Description: Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood. The fate of a replication-blocking lesion is generally explored by means of plasmid-based assays. Although such assays represent efficient tools to analyse TLS, we show here that plasmid-borne lesions are inappropriate models to study DA pathways due to extensive replication fork uncoupling. This observation prompted us to develop a method to graft, site-specifically, a single lesion in the genome of a living cell. With this novel assay, we show that in Escherichia coli DA events massively outweigh TLS events and that in contrast to plasmid, chromosome-borne lesions partially require RecA for tolerance.

Description: The SK3 channel, a potassium channel, was recently shown to control cancer cell migration, a critical step in metastasis outgrowth. Here, we report that expression of the SK3 channel was markedly associated with bone metastasis. The SK3 channel was shown to control constitutive Ca2+ entry and cancer cell migration through an interaction with the Ca2+ channel Orai1. We found that the SK3 channel triggers an association with the Orai1 channel within lipid rafts. This localization of an SK3–Orai1 complex seemed essential to control cancer cell migration. This suggests that the formation of this complex in lipid rafts is a gain-of-function, because we showed that none of the individual proteins were able to promote the complete phenotype. We identified the alkyl-lipid Ohmline as a disrupting agent for SK3–Orai1 lipid raft localization. Upon Ohmline treatment, the SK3–Orai1 complex moved away from lipid rafts, and SK3-dependent Ca2+ entry, migration, and bone metastases were subsequently impaired. The colocalization of SK3 and Orai1 in primary human tumors and bone metastases further emphasized the clinical relevance of our observations. Targeting SK3–Orai1 in lipid rafts may inaugurate innovative approaches to inhibit bone metastases. Cancer Res; 73(15); 4852–61. ©2013 AACR.

Description: Purpose: If immune cells are involved in tumor surveillance and have a prognostic impact in most primary tumors, little is known about their significance in metastases. Because patients' survival is heterogeneous, even at metastatic stages, we hypothesized that immune cells may be involved in the control of metastases. We therefore characterized the tumor immune microenvironment and its prognostic value in colorectal and renal cell carcinoma (RCC) metastases, and compared it to primary tumors. Experimental Design: We analyzed by immunohistochemistry ( n = 192) and qPCR ( n = 32) the immune environments of colorectal carcinoma and RCC lung metastases. Results: Metastases from colorectal carcinoma and RCC have different immune infiltrates. Higher densities of DC-LAMP + mature dendritic cells ( P 〈 0.0001) and lower densities of NKp46 + NK cells ( P 〈 0.0001) were observed in colorectal carcinoma as compared to RCC metastases, whereas densities of T cells were similar. High densities of CD8 + and DC-LAMP + cells correlated with longer overall survival (OS) in colorectal carcinoma ( P = 0.008) and shorter OS in RCC ( P 〈 0.0001). High NK-cell densities were associated with improved survival in RCC ( P = 0.002) but not in colorectal carcinoma. Densities of immune cells correlated significantly from primary to relapsing metastases for the same patient. A T H 1 orientation was found in colorectal carcinoma metastases, whereas a heterogeneous immune gene expression was found in RCC metastases. Conclusions: Our results show a major prognostic value of the immune pattern (CD8 + /DC-LAMP + cell densities) in colorectal carcinoma and RCC, reproducible from primary to metastatic tumors, although with opposite clinical impacts, and highlight the role of the tumor cell in shaping its immune environment. Clin Cancer Res; 19(15); 4079–91. ©2013 AACR .

Description: Background Only patients with wild-type (WT) KRAS tumors benefit from anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (Mabs) in metastatic colorectal cancer (mCRC). Pyrosequencing is now widely used for the determination of KRAS mutation burden and a conservative cut-off point of 10% has been defined. Up until now, the impact of low-frequency KRAS mutations (〈10%) on the response to anti-EGFR Mabs has yet to be evaluated. Patients and methods Tumors from patients receiving anti-EGFR Mabs based on a WT genotype for KRAS , as determined using direct sequencing, have been retrospectively analyzed by pyrosequencing. Patients were categorized as WT (no KRAS mutation) or low-frequency mutation when KRAS mutation was 〈10% ( KRAS low MT). Results A total of 168 patients treated by anti-EGFR Mabs for mCRC were analyzed. According to pyrosequencing, 138 tumors remained KRAS WT, while 30 tumors were KRAS low MT. In the KRAS low MT and KRAS WT groups, the response rates were 6.7% and 37.0%, respectively, while stabilization amounted to 23.3% versus 32.6% and progression to 70% versus 29% ( P 〈 0.01). Progression-free survival (PFS) was 2.7 ± 0.5 months for KRAS low MT and was 6.0 ± 0.3 months for KRAS WT ( P 〈 0.01). Conclusions These results appear to validate consideration of low-frequency KRAS mutation tumors as positive, and justify a large-scale prospective study.

