Targeting focal adhesion kinase (FAK) in cancer therapy: A recent update on inhibitors and PROTAC degraders.

Focal adhesion kinase (FAK) is considered as a pivotal intracellular non-receptor tyrosine kinase, and has garnered significant attention as a promising target for anticancer drug development. As of early 2024, a total of 12 drugs targeting FAK have been approved for clinical or preclinical studies worldwide, including three PROTAC degraders. In recent three years (2021-2023), significant progress has been made in designing targeted FAK anticancer agents, including the development of a novel benzenesulfofurazan type NO-releasing FAK inhibitor and the first-in-class dual-target inhibitors simultaneously targeting FAK and HDACs. Given the pivotal role of FAK in the discovery of anticancer drugs, as well as the notable advancements achieved in FAK inhibitors and PROTAC degraders in recent years, this review is underbaked to present a comprehensive overview of the function and structure of FAK. Additionally, the latest findings on the inhibitors and PROTAC degraders of FAK from the past three years, along with their optimization strategies and anticancer activities, were summarized, which might help to provide novel insights for the development of novel targeted FAK agents with promising anticancer potential and favorable pharmacological profiles.

The dual FAK-HDAC inhibitor MY-1259 displays potent activities in gastric cancers in vitro and in vivo.

Epigenetic regulation and Focal adhesion kinase (FAK) are considered to be two important targets for the development of antitumor drugs. Studies have shown that the combination of FAK and HDAC inhibitors could exhibit synergistic effects in a subset of cancer cells in vitro and in vivo. At present, there are few reports on dual target inhibitors of FAK and HDAC. Here, we first reported a new compound MY-1259 as a dual FAK and HDAC6 inhibitor, which exhibited efficient treatment effects on gastric cancers in vitro and in vivo. MY-1259 exhibited potent inhibitory activities against FAK (IC50 = 132 nM) and HDAC6 (IC50 = 16 nM). Notably, MY-1259 showed selective inhibitory potency on HDAC6 over HDAC1, HDAC2 and HDAC3. In addition, MY-1259 could potently inhibit the proliferative activities of MGC-803 and BGC-823 cells (IC50 = 3.91 and 15.46 nM, respectively, using flow cytometry counting), induce cell apoptosis, and cellular senescence. MY-1259 could effectively down-regulate the levels of Ac-Histone H3 and Ac-α-tubulin, and also inhibit the phosphorylation of FAK at three phosphorylation sites Y397, Y576/577 and Y925, thereby inhibiting the activation of ERK and AKT/mTOR. MY-1259 exhibited more effective antitumor effect in vivo than the HDAC inhibitor SAHA and FAK inhibitor TAE-226 alone or in combination, showing the advantages of FAK/HDAC dual inhibitors in the treatment of gastric cancers. Therefore, the results in this work suggested that inhibition of FAK and HDAC by MY-1259 might represent a promising strategy for the treatment of gastric cancers.

A Novel CpG Methylation Risk Indicator for Predicting Prognosis in Bladder Cancer.

PURPOSE: Bladder cancer (BLCA) is one of the most common cancers worldwide. In a large proportion of BLCA patients, disease recurs and/or progress after resection, which remains a major clinical issue in BLCA management. Therefore, it is vital to identify prognostic biomarkers for treatment stratification. We investigated the efficiency of CpG methylation for the potential to be a prognostic biomarker for patients with BLCA. PATIENTS AND METHODS: Overall, 357 BLCA patients from The Cancer Genome Atlas (TCGA) were randomly separated into the training and internal validation cohorts. Least absolute shrinkage and selector operation (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) were used to select candidate CpGs and build the methylation risk score model, which was validated for its prognostic value in the validation cohort by Kaplan-Meier analysis. Hazard curves were generated to reveal the risk nodes throughout the follow-up. Gene Set Enrichment Analysis (GSEA) was used to reveal the potential biological pathways associated with the methylation model. Quantitative real-time polymerase chain reaction (PCR) and western blotting were performed to verify the expression level of the methylated genes. RESULTS: After incorporating the CpGs obtained by the two algorithms, CpG methylation of eight genes corresponding to TNFAIP8L3, KRTDAP, APC, ZC3H3, COL9A2, SLCO4A1, POU3F3, and ADARB2 were prominent candidate predictors in establishing a methylation risk score for BLCA (MRSB), which was used to divide the patients into high- and low-risk progression groups (p < 0.001). The effectiveness of the MRSB was validated in the internal cohort (p < 0.001). In the MRSB high-risk group, the hazard curve exhibited an initial wide, high peak within 10 months after treatment, whereas some gentle peaks around 2 years were noted. Furthermore, a nomogram comprising MRSB, age, sex, and tumor clinical stage was developed to predict the individual progression risk, and it performed well. Survival analysis implicated the effectiveness of MRSB, which remains significant in all the subgroup analysis based on the clinical features. A functional analysis of MRSB and the corresponding genes revealed potential pathways affecting tumor progression. Validation of quantitative real-time PCR and western blotting revealed that TNFAIP8L3 was upregulated in the BLCA tissues. CONCLUSION: We developed the MRSB, an eight-gene-based methylation signature, which has great potential to be used to predict the post-surgery progression risk of BLCA.

In vitro and in vivo antiviral activity of a fluoronucleoside analog, NCC, against Coxsackie virus B3.

Coxsackie virus B3 (CVB3) is believed to be a major cause of viral myocarditis, with virus-induced apoptosis playing an important role in pathogenesis. The purpose of this study was to characterize the antiviral activity of a novel fluoronucleoside analogue, N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-ß-D-cytidine (NCC), against CVB3 in vitro and in vivo, and to establish whether NCC inhibits apoptosis in infected cells. In this study, HeLa cells infected with CVB3 were treated with NCC. Cell viability and apoptosis were examined. Caspase-3 and Bcl-2 levels were monitored by real-time RT PCR and Western blot analysis. For in vivo studies, BALB/c mice infected with CVB3 were treated with NCC daily. Serum markers of myocardial injury and histological studies were measured to examine myocardial injury on day 8 post-infection. To measure apoptosis, levels of Bcl-2 and caspase-3 were examined by immunohistochemistry and real-time RT-PCR. We found that NCC inhibited virus-mediated cytopathic effects in HeLa cells with an EC50 of 116.60 ± 0.32 µM. In infected mice, administration of NCC (2 mg/kg) decreased the activities of serum creatine kinase and lactic dehydrogenase, inhibited the replication of CVB3 and alleviated damage to the heart. Importantly, NCC suppressed CVB3-induced apoptosis in HeLa cells and affected the expression of apoptosis-related factors in infected mice. Together, our results demonstrate that NCC exerts significant antiviral activities against CVB3. We conclude that NCC is a potential therapeutic agent for the treatment of viral myocarditis. Keywords: coxsackie virus B3; viral myocarditis; N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-ß-D-cytidine; apoptosis.

Activation of NLRP3 inflammasome promotes the proliferation and migration of esophageal squamous cell carcinoma.

Inflammasomes can identify endogenous danger signals as an inflammatory immune response. As the most common inflammasome, the NLR pyrin family domain containing 3 (NLRP3) inflammasome is associated with the pathogenesis of different tumors. However, the function of the NLRP3 inflammasome in esophageal cancer (EC) has rarely been reported. Herein, the expression levels of the components of NLRP3 inflammasome and Ki­67 were analyzed by immunohistochemistry. Furthermore, correlations between the NLRP3 inflammasome and Ki­67 along with the clinicopathological features of EC patients were evaluated. The components of the NLRP3 inflammasome were also assessed by western blot analysis and quantitative PCR. NLRP3 was silenced or overexpressed in different esophageal squamous cell carcinoma (ESCC) cell lines, and cell viability, migration and invasion were assessed by CCK­8 and Transwell assays. The present results showed that high NLRP3 expression in the tumor specimens was significantly associated with TNM stage and T category. Spearman's correlation analysis revealed a positive correlation between NLRP3 and the Ki­67 proliferation index. The mRNA and protein levels of NLRP3, apoptosis­associated speck­like protein containing a CARD (ASC), cleaved caspase­1, and interleukin (IL)­1ß in tumor tissues were higher than those in non­cancerous tissues. The level of secreted IL­1ß in tumor tissues was also increased, as compared to that in normal tissues. Silencing of NLRP3 in KYSE­70 and TE13 cells strongly attenuated cell viability, decreased cell mobility in wound­healing assays and greatly diminished the ability of cell migration and invasion in the Transwell system. Overexpression of NLRP3 in KYSE­510 and EC9706 cells markedly promoted the proliferation, migration and invasion. Collectively, these results revealed that the the NLRP3 inflammasome is upregulated in human ESCC tissues and promotes ESCC progression. Hence, NLRP3 could be a promising new candidate diagnostic and prognostic target.

MicroRNAs: Emerging biomarkers for atrial fibrillation.

Atrial fibrillation (AF) causes severe cardiac dysrhythmia among patients with cardiovascular diseases. AF increases the risk of stroke and heart failure and is a growing public health concern. AF is also associated with various disease conditions such as hypertension, coronary artery disease, aging, and diabetes mellitus. The mechanism underlying AF is not completely understood due to its complexity. However, experimental and clinical data have revealed that the prevalence of this disease is associated with atrial arrhythmogenic remodeling. Currently, there are no biomarkers that are available for the early diagnosis of AF. Several studies have proposed microRNAs (miRNAs) as useful biomarkers for the diagnosis of AF due to their stability and easy availability both in atrial tissue and circulating blood. miRNAs play an important role in the development of the heart. The dysregulation of miRNA expression is associated with cardiac remodeling. Genetic factors strongly contribute to the pathogenesis of AF. Recently, single nucleotide polymorphisms (SNPs) in various genes and miRNAs have been reported to be associated with AF. The aim of this review was to discuss the correlation between SNPs in miRNAs and AF, including those miRNAs that are commonly reported as potential biomarkers for AF.

Cepharanthine hydrochloride reverses P­glycoprotein-mediated multidrug resistance in human ovarian carcinoma A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Ovarian cancer has the highest mortality rate among gynecologic malignant tumors. The major obstacle to treatment success is multidrug resistance (MDR) to chemotherapy drugs. Cepharanthine hydrochloride (CH), a natural alkaloid-derived compound, has shown MDR reversal potency in several tumor cell lines; however, the molecular mechanism is not entirely known. In the present study, we assessed whether CH sensitized malignant cells to chemotherapy drugs in ovarian cancer and explored the relevant mechanism. We found that CH reduced the IC50 value of paclitaxel and increased intracellular rhodamine-123 accumulation in human ovarian cancer A2780/Taxol cells in a concentration-dependent manner. Reverse transcription polymerase chain reaction and western blot assay demonstrated that CH inhibited MDR1 expression as indicated by reduced mRNA and protein levels in A2780/Taxol cells. In addition, the inhibitory effect was strengthened after CH was combined with the specific PI3K/Akt signaling pathway inhibitor LY294002. Furthermore, p­Akt expression decreased gradually with the concentration of CH (2, 4 and 8 µM). Taken together, these findings indicated that CH reversed P­glycoprotein-mediated MDR in A2780/Taxol cells by inhibiting the PI3K/Akt signaling pathway.

Intestinal absorption mechanisms of 2'-deoxy-2'-ß-fluoro-4'-azidocytidine, a cytidine analog for AIDS treatment, and its interaction with P-glycoprotein, multidrug resistance-associated protein 2 and breast cancer resistance protein.

2'-Deoxy-2'-ß-fluoro-4'-azidocytidine (FNC), a cytidine analog, has attracted great interest because of its potent activity against wild-type and multidrug-resistant HIV. The purpose of current study was to investigate the absorption mechanisms of FNC in the small intestine, as well as the interactions between FNC and P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). The experiments were performed using Caco-2 cells and the rat small intestine. The uptake experiment indicated that FNC concentration, extracellular pH and the incubated temperature could influence the uptake of FNC in Caco-2 cells. NaN3, verapamil, probenecid, MK571 and GF120918 could significantly increase the FNC uptake in Caco-2 cells. The transport experiment showed that both the absorption and secretion of FNC were concentration dependent. The secretion of FNC was approximately 2-fold greater than the absorption. In the presence of verapamil, probenecid, MK571 or GF120918, the efflux ratio decreased by >50%. In everted rat intestine, the absorption of FNC also depended on its concentration and was not significantly different in the different segments of the small intestine. Real-time RT-PCR results indicated that the gene expressions of P-gp, MRP2 and BCRP were up-regulated after exposure to FNC. The reduction in accumulation of rhodamine 123 after treatment with FNC revealed its ability to up-regulate P-gp activity. In conclusion, FNC was completely absorbed by passive diffusion and active transport mechanisms. P-gp, MRP2 and BCRP could influence the absorption of FNC in the small intestine. FNC could modulate the gene expressions of P-gp, MRP2 and BCRP, and increase the activity of P-gp.

Identification and functional characterization of the human ether-a-go-go-related gene Q738X mutant associated with hereditary long QT syndrome type 2.

QT interval prolongation, a risk factor for arrhythmias, may be associated with genetic variants in genes governing cardiac repolarization. Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go­go-related gene (hERG). This gene encodes a voltage-gated potassium channel comprised of 4 subunits, and the formation of functional channels requires the proper assembly of these 4 subunits. In the present study, we investigated the role of the LQT2 mutation, Q738X, which causes truncation of the C-terminus of hERG channels, in the assembly and function of hERG channels. When expressed in HEK293 cells, Q738X did not generate an hERG current. The co-expression of Q738X with wild-type (WT)-hERG did not cause the dominant-negative suppression of the WT-hERG current. Western blot analysis and confocal microscopy revealed that the Q738X mutation caused defective trafficking of hERG channel proteins. Co-immunoprecipitation demonstrated that Q738X did not exhibit dominant-negative effects due to the failure of the mutant and WT subunits to co-assemble. In conclusion, the functional loss caused by the Q738X mutation in hERG K+ channels may be attributed to the disruption of tetrameric assembly.

Blockage of hERG current and the disruption of trafficking as induced by roxithromycin.

Roxithromycin is an oral macrolide antibiotic agent that has been repeatedly reported to provoke excessive prolongation of the Q-T interval and torsades de pointes in clinical settings. To investigate the mechanisms underlying the arrhythmogenic side effects of roxithromycin, we studied the molecular mechanisms of roxithromycin on human ether-à-go-go-related gene (hERG) K(+) channels expressed in human embryonic kidney (HEK293) cells. Roxithromycin was found to inhibit wild-type (WT) hERG currents in a concentration-dependent manner with a half-maximum block concentration (IC50) of 55.8 ± 9.1 µmol/L. S6 residue hERG mutants (Y652A and F656C) showed reduced levels of hERG current blockage attributable to roxithromycin. Roxithromycin also inhibited the trafficking of hERG protein to the cell membrane, as confirmed by Western blot analysis and confocal microscopy. These findings indicate that roxithromycin may cause acquired long-QT syndrome via direct inhibition of hERG current and by disruption of hERG protein trafficking. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by roxithromycin, but did not significantly alter the disruption of trafficking.

Polymorphisms of STAT6 and specific serum IgE levels in patients with penicillin allergy.

OBJECTIVE: Signal transducer and activator of transcription (STAT) 6 signaling pathway is required for mediating the biologic functions of interleukin (IL)-4 which is proved to play an important role in the pathogenesis of allergic diseases and the induction of specific immunoglobulin (Ig) E synthesis. The current studies are conducted to evaluate the relationship between polymorphisms of STAT6, specific IgE and penicillin allergy. MATERIALS AND METHODS: In 242 patients with penicillin allergy and 220 controls, polymorphisms of STAT6 in2SNP3 and 3'UTRSNP3 were genotyped by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). Eight kinds of specific IgE to penicillins were detected with radioallergosorbent test (RAST). RESULTS: For STAT6 in2SNP3, the frequency of the CC genotype was significantly higher in all patients than in controls (60 vs. 47%, p = 0.005). A similar result was observed between patients of the urticaria group and controls (70 vs. 47%, p = 0.005). With respect to allele frequency, the C-allele occurred more frequently in all patients and in the urticaria group than in controls (78 vs. 69%, p = 0.002; 83 vs. 69%, p = 0.007). CONCLUSIONS: Our finding suggests that STAT6 in2SNP3 is associated with penicillin allergy, but has no effect on the specific IgE levels of patients with penicillin allergy.

Polymorphisms and haplotype analysis of IL-4Ralpha Q576R and I75V in patients with penicillin allergy.

BACKGROUND: The interleukin (IL)-4 and IL-4R genes are linked to allergic diseases and atopy and elevated serum IgE levels closely follow the presentation of penicillin allergy. We have evaluated the association between specific immunoglobulin (Ig)E, polymorphisms of IL-4Ralpha, and penicillin allergy. METHODS: Eight types of specific IgE to penicillins were detected with the radioallergosorbent test (RAST) in 242 patients with penicillin allergy and 220 control subjects. Polymorphisms of IL-4Ralpha Q576R and IL-4Ralpha I75V were genotyped by PCR-restriction fragment length polymorphism (RFLP). The polymorphisms and haplotype of IL-4Ralpha Q576R and I75V were analyzed. RESULTS: For IL-4Ralpha Q576R, the frequency of the AA genotype was higher in patients with penicillin allergy (76 vs. 64%, P = 0.005). The A allele occurred more frequently in the penicillin-allergic patients than in the controls (87 vs. 80%, P = 0.003). For IL-4Ralpha I75V, the AA genotype was more likely to be detected in the urticaria group than in the control group (32 vs. 19%, P = 0.049). Haplotype analysis revealed that Q576/I75 was more frequently found in patients with penicillin allergy than in controls (42 vs. 35%, P = 0.037). CONCLUSIONS: The IL4Ralpha Q576 allele is related to penicillin allergy, and the IL-4Ralpha I75 allele is associated with the symptom of urticaria. The Q576/I75 haplotype may be related with an allergy to penicillin.