Chemoresistance is a serious limitation of cancer treatment1. Until recently, almost all the work done to study this limitation has been restricted to tumour cells2. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

-Four sheep were fed an alfalfa hay diet. Rumen content samples were collected three hours after feeding in order to total microorganism population (TP), solid attached population (SAP) and solid attached firmly population (SAFP). Fibrolytic specific activities (xylanase, CMCase and β-glycosidases) were estimated by the amount of reducing sugars or p-nitrophenol released from the appropriate substrate. The distribution of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens) was quantified by dot-blot hybridisation using specific 16S-rRNA-targeting probes. Specific activities of polysaccharidase enzymes were higher in SAP than in TP, and in SAFP than in SAP. The sum of RNA of the three cellulolytic bacterial species represented on average 9% of the total bacterial RNA, and increased after filtration. In all samples, the relative population size of F. succinogenes was higher than that of R. albus and of R. flavefaciens. These results demonstrate that the most active enzymes are secreted by the particle-associated microorganisms. The differences in composition of the microflora between the solid and liquid phase suggest that bacteria are not equally distributed throughout the rumen content: the cellulolytic species are present in a higher proportion in the solid phase of rumen contents.rumen content / fibrolytic activity / cellulolytic bacteria / oligonucleotide probe Résumé -Répartition de l'activité fibrolytique microbienne et des bactéries cellulolytiques entre phases solides et liquides du contenu ruminal. Quatre moutons, munis d'une canule du rumen, ont reçu un régime à base de foin. Des échantillons de contenus ruminaux étaient prélevés 3 h après la distribution du repas afin d'isoler la population totale de micro-organismes (TP), la population de microorganismes associés à la phase solide du contenu (SAP) et celle fermement associée à cette phase solide (SAFP). Les activités fibrolytiques spécifiques (xylanase, CMCase et β-glycosidases) étaient estimées Reprod. Nutr. Dev. 41 (2001) 187-194 187

We used RNA probes and enzyme activities to compare the cellulolytic microbial ecosystems of the rumen and the cecum. Four rumen- and cecum-cannulated wethers were fed a diet of barley plus hay (60:40). Digesta samples were collected 1 h before feeding and 3, 6, and 9 h after feeding for measurements on microbial populations, and 1 h before feeding and 3 and 6 h after feeding for digestion measurements, pH, and VFA. Polysaccharidase and glycosidase specific activities of solid-adherent microorganisms were measured respectively by the amount of reducing sugars released from xylan or avicel or p-nitrophenol from the p-nitrophenol derivatives of xylose and glucose. The distribution and amounts of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) were determined by dot-blot hybridization using specific 16SrRNA-targeting probes. Enzyme activities were higher in the rumen than in the cecum and before feeding than at 3 h after feeding. The sum of the three cellulolytic bacterial species represented, on average, 4.5% of the total bacterial RNA in the two compartments and did not vary with sampling time. The cellulolytic bacterial community structure was different in the two compartments, with F. succinogenes as the main species in the rumen and R. flavefaciens in the cecum. The lower cellulolytic activity in the cecum than in the rumen could not be ascribed to any difference in the structure of the cellulolytic bacterial community between these two compartments, and other hypotheses related to digestion are proposed.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCreþ;FAK Y397F/Y397F-mutant mice. Conversely, ECCreþ;FAK Y861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased b1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in b1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in VegfaþAng2-or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to VegfaþAng2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. Significance: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

The involvement of prolactin in human tumourogenesis has been long debated. The reason is that the evidence supporting the role of circulating prolactin in promoting breast cancer was mainly obtained using rodent models, whereas most of the studies performed in human species in the 1980s have remained inconclusive. Things have now started to change because two alternative mechanisms of prolactin actions in tumour growth have emerged since the beginning of the 21st Century. The first involves locally‐produced prolactin, which acts by an autocrine/paracrine mechanism. Genetically‐modified mouse models have demonstrated the tumourigenic potential of local prolactin on the prostate and the mammary gland, and arguments are now emerging in humans also. The second mechanism involves genetic variants of the receptor. Although no genetic disorder has been reported for prolactin or its receptor, a variant of the prolactin receptor exhibiting constitutive activity has been recently identified in patients presenting with breast tumours, suggesting that sustained prolactin signalling may participate in breast tumourogenesis. Recent data regarding these two nonclassical mechanisms of prolactin action are discussed. Finally, we address the question of their inhibition in future cancer therapy, both in light of other findings that have revealed novel actions of prolactin in breast cancer cells, and with respect to the compounds currently available to target prolactin receptor signalling.

Cathepsins are lysosomal enzymes that were shown to release the antiangiogenic fragments 16K prolactin (PRL), endostatin, and angiostatin by processing precursors at acidic pH in vitro. However, the physiological relevance of these findings is questionable because the neutral pH of physiological fluids is not compatible with the acidic conditions required for the proteolytic activity of these enzymes. Here we show that cathepsin D secreted from various tissues is able to process PRL into 16K PRL outside the cell. To specifically target extracellular proteolysis, we used tissues from PRL receptor-deficient mice, which are unable to internalize PRL. As assessed by the use of specific inhibitors of proton extruders, we show that the proteolytic activity of cathepsin D requires local acid secretion driven by Na(+)/H(+) exchangers and H(+)/ATPase. Although it is usually assumed that cathepsin-mediated generation of antiangiogenic peptides occurs in the moderately acidic pericellular milieu found in malignant tumors, we propose a new mechanism explaining the extracellular activity of this acidic protease under physiological pH. Our data support the concept that secreted lysosomal enzymes could be involved in the maintenance of angiogenesis dormancy via the generation of active antiangiogenic peptides in nonpathological contexts.

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase‐dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb‐iCreert‐driven endothelial cell (EC)‐specific, tamoxifen‐inducible homozygous mutant mouse lines: FAK wild‐type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT / WT, ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT / WT and ECCre−;FAKWT / WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice, as compared with Cre − littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD / KD and ECCre+;FAKDM / DM mice. Furthermore, although ECCre+;FAKKD / KD mice showed reduced blood vessel leakage, ECCre+;FAKDM / DM and ECCre−;FAKDM / DM mice showed no difference in leakage. Mechanistically, fibronectin‐stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD / KD ECs as compared with Cre+;FAKWT / WT cells, with no change in phosphorylation of the known Src targets FAK‐Y577, FAK‐Y861, FAK‐Y925, paxillin‐Y118, p130Cas‐Y410. Cre+;FAKDM / DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE‐cadherin‐pY658 levels in Cre+;FAKKD / KD ECs were rescued in Cre+FAKDM / DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM / DM mice. We show that EC‐specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD‐dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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