Description: Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers. Experimental design: The influence of the immune infiltrate on patient's outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3 + ) and cytotoxic (CD8 + ) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer ( n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patient's survival, and response to pCRT. Results: The densities of CD3 + and CD8 + lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P 〈 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P 〈 0.001). Lymph node ratio added information in a prognostic model (all P 〈 0.05). In addition, high infiltration of CD3 + and CD8 + lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3 + cells; Fisher exact test P = 0.01). Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR .

Description: Mutations in the von Hippel–Lindau gene upregulate expression of the central angiogenic factor VEGF, which drives abnormal angiogenesis in clear cell renal cell carcinomas (ccRCC). However, the overexpression of VEGF in these tumors was not found to correlate with overall survival. Here, we show that the proangiogenic, proinflammatory cytokine CXCL7 is an independent prognostic factor for overall survival in this setting. CXCL7 antibodies strongly reduced the growth of ccRCC tumors in nude mice. Conversely, conditional overexpression of CXCL7 accelerated ccRCC development. CXCL7 promoted cell proliferation in vivo and in vitro, in which expression of CXCL7 was induced by the central proinflammatory cytokine interleukin (IL)-1β. ccRCC cells normally secrete low amounts of CXCL7; it was more highly expressed in tumors due to high levels of IL-1β there. We found that a pharmacological inhibitor of the CXCL7 receptors CXCR1 and CXCR2 (SB225002) was sufficient to inhibit endothelial cell proliferation and ccRCC growth. Because CXCR1 and CXCR2 are present on both endothelial and ccRCC cells, their inhibition affected both the tumor vasculature and the proliferation of tumor cells. Our results highlight the CXCL7/CXCR1/CXCR2 axis as a pertinent target for the treatment of ccRCC. Cancer Res; 74(3); 873–83. ©2013 AACR.

Description: Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema . This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Description: In deciduous trees, shoot development in early spring is assumed to be achieved mainly at the expense of nitrogen (N) stores. Indeed, the possible compensation for poor autumn N storage by early spring N uptake has been little studied. We therefore determined the dynamics of spring N uptake in relation to spring N supply, carbon and N storage and shoot development. Young peach trees ( Prunus persica L. Batsch, cv. ‘GF305 ’ ) were raised outdoors in a hydroponic set-up during the spring and summer, with an excessive N supply. During the autumn, half of the trees were then N limited. The following spring, the N supply remained either high or low, or changed from high to low or low to high. Between 6 March and 13 May, N uptake was measured automatically on an hourly basis, while shoot growth was monitored once a week. These in situ measurements were completed by three destructive harvests to assess organ composition in N and total non-structural carbohydrates (TNC). Until the end of April, N uptake was dependent on the autumn N treatment, being higher in trees that had been N limited in the autumn. Total non-structural carbohydrate mobilization was also higher in those trees that had lost at least 17 g TNC by 24 April, while TNC levels in non-limited trees remained stable or even rose. Shoot development, estimated by the number of elongated axes and leaves per axis, was also slightly delayed by an N limitation in autumn. After 24 April, N uptake rates increased notably under all treatments and was determined by the spring N supply. In trees receiving a high N supply in the spring, the uptake rates also displayed marked short-term variations. That reduced the differences between treatments and by 13 May no differences could be evidenced between the trees in terms of organ biomass and TNC and N contents, whatever the treatment. We concluded that in the early spring, N uptake may compensate for a deficit of N storage insofar as large quantities of TNC can be mobilized for that purpose.

Description: The classic model of tumor suppression implies that malignant transformation requires full “two-hit” inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a “continuum” model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the “continuum” model of tumor suppression. Cancer Res; 74(22); 6554–64. ©2014 AACR.

Description: Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in -Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis , a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5'-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo . L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity.