIF-1 transcriptional activity were evidenced under intermittent hypoxia. On the other hand, NRF2 and NF-kappaB transcription factors were not activated. Finally, an increase in endothelial cell migration and in tubulogenesis in the course of hypoxia-reoxygenation cycles was evidenced, which was inhibited by HIF-1alpha siRNA. All together, these results demonstrate a clear pro-angiogenic effect of intermittent hypoxia.

Vascularized tumors are exposed to intermittent hypoxia, that is, hypoxia followed by periods of reoxygenation. Abnormal structure and dysfunction of tumor blood vessels are responsible for these conditions. These repeated short periods of hypoxia concern tumor cells as well as endothelial cells. However, the effects of intermittent hypoxia are poorly understood. The aim of this study was to investigate the effects of intermittent hypoxia on endothelial cells and particularly on HIF-1alpha, a central actor in adaptive response to hypoxia. For that, endothelial cells were exposed to four repeated cycles of 1-h hypoxia followed by 30 min of reoxygenation. We showed that repeated cycles of hypoxia/reoxygenation induced a modification in HIF-l alpha phosphorylation pattern: a progressive increase in HIF-1alpha phosphorylated form was observed during the hypoxic periods. Activation of p42/p44, Akt and PKA was observed in parallel. PKA was shown to be involved in the phosphorylation of HIF-lalpha under intermittent hypoxia, while p42/p44 and Akt were not. As HIF-1 activity is often associated with enhanced cell survival, a better knowledge of the effects of intermittent hypoxia on endothelial cells and the highlight of particular mechanisms induced by intermittent hypoxia are essential to understand the behavior of endothelial cells during neo-angiogenesis.

A low oxygen level is a characteristic feature of solid tumours and a negative prognostic factor for the survival of cancer patients. The response of cancer cells to hypoxia not only drives neo-angiogenesis, but also enhances cancer cell survival and malignant phenotype. Hypoxia-inducible factor-1 (HIF-1) is the major regulator of the adaptive responses of cells to hypoxia [1]. It is a Bcl2 ⁄ adenovirus E1B 19 kDa interacting protein (bHLH-PAS) transcription factor composed of two subunits: aryl hydrocarbon receptor nuclear translocator (ARNT), which is constitutively expressed in the nucleus, and HIF-1a, whose level and activity are regulated by the oxygen level. In the presence of oxygen, HIF-1a is post-translationally modified by prolyl hydroxylases, targeting the protein for proteasomal degradation, and by an asparagine hydroxylase, preventing its interactions with transcription coactivators [2]. Limiting oxygen availability prevents these modifications, leading to HIF-1a accumulation, translocation into the nucleus and interaction with coactivators. On activation, the active dimer binds to target gene promoters containing the core recognition sequence 5¢-RCGTC-3¢ (HRE, hypoxia response element), leading to overexpression of the genes involved in glucose metabolism, angiogenesis and cell survival [1]. This transcriptional response mediates cell adaptation to low oxygen level, but also contributes to tumour progression, neo-angiogenesis and metastasis [3,4]. Hypoxia-inducible factor-1 (HIF-1) is now recognized as a possible target for cancer treatment. This transcription factor is responsible for the overexpression of several genes favouring cancer cell survival and inducing neoangiogenesis. Echinomycin has recently been described to inhibit HIF-1 DNA binding and transcriptional activity. In this work, it is shown that echinomycin strongly inhibits the activity of HIF-1 under hypoxic conditions, and also interferes with the activity of other transcription factors. These results demonstrate the lack of specificity of this molecule. Moreover, it is demonstrated that echinomycin induces an increase in HIF-1 activity under normoxic conditions, parallel to an increase in the expression of HIF-1 target genes. This effect is caused by an increase in HIF-1a protein level, resulting from an increase in the transcription of the HIF-1A gene in the presence of a low concentration of echinomycin. Transfection experiments with HIF-1a promoter constructs revealed the presence of an Sp1 binding element responsive to echinomycin. Furthermore, echinomycin enhanced Sp1 activity, as measured by the use of a specific reporter system. These findings show, for the first time, that echinomycin has a dual effect on HIF-1 activity under normoxic and hypoxic conditions, demonstrating that this molecule cannot be used in cancer treatment.Abbreviations AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; DHG, DMEM high glucose; HB, hypotonic buffer; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia response elem...

IntroductionTriple Negative Breast Cancers (TNBC) represent about 12% to 20% of all breast cancers (BC) and have a worse outcome compared to other BC subtypes. TNBC often show a deficiency in DNA double-strand break repair mechanisms. This is generally related to the inactivation of a repair enzymatic complex involving BRCA1 caused either by genetic mutations, epigenetic modifications or by post-transcriptional regulations.The identification of new molecular biomarkers that would allow the rapid identification of BC presenting a BRCA1 deficiency could be useful to select patients who could benefit from PARP inhibitors, alkylating agents or platinum-based chemotherapy.MethodsGenomic DNA from 131 formalin-fixed paraffin-embedded (FFPE) tumors (luminal A and B, HER2+ and triple negative BC) with known BRCA1 mutation status or unscreened for BRCA1 mutation were analysed by array Comparative Genomic Hybridization (array CGH). One highly significant and recurrent gain in the 17q25.3 genomic region was analysed by fluorescent in situ hybridization (FISH). Expression of the genes of the 17q25.3 amplicon was studied using customized Taqman low density arrays and single Taqman assays (Applied Biosystems).ResultsWe identified by array CGH and confirmed by FISH a gain in the 17q25.3 genomic region in 90% of the BRCA1 mutated tumors. This chromosomal gain was present in only 28.6% of the BRCA1 non-mutated TNBC, 26.7% of the unscreened TNBC, 13.6% of the luminal B, 19.0% of the HER2+ and 0% of the luminal A breast cancers. The 17q25.3 gain was also detected in 50% of the TNBC with BRCA1 promoter methylation. Interestingly, BRCA1 promoter methylation was never detected in BRCA1 mutated BC. Gene expression analyses of the 17q25.3 sub-region showed a significant over-expression of 17 genes in BRCA1 mutated TNBC (n = 15) as compared to the BRCA1 non mutated TNBC (n = 13).ConclusionsIn this study, we have identified by array CGH and confirmed by FISH a recurrent gain in 17q25.3 significantly associated to BRCA1 mutated TNBC. Up-regulated genes in the 17q25.3 amplicon might represent potential therapeutic targets and warrant further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0466-y) contains supplementary material, which is available to authorized users.

Intermittent Hypoxia (IH) that develops in neovascularized solid tumours has been described to positively influence the tumour growth by modulating the behaviour of cancer cells as well as of endothelial cells. However, the molecular mechanisms regulated by IH still remain poorly understood. In this work, the effects of IH were investigated on endothelial cells by a proteomic approach. Protein abundance variations were studied using fluorescent 2D-Differential in Gel Electrophoresis (2D-DIGE). Amongst the proteins of which the abundance varied under IH, NDRG1 and CRK-I/II were identified by mass spectrometry. These proteins have already been described to influence cancer cell migration as well as the angiogenic processes in solid tumours. Since an increase in endothelial cell migration under IH was evidenced in our previous work, the involvement of NDRG1 and CRK-I/II proteins in endothelial cell migration under IH was determined by silencing the expression of both proteins using siRNA. The results revealed that NDRG1 and CRK-I/II are indeed regulators of endothelial cell migration under intermittent hypoxia: silencing of CRK-I/II resulted in an increase in endothelial cell migration, whereas the invalidation of NDRG1 decreased it. These results give news insight regarding the effects of IH on endothelial cell migration and hence on neoangiogenesis.