An Epithelial-to-Mesenchymal Transcriptional Switch Triggers Evolution of Pulmonary Sarcomatoid Carcinoma (PSC) and Identifies Dasatinib as New Therapeutic Option.

PURPOSE: Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive form of NSCLC. Rarity and poor characterization have limited the development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment. EXPERIMENTAL DESIGN: A training set of 17 biphasic PSCs was selected and tested for the expression of a large panel of 770 genes related to cancer progression using NanoString technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype. RESULTS: We demonstrated that the transcriptional activation of an epithelial mesenchymal transition (EMT) program drives PSC phylogeny in vivo. We showed that loss of the epithelial-associated transcription factor (TF) OVOL2 characterizes the transition to sarcomatoid phenotype triggering the expression of EMT promoting TFs, including TWIST and ZEB and the expression of the membrane kinase DDR2. Finally, using a drug repurposing approach, we identified dasatinib as potential inhibitor of the PSC-gene expression signature and we confirmed in vitro that this drug efficiently restrains proliferation and reverts the sarcomatoid-associated phenotype. CONCLUSIONS: Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.

Blood coagulation changes in mice bearing Lewis lung carcinoma, a metastasizing tumor.

In view of the possible role of platelets and coagulation mechanisms in the growth and dissemination of solid tumors, a number of hematological parameters were followed during development of an experimental syngeneic tumor in mice, Lewis lung carcinoma. This tumor, when transplanted i.m. in C57BL/6 mice, grows locally and spontaneously metastasizes to the lungs. The transplanted animals survive for about 4 weeks. Metastases are visible from the third week. A slight but constant increase in plasma fibrinogen level and marked thrombocytopenia were first observed during the second week after tumor implantation. No other significant changes in coagulation and fibrinolysis parameters were detected. Moreover, the animals developed marked hemolytic anemia, possibly microangiopathic in origin. 125I-labelled fibrinogen survival was decreased by about 20% during the second week after tumor implantation and was not further reduced later. Fibrinogen turnover was progressively accelerated, being more than doubled by the end of the third week. Labeled fibrinogen accumulated in the primary tumor and in the lungs (its rate of disappearance from the tumor was much slower than that from lungs or blood). 51Cr-labeled platelet survival did not change throughout the observation period, whereas platelet turnover was markedly reduced from the end of the second week, suggesting defective platelet production. 51Cr-labeled RBC survival was drastically reduced to about 30% of the controls starting from the second week. The occurence of low-grade, localized intravascular coagulation could be suggested on the basis of these data. Moreover, when Lewis lung carcinoma cells were abruptly injected i.v. through the tall vein, more impressive signs of intravascular coagulation could be seen. Indeed, there was a rapid decrease in the number of platelets, a reduction in fibrinogen, and an increase in fibrin-fibrinogen degradation products. The effects of i.v. injection of Lewis lung carcinoma cells indicate a relevant interference of cancer cells with the hematostatic system. In contrast, the tenuous evidence fo coagulation disorders in animals receiving injections of tumor cells i.m. seems to indicate a limited effect on hemostasis of the same cells during i.m. tumor growth.

Platelet glycoprotein IIb/IIIa polymorphism and coronary artery disease: implications for clinical practice.

