Promote Non-small Cell Lung Cancer Metastasis Research Articles

CD73 promotes non–small cell lung cancer metastasis by regulating Axl signaling independent of GAS6

As catabolic enzyme, CD73 dephosphorylates adenosine monophosphate (AMP) and can also regulate tumor cell proliferation and metastasis. To date, very few studies have explored the role of CD73 in mediating non-small cell lung cancer (NSCLC) metastasis, and the underlying transducing signal has not been elucidated. In the present study, we demonstrated that the CD73/Axl axis could regulate Smad3-induced epithelial-to-mesenchymal transition (EMT) to promote NSCLC metastasis. Mechanically, CD73 can be secreted via the Golgi apparatus transport pathway. Then secreted CD73 may activate AXl by directly bind with site R55 located in Axl extracellular domain independently of GAS6. In addition, we proved that CD73 can stabilize Axl expression via inhibiting CBLB expression. We also identified the distinct function of CD73 activity in adenocarcinoma and squamous cell carcinoma. Our findings indicated a role of CD73 in mediating NSCLC metastasis and propose it as a therapeutic target for NSCLC.

CD248-expressing cancer-associated fibroblasts induce non-small cell lung cancer metastasis via Hippo pathway-mediated extracellular matrix stiffness.

Metastasis is a crucial stage in tumour progression, and cancer-associated fibroblasts (CAFs) support metastasis through their participation in extracellular matrix (ECM) stiffness. CD248 is a possible biomarker for non-small cell lung cancer (NSCLC)-derived CAFs, but its role in mediating ECM stiffness to promote NSCLC metastasis is unknown. We investigated the significance of CD248+ CAFs in activating the Hippo axis and promoting connective tissue growth factor (CTGF) expression, which affects the stromal collagen I environment and improves ECM stiffness, thereby facilitating NSCLC metastasis. In this study, we found that higher levels of CD248 in CAFs induced the formation of collagen I, which in turn increased extracellular matrix stiffness, thereby enabling NSCLC cell infiltration and migration. Hippo axis activation by CD248+ CAFs induces CTGF expression, which facilitates the formation of the collagen I milieu in the stromal matrix. In a tumour lung metastasis model utilizing fibroblast-specific CD248 gene knockout mice, CD248 gene knockout mice showed a significantly reduced ability to develop tumour lung metastasis compared to that of WT mice. Our findings demonstrate that CD248+ CAFs activate the Hippo pathway, thereby inducing CTGF expression, which in turn facilitates the collagen I milieu of the stromal matrix, which promotes NSCLC metastasis.

RBMS1 Coordinates with the m6A Reader YTHDF1 to Promote NSCLC Metastasis through Stimulating S100P Translation.

Metastasis is the leading cause for the high mortality of lung cancer, however, effective anti-metastatic drugs are still limited. Here it is reported that the RNA-binding protein RBMS1 is positively associated with increased lymph node metastasis in non-small cell lung cancer (NSCLC). Depletion of RBMS1 suppresses cancer cell migration and invasion in vitro and inhibits cancer cell metastasis in vivo. Mechanistically, RBMS1 interacts with YTHDF1 to promote the translation of S100P, thereby accelerating NSCLC cell metastasis. The RRM2 motif of RBMS1 and the YTH domain of YTHDF1 are required for the binding of RBMS1 and YTHDF1. RBMS1 ablation inhibits the translation of S100P and suppresses tumor metastasis. Targeting RBMS1 with NTP, a small molecular chemical inhibitor of RBMS1, attenuates tumor metastasis in a mouse lung metastasis model. Correlation studies in lung cancer patients further validate the clinical relevance of the findings. Collectively, the study provides insight into the molecular mechanism by which RBMS1 promotes NSCLC metastasis and offers a therapeutic strategy for metastatic NSCLC.

SYT7 is a key player in increasing exosome secretion and promoting angiogenesis in non-small-cell lung cancer

Lung cancer is the leading cause of cancer-related mortality, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Our previous study confirmed that synaptotagmin 7 (SYT7) promoted NSCLC metastasis in vivo and in vitro. Studies have shown that SYT7 is an important regulatory molecule of exocytosis in various cells. However, the characteristics of SYT7 across cancers and the function of SYT7 in tumor exosome secretion remain unclear. In this study, we conducted systematic pancancer analyses of SYT7, namely, analyses of expression patterns, diagnostic and prognostic values, genetic alterations, methylation, immune infiltration, and potential biological pathways. Furthermore, we demonstrated that SYT7 increased the secretion of exosomes from A549 and H1299 cells, promoting the migration, proliferation, and tube formation of human umbilical vein endothelial cells (HUVECs). Notably, SYT7 promoted angiogenesis by transferring exosomes containing the molecule centrosomal protein of 55 kDa (CEP55) protein to HUVECs. The CEP55 protein levels was downregulated in STAT1 inhibitor-treating SYT7-overexpresion NSCLC cells. We further found that SYT7 activated the mTOR signaling pathway through the downstream molecule CEP55, thereby promoting the invasion and metastasis of NSCLC cells. SYT7 promoted exosome secretion by NSCLC cells through upregulating syntaxin-1a and syntaxin-3. In vivo, SYT7 promoted the tumorigenesis, angiogenesis and metastasis of A549 cells through the exosome pathway. Our study is of great importance for understanding the mechanism of tumor exosome secretion and the role of exosomes in tumor progression.

BMP2 promotes lung adenocarcinoma metastasis through BMP receptor 2-mediated SMAD1/5 activation

Bone morphogenetic protein 2 (BMP2) is highly overexpressed in human non-small cell lung cancer (NSCLC) and correlates with tumor stage and metastatic burden. Although several lines of evidence suggest that BMP2 promotes cell migration and invasiveness in vitro, the in vivo role of BMP2 in the metastasis of lung adenocarcinoma cells remains less well understood. Here, we revealed that BMP2 is highly overexpressed in lung adenocarcinoma patients with lymph node metastasis compared with patients without lymph node metastasis. Using an in vivo orthotopic mouse model, we clearly demonstrated that BMP2 promotes lung adenocarcinoma metastasis. The depletion of BMP2 or its receptor BMPR2 significantly reduced cell migration and invasiveness. We further identified that BMP2/BMPR2-mediated cell migration involves the activation of the SMAD1/5/8 signaling pathway, independent of the KRAS signaling pathway. Significantly, the depletion of SMAD1/5/8 or the inhibition of SMAD1/5/8 by LDN193189 inhibitor significantly reduced cell migration. These findings show that BMP2 promotes NSCLC metastasis, indicating that targeting the BMP2 signaling pathway may represent a potential therapeutic strategy for treating patients with metastatic NSCLC.

MPZL1 upregulation promotes tumor metastasis and correlates with unfavorable prognosis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancer, is one of the leading causes of cancer-related death worldwide. Previously, we demonstrated that MPZL1 gene amplification promotes liver cancer metastasis through activating Src/Cortactin pathway. However, the clinical relevance and biological roles of the MPZL1 gene in lung cancer are still unknown. Here, we found that MPZL1 expression upregulates in human NSCLC, which is partly due to the copy number amplification of this gene. Next, we observed that high MPZL1 expression correlates with unfavorable prognosis of NSCLC patients. We further demonstrated that ectopic MPZL1 overexpression promotes in vitro migratory but not proliferation and colony formation abilities of both H1299 and H460 cells. Consistently, we found that MPZL1 knockdown impairs the migratory abilities of A549 and H1775 cells. Moreover, we found that MPZL1 knockdown inhibits in vivo metastatic but not tumor growth abilities of the A549 cells. Additionally, a total of 297 differentially expressed genes (DEGs) were identified by RNA sequencing in A549 cells upon MPZL1 knockdown. By integrative analysis of DEGs regulated by MPZL1 in A549 cells and human NSCLC tissues, we revealed that COL11A1 is the potential effector gene that positively regulated by MPZL1 and correlates with poor prognosis of NSCLC patients. In conclusion, our work indicates that one of the mechanisms by which MPZL1 promotes NSCLC metastasis is through upregulating the COL11A1, and MPZL1 can be used as a biomarker to predict the prognosis of NSCLC patients.

Abstract 1581: Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC)

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, which could be attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for developing effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are small extracellular vesicles (sEVs) of approximately 100-150 nm in diameter, often referred as exosomes. Previous report revealed that increased levels of exosomes in NSCLC patients correlated with lower overall survival. Indeed, secretion of sEVs by cancer cells can induce malignant transformation of non-cancerous cells nearby or at distant locations through influencing pre-metastatic niche formation. Therefore, biogenesis and release of cancer derived sEVs are determinant steps for the development of tumor metastasis. However, the mechanisms through which sEVs are produced and secreted by cancer cells are yet to be determined. Here, we isolated and characterized sEVs from a panel of NSCLC cell lines (H358, Calu6, H460, SKMES-1) and non-tumorigenic cells (BEAS-2B and HBEC) to identify mechanisms involved in the elevated secretion of EVs by cancer cells. Contrary to what has been previously reported in literature, our data demonstrated that not all cancer cell lines release high levels of sEVs. Our data suggests that elevated secretion of sEVs could be driven by specific mutations and oncogenic signaling in cancer cells. Among all the cell lines tested, H358 cells which harbor the infamous G12C mutation in KRAS, had the highest sEV release. Therefore, we hypothesize that the KRASG12C can drive high sEV production and secretion in H358 cells. Our preliminary data suggests that siRNA mediated knockdown of oncogenic KRAS signaling in H358 cells significantly decreases secretion of sEVs. Ongoing studies aim to pinpoint the detailed mechanisms by which cancer cells utilize KRAS oncogenic signaling to promote EV biogenesis pathways and tumorigenesis. The current study will provide the foundation to developed new therapeutic strategies against tumor sEVs-driven metastasis. Citation Format: Zulaida M. Soto Vargas, Ikjot Singh Sohal, Humna Hasan, David Arteaga, Andrea L. Kasinski. Uncovering mechanisms of biogenesis and secretion of small extracellular vesicles in non small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1581.

Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer.

Profilin 1 (PFN1), an actin-binding protein, plays contrasting roles in the metastasis of several cancers; however, its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, PFN1 expression was upregulated in metastatic NSCLC tissues. PFN1 overexpression significantly promotes NSCLC metastasis in vitro and in vivo. Proteomics analysis revealed PFN1 involvment in microvesicles (MVs) secretion. In vitro experiments confirmed that PFN1 overexpression increased secretion of MVs. MVs are important mediators of metastasis. Here, we show an increased abundance of MVs in the sera of patients with metastatic NSCLC compared to that in the sera of patients with non-metastatic NSCLC. Both in vitro and in vivo experiments revealed that PFN1 could increase MV secretion, and MVs derived from PFN1-overexpressing cells markedly promoted NSCLC metastasis. We then elucidated the mechanisms underlying PFN1-mediated regulation of MVs and found that PFN1 could interact with ROCK1 and enhance its kinase activity to promote myosin light chain (MLC) phosphorylation for MV secretion. Inhibition of ROCK1 decreased MV secretion and partially reversed the PFN1-induced promotion of NSCLC metastasis. Collectively, these findings show that PFN1 regulates MV secretion to promote NSCLC metastasis. PFN1 and MVs represent potential predictors or therapeutic targets for NSCLC metastasis.

LncRNA OXCT1-AS1 Promotes Metastasis in Non-Small-Cell Lung Cancer by Stabilizing LEF1, In Vitro and In Vivo.

Long noncoding RNAs (lncRNAs) play nonnegligible roles in the metastasis of non-small-cell lung cancer (NSCLC). This study is aimed at investigating the biological role of lncRNA OXCT1-AS1 in NSCLC metastasis and the underlying regulatory mechanisms. The expression profiles of lncRNA OXCT1-AS1 in different NSCLC cell lines were examined. Then, the biological function of lncRNA OXCT1-AS1 in NSCLC metastasis was explored by loss-of-function assays in vitro and in vivo. Further, the protective effect of lncRNA OXCT1-AS1 on lymphoid enhancer factor 1 (LEF1) was examined using RNA pull-down and RNA immunoprecipitation assays. Additionally, the role of LEF1 in NSCLC metastasis was investigated. Results indicated that lncRNA OXCT1-AS1 expression was significantly increased in NSCLC cell lines. Functional analysis revealed that knockdown of lncRNA OXCT1-AS1 impaired invasion and migration in vitro. Additionally, the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis was also confirmed in vivo. Mechanistically, through direct interaction, lncRNA OXCT1-AS1 maintained LEF1 stability by blocking NARF-mediated ubiquitination. Furthermore, LEF1 knockdown impaired invasion and migration of NSCLC in vitro and in vivo. Collectively, these data highlight the ability of lncRNA OXCT1-AS1 to promote NSCLC metastasis by stabilizing LEF1 and suggest that lncRNA OXCT1-AS1 represents a novel therapeutic target in NSCLC.

Tumor-associated macrophages secret exosomal miR-155 and miR-196a-5p to promote metastasis of non-small-cell lung cancer.

BackgroundUnderstanding the molecular basis underlying metastasis of non-small cell lung cancer (NSCLC) may provide a new therapeutic modality for the treatment of NSCLC. However, the mechanisms by which tumor-associated macrophages (TAMs) affect NSCLC metastasis remain undefined. In this study, we aimed to discover a novel regulatory pathway involved in NSCLC metastasis.MethodsCell Counting Kit-8 (CCK-8), Transwell, western blot assays were used to assess cell viability, migration, invasion and epithelial-mesenchymal transition (EMT). Exosomes from macrophages medium were characterized, and in vitro cell coculture was further conducted to investigate M2 derived exosomes mediated crosstalk between TAMs and tumor cells. Besides, miRNA microarray was used to analyze miRNA expression profiles of M0 and M2 derived exosomes. Luciferase reporter assay was used to verify the potential binding between miRNA and mRNA. Moreover, 6-week-old male BALB/c nude mice were performed to establish transplantation tumor model using tail vein injection. Hematoxylin & eosin staining was used to detect the metastasis of tumor tissues.ResultsWe found that M2 TAMs were the main TAMs in metastatic tissues of NSCLC patients and exosomes derived from M2 TAMs were able to promote cell viability, cell migration, cell invasion and EMT in NSCLC. We demonstrated that miR-155 and miR-196a-5p were abundant in M2 TAMs and exosomes secreted by M2 TAMs. Functional experiments demonstrated that the deletion of miR-155 and miR-196a-5p in M2 TAMs significantly prevented NSCLC metastasis in vitro and in vivo. To clarify the mechanism governing miR-155 and miR-196a-5p from M2 TAMs, we carried out bioinformatics analysis to predict potential target genes. Mechanistically, miR-155 and miR-196a-5p directly bound to the 3'-UTR of Ras association domain family member 4 (RASSF4), and negatively regulating RASSF4 expression. At last, rescue assays demonstrated that miR-155 and miR-196a-5p exerted its performance by RASSF4.ConclusionsOverall, we revealed a new regulatory pathway that was M2 TAMs secreted exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis. This dynamic and reciprocal cross-talk between NSCLC and macrophages innovatively provided a potential opportunity for diagnosis and treatment of NSCLC.

Myeloid-derived suppressor cells promote lung cancer metastasis by CCL11 to activate ERK and AKT signaling and induce epithelial-mesenchymal transition in tumor cells.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune activities and facilitate cancer progression. Although the concept of immunosuppressive MDSCs is well established, the mechanism that MDSCs regulate non-small cell lung cancer (NSCLC) progression through the paracrine signals is still lacking. Here, we reported that the infiltration of MDSCs within NSCLC tissues was associated with the progression of cancer status, and was positively correlated with the Patient-derived xenograft model establishment, and poor patient prognosis. Intratumoral MDSCs directly promoted NSCLC metastasis and highly expressed chemokines that promote NSCLC cells invasion, including CCL11. CCL11 was capable of activating the AKT and ERK signaling pathways to promote NSCLC metastasis through the epithelial-mesenchymal transition (EMT) process. Moreover, high expression of CCL11 was associated with a poor prognosis in lung cancer as well as other types of cancer. Our findings underscore that MDSCs produce CCL11 to promote NSCLC metastasis via activation of ERK and AKT signaling and induction of EMT, suggesting that the MDSCs-CCL11-ERK/AKT-EMT axis contains potential targets for NSCLC metastasis treatment.

Stromal-derived miR-486-5p promotes metastasis of non-small-cell lung cancer cells by targeting the CADM1/tight junctions axis in vascular endothelial cells.

Serum microRNA has been demonstrated as a noninvasive predictor for the progression of non-small-cell lung cancer (NSCLC). The role of microRNA-486-5p (miR-486-5p) in NSCLC seems to be paradoxical. On the one hand, elevated expression of miR-486-5p in serum is associated with unfavorable survival; on the other hand, miR-486-5p was notably reduced in NSCLC tissues and acted as a tumor-suppressor to inhibit NSCLC metastasis. The expression of miR-486-5p was analyzed in serum and tissue samples and their relationship was explored. The miR-486-5p-expressing cells were isolated by fluorescent-activated cell sorting. The downstream target of miR-486-5p was identified by bioinformatics prediction and experimental confirmation. Functional studies of miR-486-5p on NSCLC metastasis were determined by endothelial permeability assay and trans-endothelial invasion assay. We found that the expression of miR-486-5p was remarkably increased in serum, while dramatically downregulated in tumor tissues of NSCLC. However, the level of miR-486-5p in serum was positively correlated with that in tumor tissues. Next, we identified CD31+ vascular endothelial cells in the lung stroma as miR-486-5p-expressing cells. According to bioinformatics prediction, quantitative real-time reverse transcription PCR, luciferase reporter assay, and western blot, miR-486-5p directly targeted the cell adhesion molecule 1/tight junctionsaxis in vascular endothelial cells. In addition, endothelial permeability assay and trans-endothelial invasion assay confirmed that miR-486-5p promoted NSCLC metastasis. Highly elevated expression of miR-486-5p in CD31+ vascular endothelial cells increased vascular permeability and promoted NSCLC metastasis. In conclusion, stromal-derived miR-486-5p is responsible for the paradoxical effect of miR-486-5p in serum and tumor tissue.

LncRNA PVT1 Promotes Metastasis of Non-Small Cell Lung Cancer Through EZH2-Mediated Activation of Hippo/NOTCH1 Signaling Pathways.

Objective:Although growing evidences have showed that long non-coding RNA (lncRNAs) plasmacytoma variant translocation 1 (PVT1) plays a critical role in the progression of non-small cell lung cancer (NSCLC), there are still many unsolved mysteries remains to be deeply elucidated. This study aimed to find a new underlying mechanism of PVT1 in regulating the tumorigenesis and development of NSCLC.Materials and Methods:In this experimental study, Quantitative reverse transcription polymerase chain reaction (qRTPCR) was used to profile the expression of PVT1 in NSCLC tissues and cells. The effects of PVT1 on cell growth, migration and invasion were detected by colony formation assay, Matrigel-free transwell and Matrigel transwell assays, respectively. Changes of the key protein expression in Hippo and NOTCH signaling pathways, as well as epithelialmesenchymal transition (EMT) markers, were analyzed using western blot. Interaction of PVT1 with enhancer of zeste homolog 2 (EZH2) was verified by RNA pull-down, and their binding to the downstream targets was detected by Chromatin Immunoprecipitation (ChIP) assays.Results:These results showed that PVT1 was up-regulated in NSCLC tissue and cell lines, promoting NSCLC cell proliferation, migration and invasion. Knockdown of PVT1 inhibited the expression of Yes-associated protein 1 (YAP1) and NOTCH1 signaling activation. Further, we have confirmed that PVT1 regulated expression of YAP1 through EZH2-mediated miR-497 promoter methylation resulting in the inhibition of miR-497 transcription and its target YAP1 upregulation, and finally NOTCH signaling pathway was activated, which promoted EMT and invasion and metastasis.Conclusion:These results suggested that lncRNA PVT1 promotes NSCLC metastasis through EZH2-mediated activation of Hippo/NOTCH1 signaling pathways. This study provides a new opportunity to advance our understanding in the potential mechanism of NSCLC development.

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.

Estrogen promotes the metastasis of non‑small cell lung cancer via estrogen receptor β by upregulation of Toll‑like receptor4 and activation of the myd88/NF‑κB/MMP2 pathway.

Estrogen promotes non‑small cell lung cancer (NSCLC) metastasis via estrogen receptorβ (ERβ)‑mediated invasiveness‑associated matrix metalloprotease2 (MMP2) upregulation. However, how ERβ increases the aggressiveness of NSCLC cells remains unclear. Recently, MMP2 was found to be upregulated by Toll‑like receptor4 (TLR4) signaling activation and to promote NSCLC metastasis. Our present study aimed to examine the role of ERβ in the activation of TLR4 signaling and in tumor progression and metastasis, and to explore the synergistic metastatic effect of a combination of ERβ and TLR4 activation on human NSCLC cells invitro and invivo. Here, we found that ERβ is associated with TLR4 in metastatic lymph nodes. Western blot analysis and immunofluorescence revealed that ERβ overexpression upregulated TLR4 protein expression and activated downstream targets, myeloid differentiation primary response88 (myd88)/nuclear factor (NF)‑κB/MMP2, enhancing NSCLC cell migration and invasion invitro. A novel ERβ‑TLR4 interaction in cell plasma was identified by co‑immunoprecipitation and confocal immunofluorescence. The combination of estradiol and specific TLR4 agonist lipopolysaccharide (LPS) synergistically promoted metastatic behaviors in NSCLC cells. In cell culture and murine lung metastasis models, exposure to estradiol and LPS induced increased matrix degradation and accelerated invadopodia and metastasis formation in NSCLC cells compared with that in cells treated with estradiol or LPS alone. Together, we showed that estrogen promoted NSCLC metastasis via ERβ by upregulating TLR4 and activating its downstream signaling axis myd88/NF‑κB/MMP2. The combined targeting of ERβ and TLR4 may be a novel therapeutic strategy against advanced metastatic lung cancer.

Neuropilin 1 modulates TGF‑β1‑induced epithelial‑mesenchymal transition in non‑small cell lung cancer.

Previously, the authors reported that neuropilin-1 (NRP1) was significantly increased and acted as a vital promoter in the metastasis of non-small cell lung cancer (NSCLC). However, the regulatory mechanism of NRP1 in NSCLC cell migration and invasion remained unclear. The present study aimed to explore the regulatory mechanism of NRP1 in the transforming growth factor-β (TGF-β) 1-induced migration and invasion of NSCLC cells. The expression level of NRP1 was determined by RT-qPCR analysis in human tissue samples with or without lymph node metastasis. Transwell assay and wound healing assay were conducted to determine the cell migration. Lentivirus-mediated stable knockdown and overexpression of NRP1 cell lines were constructed. Exogenous TGF-β1 stimulation, SIS3 treatment, western blot analysis and in vivo metastatic model were utilized to clarify the underlying regulatory mechanisms. The results demonstrated that the expression of NRP1 was increased in metastatic NSCLC tissues. NRP1 promoted NSCLC metastasis in vitro and in vivo. The Transwell assays, wound healing assays and western blot analysis revealed that the knockdown of NRP1 significantly inhibited TGF-β1-mediated EMT and migratory and invasive capabilities of NSCLC. Furthermore, the overexpression of NRP1 weakened the inhibitory effect of SIS3 on the NSCLC migration and invasion. Co-IP assay revealed that NRP1 interacted with TGFβRII to induce EMT. On the whole, the findings of this study demonstrated that NRP1 was overexpressed in metastatic NSCLC tissues. NRP1 could contributes to TGF-β1-induced EMT and metastasis in NSCLC by binding with TGFβRII.

Cyclophilin A promotes non-small cell lung cancer metastasis via p38 MAPK.

BackgroundCyclophilin A (CypA) is associated with metastasis in diverse cancers; however, its role in lung cancer metastasis and the underlying mechanisms remain poorly understood. Our study investigated the effect of CypA on non‐small cell lung cancer (NSCLC) metastasis in vitro and in vivo to determine its mechanisms.MethodsIn this study, A549 and H1299 cell lines with downregulated and overexpressed CypA, respectively, were constructed by lentivirus transfection of NSCLC cells. in vitro experiments, including wound healing and transwell assays and Western blotting, showed that CypA promoted cancer cell migration and epithelial‐mesenchymal transition in NSCLC. Lung metastasis mouse models were used for the first time to confirm that CypA promoted NSCLC metastasis in vivo. The p38 inhibitor SB203580 was used to show that p38 MAPK is involved in CypA‐mediated NSCLC metastasis.ResultsWound healing and transwell assays showed that the migration of both A549 and H1299 cells decreased in the CypA downregulated group and increased in the CypA overexpressed group. CypA also positively promoted the expression of epithelial‐mesenchymal transition‐relevant proteins. Results of mouse models confirmed that the tumor metastasis rate was much higher in the CypA overexpressed than in the CypA downregulated group. In addition, SB203580 inhibited NSCLC cell migration significantly in the CypA overexpressed group, while the difference in the CypA downregulated group was not significant.ConclusionsIn conclusion, this study demonstrated that CypA promotes NSCLC cancer metastasis via p38 MAPK.

PLOD2 regulated by transcription factor FOXA1 promotes metastasis in NSCLC

In multiple types of tumors, fibrotic collagen is regarded as the 'highway' for cancer cell migration, which is mainly modified by lysyl hydroxylase 2 (PLOD2). The previous findings have demonstrated that the expression of PLOD2 was regulated by multiple factors, including HIF-1α, TGF-β and microRNA-26a/b. Although PLOD2 was confirmed to be related to poor prognosis in lung adenocarcinoma, the regulatory mechanism and function of PLOD2 in human lung adenocarcinoma is poorly understood. On the other hand, upregulation or hyperactivation of epidermal growth factor receptor is considered as a prognostic marker in many cancers, especially in non-small-cell lung cancer (NSCLC). In this study, we found that PLOD2 was elevated in NSCLC specimens and positively links to NSCLC poor prognosis. Gain- and loss-of-function studies and orthotopic implantation metastasis model pinpointed that PLOD2 promotes NSCLC metastasis directly by enhancing migration and indirectly by inducing collagen reorganization. In addition, we revealed that PLOD2 was regulated by PI3K/AKT-FOXA1 axis. The transcription factor FOXA1 directly bound to the PLOD2 promoter, and turned on PLOD2 transcription. In summary, our findings revealed a regulatory mechanism of NSCLC metastasis through EGFR-PI3K/AKT-FOXA1-PLOD2 pathway, and provided PLOD2 as a therapeutic target for NSCLC treatment.

Estrogen promotes tumor metastasis via estrogen receptor beta-mediated regulation of matrix-metalloproteinase-2 in non-small cell lung cancer.

In non–small cell lung cancer (NSCLC), estrogen significantly promotes NSCLC cell growth via estrogen receptor beta (ERβ). However, the effects by which ERβ contributes to metastasis in NSCLC have not been previously reported. This study aims at defining whether the stimulation of ERβ promotes NSCLC metastasis in vitro and in vivo. Here, Our results showed that estrogen and ERβ agonist enhanced aggressiveness of two lung cancer cell lines (A549 and H1793) and promoted murine lung metastasis formation. ER-inhibitor Fulvestrant treatment or ERβ-knockdown significantly suppressed the migration, invasion and nodule formation of NSCLC cells. The expression level of ERβ protein was analyzed in matched samples of metastatic lymph node and primary tumor tissues from the same individuals, and we found significantly higher levels of ERβ were expressed in lymph node compared to primary tumor tissues. Moreover, Studies on both surgical biopsies and on lung cancer cells revealed that the expression level of ERβ and matrix-metalloproteinase-2 (MMP-2) were associated. Furthermore, inhibition of ERβ resulted in down-regulation of MMP-2 expression. Taken together, our results demonstrate that activation of ERβ in lung cancer cells promotes tumor metastasis through increasing expression of invasiveness-associated MMP-2. These results also highlight the therapeutic potential of inhibition of ERβin the treatment of advanced NSCLC.

Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer

In a substantial population of non-small cell lung cancer (NSCLC), expression and activation of EGF receptor (EGFR) have been reported and is regarded as a novel molecular target. A growing body of evidence has shown the signaling crosstalk between EGFR and integrins in cellular migration and invasion. NEDD9 is an integrin signaling adaptor protein composed of multiple domains serving as substrate for a variety of tyrosine kinases. In the present study, we aimed at elucidating a role of NEDD9 in the signaling crosstalk between EGFR and integrins. Using NSCLC cell lines, we conducted immunoblotting and cellular migration/invasion assay in vitro. Next, we analyzed metastasis assays in vivo by the use of xenograft transplantation model. Finally, we retrospectively evaluated clinical samples and records of patients with NSCLCs. We showed that tyrosine phosphorylation of NEDD9 was reduced by the inhibition of EGFR in NSCLC cell lines. Overexpression of constitutively active EGFR caused tyrosine phosphorylation of NEDD9 in the absence of integrin stimulation. By gene transfer and gene knockdown, we showed that NEDD9 plays a pivotal role in cell migration and invasion of those cells in vitro. Furthermore, overexpression of NEDD9 promoted lung metastasis of an NSCLC cell line in NOD/Shi-scid, IL-2Rγ(null) mice (NOG) mice. Finally, univariate and multivariate Cox model analysis of NSCLC clinical specimens revealed a strong correlation between NEDD9 expression and recurrence-free survival as well as overall survival. Our data thus suggest that NEDD9 is a promising biomarker for the prognosis of NSCLCs and its expression can promote NSCLC metastasis.