sphingosine-kinase and Lung-Neoplasms

Reciprocal interactions between breast cancer cells and the tumor microenvironment (TME) are important for cancer progression and metastasis. We report here that the deletion or inhibition of sphingosine kinase 2 (SphK2), which produces sphingosine-1-phosphate (S1P), markedly suppresses syngeneic breast tumor growth and lung metastasis in mice by creating a hostile microenvironment for tumor growth and invasion. SphK2 deficiency decreased S1P and concomitantly increased ceramides, including C16-ceramide, in stromal fibroblasts. Ceramide accumulation suppressed activation of cancer-associated fibroblasts (CAF) by upregulating stromal p53, which restrained production of tumor-promoting factors to reprogram the TME and to restrict breast cancer establishment. Ablation of p53 in SphK2-deficient fibroblasts reversed these effects, enabled CAF activation and promoted tumor growth and invasion. These data uncovered a novel role of SphK2 in regulating non-cell-autonomous functions of p53 in stromal fibroblasts and their transition to tumor-promoting CAFs, paving the way for the development of a strategy to target the TME and to enhance therapeutic efficacy.. Sphingosine kinase 2 (SphK2) facilitates the activation of stromal fibroblasts to tumor-promoting cancer-associated fibroblasts by suppressing host p53 activity, revealing SphK2 as a potential target to reprogram the TME.

Topics: Animals; Cancer-Associated Fibroblasts; Fibroblasts; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Phosphotransferases (Alcohol Group Acceptor); Tumor Microenvironment; Tumor Suppressor Protein p53

Lung adenocarcinoma (LUAD) is a fatal threat to human health, while the mechanism remains unclear, and the therapy brings limited therapeutic effects. Transcription factor Homeobox C11 (HOXC11) was previously proved to be related to hind limbs and metanephric development during the embryonic phase, and its role in tumors has been gradually recognized. Our study found that HOXC11 overexpressed in LUAD and was associated with worse overall survival. Moreover, its expression in lung cancer was regulated by IκB kinase α (IKKα), a pivotal kinase in NF-κB signaling, which was related to the ubiquitination of HOXC11. We further proved that HOXC11 could enhance the ability of proliferation, migration, invasion, colony formation, and the progression of the cell cycle in LUAD cells. Meanwhile, it also accelerated the formation of subcutaneous and lung metastases tumors. In contrast, loss of HOXC11 in LUAD cells significantly inhibited these malignant phenotypes. At the same time, HOXC11 regulated the expression of sphingosine kinase 1 (SPHK1) by directly binding to its promoter region. Therefore, we conclude that HOXC11 impacts the development of LUAD and facilitates lung cancer progression by promoting the expression of SPHK1.

Topics: Adenocarcinoma of Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor)

Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Its role in LUAD has gained attention, but the detailed function and mechanism have not been clarified completely at present.. Tumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, we detected the influence of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, we comprehensively inspected the different changed proteins influenced by TIM and did global bioinformatic analysis.. We found that TIM expression was elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. We also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, we found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. We further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells.. Our study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

Topics: Adenocarcinoma of Lung; Adenosine Triphosphate; AMP-Activated Protein Kinases; Cell Line, Tumor; Cell Movement; Cell Proliferation; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; RNA, Small Interfering

Long non-coding RNAs (lncRNAs) are closely associated with the development of lung adenocarcinoma (LADC). The present study focused on the role of LINC00960 in LADC. miRNA and mRNA expression levels were detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cellular functions were evaluated by MTT, colony formation, and Transwell assays, respectively. LINC00960 Luciferase and RNA pull-down assays were performed to clarify the interaction between miR-124a and LINC00960 or Recombinant Sphingosine Kinase 1 (SphK1). We observed that LINC00960 was overexpressed in LADC tumor tissues and cell lines. LINC00960 knockdown suppressed the proliferation, migration, and invasion of LADC cells. Moreover, LINC00960 sponged miR-124a to inhibit the SphK1/S1P pathway in LADC cells. LINC00960 knockdown markedly reduced the rate of tumor growth. The luciferase reporter assay results demonstrated an interaction between miR-124a and LINC00960 or SphK1. This interaction was confirmed using the RNA pull-down assay. In addition, miR-124a downregulation or SphK1 upregulation reversed the inhibitory effects of LINC00960 knockdown on cellular functions of LADC cells, suggesting that LINC00960 may be a potential therapeutic biomarker for LADC via the miR-124a/SphK1 axis. Accordingly, LINC00960 may be a potential therapeutic biomarker for LADC.

