BRD4 facilitates replication stress-induced DNA damage response.

Previous reports have demonstrated that select cancers depend on BRD4 to regulate oncogenic gene transcriptional programs. Here we describe a novel role for BRD4 in DNA damage response (DDR). BRD4 associates with and regulates the function of pre-replication factor CDC6 and plays an indispensable part in DNA replication checkpoint signaling. Inhibition of BRD4 by JQ1 or AZD5153 resulted in a rapid, time-dependent reduction in CHK1 phosphorylation and aberrant DNA replication re-initiation. Furthermore, BRD4 inhibition sensitized cancer cells to various replication stress-inducing agents, and synergized with ATR inhibitor AZD6738 to induce cell killing across a number of cancer cell lines. The synergistic interaction between AZD5153 and AZD6738 is translatable to in vivo ovarian cell-line and patient-derived xenograft models. Taken together, our study uncovers a new biological function of BRD4 and provides mechanistic rationale for combining BET inhibitors with DDR-targeted agents for cancer therapy

Dynamic clonal equilibrium and predetermined cancer risk in Barrett's oesophagus

abstract: Surveillance of Barrett’s oesophagus allows us to study the evolutionary dynamics of a human neoplasm over time. Here we use multicolour fluorescence in situ hybridization on brush cytology specimens, from two time points with a median interval of 37 months in 195 non-dysplastic Barrett's patients, and a third time point in a subset of 90 patients at a median interval of 36 months, to study clonal evolution at single-cell resolution. Baseline genetic diversity predicts progression and remains in a stable dynamic equilibrium over time. Clonal expansions are rare, being detected once every 36.8 patient years, and growing at an average rate of 1.58 cm[superscript 2] (95% CI: 0.09–4.06) per year, often involving the p16 locus. This suggests a lack of strong clonal selection in Barrett’s and that the malignant potential of ‘benign’ Barrett’s lesions is predetermined, with important implications for surveillance programs.The final version of this article, as published in Nature Communications, can be viewed online at: https://www.nature.com/articles/ncomms1215

Differential clonal evolution in oesophageal cancers in response to neo-adjuvant chemotherapy

How chemotherapy affects carcinoma genomes is largely unknown. Here we report whole-exome and deep sequencing of 30 paired oesophageal adenocarcinomas sampled before and after neo-adjuvant chemotherapy. Most, but not all, good responders pass through genetic bottlenecks, a feature associated with higher mutation burden pre-treatment. Some poor responders pass through bottlenecks, but re-grow by the time of surgical resection, suggesting a missed therapeutic opportunity. Cancers often show major changes in driver mutation presence or frequency after treatment, owing to outgrowth persistence or loss of sub-clones, copy number changes, polyclonality and/or spatial genetic heterogeneity. Post-therapy mutation spectrum shifts are also common, particularly C>A and TT>CT changes in good responders or bottleneckers. Post-treatment samples may also acquire mutations in known cancer driver genes (for example, SF3B1, TAF1 and CCND2) that are absent from the paired pre-treatment sample. Neo-adjuvant chemotherapy can rapidly and profoundly affect the oesophageal adenocarcinoma genome. Monitoring molecular changes during treatment may be clinically useful

The degree of segmental aneuploidy measured by total copy number abnormalities predicts survival and recurrence in superficial gastroesophageal adenocarcinoma

Background: Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC. Methods: We quantified copy number changes in 41 superficial EAC using Affymetrix SNP 6.0 arrays. We identified recurrent chromosomal gains and losses and calculated the total copy number abnormality (CNA) count for each tumor as a measure of aneuploidy. We correlated CNA count with overall survival and time to first recurrence in univariate and multivariate analyses. Results: Recurrent segmental gains and losses involved multiple genes, including: HER2, EGFR, MET, CDK6, KRAS (recurrent gains); and FHIT, WWOX, CDKN2A/B, SMAD4, RUNX1 (recurrent losses). There was a 40-fold variation in CNA count across all cases. Tumors with the lowest and highest quartile CNA count had significantly better overall survival (p = 0.032) and time to first recurrence (p = 0.010) compared to those with intermediate CNA counts. These associations persisted when controlling for other prognostic variables. Significance: SNP arrays facilitate the assessment of recurrent chromosomal gain and loss and allow high resolution, quantitative assessment of segmental aneuploidy (total CNA count). The non-monotonic association of segmental aneuploidy with survival has been described in other tumors. The degree of aneuploidy is a promising prognostic biomarker in a potentially curable form of EAC. © 2014 Davison et al

Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis.

Cancer genome sequencing studies have identified numerous driver genes, but the relative timing of mutations in carcinogenesis remains unclear. The gradual progression from premalignant Barrett's esophagus to esophageal adenocarcinoma (EAC) provides an ideal model to study the ordering of somatic mutations. We identified recurrently mutated genes and assessed clonal structure using whole-genome sequencing and amplicon resequencing of 112 EACs. We next screened a cohort of 109 biopsies from 2 key transition points in the development of malignancy: benign metaplastic never-dysplastic Barrett's esophagus (NDBE; n=66) and high-grade dysplasia (HGD; n=43). Unexpectedly, the majority of recurrently mutated genes in EAC were also mutated in NDBE. Only TP53 and SMAD4 mutations occurred in a stage-specific manner, confined to HGD and EAC, respectively. Finally, we applied this knowledge to identify high-risk Barrett's esophagus in a new non-endoscopic test. In conclusion, mutations in EAC driver genes generally occur exceptionally early in disease development with profound implications for diagnostic and therapeutic strategies

Spheroid-plug model as a tool to study tumor development, angiogenesis, and heterogeneity in vivo

Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit+ and Sca-1+). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research

FGFR2 amplification has prognostic significance in gastric cancer: results from a large international multicentre study

Background: In preclinical gastric cancer (GC) models, FGFR2 amplification was associated with increased tumour cell proliferation and survival, and drugs targeting this pathway are now in clinical trials. Methods: FGFR2 FISH was performed on 961 GCs from the United Kingdom, China and Korea, and the relationship with clinicopathological data and overlap with HER2 amplification were analysed. Results: The prevalence of FGFR2 amplification was similar between the three cohorts (UK 7.4%, China 4.6% and Korea 4.2%), and intratumoral heterogeneity was observed in 24% of FGFR2 amplified cases. FGFR2 amplification was associated with lymph node metastases (Po0.0001). FGFR2 amplification and polysomy were associated with poor overall survival (OS) in the Korean (OS: 1.83 vs 6.17 years, P ¼ 0.0073) and UK (OS: 0.45 vs 1.9 years, Po0.0001) cohorts, and FGFR2 amplification was an independent marker of poor survival in the UK cohort (P ¼ 0.0002). Co-amplification of FGFR2 and HER2 was rare, and when high-level amplifications did co-occur these were detected in distinct areas of the tumour. Conclusion: A similar incidence of FGFR2 amplification was found in Asian and UK GCs and was associated with lymphatic invasion and poor prognosis. This study also shows that HER2 and FGFR2 amplifications are mostly exclusive

Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4–9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P=0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in non-neoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (P<0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy