Mechanism Of Lung Cancer Metastasis Research Articles

Impact of surgery and adjuvant chemotherapy on the survival of stage I lung adenocarcinoma patients with tumor spread through air spaces

ObjectivesTumor spread through air spaces (STAS) is a unique mechanism of lung cancer metastasis; however, its clinical value for stage I lung adenocarcinoma (ADC) remains unclear at present. We investigated the (1) prognosis of patients after sublobar resection compared with lobectomy for stage I lung adenocarcinoma with STAS; and (2) potential benefits of adjuvant chemotherapy (ACT) for patients with stage I ADC and STAS. MethodsA total of 3328 consecutive patients with stage I ADC were retrospectively identified between 2014 and 2018 at our institution; among them, 600 were diagnosed with STAS. Kaplan–Meier analysis and Cox proportional hazard regression models were used to evaluate the impact of STAS on overall survival (OS) and recurrence-free survival (RFS). ResultsAmong stage IA patients with STAS, there was no significant difference between those who underwent sublobar resection and lobectomy in OS (P = 0.919) and RFS (P = 0.066). Multivariate analysis confirmed this result (sublobar resection versus lobectomy, OS: HR = 0.523, 95 % CI, 0.056–18.458, P = 0.714; RFS, HR = 0.360, 95 % CI, 0.115–1.565, P = 0.897). ACT did not improve the prognosis of stage IA patients but did improve the RFS of stage IB patients with high-risk recurrence factors, including poorly differentiated tumors, lymphovascular invasion and visceral pleural invasion (P = 0.046). ConclusionsSublobar and lobectomy resection provided a comparable prognosis for stage IA ADC patients with STAS. When STAS was confirmed postoperatively, ACT should be considered for patients with stage IB with high-risk recurrence factors but not for those with stage IA disease.

Elevation of CD109 promotes metastasis and drug resistance in lung cancer via activation of EGFR-AKT-mTOR signaling.

Lung cancer is the most commonly diagnosed cancer worldwide, and metastasis in lung cancer is the leading cause of cancer‐related deaths. Thus, understanding the mechanism of lung cancer metastasis will improve the diagnosis and treatment of lung cancer patients. Herein, we found that expression of cluster of differentiation 109 (CD109) was correlated with the invasive and metastatic capacities of lung adenocarcinoma cells. CD109 is upregulated in tumorous tissues, and CD109 overexpression was associated with tumor progression, distant metastasis, and a poor prognosis in patient with lung adenocarcinoma. Mechanistically, expression of CD109 regulates protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling via its association with the epidermal growth factor receptor (EGFR). Inhibition of CD109 decreases EGFR phosphorylation, diminishes EGF‐elicited activation of AKT/mTOR, and sensitizes tumor cells to an EGFR inhibitor. Taken together, our results show that CD109 is a potential diagnostic and therapeutic target in lung cancer patients.

Proteasome-dependent degradation of Smad7 is critical for lung cancer metastasis.

Lung cancer is one of the cancers with highest morbidity and mortality rates and the metastasis of lung cancer is a leading cause of death. Mechanisms of lung cancer metastasis are yet to be fully understood. Herein, we demonstrate that mice deficient for REGγ, a proteasome activator, exhibited a significant reduction in tumor size, numbers, and metastatic rate with prolonged survival in a conditional Kras/p53 mutant lung cancer model. REGγ enhanced the TGFβ-Smad signaling pathway by ubiquitin-ATP-independent degradation of Smad7, an inhibitor of the TGFβ pathway. Activated TGFβ signaling in REGγ-positive lung cancer cells led to diminished expression of E-cadherin, a biomarker of epithelial-mesenchymal transitions (EMT), and elevated mesenchymal markers compared with REGγ-deficient lung cancer cells. REGγ overexpression was found in lung cancer patients with metastasis, correlating with the reduction of E-Cadherin/Smad7 and a poor prognosis. Overall, our study indicates that REGγ promotes lung cancer metastasis by activating TGF-β signaling via degradation of Smad7. Thus, REGγ may serve as a novel therapeutic target for lung cancers with poor prognosis.

MiR-34b inhibits the migration/invasion and promotes apoptosis of non-small-cell lung cancer cells by YAF2.

Lung cancer is the leading cause of cancer death in the world and microRNAs (miRNA) have been found to be involved in the initiation and development of cancer by acting as potential oncogenes or tumor suppressor genes. In this study, we investigated the expression of miR-34b in non-small cell lung cancer (NSCLC) patients and discussed the molecular mechanism of miR-34b in the invasion and migration of A549 cells in vitro. Our results showed that miR-34b was significantly down-regulated in primary cancer tissues when compared with the normal lung tissues. The over-expression of miR-34b inhibited migration and invasion, and promoted apoptosis of A549 lung cancer cells. Furthermore, Luciferase reporter assay validated YY1-associated factor 2 (YAF2) as a direct target of miR-34b and YAF2 expression was significantly increased in clinical NSCLC tissue samples. In addition, the over-expression of miR-34b inhibited YAF2, p-Jak2, p-STAT3 and MMP2 protein expression and promoted caspase 3 protein expression in cancer cells. Our results suggest that miR-34b may inhibit migration and invasion of NSCLC cells by targeting YAF2. Thus, our findings provide new insight into the molecular mechanisms of lung cancer metastasis and miR-34b may serve as a potential target in the treatment of human lung cancer.

Establishment and characterization of a patient-derived circulating lung tumor cell line in vitro and in vivo

BackgroundCirculating tumor cells (CTCs) have been described as a population of cells that may seed metastasis, which is a reliable target for the prevention of metastases in lung cancer patients at the early stage. The culturing of CTCs in vitro can be used to study the mechanism of lung cancer metastasis and to screen antimetastasis drugs. This study aims to establish CTC cell line in vitro and explore the potential mechanism of its metastasis.MethodsA mixture of EpCAM- and EGFR-coated immunomagnetic microbeads in microfluidic Herringbone-Chip was used to capture CTCs. The CTCs, 95-D and A549 cells was evaluated by cell proliferation assays, clonal formation assays, migration assays and drug resistance. Flow cytometry and cytokine protein chip were used to detect the difference in phenotype and cytokine secretion between CTCs, 95-D and A549 cells. The NOD/SCID mice were used to study tumorigenicity, lung organ colonization and metastasis of CTCs. The H&E staining, immunohistochemistry and immunofluorescence assay were used to detect the pathological status of CTCs.ResultsThe number of EpCAM(+)/EGFR(+)/CK(+)/CD45(−) lung CTCs showed a weak negative correlation with clinical stages in patients with non-small cell lung cancer (NSCLC). In a phase IIa lung cancer patient, we successfully establish a permanent CTC cell line, named CTC-TJH-01. In vitro studies showed the CTC-TJH-01 cells were in the intermediate stage of epithelial to mesenchymal transition (EMT), had stem cell characteristics and were drug resistant. In vivo studies showed that CTC-TJH-01 cells can induce tumorigenesis, lung organ colonization and metastasis after xenografting in immunodeficient mice. In addition, the low expression level of CX3CL1 and high expression level of CXCL5 in the CTC-TJH-01 cells may be an important mechanism for their metastasis.ConclusionsWe successfully established a permanent CTC cell line with metastatic ability, which can be used to screen antimetastatic drugs and study the mechanism of lung cancer metastasis.

Expression of microRNA-338 in lung adenocarcinoma and its inhibition mechanism of lung cancer metastasis

Objective To study the expression of microRNA (miRNA, miR)-338 in lung adenocarcinoma cell line and lung adenocarcinoma tissues, and to study the mechanism of lung cancer metastasis. Methods The expression of miR-338 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) in lung adenocarcinoma cell line A549, and 115 cases of lung adenocarcinoma tissues and normal lung tissues adjacent to tumor. To construct miR-338 over expressing lung cancer cell line and explore the function of miR-338 in the proliferation, invasion and metastasis of lung cancer cells. To detect the expression of integrin β3 in miR-338 over expression lung cancer cell line, and to study the mechanism of miR-338 inhibiting lung cancer metastasis. Results (1)The expression of miR-338 was down regulated in lung adenocarcinoma cell line and lung adenocarcinoma tissues (1.00 vs. 0.15±0.01, P=0.035), (0.00 vs. -2.99±3.69, P=0.001). The expression of miR-338 was correlated with tumor embolus, tumor recurrence, TNM stage (P=0.005, 0.004, 0.025). The 5 years overall survival rate of the low expression group was significantly lower than that of the high expression group (median survival time 44 months vs. 53 months, P=0.001). (2) Up-regulating the expression of miR-338 can inhibit the proliferation of lung cancer cells, migration [average cell count (347±41) cells vs. (190±8) cells, P=0.029] and invasion [average cell count (197±12) cells vs. (68±14) cells, P=0.008]. Xenograft tumor model in nude mice showed that the high expression of miR-338 can effectively inhibit the growth of transplanted tumor [average tumor clone number (20±7) cells vs. (4±2) cells, P=0.004]. (3) In the miR-338 over expression lung adenocarcinoma cell line, the expression of integrin β3 was significantly lower than that in control cells (1.00 vs. 0.35±0.04, P=0.001). Dual luciferase activity assay revealed that the miR-338 over expression group was reduced in the luciferase activity (1.00 vs. 0.75±0.08, P=0.030). Conclusion (1) MiR-338 can inhibit the proliferation, invasion and metastasis of lung cancer cells; (2) MiR-338 play a negative regulation post transcription by targeting the integrin β3 3’untranslated regions (3’UTR) mRNA region. integrin β3 is a new miR-338 lung cancer target gene. Key words: Lung adenocarcinoma; Metastasis; MicroRNA-338; Integrin β3

COL5A1 may contribute the metastasis of lung adenocarcinoma

BackgroundLung cancer leads to the largest number of cancer-related deaths worldwide and is usually accompanied with metastasis which is the primary cause of those death and correlated with poor prognosis. However, the mechanism of lung cancer metastasis is still lack of definition. MethodsWe compared the primary lung adenocarcinoma (AD) and its metastasis tissues induced by overexpression of KrasG12D and inactivation of P53 in mouse lungs by analyzing GSE40222 about the differentially expressed genes (DEGs), pathways and hub genes. And human lung AD databases are used to verify the conversed changes of identified key gene and then followed functional studies are performed to explore the functions of key gene. ResultsWe identified 165 genes differentially expressed in lung AD metastasis compared to primary AD. Pathway analysis identified 649 GO biological processes and 8 KEGG pathways, such as ECM-receptor interaction. Biological network interaction identified the hub genes during lung adenocarcinoma metastasis, such as the up-regulated COL5A1, a novel gene in AD metastasis. We found it's also increased in human AD and advanced stage. Knockdown of COL5A1 in human AD metastatic cells inhibited cell growth and invasion, and induced cell apoptosis. Notably, higher expression of COL5A1 was observed in the lung AD patients with recurrence and short survive. ConclusionBy analyzing mouse lung AD and its metastases, we identified the potential key genes and pathways regulating lung AD metastasis, such as COL5A1. The following analysis of COL5A1 in human AD database and cells explores its functions, holding the implications of target therapy in AD metastasis and also providing more clues for future studies.

MiRNA-148a serves as a prognostic factor and suppresses migration and invasion through Wnt1 in non-small cell lung cancer.

Lung cancer is the leading cause of cancer death in the world, and aberrant expression of miRNA is a common feature during the cancer initiation and development. Our previous study showed that levels of miRNA-148a assessed by quantitative real-time polymerase chain reaction (qRT-PCR) were a good prognosis factor for non-small cell lung cancer (NSCLC) patients. In this study, we used high-throughput formalin-fixed and paraffin-embedded (FFPE) lung cancer tissue arrays and in situ hybridization (ISH) to determine the clinical significances of miRNA-148a and aimed to find novel target of miRNA-148a in lung cancer. Our results showed that there were 86 of 159 patients with low miRNA-148a expression and miRNA-148a was significantly down-regulated in primary cancer tissues when compared with their adjacent normal lung tissues. Low expression of miRNA-148a was strongly associated with high tumor grade, lymph node (LN) metastasis and a higher risk of tumor-related death in NSCLC. Lentivirus mediated overexpression of miRNA-148a inhibited migration and invasion of A549 and H1299 lung cancer cells. Furthermore, we validated Wnt1 as a direct target of miRNA-148a. Our data showed that the Wnt1 expression was negatively correlated with the expression of miRNA-148a in both primary cancer tissues and their corresponding adjacent normal lung tissues. In addition, overexpression of miRNA-148a inhibited Wnt1 protein expression in cancer cells. And knocking down of Wnt-1 by siRNA had the similar effect of miRNA-148a overexpression on cell migration and invasion in lung cancer cells. In conclusion, our results suggest that miRNA-148a inhibited cell migration and invasion through targeting Wnt1 and this might provide a new insight into the molecular mechanisms of lung cancer metastasis.

Lung squamous cell carcinoma metastasizing to the nasopharynx following bronchoscopy intervention therapies: a case report

Metastatic carcinoma to the nasopharynx is extremely rare, and few cases have been reported in the literature. In the present report, we describe the case of a patient with a mass in the nasopharynx found by bronchoscopy. Our patient was a 61-year-old man receiving multiple bronchoscopy intervention therapies for advanced lung squamous cell carcinoma (SCC), which was histopathologically confirmed. The SCC metastasized to the nasopharynx following the bronchoscopy intervention therapies. The lesion was considered metastatic from lung cancer on the basis of clinical and histological clues. The exact mechanism of lung cancer metastasis to the nasopharynx in this case remains unclear because either implantation or hematogenous and lymphatic spread is possible. A thorough head and neck examination should be undertaken during bronchoscopic evaluation, especially in patients receiving bronchoscopy intervention therapies. The early detection of a silent nasopharyngeal metastasis is important to choosing from among the multiple treatment options available.

Molecular determinants of lung cancer metastasis to the central nervous system.

Lung cancer remains the leading cause of cancer-related mortality worldwide. The propensity for metastasis to the central nervous system (CNS) is a major clinical hurdle contributing to the low five-year survival rate of advanced disease. CNS metastases significantly outnumber primary brain tumors and carry a dismal prognosis in part due to the inability of therapeutic agents to cross the blood brain barrier. Standard treatment using radiation has been largely ineffective in improving mortality, suggesting the need for new agents targeting the critical metastatic drivers. The genetic and molecular events governing CNS metastasis from the lung are poorly understood at this time. This review highlights genetic events associated with CNS dissemination from the lung and molecular mechanisms associated with CNS metastasis. In vivo model systems that faithfully recapitulate escape from the lung and colonization of the CNS are described as tools for understanding the metastatic phenotype and for testing new therapeutic agents. A deeper understanding of the mechanisms of lung cancer metastasis to the CNS is needed to elucidate novel therapeutic avenues towards the improvement of the mortality associated with advanced stage lung cancer.

Transforming growth factor-β1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition

Lung cancer is a highly malignant carcinoma, and most deaths of lung cancer are caused by metastasis. The alterations associated with epithelial-to-mesenchymal transition (EMT) may be related to the cancer cell metastasis. Nevertheless, the mechanism of lung cancer metastasis remains unclear. We conducted a study in vitro to investigate whether transforming growth factor-β1 (TGF-β1) could induce changes of, such as cell morphology, expression of relative protein markers, and cellular motile and invasive activities. In this research, the changes of cell morphology were first investigated under a phase contrast microscope, then western blotting was employed to detect the expression of E-cadherin, vimentin, and fibronectin, and finally cell motility and invasion were evaluated by cell wound-healing as well as invasion assays. The data indicated that human lung adenocarcinoma cell lines, A-549 and PC-9 cells of epithelial cell characteristics, were induced to undergo EMT by TGF-β1. Following TGF-β1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker E-cadherin and increased mesenchymal markers vimentin and fibronectin. More importantly, cell motility and invasion were also enhanced in the EMT process. These results indicated that TGF-β1 may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT.

Development of a highly metastatic model that reveals a crucial role of fibronectin in lung cancer cell migration and invasion

BackgroundThe formation of metastasis is the most common cause of death in patients with lung cancer. A major implement to understand the molecular mechanisms involved in lung cancer metastasis has been the lack of suitable models to address it. In this study, we aimed at establishing a highly metastatic model of human lung cancer and characterizing its metastatic properties and underlying mechanisms.MethodsThe human lung adeno-carcinoma SPC-A-1 cell line was used as parental cells for developing of highly metastatic cells by in vivo selection in NOD/SCID mice. After three rounds of selection, a new SPC-A-1sci cell line was established from pulmonary metastatic lesions. Subsequently, the metastatic properties of this cell line were analyzed, including optical imaging of in vivo metastasis, immunofluorescence and immunohistochemical analysis of several epithelial mesenchymal transition (EMT) makers and trans-well migration and invasion assays. Finally, the functional roles of fibronectin in the invasive and metastatic potentials of SPC-A-1sci cells were determined by shRNA analysis.ResultsA spontaneously pulmonary metastatic model of human lung adeno-carcinoma was established in NOD/SCID mice, from which a new lung cancer cell line, designated SPC-A-1sci, was isolated. Initially, the highly metastatic behavior of this cell line was validated by optical imaging in mice models. Further analyses showed that this cell line exhibit phenotypic and molecular alterations consistent with EMT. Compared with its parent cell line SPC-A-1, SPC-A-1sci was more aggressive in vitro, including increased potentials for cell spreading, migration and invasion. Importantly, fibronectin, a mesenchymal maker of EMT, was found to be highly expressed in SPC-A-1sci cells and down-regulation of it can decrease the in vitro and in vivo metastatic abilities of this cell line.ConclusionsWe have successfully established a new human lung cancer cell line with highly metastatic potentials, which is subject to EMT and possibly mediated by increased fibronectin expression. This cell line and its reproducible s.c. mouse model can further be used to identify underlying mechanisms of lung cancer metastasis.

Clinical significance of VEGF-C and VEGFR-3 expression in non-small cell lung cancer

The relationship between vascular endothelial growth factor-C (VEGF-C)/vascular endothelial growth factor receptor 3 (VEGFR-3) expression and clinicopathologic features of the patients with non-small cell lung cancer (NSCLC) was investigated. The expression of VEGF-C and VEGFR-3 was assessed in 65 patients with NSCLC by immunohistochemistry. The significance of VEGF-C and VEGFR-3 expression was analyzed statistically. The results showed that VEGF-C and VEGFR-3 were highly expressed in cytoplasm and membrane in lung cancer tissues with the positive rate being 55.4 % and 52.3 % respectively, while there was no expression in the normal lung tissues. The expression of VEGF-C was significantly increased in adenocacinoma as compared to other types of NSCLC (P<0.05). The VEGFR-3 expression was closely related with lymph node metastasis (P<0.01) and TNM stage (P<0.05). There was a positive correlation between the VEGF-C and VEGFR-3 expression in NSCLC patients (r=0.658, P<0.01). It is suggested that VEGFR-3 plays an important role in the lymphatic metastasis of NSCLC. The interaction between VEGF-C and VEGFR-3 may be deeply involved in the mechanism of lung cancer metastasis.

Analysis of two-dimension gel electrophoresis of human large cell lung cancer cell lines with different metastasis potentials

Metastasis is not only the malignant characteristics of lung can- cer, but also the chief cause of failure to cure and high mortality of lung cancer. To better explore and understand the mechanism of lung cancer metastasis and to search for potential markers for early diagnosing and reversing lung cancer metastasis, differential proteomic analysis is conducted in two human large cell lung cancer cell lines with high metastasis potentials (L9981) and low metastasis potentials (NL9980) by two-dimension gel electrophoresis (2-DE). The total proteins of the two cell lines were separated by immobilized pH gradient (IPG)-based 2-DE. The differentially expressed proteins of the two cell lines were analyzed using image analysis software. A high resolution and reproducible 2-DE image was successfully obtained. Average deviations for protein position in IEF direction were (0.858±0.076)mm and (1.514±0.127)mm in SDS-PAGE direction. The relative standard deviation for protein volume was (12.06±0.580)% in L9981 and (12.22±0.640)% in NL9980. The average total number of protein spots was 902±169 in L9981 cells and 941±173 in NL9980 cells in three repeated experiments. Image analysis of siliver-stained 2-DE image revealed that 4 protein spots had significant differential expressions in L9981 and NL9980 (student's t-test, P < 0.05). Fifteen protein spots were only detected in L9981, and 27 protein spots were only detected in NL9980. The results in this study suggest that an obviously differential proteomic expression exists between the human high- and low-metastatic large cell lung cancer cell lines. It will be helpful to further understand the molecular mechanisms of lung cancer invasion and metastasis, and provide new experimental evidence for searching metastatic-related molecule of lung cancer.

Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo.

Most lung cancer patients are unfortunately uncurable and die because of widespread metastases, thus indicating the importance of identification of molecules with a crucial role in this process. Our previous expression profiling analysis of a highly metastatic lung cancer cell line, NCI-H460-LNM35, and its parental low metastatic line, NCI-H460-N15, revealed significant up-regulation of both known and unknown genes in LNM35. In this study, we describe the isolation and detailed characterizations of a novel gene, named CLCP1, which corresponds to one of such expression sequence tags with up-regulated expression in LNM35. The CLCP1 gene was found to encode a protein with 775 amino acids with structural similarities to, but distinct from neuropilins, cell surface receptors for VEGF165 and semaphorins. Notably, CLCP1 was shown to be up-regulated not only in LNM35 in association with its acquisition of metastatic phenotype during in vivo selection, but also in a significant fraction of lung cancers in vivo with high frequency in metastatic lesions, warranting future studies for a better understanding of the molecular mechanisms of lung cancer metastasis.

Heterogeneity of multiorgan metastases of human lung cancer cells genetically engineered to produce cytokines and reversal using chimeric monoclonal antibodies in natural killer cell-depleted severe combined immunodeficient mice.

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.