Analysis of PIK3CA mutations and activation pathways in <i>triple negative</i> breast cancer

Background: Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods: PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results: PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions: Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies

Genetic determinants in a critical domain of ns5a correlate with hepatocellular carcinoma in cirrhotic patients infected with hcv genotype 1b

HCV is an important cause of hepatocellular carcinoma (HCC). HCV NS5A domain‐1 interacts with cellular proteins inducing pro‐oncogenic pathways. Thus, we explore genetic variations in NS5A domain‐1 and their association with HCC, by analyzing 188 NS5A sequences from HCV genotype‐1b infected DAA‐naïve cirrhotic patients: 34 with HCC and 154 without HCC. Specific NS5A mutations significantly correlate with HCC: S3T (8.8% vs. 1.3%, p = 0.01), T122M (8.8% vs. 0.0%, p &lt; 0.001), M133I (20.6% vs. 3.9%, p &lt; 0.001), and Q181E (11.8% vs. 0.6%, p &lt; 0.001). By multivariable analysis, the presence of &gt;1 of them independently correlates with HCC (OR (95%CI): 21.8 (5.7–82.3); p &lt; 0.001). Focusing on HCC‐group, the presence of these mutations correlates with higher viremia (median (IQR): 5.7 (5.4–6.2) log IU/mL vs. 5.3 (4.4–5.6) log IU/mL, p = 0.02) and lower ALT (35 (30–71) vs. 83 (48–108) U/L, p = 0.004), suggesting a role in enhancing viral fitness without affecting necroinflammation. Notably, these mutations reside in NS5A regions known to interact with cellular proteins crucial for cell‐cycle regulation (p53, p85‐PIK3, and β‐ catenin), and introduce additional phosphorylation sites, a phenomenon known to ameliorate NS5A interaction with cellular proteins. Overall, these results provide a focus for further investigations on molecular bases of HCV‐mediated oncogenesis. The role of these NS5A domain‐1 mutations in triggering pro‐oncogenic stimuli that can persist also despite achievement of sustained virological response deserves further investigation

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

A comparative study of thermodynamic properties of binary mixtures containing perfluoroalkanes

Literature data on molar excess enthalpies and molar excess Gibbs energies, liquid-liquid equilibria, activity coefficients at infinite dilution and partial molar enthalpies at infinite dilution of binary mixtures of n-perfluoroalkanes (C-5-C-8)+n-alkanes (C5-C8) and of n-perfluorohexane+linear monoethers of general formula, CH3(CH2)(m)-O-(CH2)(n)-CH3 (m,n=1-4), are treated in the framework of DISQUAC, an extended quasichemical group contribution theory. The systems are characterized by two or three types of contact surfaces: aliphatic (CH3, CH2, CH and C groups), fluorine (F group) and oxygen (O group). Using a limited number of adjusted contact interchange energies parameters, structure dependent, the model provides a fairly consistent description of the thermodynamic properties as a function of concentration. The model may serve to predict missing data

2-[N-Alkyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines as anticancer agents inhibitors of folate enzymes

Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60 human tumors cell lines at National Cancer Institute of Bethesda (NCI), we have synthesized a novel series of thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1-31). The lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR enzyme, with a Ki of 0.2 μM. Docking studies were performed on compound 1 and here reported to predict its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1-31) were assayed versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds inhibited hDHFR with Ki values included between 0.2 and 11 μM, while only a few (6, 21, 24, 27, 29) showed great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50 equal to -5.49 and log LC50 equal to -4.19 and maintained significant percent of growth inhibition on seven cancer cell lines at the concentration of 1 μM. Compound 17 was the second most active and moreover showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257, Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100-0.01 μM)

2-[<i>N</i>-Alkyl(<i>R</i>-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines as anticancer agents inhibitors of folate enzymes

Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60 human tumors cell lines at National Cancer Institute of Bethesda (NCI), we have synthesized a novel series of thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1–31). The lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR enzyme, with a Ki of 0.2 μM. Docking studies were performed on compound 1 and here reported to predict its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1–31) were assayed versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds inhibited hDHFR with Ki values included between 0.2 and 11 μM, while only a few (6, 21, 24, 27, 29) showed great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50 equal to −5.49 and log LC50 equal to −4.19 and maintained significant percent of growth inhibition on seven cancer cell lines at the concentration of 1 μM. Compound 17 was the second most active and moreover showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257, Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100–0.01 μM)

Protective effect of deferoxamine on sodium nitroprusside-induced apoptosis in PC12 cells

Reportedly, the generation of nitric oxide (NO) may lead to iron mobilization from ferritin disrupting intracellular iron homeostasis and increasing levels of reactive oxygen species. In the present study, we evaluated the role of endogenous iron in NO-induced apoptosis in PC12 cells. Apoptosis was tested by flow cytometry, fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated 2′-deoxy-uridine 5′-triphosphate nick end labeling (TUNEL) technique. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. When incubated with 0.5–0.75 mM sodium nitroprusside (SNP, a chemical NO donor), PC12 cells were shown to undergo apoptosis. In addition, SNP induced a time-dependent decrease in cell viability. Since deferoxamine (0.05–0.1 mM), a powerful iron chelator, inhibited both SNP-induced apoptosis and the decrease in cell viability, we suggest that these NO effects may be dependent upon iron mobilization within the cell

Collateral sensitivity to novel thymidylate synthase inhibitors correlates with folate cycle enzymes impairment in cisplatin-resistant human ovarian cancer cells

The cytotoxicity of two novel folate cycle inhibitors with quinoxalinic structure, 3-methyl-7-trifluoromethyl-2(R)-[3,4,5-trimethoxyanilino]-quinoxaline (453R) and 3-piperazinilmethyl-2[4(oxymethyl)-phenoxy]quinoxaline (311S), was tested against a panel of both cisplatin(cDDP)-sensitive and -resistant carcinoma cell lines. Interestingly, the cisplatin-resistant human ovarian line, C13 cells, exhibited collateral sensitivity towards the two compounds when compared to its sensitive parental 2008 cells. In this resistant line, which showed elevated expression of the folate cycle enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), due to cisplatin-resistance phenotype, collateral sensitivity correlated with the greater reduction of enzyme expression. In addition, TS and DHFR expression of the other resistant lines, the human ovarian carcinoma A2780/CP cells and the human breast cancer MDA/CH cells, were decreased in accordance with the similar sensitivity or the low level of cross-resistance to these compounds in comparison to their respective parental lines. Noteworthy, unlike 5-fluorouracil, both drugs reduced the level of TS without inducing ternary complex formation with the co-substrate and the nucleotide analogue. Median effect analysis of the interactive effects of cisplatin with the two quinoxalines mainly showed additive or synergistic cell killing, depending on schedules of drug combinations. In particular, synergistic effects were more often obtained, even on the resistant cells, when cisplatin was added at the beginning of the treatment. These results indicate that, despite the possibility of other mechanisms being involved, inhibition of TS cycle enzymes plays an important role in the pharmacology of these compounds, which might also represent a useful component in drug treatment protocols against cDDP-resistant cells