Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion

<p>Abstract</p> <p>Background</p> <p>Focal adhesion kinase (FAK) and Src are protein tyrosine kinases that physically and functionally interact to facilitate cancer progression by regulating oncogenic processes such as cell motility, survival, proliferation, invasiveness, and angiogenesis.</p> <p>Method</p> <p>To understand how FAK affects oncogenesis through the phosphorylation of cellular substrates of Src, we analyzed the phosphorylation profile of a panel of Src substrates in parental and v-Src-expressing FAK+/+ and FAK-/- mouse embryo fibroblasts, under conditions of anchorage-dependent (adherent) and -independent (suspension) growth.</p> <p>Results</p> <p>Total Src-induced cellular tyrosine phosphorylation as well as the number of phosphotyrosyl substrates was higher in suspension versus adherent cultures. Although the total level of Src-induced cellular phosphorylation was similar in FAK+/+ and FAK-/- backgrounds, the phosphorylation of some substrates was influenced by FAK depending on adherence state. Specifically, in the absence of FAK, Src induced higher phosphorylation of p190RhoGAP, paxillin (poY118) and Crk irrespective of adhesion state, PKC-δ (poY311), connexin-43 (poY265) and Sam68 only under adherent conditions, and p56Dok-2 (poY351) and p120catenin (poY228) only under suspension conditions. In contrast, FAK enhanced the Src-induced phosphorylation of vinculin (poY100 and poY1065) and p130CAS (poY410) irrespective of adherence state, p56Dok-2 (poY351) and p120catenin (poY228) only under adherent conditions, and connexin-43 (poY265), cortactin (poY421) and paxillin (poY31) only under suspension conditions. The Src-induced phosphorylation of Eps8, PLC-γ1 and Shc (poY239/poY240) were not affected by either FAK or adherence status. The enhanced anchorage-independent growth of FAK-/-[v-Src] cells was selectively decreased by expression of paxillin^Y118F, but not by WT-paxillin, p120catenin^Y228For Shc^Y239/240F, identifying for the first time a role for paxillin^poY118in Src-induced anchorage-independent growth. Knockdown of FAK by siRNA in the human colon cancer lines HT-25 and RKO, resulted in increased paxillin^poY118levels under suspension conditions as well as increased anchorage-independent growth, supporting the notion that FAK attenuates anchorage-independent growth by suppressing adhesion-dependent phosphorylation of paxillin^Y118.</p> <p>Conclusion</p> <p>These data suggest that phosphorylation of Src substrates is a dynamic process, influenced temporally and spatially by factors such as FAK and adhesion.</p

FMT: Removing Backdoor Feature Maps via Feature Map Testing in Deep Neural Networks

Deep neural networks have been widely used in many critical applications, such as autonomous vehicles and medical diagnosis. However, their security is threatened by backdoor attack, which is achieved by adding artificial patterns to specific training data. Existing defense strategies primarily focus on using reverse engineering to reproduce the backdoor trigger generated by attackers and subsequently repair the DNN model by adding the trigger into inputs and fine-tuning the model with ground-truth labels. However, once the trigger generated by the attackers is complex and invisible, the defender can not successfully reproduce the trigger. Consequently, the DNN model will not be repaired since the trigger is not effectively removed. In this work, we propose Feature Map Testing~(FMT). Different from existing defense strategies, which focus on reproducing backdoor triggers, FMT tries to detect the backdoor feature maps, which are trained to extract backdoor information from the inputs. After detecting these backdoor feature maps, FMT will erase them and then fine-tune the model with a secure subset of training data. Our experiments demonstrate that, compared to existing defense strategies, FMT can effectively reduce the Attack Success Rate (ASR) even against the most complex and invisible attack triggers. Second, unlike conventional defense methods that tend to exhibit low Robust Accuracy (i.e., the model's accuracy on the poisoned data), FMT achieves higher RA, indicating its superiority in maintaining model performance while mitigating the effects of backdoor attacks~(e.g., FMT obtains 87.40\% RA in CIFAR10). Third, compared to existing feature map pruning techniques, FMT can cover more backdoor feature maps~(e.g., FMT removes 83.33\% of backdoor feature maps from the model in the CIFAR10 \& BadNet scenario).Comment: 12 pages, 4 figure

Feature Map Testing for Deep Neural Networks

Due to the widespread application of deep neural networks~(DNNs) in safety-critical tasks, deep learning testing has drawn increasing attention. During the testing process, test cases that have been fuzzed or selected using test metrics are fed into the model to find fault-inducing test units (e.g., neurons and feature maps, activating which will almost certainly result in a model error) and report them to the DNN developer, who subsequently repair them~(e.g., retraining the model with test cases). Current test metrics, however, are primarily concerned with the neurons, which means that test cases that are discovered either by guided fuzzing or selection with these metrics focus on detecting fault-inducing neurons while failing to detect fault-inducing feature maps. In this work, we propose DeepFeature, which tests DNNs from the feature map level. When testing is conducted, DeepFeature will scrutinize every internal feature map in the model and identify vulnerabilities that can be enhanced through repairing to increase the model's overall performance. Exhaustive experiments are conducted to demonstrate that (1) DeepFeature is a strong tool for detecting the model's vulnerable feature maps; (2) DeepFeature's test case selection has a high fault detection rate and can detect more types of faults~(comparing DeepFeature to coverage-guided selection techniques, the fault detection rate is increased by 49.32\%). (3) DeepFeature's fuzzer also outperforms current fuzzing techniques and generates valuable test cases more efficiently.Comment: 12 pages, 5 figures. arXiv admin note: text overlap with arXiv:2307.1101

Thiazolyl N-Benzyl-Substituted Acetamide Derivatives: Synthesis, Src Kinase Inhibitory and Anticancer Activities

KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of pyridine ring and N-benzylsubstitution in KX2-391 and establish the structure-activity relationship, a number of N-benzyl substituted (2-morpholinoethoxy)phenyl)thiazol-4-yl)acetamide derivatives containing thiazole instead of pyridine were synthesized and evaluated for Src kinase inhibitory activities. The unsubstituted N-benzyl derivative (8a) showed the inhibition of c-Src kinase with GI50 values of 1.34 μM and 2.30 M in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), breast carcinoma (BT-20), and leukemia (CCRF-CEM) cells. 4-Fluorobenzylthiazolyl derivative 8b exhibited 64-71% inhibition in the cell proliferation of BT-20 and CCR5 cells at concentration of 50 μM

Discovery and Characterization of Potent Dual P-Glycoprotein and CYP3A4 Inhibitors: Design, Synthesis, Cryo-EM Analysis, and Biological Evaluations

Targeted concurrent inhibition of intestinal drug efflux transporter P-glycoprotein (P-gp) and drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4) is a promising approach to improve oral bioavailability of their common substrates such as docetaxel, while avoiding side effects arising from their pan inhibitions. Herein, we report the discovery and characterization of potent small molecule inhibitors of P-gp and CYP3A4 with encequidar (minimally absorbed P-gp inhibitor) as a starting point for optimization. To aid in the design of these dual inhibitors, we solved the high-resolution cryo-EM structure of encequidar hound to human P-gp. The structure guided us to prudently decorate the encequidar scaffold with CYP3A4 pharmacophores, leading to the identification of several analogues with dual potency against P-gp and CYP3A4. In vivo, dual P-gp and CYP3A4 inhibitor 3a improved the oral absorption of docetaxel by 3-fold as compared to vehicle, while 3a itself remained poorly absorbed.ISSN:1520-4804ISSN:0022-262

Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation

Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (<i>n</i> = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics