Rab3B enhances the stabilization of DDX6 to promote lung adenocarcinoma aggressiveness.

BACKGROUND: Liver kinase B1 (LKB1) is frequently mutated in lung adenocarcinoma, and its loss contributes to tumor progression. METHODS: To identify LKB1 downstream genes that promote lung adenocarcinoma aggressiveness, we performed bioinformatical analysis using publicly available datasets. RESULTS: Rab3B was upregulated in LKB1-depleted lung adenocarcinoma cells and suppressed by LKB1 overexpression. CREB protein was enriched at the promoter of Rab3B in lung cancer cells. Silencing of CREB abrogated the upregulation of Rab3B upon LKB1 loss. Immunohistochemistry revealed the elevated expression of Rab3B in lung adenocarcinomas relative to adjacent normal tissues. Upregulation of Rab3B was significantly associated with lymph node metastasis, advanced tumor stage, and reduced overall survival in lung adenocarcinoma patients. Knockdown of Rab3B suppressed and overexpression of Rab3B promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells in vitro. In a mouse xenograft model, Rab3B depletion restrained and Rab3B overexpression augmented the growth of lung adenocarcinoma tumors. Mechanistically, Rab3B interacted with DDX6 and enhanced its protein stability. Ectopic expression of DDX6 significantly promoted the proliferation, colony formation, and migration of lung adenocarcinoma cells. DDX6 knockdown phenocopied the effects of Rab3B depletion on lung adenocarcinoma cells. Additionally, DDX6 overexpression partially rescued the aggressive phenotype of Rab3B-depleted lung adenocarcinoma cells. CONCLUSION: LKB1 deficiency promotes Rab3B upregulation via a CREB-dependent manner. Rab3B interacts with and stabilizes DDX6 protein to accelerate lung adenocarcinoma progression. The Rab3B-DDX6 axis may be potential therapeutic target for lung adenocarcinoma.

Hsa_circ_0096157 silencing suppresses autophagy and reduces cisplatin resistance in non-small cell lung cancer by weakening the Nrf2/ARE signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality worldwide, and new diagnostic markers are urgently needed. We aimed to investigate the mechanism by which hsa_circ_0096157 regulates autophagy and cisplatin (DDP) resistance in NSCLC. METHODS: A549 cells were treated with DDP (0 µg/mL or 3 µg/mL). Then, the autophagy activator rapamycin (200 nm) was applied to the A549/DDP cells. Moreover, hsa_circ_0096157 and Nrf2 were knocked down, and Nrf2 was overexpressed in A549/DDP cells. The expression of Hsa_circ_0096157, the Nrf2/ARE pathway-related factors Nrf2, HO-1, and NQO1, and the autophagy-related factors LC3, Beclin-1, and p62 was evaluated by qRT‒PCR or western blotting. Autophagosomes were detected through TEM. An MTS assay was utilized to measure cell proliferation. The associated miRNA levels were also tested by qRT‒PCR. RESULTS: DDP (3 µg/mL) promoted hsa_circ_0096157, LC3 II/I, and Beclin-1 expression and decreased p62 expression. Knocking down hsa_circ_0096157 resulted in the downregulation of LC3 II/I and Beclin-1 expression, upregulation of p62 expression, and decreased proliferation. Rapamycin reversed the effect of interfering with hsa_circ_0096157. Keap1 expression was lower, and Nrf2, HO-1, and NQO1 expression was greater in the A549/DDP group than in the A549 group. HO-1 expression was repressed after Nrf2 interference. In addition, activation of the Nrf2/ARE pathway promoted autophagy in A549/DDP cells. Moreover, hsa_circ_0096157 activated the Nrf2/ARE pathway. The silencing of hsa_circ_0096157 reduced Nrf2 expression by releasing miR-142-5p or miR-548n. Finally, we found that hsa_circ_0096157 promoted A549/DDP cell autophagy by activating the Nrf2/ARE pathway. CONCLUSION: Knockdown of hsa_circ_0096157 inhibits autophagy and DDP resistance in NSCLC cells by downregulating the Nrf2/ARE signaling pathway.

Metabolic reprogramming-driven homologous recombination and TCA cycle dysregulation contribute to poor prognoses in lung adenocarcinoma.

Increasing evidence has shown that homologous recombination (HR) and metabolic reprogramming are essential for cellular homeostasis. These two processes are independent as well as closely intertwined. Nevertheless, they have rarely been reported in lung adenocarcinoma (LUAD). We analysed the genomic, immune microenvironment and metabolic microenvironment features under different HR activity states. Using cell cycle, EDU and cell invasion assays, we determined the impacts of si-SHFM1 on the LUAD cell cycle, proliferation and invasion. The levels of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH) were determined by ELISA in the NC and si-SHFM1 groups of A549 cells. Finally, cell samples were used to extract metabolites for HPIC-MS/MS to analyse central carbon metabolism. We found that high HR activity was associated with a poor prognosis in LUAD, and HR was an independent prognostic factor for TCGA-LUAD patients. Moreover, LUAD samples with a high HR activity presented low immune infiltration levels, a high degree of genomic instability, a good response status to immune checkpoint blockade therapy and a high degree of drug sensitivity. The si-SHFM1 group presented a significantly higher proportion of cells in the G0/G1 phase, lower levels of DNA replication, and significantly lower levels of cell migration and both TCA enzymes. Our current results indicated that there is a strong correlation between HR and the TCA cycle in LUAD. The TCA cycle can promote SHFM1-mediated HR in LUAD, raising their activities, which can finally result in a poor prognosis and impair immunotherapeutic efficacy.

Dysfunctional circadian clock accelerates cancer metastasis by intestinal microbiota triggering accumulation of myeloid-derived suppressor cells.

Circadian homeostasis in mammals is a key intrinsic mechanism for responding to the external environment. However, the interplay between circadian rhythms and the tumor microenvironment (TME) and its influence on metastasis are still unclear. Here, in patients with colorectal cancer (CRC), disturbances of circadian rhythm and the accumulation of monocytes and granulocytes were closely related to metastasis. Moreover, dysregulation of circadian rhythm promoted lung metastasis of CRC by inducing the accumulation of myeloid-derived suppressor cells (MDSCs) and dysfunctional CD8+ T cells in the lungs of mice. Also, gut microbiota and its derived metabolite taurocholic acid (TCA) contributed to lung metastasis of CRC by triggering the accumulation of MDSCs in mice. Mechanistically, TCA promoted glycolysis of MDSCs epigenetically by enhancing mono-methylation of H3K4 of target genes and inhibited CHIP-mediated ubiquitination of PDL1. Our study links the biological clock with MDSCs in the TME through gut microbiota/metabolites in controlling the metastatic spread of CRC, uncovering a systemic mechanism for cancer metastasis.

Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer.

BACKGROUND: Studies have shown that oxidative stress and its resistance plays important roles in the process of tumor metastasis, and mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage is an important molecular event in oxidative stress. In lung cancer, the normal fibroblasts (NFs) are activated as cancer-associated fibroblasts (CAFs), and act in the realms of the tumor microenvironment (TME) with consequences for tumor growth and metastasis. However, its activation mechanism and whether it participates in tumor metastasis through antioxidative stress remain unclear. METHODS: The role and signaling pathways of tumor cell derived extracellular vesicles (EVs) activating NFs and the characteristic of induced CAFs (iCAFs) were measured by the transmission electron microscopy, nanoparticle tracking analysis, immunofluorescence, collagen contraction assay, quantitative PCR, immunoblotting, luciferase reporter assay and mitochondrial membrane potential detection. Mitochondrial genome and single nucleotide polymorphism sequencing were used to investigate the transport of mtDNA from iCAFs to ρ0 cells, which were tumor cells with mitochondrial dysfunction caused by depletion of mtDNA. Further, the effects of iCAFs on mitochondrial function, growth and metastasis of tumor cells were analysed in co-culture models both in vitro and in vivo, using succinate dehydrogenase, glutathione and oxygen consumption rate measurements, CCK-8 assay, transwell assay, xenotransplantation and metastasis experiments as well as in situ hybridization and immunohistochemistry. RESULTS: Our findings revealed that EVs derived from high-metastatic lung cancer cells packaged miR-1290 that directly targets MT1G, leading to activation of AKT signaling in NFs and inducing NFs conversion to CAFs. The iCAFs exhibit higher levels of autophagy and mitophagy and more mtDNA release, and reactive oxygen species (ROS) could further promote this process. After cocultured with the conditioned medium (CM) of iCAFs, the ρ0 cells may restore its mitochondrial function by acquisition of mtDNA from CAFs, and further promotes tumor metastasis. CONCLUSIONS: These results elucidate a novel mechanism that CAFs activated by tumor-derived EVs can promote metastasis by transferring mtDNA and restoring mitochondrial function of tumor cells which result in resistance of oxidative stress, and provide a new therapeutic target for lung cancer metastasis.

Heterogeneity in PD-L1 expression between primary and metastatic lymph nodes: a predictor of EGFR-TKI therapy response in non-small cell lung cancer.

BACKGROUND: There is inconclusive evidence to suggest that the expression of programmed cell death ligand 1 (PD-L1) is a putative predictor of response to EGFR-TKI therapy in advanced EGFR-mutant non-small cell lung cancer (NSCLC). We evaluated the heterogeneity in PD-L1 expression in the primary lung site and metastatic lymph nodes to analyze the association between PD-L1 expression and response for patients treated with EGFR-TKI. METHODS: This study reviewed 184 advanced NSCLC patients with EGFR mutations who received first-generation EGFR-TKI as first-line treatment from 2020 to 2021 at Shanghai Chest Hospital. The patients were divided into the primary lung site group (n = 100) and the metastatic lymph nodes group (n = 84) according to the biopsy site. The patients in each group were divided into TPS < 1%, TPS 1-49%, and TPS ≥ 50% groups according to PD-L1 expression. RESULTS: The median PFS was 7 (95% CI: 5.7-8.3) months, and the median OS was 26 (95% CI: 23.5-28.5) months for all patients. No correlation existed between PFS or OS and PD-L1 expression. The median PFS in the primary lung site group was 11 months (95% CI: 9.6-12.4) in the TPS < 1% group, 8 months (95% CI: 6.6-9.4) in TPS 1-49% group, and 4 months (95% CI: 3.2-4.8) in TPS ≥ 50% group, with statistically significant differences (p = 0.000). The median OS of the TPS < 1% group and TPS ≥ 50% group showed a statistically significant difference (p = 0.008) in the primary lung site group. In contrast, PD-L1 expression in the lymph nodes of EGFR-mutant patients was unrelated to PFS or OS after EGFR-TKI therapy. CONCLUSION: PD-L1 expression from the primary lung site might predict clinical benefit from EGFR-TKI, whereas PD-L1 from metastatic lymph nodes did not. TRIAL REGISTRATION: This retrospective study was approved by the Ethics Committee of Shanghai Chest Hospital (ID: IS23060) and performed following the Helsinki Declaration of 1964 (revised 2008).

PURPL Promotes M2 Macrophage Polarization in Lung Cancer by Regulating RBM4/xCT Signaling.

Lung cancer is the most common malignancy worldwide. Long non-coding RNA (lncRNA) p53 upregulated regulator of P53 levels (PURPL) is abnormally in various cancers. However, the reports on its roles in lung cancer are limited. The purpose of present study is to investigate the potentials of lncRNA PURPL in lung cancer. PURPL and mRNA expression was determined using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). The location of PURPL was detected using RNA fluorescence in situ hybridization (FISH) assay. Protein expression was detected using western blot. Cellular functions were determined using flow cytometry. The interaction between PURPL and RNA-binding motif 4 (RBM4) was confirmed using RNA immunoprecipitation (RIP) assay. PURPL was overexpressed in lung cancer cells and patients. Overexpressed PURPL promoted M2 macrophage polarization and suppressed ferroptosis. Additionally, PURPL maintained the mRNA stability of cystine glutamate reverse transporter (xCT) via regulating RBM4. xCT knockdown antagonized the effects of overexpressed PURPL and inhibited M2 macrophage polarization via inducing macrophage ferroptosis. PURPL/RBM4/xCT axis promoted M2 macrophage polarization in lung cancer. Therefore, PURPL may be a potential target of lung cancer.

Oridonin represses lung cancer cell proliferation and migration by modulating AFAP1-AS1/IGF2BP1.

Oridonin belongs to a small molecule from the Chinese herb Rabdosia rubescens with potent anticancer activity. In spite of the lncRNA AFAP1-AS1 has been proven to exert promoting function in lung cancer, its relationship with oridonin in lung cancer is obscure. Therefore, our study planned to explore the potential of oridonin in lung cancer as well as unveil the regulatory mechanism of oridonin on AFAP1-AS1 in lung cancer cells. In the present study, oridonin inhibited lung cancer cell proliferation, migration, as well as invasion, as evidenced by MTT, wound healing, as well as transwell assays. Besides, we observed that oridonin could downregulate AFAP1-AS1 expression, and overexpressed AFAP1-AS1 could reverse the repressive effects of oridonin on lung cancer cell proliferation, migration, as well as invasion. More importantly, we found that AFAP1-AS1 could bind to IGF2BP1 through starBase prediction and RIP assay. The expression level of IGF2BP1 was also reduced by oridonin treatment but reversed after AFAP1-AS1 overexpression. Additionally, we proved that overexpressed IGF2BP1 could reverse the repressive impacts of oridonin on lung cancer cell proliferation, migration, as well as invasion. Further, in vivo experiments validated the repressive role of oridonin on tumor growth of lung cancer. Together, oridonin inhibits lung cancer cell proliferation as well as migration by modulating AFAP1-AS1/IGF2BP1, and AFAP1-AS1/IGF2BP1 possesses the potential to be a promising therapy targeting for lung cancer, especially in oridonin treatment.

LINC01133 contributes to the malignant phenotypes of non-small cell lung cancer by targeting miR-30b-5p/FOXA1 pathway.

This study aimed to explore the mechanism of action of LINC01133 in non-small cell lung cancer. LINC01133 expression in NSCLC patient tissues and cells was detected by qRT-PCR. After transfecting siRNA-LINC01133 in NSCLC cells, the proliferation and invasive migration ability of the cells were assessed via CCK-8 and Transwell assay, respectively. The sublocalization of LINC01133 in NSCLC cells was analyzed by bioinformatics prediction and nucleoplasm separation assay and RNA-FISH assay. Analysis of the binding relationship between LINC01133, FOXA1 and miR-30b-5p was all through bioinformatics website analysis, dual-luciferase reporter and RNA Pulldown assay. Functional rescue experiments confirmed the character of miR-30b-5p and FOXA1 in LINC01133 regulating the NSCLC cells biological behavior. LINC01133 high expressions were found in NSCLC tissues and cells. siRNA-LINC01133 treatment inhibited NSCLC cells malignant behavior. Mechanistically: LINC01133 promoted FOXA1 expression through adsorption binding of miR-30b-5p. Knocking down miR-30b-5p expression or up-regulating FOXA1 expression was able to reverse siRNA-LINC01133 inhibitory effect of tumor cell malignant behavior. LINC01133 promoted FOX1 expression by competitively binding miR-30b-5p, which attenuated the targeting inhibitory effect of miR-30b-5p on FOXA1 and ultimately promoted proliferation and invasive migration of NSCLC cells.

LINC00472 suppresses non-small cell lung cancer progression via regulating miR-23a-3p/CCL22 axis.

Long non-coding RNA (lncRNA) LINC00472 has a close connection with the development of tumors. The aim was to explore the role of LINC00472 on NSCLC cell biological function in vivo and its potential mechanisms. The mRNA levels of LncRNA 00472 and microRNA-23a-3p, were determined by RT-qPCR. Cell Counting Kit-8, cell scratches and western blot assays were used to analyze the proliferation, migration and level of apoptosis-associated proteins. Luciferase reporter assay validates the binding between LINC00472/CCL22 and miR-23a-3p. LINC00472 and CCL22 were lowly expressed in NSCLC tissues and cells, while miR-23a-3p expression was upregulated. LINC00472 overexpression significantly depressed NSCLC cell cellular behavior, whereas promoting cell death. MiR-23a-3p could reverse these above-mentioned biological behavior changes caused by LINC00472 overexpression. Additionally, LINC00472 increased CCL22 expression through sponging miR-23a-3p. Knocking down CCL22 antagonized the inhibitory effect of LINC00472 on NSCLC cell survival. LINC00472 may reduce the cellular growth, and accelerate death of NSCLC through increasing CCL22 expression by targeting miR-23a-3p.

Jianpi-Tiaoqi decoction inhibits tumour proliferation and lung metastasis in tumour-bearing mice with triple-negative breast cancer.

Traditional Chinese medicine, specifically the Jianpi Tiaoqi (JPTQ) decoction, has been explored for its role in treating breast cancer, particularly in inhibiting lung metastasis in affected mice. Our study evaluated the effects of JPTQ on several factors, including tumour growth, apoptosis, angiogenesis, epithelial-to-mesenchymal transition (EMT) and immune microenvironment regulation. We used bioluminescence imaging to observe in situ tumour growth and potential lung metastasis. Transcriptomic analysis provided insights into gene expression, whereas flow cytometry was used to examine changes in specific immune cells, such as CD4+ T cells and myeloid-derived suppressor cells. Several essential proteins and genes, including vascular endothelial growth factor (VEGF), matrix metalloprotein-9 (MMP-9) and B-cell lymphoma 2 (Bcl-2), were assessed through quantitative real-time polymerase chain reaction, western blotting and immunohistochemistry. Our findings showed that JPTQ treatment inhibited tumour proliferation in cancer-bearing mice. Bioluminescence imaging and pathological analysis indicated a reduction in lung metastasis. Transcriptome analysis of lung and tumour tissues indicated that the genes associated with EMT, angiogenesis, proliferation and apoptosis were regulated in the JPTQ-treated group. Kyoto Encyclopedia of Genes and Genomes analysis suggested enrichment of immune-related pathways. Flow cytometry indicated that JPTQ treatment reduced the proportion of monocyte-myeloid-derived suppressor cells in the lung and increased the number of CD4+ T cells in the peripheral blood and the number of T helper 1 (Th1) cells in the spleen (P < 0.05). E-cadherin and cleaved caspase 3 were upregulated, whereas Snail, Bcl-2, Ki67 and VEGF were downregulated in the lung and tumour tissues; moreover, the expression of MMP-9 was downregulated in the lung tissue (P < 0.05). In essence, JPTQ not only inhibits tumour growth in affected mice, but also promotes positive immune responses, reduces angiogenesis, boosts tumour cell apoptosis, reverses EMT and decreases breast cancer lung metastasis.

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

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.

POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway.

AIM: The most common type of cancer that leads to death worldwide is lung cancer. Despite significant surgery and chemotherapy improvements, lung cancer patient's survival rate is still poor. The RNA polymerase I subunit D (POLR1D) gene can induce various cancers. A current study reported that POLR1D plays a vital role in cancer prognosis. However, its biological function in the development of lung cancer remains unclear. METHODS: Reverse transcription PCR (RT-PCR) measured the relative POLR1D protein expression level in lung cancer cell lines. Lung cancer cell proliferation, migration, and invasion were analyzed by performing cell counting kit-8 (CCK-8), and transwell. The phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/AKT) signaling pathway-related protein expressions were examined by Western blotting assay. RESULTS: POLR1D protein expression was elevated in lung cancer. Lung cancer cell loss-of-function tests showed that POLR1D silencing could attenuate cell viability both in SK-MES-1 and in H2170 cells. Furthermore, silencing POLR1D inhibited SK-MES-1 and H2170 cells proliferation, migration, and invasion. Moreover, SK-MES-1 and H2170 cells' migration and invasion capacity were potentially suppressed by the knockdown of POLR1D. The progression of multiple cancers has been implicated in the PI3K/AKT pathway. Here, we observed that POLR1D silencing suppressed lung cancer progression by inhibition of the PI3K-Akt pathway. CONCLUSIONS: The study speculated that POLR1D might provide a new potential therapeutic possibility for treating lung cancer patients via targeting PI3K/AKT.

Knockdown of HE4 suppresses tumor growth and invasiveness in lung adenocarcinoma through regulation of EGFR signaling.

It has been shown that the high expression of human epididymis protein 4 (HE4) in most lung cancers is related to the poor prognosis of patients, but the mechanism of pathological transformation of HE4 in lung cancer is still unclear. The current study is expected to clarify the function and mechanism of HE4 in the occurrence and metastasis of lung adenocarcinoma (LUAD). Immunoblotting evaluated HE4 expression in lung cancer cell lines and biopsies, and through analysis of The Cancer Genome Atlas (TCGA) dataset. Frequent HE4 overexpression was demonstrated in LUAD, but not in lung squamous cell carcinoma (LUSC), indicating that HE4 can serve as a biomarker to distinguish between LUAD and LUSC. HE4 knockdown significantly inhibited cell growth, colony formation, wound healing, and invasion, and blocked the G1-phase of the cell cycle in LUAD cell lines through inactivation of the EGFR signaling downstream including PI3K/AKT/mTOR and RAF/MAPK pathways. The first-line EGFR inhibitor gefitinib and HE4 shRNA had no synergistic inhibitory effect on the growth of lung adenocarcinoma cells, while the third-line EGFR inhibitor osimertinib showed additive anti-proliferative effects. Moreover, we provided evidence that HE4 regulated EGFR expression by transcription regulation and protein interaction in LUAD. Our findings suggest that HE4 positively modulates the EGFR signaling pathway to promote growth and invasiveness in LUAD and highlight that targeting HE4 could be a novel strategy for LUAD treatment.

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

BACKGROUND: Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. METHODS: We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. RESULTS: Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. CONCLUSIONS: Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

PPIA dictates NRF2 stability to promote lung cancer progression.

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.

MNT inhibits lung adenocarcinoma ferroptosis and chemosensitivity by suppressing SAT1.

Ferroptosis, a type of iron-dependent non-apoptotic cell death, plays a vital role in both tumor proliferation and resistance to chemotherapy. Here, our study demonstrates that MAX's Next Tango (MNT), by involving itself in the spermidine/spermine N1-acetyltransferase 1 (SAT1)-related ferroptosis pathway, promotes the proliferation of lung adenocarcinoma (LUAD) cells and diminishes their sensitivity to chemotherapy. Initially, an RNA-sequence screen of LUAD cells treated with ferroptosis inducers (FINs) reveals a significant increase in MNT expression, suggesting a potential link between MNT and ferroptosis. Overexpression of MNT in LUAD cells hinders changes associated with ferroptosis. Moreover, the upregulation of MNT promotes cell proliferation and suppresses chemotherapy sensitivity, while the knockdown of MNT has the opposite effect. Through the intersection of ChIP-Seq and ferroptosis-associated gene sets, and validation by qPCR and western blot, SAT1 is identified as a potential target of MNT. Subsequently, we demonstrate that MNT binds to the promoter sequence of SAT1 and suppresses its transcription by ChIP-qPCR and dual luciferase assays. Restoration of SAT1 levels antagonizes the efficacy of MNT to inhibit ferroptosis and chemosensitivity and promote cell growth in vitro as well as in vivo. In the clinical context, MNT expression is elevated in LUAD and is inversely connected with SAT1 expression. High MNT expression is also associated with poor patient survival. Our research reveals that MNT inhibits ferroptosis, and impairing chemotherapy effectiveness of LUAD.

GLUT1 promotes cell proliferation via binds and stabilizes phosphorylated EGFR in lung adenocarcinoma.

BACKGROUND: While previous studies have primarily focused on Glucose transporter type 1 (GLUT1) related glucose metabolism signaling, we aim to discover if GLUT1 promotes tumor progression through a non-metabolic pathway. METHODS: The RNA-seq and microarray data were comprehensively analyzed to evaluate the significance of GLUT1 expression in lung adenocarcinoma (LUAD). The cell proliferation, colony formation, invasion, and migration were used to test GLUT1 's oncogenic function. Co-immunoprecipitation and mass spectrum (MS) were used to uncover potential GLUT1 interacting proteins. RNA-seq, DIA-MS, western blot, and qRT-PCR to probe the change of gene and cell signaling pathways. RESULTS: We found that GLUT1 is highly expressed in LUAD, and higher expression is related to poor patient survival. GLUT1 knockdown caused a decrease in cell proliferation, colony formation, migration, invasion, and induced apoptosis in LUAD cells. Mechanistically, GLUT1 directly interacted with phosphor-epidermal growth factor receptor (p-EGFR) and prevented EGFR protein degradation via ubiquitin-mediated proteolysis. The GLUT1 inhibitor WZB117 can increase the sensitivity of LUAD cells to EGFR-tyrosine kinase inhibitors (TKIs) Gefitinib. CONCLUSIONS: GLUT1 expression is higher in LUAD and plays an oncogenic role in lung cancer progression. Combining GLUT1 inhibitors and EGFR-TKIs could be a potential therapeutic option for LUAD treatment.