Increased fatty acid synthesis benefits glioblastoma malignancy. However, the coordinated regulation of cytosolic acetyl-CoA production, the exclusive substrate for fatty acid synthesis, remains unclear. Here, we show that proto-oncogene tyrosine kinase c-SRC is activated in glioblastoma and remodels cytosolic acetyl-CoA production for fatty acid synthesis. Firstly, acetate is an important substrate for fatty acid synthesis in glioblastoma. c-SRC phosphorylates acetyl-CoA synthetase ACSS2 at Tyr530 and Tyr562 to stimulate the conversion of acetate to acetyl-CoA in cytosol. Secondly, c-SRC inhibits citrate-derived acetyl-CoA synthesis by phosphorylating ATP-citrate lyase ACLY at Tyr682. ACLY phosphorylation shunts citrate to IDH1-catalyzed NADPH production to provide reducing equivalent for fatty acid synthesis. The c-SRC-unresponsive double-mutation of ACSS2 and ACLY significantly reduces fatty acid synthesis and hampers glioblastoma progression. In conclusion, this remodeling fulfills the dual needs of glioblastoma cells for both acetyl-CoA and NADPH in fatty acid synthesis and provides evidence for glioma treatment by c-SRC inhibition.

OBJECTIVE: This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism.
METHODS: FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining.
RESULTS: GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch.
CONCLUSIONS: GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.
目的: 探讨在周期性牵张力作用下葡萄糖调节蛋白78（GRP78）对牙周膜成纤维细胞成骨分化的影响，并阐述其机制。方法: 应用FlexCell 5000细胞应力装置模拟牙齿正畸受力环境；应用流式细胞术细胞分选技术获得牙周膜成纤维细胞GRP78高表达细胞和GRP78低表达细胞；采用基因转染技术敲减GRP78、c-Src的表达以及过表达c-Src；蛋白质印迹实验检测成骨转录因子Runt相关基因2（RUNX2）、锌指结构转录因子（Osterix）以及成骨标志蛋白骨钙蛋白（OCN）、骨桥蛋白（OPN）的表达；免疫共沉淀实验检测GRP78与c-Src激酶的相互作用；茜素红染色实验检测细胞矿化结节的形成。结果: GRP78在牙周膜成纤维细胞呈异质性表达，流式分选实验获得GRP78高表达和GRP78低表达细胞。周期性牵张力处理后，与GRP78低表达细胞相比，GRP78高表达细胞成骨分化能力及c-Src激酶磷酸化水平均升高，差异具有统计学意义（P<0.05）；GRP78与c-Src激酶存在相互作用；与对照组相比，过表达c-Src组细胞成骨分化能力升高（P<0.05），sic-Src组细胞成骨分化能力降低（P<0.05）。结论: GRP78通过与c-Src激酶相互作用并上调其表达，进而促进周期性牵张力诱导的牙周膜成纤维细胞成骨分化。.

Oncogenic overexpression or activation of C-terminal Src kinase (CSK) has been shown to play an important role in triple-negative breast cancer (TNBC) progression, including tumor initiation, growth, metastasis, drug resistance. This revelation has pivoted the focus toward CSK as a potential target for novel treatments. However, until now, there are few inhibitors designed to target the CSK protein. Responding to this, our research has implemented a comprehensive virtual screening protocol. By integrating energy-based screening methods with AI-driven scoring functions, such as Attentive FP, and employing rigorous rescoring methods like Glide docking and molecular mechanics generalized Born surface area (MM/GBSA), we have systematically sought out inhibitors of CSK. This approach led to the discovery of a compound with a potent CSK inhibitory activity, reflected by an IC50 value of 1.6 nM under a homogeneous time-resolved fluorescence (HTRF) bioassay. Subsequently, molecule 2 exhibits strong growth inhibition of MD anderson - metastatic breast (MDA-MB) -231, Hs578T, and SUM159 cells, showing a level of growth inhibition comparable to that observed with dasatinib. Treatment with molecule 2 also induced significant G1 phase accumulation and cell apoptosis. Furthermore, we have explored the explicit binding interactions of the compound with CSK using molecular dynamics simulations, providing valuable insights into its mechanism of action.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs\' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 μM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C-F···C═O interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.

OBJECTIVE: C-terminal Src kinase (CSK), a sarcoma (Src) homologous family kinase, is one of the most important negative regulators. It acts as a tumor suppressor by inhibiting the activity of Src family tyrosine kinases. Paradoxically, CSK is highly expressed in a variety of common tumors. Therefore, we report the expression profile of CSK in pan-cancer patients, focusing on the prognostic value, immune infiltration pattern, and biological function of CSK in gastric cancer.
METHODS: We used the TCGA database to analyze CSK expression, clinical relevance, prognostic significance, assessment of the tumor immune microenvironment, and GO and Kegg enrichment analysis based on co-expressed genes using a bioinformatics approach.
RESULTS: CSK is a protective factor in gastric cancer, and its expression correlates with the level of immune cell infiltration and immune checkpoint molecules.
CONCLUSIONS: Our findings suggest that CSK is an independent prognostic factor in gastric cancer and may predict molecular targeting and immunotherapy and provide ideas for its therapeutic strategy.

To observe the effects of oxidative stress on vascular endothelial growth factor (VEGF) and connections of lens epithelial cells.
Human lens epithelium of patients with age-related cataract (ARC), both SRA01/04 cells and whole mice lens stimulated by H2O2 were employed. VEGF in human aqueous humor of ARC-patients and the supernatant of SRA01/04 cells was determined by ELISA. The expressions of VEFG in human lens epithelium were detected by immunofluorescence staining. Multiple linear regression analysis and spearman rank-order correlation were used to determine the associations between VEGF and parameters of ARC individuals. In H2O2-induced SRA01/04 cells, Catalase (CAT), PP1 (inhibitor of c-Src kinase) and Avastin (VEGF antibody) were used to inhibit the effects of H2O2, activation of c-Src kinase and VEGF, which were detected by Western blot. The alterations of ZO-1 and N-cadherin were tested by immunofluorescence staining and Western blot. In H2O2-induced whole lens, the changes of opacification area in different treatment of inhibitors were observed.
The secretion of VEGF in aqueous humor and expression of VEGF in the lens epithelium of ARC patients increased significantly with age. In H2O2-induced SRA01/04 cells, the VEGF in the supernatant was increased with the culture duration and the dose of H2O2. The expressions of p-Src418 and VEGF were also up-regulated, whereas the expressions of ZO-1 and N-cadherin were down-regulated. CAT effectively prevented these changes induced by H2O2, while PP1 inhibited not only p-Src418 but also up-regulation of VEGF, Avastin partially inhibited VEGF up-regulation. Both PP1 and Avastin prevented down-regulation of ZO-1 and N-cadherin, respectively, but Avastin combined with PP1 had no significant synergistic effects. In H2O2-induced cataract, CAT prevented development of opacification area effectively, and PP1 and Avastin did partially.
Oxidative stress disrupts connections of lens epithelial cells by activating c-Src/VEGF, inhibiting which may prevent cataract.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Triple-negative breast cancer (TNBC) is recognized for its poor prognosis and limited options for treatment. Circular RNA KIF4A (circKIF4A) was documented to be abnormally overexpressed in TNBC and was correlated with a poor survival rate. The objective of this study is to further examine the functional role of circKIF4A and its underlying mechanism. CircKIF4A was significantly upregulated in TNBC and the knockdown of circKIF4A suppressed TNBC cell proliferation, migration, and invasion. CircKIF4A was directly bound to EIF4A3, which interacted with SDC1. Knockdown of circKIF4A reduced interaction between EIF4A3 and SDC1 as well as SDC1 mRNA stability. SDC1 activated the c-src/FAK signaling pathways and finally promoted TNBC progression. circKIF4A induced TNBC progress in the in vivo mouse model via SDC1. CircKIF4A interacts with EIF4A3 to stabilize SDC1 mRNA, which activates the c-src/FAK signaling pathways and promotes TNBC progression. This may provide a potential therapy for TNBC treatment.