Isotoosendanin exerts anti-tumor effects in NSCLC by enhancing the stability of SHP-2 and inhibiting the JAK/STAT3 pathway.

BACKGROUND: Lung cancer has been considered as a serious problem for the public health system. NSCLC is the main type of lung cancer, and finding improved treatments for NSCLC is a pressing concern. In this study, we have explored the efficacy of isotoosendanin (ITSN) for the treatment of NSCLC, and also explored the potential underlying mechanisms. METHODS: NSCLC cells were cultured, and colony formation, cell cycle as well as apoptosis assays have been conducted for investigating the biological functions of ITSN on NSCLC cells. Furthermore, target genes of ITSN have been predicted via PharmMapper and SuperPred database, subsequently validated using the drug affinity responsive target stability (DARTS) approach, a cellular thermal shift assay (CETSA) as well as surface plasmon resonance (SPR) analysis. Additionally, ubiquitination experiments have been conducted for the level of ubiquitination of the NSCLC cells. Finally, a nude mouse xenograft model has been established for evaluating the anti-tumor effects of ITSN in vivo. RESULTS: ITSN has shown anti-NSCLC activities both in vitro and in vivo. Mechanistically, ITSN interacts with SHP-2 through enhancing its stability and decreases the level of ubiquitination. Notably, ITSN may regulate the behaviors of NSCLC cells via affecting the JAK/STAT3 signaling, and finally, the anti-tumor effects of ITSN was partially reversed by the application of SHP-2 inhibitor or siRNA of SHP-2. CONCLUSIONS: ITSN may exert its anti-tumor effects by directly targeting SHP-2, increasing its stability and minimizing its ubiquitination. These results imply that ITSN could be a revolutionary component for treating NSCLC.

Integrin αVß1-activated PYK2 promotes the progression of non-small-cell lung cancer via the STAT3-VGF axis.

BACKGROUND: Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer and is the leading cause of cancer-related deaths globally. Although various treatment strategies have been introduced, the 5-year survival rate of patients with NSCLC is only 20-30%. Thus, it remains necessary to study the pathogenesis of NSCLC and develop new therapeutic drugs. Notably, PYK2 has been implicated in the progression of many tumors, including NSCLC, but its detailed mechanism remains unclear. In this study, we aimed to elucidate the mechanisms through which PYK2 promotes NSCLC progression. METHODS: The mRNA and protein levels of various molecules were measured using qRT-PCR, western blot (WB), and immunohistochemistry (IHC), respectively. We established stable PYK2 knockdown and overexpression cell lines, and CCK-8, EdU, and clonogenic assays; wound healing, transwell migration, and Matrigel invasion assays; and flow cytometry were employed to assess the phenotypes of tumor cells. Protein interactions were evaluated with co-immunoprecipitation (co-IP), immunofluorescence (IF)-based colocalization, and nucleocytoplasmic separation assays. RNA sequencing was performed to explore the transcriptional regulation mediated by PYK2. Secreted VGF levels were examined using ELISA. Dual-luciferase reporter system was used to detect transcriptional regulation site. PF4618433 (PYK2 inhibitor) and Stattic (STAT3 inhibitor) were used for rescue experiments. A public database was mined to analyze the effect of these molecules on NSCLC prognosis. To investigate the role of PYK2 in vivo, mouse xenograft models of lung carcinoma were established and examined. RESULTS: The protein level of PYK2 was higher in human NSCLC tumors than in the adjacent normal tissue, and higher PYK2 expression was associated with poorer prognosis. PYK2 knockdown inhibited the proliferation and motility of tumor cells and caused G1-S arrest and cyclinD1 downregulation in A549 and H460 cells. Meanwhile, PYK2 overexpression had the opposite effect in H1299 cells. The siRNA-induced inhibition of integrins alpha V and beta 1 led to the downregulation of p-PYK2(Tyr402). Activated PYK2 could bind to STAT3 and enhance its phosphorylation at Tyr705, regulating the nuclear accumulation of p-STAT3(Tyr705). This further promoted the expression of VGF, as confirmed by RNA sequencing in a PYK2-overexpressing H1299 cell line and validated by rescue experiments. Two sites in promoter region of VGF gene were confirmed as binding sites of STAT3 by Dual-luciferase assay. Data from the TGCA database showed that VGF was related to the poor prognosis of NSCLC. IHC revealed higher p-PYK2(Tyr402) and VGF expression in lung tumors than in adjacent normal tissues. Moreover, both proteins showed higher levels in advanced TNM stages than earlier ones. A positive linear correlation existed between the IHC score of p-PYK2(Tyr402) and VGF. Knockdown of VGF inhibited tumor progression and reversed the tumor promoting effect of PYK2 overexpression in NSCLC cells. Finally, the mouse model exhibited enhanced tumor growth when PYK2 was overexpressed, while the inhibitors PF4618433 and Stattic could attenuate this effect. CONCLUSIONS: The Integrin αVß1-PYK2-STAT3-VGF axis promotes NSCLC development, and the PYK2 inhibitor PF4618433 and STAT3 inhibitor Stattic can reverse the pro-tumorigenic effect of high PYK2 expression in mouse models. Our findings provide insights into NSCLC progression and could guide potential therapeutic strategies against NSCLC with high PYK2 expression levels.

Tea intake and lung diseases: a Mendelian randomization study.

Background: Existing studies on the relationship between tea intake and lung diseases have yielded inconsistent results, leading to an ongoing dispute on this issue. The impact of tea consumption on the respiratory system remained elucidating. Materials and methods: We conducted a two-sample Mendelian randomization (MR) study to evaluate the associations between five distinct tea intake phenotypes and 15 different respiratory outcomes using open Genome-wide association study (GWAS) data. The inverse variance weighted (IVW) was used for preliminary screening and a variety of complementary methods were used as sensitivity analysis to validate the robustness of MR estimates. Pathway enrichment analysis was used to explore possible mechanisms. Results: IVW found evidence for a causal effect of standard tea intake on an increased risk of lung squamous cell cancer (LSCC) (OR = 1.004; 95% CI = 1.001-1.007; P = 0.00299). No heterogeneity or pleiotropy was detected. After adjustment for potential mediators, including smoking, educational attainment, and time spent watching television, the association was still robust in multivariable MR. KEGG and GO enrichment predicted proliferation and activation of B lymphocytes may play a role in this causal relation. No causalities were observed when evaluating the effect of other kinds of tea intake on various pulmonary diseases. Conclusion: Our MR estimates provide causal evidence of the independent effect of standard tea intake (black tea intake) on LSCC, which may be mediated by B lymphocytes. The results implied that the population preferring black tea intake should be wary of a higher risk of LSCC.