Membrane glycoprotein (GP) IIb/IIIa plays a major role in platelet function; indeed it enables stimulated platelets to bind fibrinogen and related adhesive proteins, a process that is considered key in the development of thrombosis. The gene encoding GPIIIa (ITGB3, also known as GP3A) shows a common platelet antigen polymorphism [PL(A1)/PL(A2); expressed by alleles ITGB3*001 and ITGB3*002] that was variably associated with vascular disease. In 1996, the presence of the PL(A2) allele (ITGB3*001) was first reported to increase the risk of coronary heart disease. Shortly after, the interest in this study was increased by the publication of a case report on the death from myocardial infarction of an Olympic athlete who was found to be homozygous for the PL(A2) allele. Overviews of the published studies on the PL(A1)/PL(A2) polymorphism and coronary risk suggest an influence of the PL(A2) allele on the clinical phenotype and the interaction with other environmental factors. In particular, the strongest effect of the ITGB3 PL(A2) allele was expressed on the risk of occlusion after revascularization procedures, mainly after stent implantation, a condition in which platelet activation is more important as compared with other stenotic mechanisms. In the future, the identification of patients who are particularly responsive to GPIIb/IIIa antagonist therapy (e.g. those with the PL(A2) allele) might help to improve the treatment efficacy in this relatively small population. In a mechanism possibly unrelated to its effect on platelet reactivity to aggregating stimuli, the presence of the PL(A2) allele might influence the antiaggregatory effect of platelet inhibitory drugs such as aspirin (acetylsalicylic acid), clopidogrel, and GPIIb/IIIa antagonists. Although interesting, current data does not yet have direct clinical implications for patient risk identification and drug therapy tailoring. Larger studies are necessary to define the role of the PL(A2) allele in more homogeneous groups where platelet GPIIb/IIIa activation might be particularly relevant.

Consumption of healthy foods at different content of antioxidant vitamins and phytochemicals and metabolic risk factors for cardiovascular disease in men and women of the Moli-sani study.

BACKGROUND/OBJECTIVES: To categorize healthy food groups into categories of low-antioxidant (LAC) or high-antioxidant vitamins and phytochemicals content (HAC) and comparatively associate them with metabolic risk factors for cardiovascular disease (CVD). SUBJECTS/METHODS: A total of 6879 women (55±12 years) and 6892 men (56 ± 12 years) were analyzed from the Moli-sani cohort, randomly recruited from the general population. The European Prospective Investigation into Cancer and Nutrition Food Frequency Questionnaire was used for dietary assessment. The antioxidant content of each food group was evaluated using Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione and United States Department of Agriculture (USDA) food composition tables. Healthy foods, according to a Mediterranean dietary pattern, were categorized into HAC or LAC; total food antioxidant content (FAC) score was constructed for a comparative evaluation of the consumption of these two groups. RESULTS: In men, an increase in FAC score, which represents an increased consumption of HAC with respect to LAC foods, was associated with a decrease in systolic blood pressure, diastolic blood pressure and C-reactive protein (CRP) (ß=-0.5, P=0.02, ß=-0.3, P=0.02 and ß=-0.03, P=0.03, respectively). Logistic regression analyses showed that in men 15% (30 units) increase in FAC score was associated with 6% decrease in the likelihood of having hypertension (odds ratio (OR)=0.94, 95% confidence interval (CI) 0.91-0.98) and 3% decrease in the likelihood of having a high CRP risk level (OR=0.97, 95% CI 0.94-0.99). No significant associations were observed in women. CONCLUSIONS: A possible greater protective role of healthy HAC as compared with healthy LAC foods on hypertension and inflammation was detected in men. These results stress the importance of studying healthy foods according to their content in antioxidant vitamins and phytochemicals, in primary prevention of CVD.

Inhibition by soya isoflavones of human polymorphonuclear leukocyte function: possible relevance for the beneficial effects of soya intake.

