Unraveling the mechanisms of RECQL4-mediated cervical cancer progression through the PI3K/AKT pathway.

BACKGROUND: RECQL4 is a member of the DNA helicase family and is critical for DNA replication, DNA damage repair, and tumor progression. However, its specific role in cervical cancer remains uncertain. METHODS: In this study, we aimed to investigate the impact of RECQL4 on cervical cancer prognosis using clinical specimens from The Cancer Genome Atlas. We evaluated the malignant effects of RECQL4 through various experimental assays including cell Cell Counting Kit-8, EdU, colony formation, cell cycle analysis, cell apoptosis, scratch, and Transwell assays. We explored the mechanisms of RECQL4-regulated malignancy using analyses of bioinformatics, RNA sequencing data, polymerase chain reaction (PCR), western blotting, and cell immunofluorescence experiments. Furthermore, we validated the effects of RECQL4 knockdown on tumor growth using subcutaneous tumor models in nude mice. RESULTS: RECQL4 was upregulated in cervical cancer and correlated with prognosis, demonstrating a positive relationship with tumor mutational burden. Knockdown of RECQL4 inhibits cervical cancer cell proliferation, migration, and invasion, suppresses epithelial-mesenchymal transition status, induces cell cycle arrest, and promotes apoptosis. Mechanistically, RECQL4 mediated malignancy through the PI3K/AKT pathway and reduced nuclear ß-catenin expression. In vivo studies further confirmed that RECQL4 knockout significantly inhibited tumor growth. CONCLUSIONS: Our findings provide novel insights into the mechanism behind RECQL4-mediated cervical cancer progression through the PI3K/AKT pathway. Furthermore, our study suggests potential therapeutic strategies for targeting RECQL4 in cervical cancer treatment.

LILRB2 inhibition enhances radiation sensitivity in non-small cell lung cancer by attenuating radiation-induced senescence.

Radiotherapy (RT) in non-small cell lung cancer (NSCLC) triggers cellular senescence, complicating tumor microenvironments and affecting treatment outcomes. This study examines the role of lymphocyte immunoglobulin-like receptor B2 (LILRB2) in modulating RT-induced senescence and radiosensitivity in NSCLC. Through methodologies including irradiation, lentivirus transfection, and various molecular assays, we assessed LILRB2's expression and its impact on cellular senescence levels and tumor cell behaviors. Our findings reveal that RT upregulates LILRB2, facilitating senescence and a senescence-associated secretory phenotype (SASP), which in turn enhances tumor proliferation and resistance to radiation. Importantly, LILRB2 silencing attenuates these effects by inhibiting the JAK2/STAT3 pathway, significantly increasing radiosensitivity in NSCLC models. Clinical data correlate high LILRB2 expression with reduced RT response and poorer prognosis, suggesting LILRB2's pivotal role in RT-induced senescence and its potential as a therapeutic target to improve NSCLC radiosensitivity.

ILT4 facilitates angiogenesis in non-small cell lung cancer.

Antiangiogenic therapy targeting VEGF-A has become the standard of first-line therapy for non-small cell lung cancer (NSCLC). However, its clinical response rate is still less than 50%, and most patients eventually develop resistance, even when using combination therapy with chemotherapy. The major cause of resistance is the activation of complex bypass signals that induce angiogenesis and tumor progression. Therefore, exploring novel proangiogenic mechanisms and developing promising targets for combination therapy are crucial for improving the efficacy of antiangiogenic therapy. Immunoglobulin-like transcript (ILT) 4 is a classic immunosuppressive molecule that inhibits myeloid cell activation. Recent studies have shown that tumor cell-derived ILT4 drives tumor progression via the induction of malignant biologies and creation of an immunosuppressive microenvironment. However, whether and how ILT4 participates in NSCLC angiogenesis remain elusive. Herein, we found that enriched ILT4 in NSCLC is positively correlated with high microvessel density, advanced disease, and poor overall survival. Tumor cell-derived ILT4 induced angiogenesis both in vitro and in vivo and tumor progression and metastasis in vivo. Mechanistically, ILT4 was upregulated by its ligand angiopoietin-like protein 2 (ANGPTL2). Their interaction subsequently activated the ERK1/2 signaling pathway to increase the secretion of the proangiogenic factors VEGF-A and MMP-9, which are responsible for NSCLC angiogenesis. Our study explored a novel mechanism for ILT4-induced tumor progression and provided a potential target for antiangiogenic therapy in NSCLC.

Inhibition of tumor intrinsic BANF1 activates antitumor immune responses via cGAS-STING and enhances the efficacy of PD-1 blockade.

BACKGROUND: BANF1 is well known as a natural opponent of cyclic GMP-AMP synthase (cGAS) activity on genomic self-DNA. However, the roles of BANF1 in tumor immunity remain unclear. Here, we investigate the possible impact of BANF1 on antitumor immunity and response to immunotherapy. METHODS: The Cancer Genome Atlas public data were analyzed to evaluate the relevance of the expression of BANF1, patients' survival and immune cell infiltration. We monitored tumor growth and explored the antitumor efficacy of targeting tumor-intrinsic BANF1 in combination with anti-programmed cell death protein-1 (PD-1) in MC38 or B16F10 tumor models in both immunocompetent and immunodeficient mice. Flow cytometry, immunofluorescence and T cells depletion experiments were used to validate the role of BANF1 in tumor immune microenvironment reprogramming. RNA sequencing was then used to interrogate the mechanisms how BANF1 regulated antitumor immunity. RESULTS: We show that upregulated expression of BANF1 in tumor tissues is significantly associated with poor survival and is negatively correlated with immune cell infiltration. Deficiency of BANF1 in tumor cells markedly antagonizes tumor growth in immunocompetent but not immunocompromised mice, and enhances the response to immunotherapy in murine models of melanoma and colon cancer. In the immunotherapy clinical cohort, patients with high BANF1 expression had a worse prognosis. Mechanistically, BANF1 knockout activates antitumor immune responses mediated by cGAS-synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in an immune-activating tumor microenvironment including increased CD8+ T cell infiltration and decreased myeloid-derived suppressor cell enrichment. CONCLUSIONS: BANF1 is a key regulator of antitumor immunity mediated by cGAS-STING pathway. Therefore, our study provides a rational that targeting BANF1 is a potent strategy for enhancing immunotherapy for cancer with BANF1 upregulation.

ILT4 reprograms glucose metabolism to promote tumor progression in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive and poorly treated subtype of breast cancer. Identifying novel drivers and mechanisms for tumor progression is essential for precise targeted therapy of TNBC. Immunoglobulin-like transcript 4 (ILT4; also known as LILRB2) is a classic myeloid suppressor for their activation and immune response. Our recent results found that ILT4 is also highly expressed in lung cancer cells, where it has a role in promoting immune evasion and thus tumor formation. However, the expression and function of ILT4 in breast cancer remains elusive. Here, using our patient cohort and public database analysis, we found that TNBC displayed the most abundant ILT4 expression among all breast cancer subtypes. Functionally, enriched ILT4 promoted TNBC cell proliferation, migration and invasion in vitro, as well as tumor growth and metastasis in vivo. Further mechanistic analysis revealed that ILT4 reprogrammed aerobic glycolysis of tumor cells via AKT-mTOR signaling-mediated glucose transporter 3 (GLUT3; also known as SLC2A3) and pyruvate kinase muscle 2 (PKM2, an isoform encoded by PKM) overexpression. ILT4 inhibition in TNBC reduced tumor progression and GLUT3 and PKM2 expression in vivo. Our study identified a novel driver for TNBC progression and proposed a promising strategy to combat TNBC by targeting ILT4.

Inhibition of CDC20 potentiates anti-tumor immunity through facilitating GSDME-mediated pyroptosis in prostate cancer.

BACKGROUND: Increasing evidence suggests that immunotherapy, especially immune checkpoint inhibitors (ICIs), has the potential to facilitate long-term survival in various cancer besides prostate cancer. Emerging evidence indicated that pyroptosis, an immunogenic form of cell death, could trigger an anti-tumor immune microenvironment and enhance the effectiveness of immunotherapy. Nevertheless, the mechanism underlying the regulation of pyroptosis signaling in prostate cancer remains unclear. METHODS: The differential expression of human E3 ligases in prostate cancer was integratedly analyzed from five independent public datasets. Moreover, the immunohistochemistry analysis of a tissue microarray derived from prostate cancer patients confirmed the results from the bioinformatic analysis. Furthermore, prostate cancer cell lines were evaluated via the next-generation RNA sequencing to assess transcriptomic profile upon CDC20 depletion. Next, qRT-PCR, Western blotting, cycloheximide assay, immunoprecipitation, and ubiquitination assay were employed to explore the correlation and interaction between CDC20 and GSDME. Both immune-deficient and immune-competent murine models were utilized to examine the anti-tumor efficacy of CDC20 inhibition with or without the anti-PD1 antibodies, respectively. To analyze the immune microenvironment of the xenografts, the tumor tissues were examined by immunohistochemistry and flow cytometry. RESULTS: The analysis of multiple prostate cancer cohorts suggested that CDC20 was the most significantly over-expressed E3 ligase. In addition, CDC20 exerted a negative regulatory effect on the pyroptosis pathway by targeting GSDME for ubiquitination-mediated proteolysis in a degron-dependent manner. Knockdown of CDC20 leads to increased GSDME abundance and a transition from apoptosis to pyroptosis in response to death signals. Furthermore, in our syngeneic murine models, we found that depletion of CDC20 significantly enhances the anti-tumor immunity by promoting the infiltration of CD8+ T lymphocytes dependent on the existence of GSDME, as well as reducing myeloid immune cells. More importantly, Apcin, a small molecular inhibitor that targets CDC20, exhibited synergistic effects with anti-PD1-based immunotherapy in murine models of prostate cancer. CONCLUSIONS: Overall, these findings provide new insights into the upstream regulation of GSDME-mediated pyroptosis by CDC20, which specifically interacts with GSDME and facilitates its ubiquitination in a degron-dependent manner. Importantly, our data highlight novel molecular pathways for targeting cellular pyroptosis and enhancing the effectiveness of anti-PD1-based immunotherapy.

[Progress in antitumor activity of diterpenoid alkaloids in plants of Aconitum].

Small-molecule compounds with rich sources have diverse structures and activities. The active ingredients in traditional Chinese medicine(TCM) provide new sources for the discovery of new antitumor drugs. Aconitum plants as Chinese medicinal plants have the effects of dispelling wind, removing dampness, warming meridian, and relieving pain. They are mainly used to treat inflammation, pain, rheumatism, and tumors, improve heart function, and dilate blood vessels in clinical practice. Diterpenoid alkaloids are the main active components of Aconitum plants, including C20-, C19-, C18-diterpenoid alkaloids and bis-diterpenoid alkaloids. Stu-dies have demonstrated that diterpenoid alkaloids can effectively treat lung cancer, liver cancer, breast cancer, colon cancer and other cancers. Diterpenoid alkaloids are considered as the most promising natural compounds against cancers. In this review, we summarized the chemical structures and antitumor activities of C20-, C19-, C18-diterpenoid alkaloids and bis-diterpenoid alkaloids extracted from plants of Aconitum, aiming to provide reference for further development of diterpenoid alkaloids from Aconitum as antitumor drugs.

Cellular Atlas of Senescent Lineages in Radiation- or Immunotherapy-Induced Lung Injury by Single-Cell RNA-Sequencing Analysis.

PURPOSE: Although the combination of immunotherapy and radiation therapy to treat various malignancies is rapidly expanding, concerns regarding increased pulmonary toxicities remain. The mechanisms of immunotherapy- and irradiation-induced lung injury involve a complex interplay of cell types and signaling pathways, much of which remains to be elucidated. METHODS AND MATERIALS: C57/BL6 mice were treated with a single fraction (20 Gy) of radiation therapy to the right lung or 200 µg anti-Programmed cell death protein 1 antibody twice a week. At 7, 30, and 60 days after treatment, the lung tissues were obtained for unbiased single-cell RNA sequencing or histologic staining. The Seurat analysis pipeline, Cellchat, Monocol, and Single-Cell rEgulatory Network Inference and Clustering were used to define cell types, mechanisms, and mediators driving pathologic remodeling in response to this lung injury. Reverse transcription polymerase chain reaction, immunofluorescent staining, and multiplex immunohistochemistry were applied to validate the key results. RESULTS: Thirty distinct cell subsets encompassing 75,396 cells were identified. A comprehensive investigation of cell-cell crosstalk revealed that monokine signals derived from senescent fibroblasts were substantially elevated after lung injury. Independent analytical strategies revealed that senescence-like subtypes of fibroblasts, alveolar epithelial cells, B cells, and myeloid immune cells were functionally pathologic, with high expression of senescence-signature proteins, especially Apolipoprotein E, during injury response. Senescence markers were also elevated in irradiated human cell lines, mouse cell lines (B3T3 and L929), and the publicly available human pulmonary fibrosis data set. CONCLUSIONS: These findings demonstrate that the accumulation of senescence-like fibroblasts, macrophages, and alveolar epithelial cells is the primary common pathologic mechanism of immunotherapy- and irradiation-induced lung injury. These high-resolution transcriptomic data provide novel insights into therapeutic opportunities to predict or prevent therapy-induced lung injury.

Radiation combined with KRAS-MEK inhibitors enhances anticancer immunity in KRAS-mutated tumor models.

KRAS mutation is a common driver in solid tumors, and KRAS-mutated tumors are relatively resistant to radiotherapy. Therefore, we investigated the combined effect of radiation and KRAS-MEK inhibitors (AMG510 and trametinib) in KRAS-mutated tumors. The expression of programmed death-ligand 1 (PD-L1), major histocompatibility complex (MHC) class I molecules, and cytokines in KRAS-mutated cell lines was assessed using flow cytometry, western blot analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. In vivo, tumor growth, T cell infiltration, and gene sequencing analyses were conducted in 2 murine KRAS-mutated models. Both AMG510 and trametinib decreased the radiation-induced increase in PD-L1 expression. Radiation and trametinib additively induced the expression of CXCL10 and CXCL11 cytokines and MHC class I in murine CT26 and LLC cell lines. The combination of trametinib and radiation controlled tumor growth and induced more infiltration of CD4+ and CD8+ T cells in vivo, wherein tumor inhibition function and the survival period of mice could be reduced by CD8+ and/or CD4+ T cell depletion. The expression levels of immune-related genes also increased in the combination therapy group. Our results indicate that KRAS-MEK inhibitors in combination with radiotherapy can enhance antitumor immunity, providing new therapeutic strategies for KRAS-mutated tumors.

Tumor-derived immunoglobulin like transcript 5 induces suppressive immunocyte infiltration in colorectal cancer.

Infiltration of immunosuppressive cells in the tumor microenvironment (TME) induced colorectal cancer (CRC) progression and its resistance to immunotherapy. Identification of tumor-specific factors to modulate inhibitory immunocyte infiltration would provide alternative and novel targets for CRC immunotherapy. Immunoglobulin-like transcript (ILT) 5 is a negative regulator of myeloid cell activation. However, its expression and functional role in solid tumors is still unknown. Using human CRC tissues and cell lines, we found that ILT5 was highly expressed in CRC cells compared with normal colorectal epithelial cells. Enriched ILT5 in tumor cells was correlated with advanced tumor stages and poor patient survival. Our subsequent in vitro and in vivo studies revealed that tumor-derived ILT5 inhibited the infiltration of T cells, especially that of CD8+ T cells in the TME, creating suppressive T-cell contexture. Furthermore, ILT5 directed M2-like polarization of tumor-associated macrophages (TAMs). Inhibition of tumor-derived ILT5 restored the immunosuppressive T-cell and TAM contexture, and restricted CRC progression. Our findings identified ILT5 expression in solid tumor cells for the first time and raised ILT5 as a potential immunotarget and prognostic predictor in CRC.

Neurturin promotes tumor cell motility and angiogenesis in colorectal cancer.

Neurturin (NRTN) is one of the glial cell line-derived neurotrophic factor family ligands crucial for neuron growth, differentiation and maintenance. Recent studies showed NRTN promotes an aggressive pancreatic cancer phenotype, and predicts shorter survival in lung cancer patients. However, its expression and function in colorectal cancer (CRC) remain unclear. Herein, we found NRTN was enriched in CRC cells, and predicted poor patients outcomes. Upregulated NRTN enhanced the migration and invasion of CRC cells and vascularization of endothelial cells. In mechanism, NRTN promoted ZEB1/N-cadherin and vascular endothelial growth factor (VEGF)-A expression in CRC cells, which were responsible for tumor cell motility and angiogenesis, respectively. More importantly, NRTN inhibition prevented CRC metastasis and angiogenesis in vivo. In conclusion, NRTN promotes CRC cells motility and tumor angiogenesis via inducing ZEB1/N-cadherin and VEGF-A overexpression. It is a potential therapeutic target and negative prognostic biomarker for CRC patients.

ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

Rationale: Immune checkpoint inhibitors (ICIs) against the PD-1/PD-L1 pathway showed limited success in non-small cell lung cancer (NSCLC) patients, especially in those with activating epidermal growth factor receptor (EGFR) mutations. Elucidation of the mechanisms underlying EGFR-mediated tumor immune escape and the development of effective immune therapeutics are urgently needed. Immunoglobulin-like transcript (ILT) 4, a crucial immunosuppressive molecule initially identified in myeloid cells, is enriched in solid tumor cells and promotes the malignant behavior of NSCLC. However, the upstream regulation of ILT4 overexpression and its function in tumor immunity of NSCLC with EGFR activation remains unclear. Methods: ILT4 expression and EGFR phosphorylation in human NSCLC tissues and cell lines were analyzed using immunohistochemistry (IHC), real-time PCR, Western blotting, immunofluorescence, and flow cytometry. The molecular signaling for EGFR-regulated ILT4 expression was investigated using mRNA microarray and The Cancer Genome Atlas (TCGA) database analyses and then confirmed by Western blotting. The regulation of tumor cell proliferation and apoptosis by ILT4 was examined by CCK8 proliferation and apoptosis assays. The impact of ILT4 and PD-L1 on tumor-associated macrophage (TAM) recruitment and polarization was evaluated using Transwell migration assay, flow cytometry, enzyme linked immunosorbent assay (ELISA) and real-time PCR, while their impact on T cell survival and cytotoxicity was analyzed by CFSE proliferation assay, apoptotic assay, flow cytometry, ELISA and cytolytic assay. Tumor immunotherapy models targeting at paired Ig-like receptor B (PIR-B, an ortholog of ILT4 in mouse)/ILT4 and/or PD-L1 were established in C57BL/6 mice inoculated with stable EGFR- overexpressing Lewis lung carcinoma (LLC) cells and in humanized NSG mice inoculated with EGFR mutant, gefitinib-resistant PC9 (PC9-GR) or EGFR-overexpressing wild type H1299 cells. PIR-B and ILT4 inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using human/mouse neutralizing antibodies. The tumor growth model was established in NSG mice injected with PIR-B-downregulated LLC cells to evaluate the effect of PIR-B on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse spleens and blood were detected by flow cytometry while those in tumor tissues were determined by IHC and immunofluorescence. Results: We found that ILT4 expression in tumor cells was positively correlated with EGFR phosphorylation in human NSCLC tissues. Using NSCLC cell lines, we demonstrated that ILT4 was upregulated by both tyrosine kinase mutation-induced and epidermal growth factor (EGF)-dependent EGFR activation and subsequent AKT/ERK1/2 phosphorylation. Overexpressed ILT4 in EGFR-activated tumor cells induced TAM recruitment and M2-like polarization, which impaired T cell function. ILT4 also directly inhibited T cell proliferation, cytotoxicity, and IFN-γ expression and secretion. In EGFR-activated cell lines in vitro and in wild-type EGFR-activated C57BL/6 and humanized NSG immunotherapy models in vivo, either ILT4 (PIR-B) or PD-L1 inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immuno-suppressive TME; the combined inhibition of both molecules showed the most dramatic tumor retraction. Surprisingly, in EGFR mutant, TKI resistant humanized NSG immunotherapy model, ILT4 inhibition alone rather than in combination with a PD-L1 inhibitor suppressed tumor growth and immune evasion. Conclusions: ILT4 was induced by activation of EGFR-AKT and ERK1/2 signaling in NSCLC cells. Overexpressed ILT4 suppressed tumor immunity by recruiting M2-like TAMs and impairing T cell response, while ILT4 inhibition prevented immunosuppression and tumor promotion. Furthermore, ILT4 inhibition enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type but not in EGFR mutant NSCLC. Our study identified novel mechanisms for EGFR-mediated tumor immune escape, and provided promising immunotherapeutic strategies for patients with EGFR-activated NSCLC.

ILT3 promotes tumor cell motility and angiogenesis in non-small cell lung cancer.

Immunoglobulin-like transcript (ILT) 3 is an immunosuppressive molecule that negatively regulates myeloid cell activation. ILT3 overexpression in tumor cells induces immune escape of solid tumors and facilitates invasion of monocytic acute myeloid leukemia cells. However, the expression and function of ILT3 in non-small cell lung cancer (NSCLC) cells remain elusive. Herein, we found that ILT3 was enriched in human NSCLC cells, and predicted advanced disease and poor overall survival. ILT3 overexpression enhanced the migration and invasion of NSCLC cells and tubule formation of human umbilical vein endothelial cells by upregulating and interacting with its ligand apolipoprotein E (ApoE) in vitro. Mechanistically, ILT3 recruited SHP2 and SHIP1, and subsequently activated ERK1/2 signaling mediating epithelial-mesenchymal transition (EMT) and increasing vascular endothelial growth factor (VEGF)-A expression in NSCLC cells, which are responsible for tumor cell motility and angiogenesis, respectively. Using murine metastasis models, we further confirmed ILT3 promoted NSCLC metastasis and explored the exact correlation of ILT3 with ApoE, EMT, and VEGF-A in vivo. These results unraveled novel mechanisms for ILT3-induced tumor progression and proposed ILT3 as a potential therapeutic target and prognostic biomarker for NSCLC patients.

Overexpressed immunoglobulin-like transcript (ILT) 4 in lung adenocarcinoma is correlated with immunosuppressive T cell subset infiltration and poor patient outcomes.

BACKGROUND: The poor response to current PD-1/PD-L1 inhibitors in lung cancer patients requires development of novel immunotargets. Immunoglobulin-like transcript (ILT)4 is an immunosuppressive molecule mainly expressed in myeloid innate cells. Recent studies showed that ILT4 was highly expressed in multiple malignant cells and regulated tumor biologies including proliferation, invasion and metastasis. However, the immunomodulatory role of tumor cell-derived ILT4 is unclear. Here we aimed to analyze the correlation of tumor cell ILT4 expression with T cell infiltration and subset distribution, illustrate ILT4-regulated immunosuppressive microenvironment, and raise tumor cell-derived ILT4 as a novel immunotherapeutic target and prognostic biomarker for lung adenocarcinoma (LUAD) patients. METHODS: We collected the tissue samples and corresponding clinicopathological data from 216 primary LUAD patients. Using immunohistochemical staining and public database analyses we investigated the relationship between ILT4 expression and different T cell subset density as well as patient outcomes. RESULTS: Enriched ILT4 expression in tumor cells of LUAD tissues indicated reduced T cell infiltration in the tumor microenvironment (TME), advanced diseases and poor patient overall survival (OS). Further T cell subset analyses revealed that ILT4 expression was correlated with decreased CD8+T cell and increased Treg frequency in both cancer nest and stroma, but not with altered CD4+T cell frequency. High ILT4 level combined with low CD8+T cell/high Treg density predicted markedly poorer clinical outcomes compared with any of these biomarkers alone. CONCLUSIONS: Tumor cell-derived ILT4 is correlated with immunosuppressive T cell subset infiltration and poor clinical outcomes, and might be a potential immunotherapeutic target and prognostic biomarker for LUAD patients. Combined ILT4 expression and CD8+ T cell/Treg frequency in tumor infiltrating lymphocytes (TILs) are stronger predictors for patient outcomes.

Forkhead box C1 promotes metastasis and invasion of non-small cell lung cancer by binding directly to the lysyl oxidase promoter.

Increasing evidence indicates that human forkhead box C1 (FOXC1) plays important roles in tumor development and metastasis. However, the underlying molecular mechanism of FOXC1 in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, we identified FOXC1 as an independent prognostic factor in NSCLC and showed clear biological implications in invasion and metastasis. FOXC1 overexpression enhanced the proliferation, migration and invasion of NSCLC cells, whereas FOXC1 silencing impaired the effects both in vitro and in vivo. Importantly, we found a positive correlation between FOXC1 expression and lysyl oxidase (LOX) expression in NSCLC cells and patient samples. Downregulation of LOX or LOX activity inhibition in NSCLC cells inhibited the FOXC1-driven effects on cellular migration and invasion. Xenograft models showed that inhibition of LOX activity by ß-aminopropionitrile monofumarate decreased the number of lung metastases. Mechanistically, we demonstrated a novel FOXC1-LOX mechanism that was involved in the invasion and metastasis of NSCLC. Dual-luciferase assay and ChIP identified that FOXC1 bound directly in the LOX promoter region and activated its transcription. Collectively, the present study offered new insight into FOXC1 in the mediation of NSCLC metastasis through interaction with the LOX promoter and further revealed that targeted inhibition of LOX protein activity could prevent lung metastasis in murine xenograft models. These data implicated FOXC1 as a potential therapeutic strategy for the treatment of NSCLC metastasis.

CUGBP1 promotes cell proliferation and suppresses apoptosis via down-regulating C/EBPα in human non-small cell lung cancers.

CUGBP1, which is involved in posttranscriptional regulatory networks, may control cell growth, activation and differentiation. Meanwhile, CCAAT/enhancer-binding protein α (C/EBPα) acts as a basic leucine zipper transcription factor which controls differentiation-dependent gene expression and inhibits cell proliferation. To date, very little is known about the association between CUGBP 1 and C/EBPα in regulating cell proliferation and apoptosis in non-small cell lung cancer (NSCLC). CUGBP1 and C/EBPα mRNA expressions were analyzed in NSCLC tumor and adjacent normal tissues, and the relationship in clinicopathological parameters was evaluated. Knockdown of CUGBP1 and C/EBPα regulated by CUGBP1 in NSCLC cell line was identified by real-time PCR and Western blot. The effect of depletion of CUGBP1 was evaluated by MTT assay and Annexin/Propidium Iodide Apoptosis assay. CUGBP1 is highly expressed and expression of C/EBPα is low in NSCLC tissues. The correlation analysis revealed that there was negative correlation between the expression of CUGBP 1 and C/EBPα. Knockdown of CUGBP1 effectively silenced the expression of CUGBP1 and up-regulated C/EBPα. Also, suppression of CUGBP1 inhibits proliferation and induces apoptosis in A549 cells. These observations suggest that the first proof the overexpression of CUGBP1 in NSCLC contributes to tumorigenesis through down-regulation of C/EBPα. Knockdown of CUGBP1 or up-regulation C/EBPα might be a potential therapeutic approach for human non-small cell lung cancers.

Prediction of lung cancer based on serum biomarkers by gene expression programming methods.

In diagnosis of lung cancer, rapid distinction between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) tumors is very important. Serum markers, including lactate dehydrogenase (LDH), C-reactive protein (CRP), carcino-embryonic antigen (CEA), neurone specific enolase (NSE) and Cyfra21-1, are reported to reflect lung cancer characteristics. In this study classification of lung tumors was made based on biomarkers (measured in 120 NSCLC and 60 SCLC patients) by setting up optimal biomarker joint models with a powerful computerized tool - gene expression programming (GEP). GEP is a learning algorithm that combines the advantages of genetic programming (GP) and genetic algorithms (GA). It specifically focuses on relationships between variables in sets of data and then builds models to explain these relationships, and has been successfully used in formula finding and function mining. As a basis for defining a GEP environment for SCLC and NSCLC prediction, three explicit predictive models were constructed. CEA and NSE are frequently- used lung cancer markers in clinical trials, CRP, LDH and Cyfra21-1 have significant meaning in lung cancer, basis on CEA and NSE we set up three GEP models-GEP 1(CEA, NSE, Cyfra21-1), GEP2 (CEA, NSE, LDH), GEP3 (CEA, NSE, CRP). The best classification result of GEP gained when CEA, NSE and Cyfra21-1 were combined: 128 of 135 subjects in the training set and 40 of 45 subjects in the test set were classified correctly, the accuracy rate is 94.8% in training set; on collection of samples for testing, the accuracy rate is 88.9%. With GEP2, the accuracy was significantly decreased by 1.5% and 6.6% in training set and test set, in GEP3 was 0.82% and 4.45% respectively. Serum Cyfra21-1 is a useful and sensitive serum biomarker in discriminating between NSCLC and SCLC. GEP modeling is a promising and excellent tool in diagnosis of lung cancer.