Lower CVD incidence is reported in Asian populations consuming soya-containing food. As polymorphonuclear leukocytes (PMN) are involved in the risk of CVD, we investigated the modulatory effect of soya isoflavones on several PMN functions and their molecular mechanisms in vitro. PMN, isolated from blood from healthy subjects, were tested upon activation with 1 microm- n-formyl-methyl-leucyl-phenylalanine (fMLP) for superoxide anion production (ferric cytochrome c reduction) and released elastase (chromogenic test). PMN homotypic aggregates stimulated by fMLP or P-selectin in dynamic conditions were detected by optical microscopy. PMN, mixed with thrombin-activated, washed platelets, formed cell aggregates, measured by flow cytometry. Phosphorylation of Pyk2, a focal adhesion kinase, was studied by immunoprecipitation and immunoblotting with specific antibodies. Genistein, daidzein and equol inhibited superoxide anion production (IC50 0.25 (sem 0.1), 21.0 (sem 4.2) and 13.0 (sem 2.8) microm, respectively); the release of elastase was prevented by genistein (IC50 63 (sem 17) microm). PMN homotypic aggregates, stimulated by fMLP, were significantly reduced (24 (sem 12) and 51 (sem 14) % of control) by 100 microm genistein and equol. P-selectin-induced aggregates were reduced to 19 (sem 6), 44 (sem 10) and 28 (sem 9) % of control by 100 microm genistein, daidzein and equol, respectively. Genistein, daidzein and equol also significantly reduced mixed platelet-PMN aggregates (IC50 4.0 (sem 0.9), 57 (sem 6) and 66 (sem 23) microm, respectively). In PMN challenged by fMLP or P-selectin, activation of Pyk2 was prevented by isoflavones. The cardioprotective effect of soya-containing food might be linked to reduction of PMN activation and PMN-platelet interaction, novel targets for the biological effects of soya isoflavones.

Genes encoding fibrinogen and cardiovascular risk.

The role of fibrinogen in cardiovascular disease has been extensively studied, and meta-analyses have definitively confirmed that high levels of fibrinogen are associated with an increased risk of the disease. In recent years, several polymorphisms have been identified in the fibrinogen chain genes that contribute to determine the levels of fibrinogen in the general population. The fibrinogen beta-chain gene has been more extensively studied because the beta-chain synthesis is the limiting step in the production of mature fibrinogen. Overall, the studies show an association between beta-fibrinogen chain polymorphisms and the levels of fibrinogen. In contrast, the majority of the studies did not find any relation with the risk of cardiovascular disease. The individual responses to gender or to environmental stimuli such as smoking, physical exercise, or infections may be genetically determined, and genetic variability underlies changes in biological reactions that contribute to differences in cardiovascular risk. In the future, gene-environment interactions should be considered in evaluating the relevance of genetic variations on the risk of cardiovascular disease.

Polymorphisms in the coagulation factor VII gene and the risk of myocardial infarction.

BACKGROUND: High blood levels of coagulation factor VII are associated with a risk of ischemic vascular disease. Although factor VII levels may be genetically determined, the relation between genetic polymorphisms of factor VII, factor VII blood levels, and the risk of myocardial infarction has not been established. METHODS: We performed a case-control study of 165 patients with familial myocardial infarction (mean [+/-SD] age, 55+/-9 years) and 225 controls without a personal or family history of cardiovascular disease (mean age, 56+/-8 years). The polymorphisms involving R353Q and hypervariable region 4 of the factor VII gene were studied. Factor VII clotting activity and antigen levels were also measured. RESULTS: Patients with the QQ or H7H7 genotype had a decreased risk of myocardial infarction (odds ratios, 0.08 [95 percent confidence interval, 0.01 to 0.9] and 0.22 [95 percent confidence interval, 0.08 to 0.63], respectively). For the R353Q polymorphism, the RR genotype was associated with the highest risk, followed by the RQ genotype and then by the QQ genotype (P<0.001). For the polymorphism involving hypervariable region 4, the combined H7H5 and H6H5 genotypes were associated with the highest risk, followed in descending order by the H6H6, H6H7, and H7H7 genotypes (P<0.001). Patients with the QQ or H7H7 genotype had lower levels of both factor VII antigen and factor VII clotting activity than those with the RR or H6H6 genotype. Patients with the lowest level of factor VII clotting activity had a lower risk of myocardial infarction than those with the highest level (odds ratio, 0.13; 95 percent confidence interval, 0.05 to 0.34). CONCLUSIONS: Our findings suggest that certain polymorphisms of the factor VII gene may influence the risk of myocardial infarction. It is possible that this effect may be mediated by alterations in factor VII levels.