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasm Transplantation; Phosphotransferases (Alcohol Group Acceptor); RNA, Long Noncoding; Up-Regulation

Epimesatines A-I, nine undescribed prenylated flavonoids, along with ten known analogues, were isolated from the aerial parts of Epimedium sagittatum Maxim. The structures and absolute configurations of epimesatines A-I were determined using a combination of spectroscopic data, Rh

Topics: Carcinoma, Non-Small-Cell Lung; Epimedium; Flavonoids; Humans; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor)

Sphingosine kinase 1 (SphK1) and sphingosine kinase (SphK2) are both important therapeutic targets of non-small cell lung cancer (NSCLC). SKI-349 is a novel, highly efficient and small molecular SphK1/2 dual inhibitor. Here in primary human NSCLC cells and immortalized cell lines, SKI-349 potently inhibited cell proliferation, cell cycle progression, migration and viability. The dual inhibitor induced mitochondrial depolarization and apoptosis activation in NSCLC cells, but it was non-cytotoxic to human lung epithelial cells. SKI-349 inhibited SphK activity and induced ceramide accumulation in primary NSCLC cells, without affecting SphK1/2 expression. SKI-349-induced NSCLC cell death was attenuated by sphingosine-1-phosphate and by the SphK activator K6PC-5, but was potentiated by the short-chain ceramide C6. Moreover, SKI-349 induced Akt-mTOR inactivation, JNK activation, and oxidative injury in primary NSCLC cells. In addition, SKI-349 decreased bromodomain-containing protein 4 (BRD4) expression and downregulated BRD4-dependent genes (Myc, cyclin D1 and Klf4) in primary NSCLC cells. At last, SKI-349 (10 mg/kg) administration inhibited NSCLC xenograft growth in nude mice. Akt-mTOR inhibition, JNK activation, oxidative injury and BRD4 downregulation were detected in SKI-349-treated NSCLC xenograft tissues. Taken together, targeting SphK1/2 by SKI-349 potently inhibits NSCLC cell growth in vitro and in vivo.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Ceramides; Humans; Lung Neoplasms; Mice; Mice, Nude; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Sphingosine; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Humans; Lung Neoplasms; Lysophospholipids; Phosphotransferases (Alcohol Group Acceptor); Pre-B-Cell Leukemia Transcription Factor 1; Proto-Oncogene Proteins c-akt; Sphingosine

The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) exerts a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1-5). Epidemiological studies proved high levels of circulating S1P in non-small cell lung cancer (NSCLC) patients. Studies in literature suggest that high levels of S1P support carcinogenesis but the exact mechanism is still elusive. The aim of this study was to understand the mechanism/s underlying S1P-mediated lung tumor cell proliferation.. We used human samples of NSCLC, a mouse model of first-hand smoking and of Benzo(a)pyrene (BaP)-induced tumor-bearing mice and A549 lung adenocarcinoma cells.. We found that the expression of S1PR3 was also into the nucleus of lung cells in vitro, data that were confirmed in lung tissues of NSCLC patients, smoking and tumor bearing BaP-exposed mice. The intranuclear, but not the membrane, localization of S1PR3 was associated to S1P-mediated proliferation of lung adenocarcinoma cells. Indeed, the inhibition of the membrane S1PR3 did not alter tumor cell proliferation after Toll Like Receptor (TLR) 9 activation. Instead, according to the nuclear localization of sphingosine kinase (SPHK) II, the inhibition of the kinase completely blocked the endogenous S1P-induced tumor cell proliferation.. These results prove that the nuclear S1PR3/SPHK II axis is involved in lung tumor cell proliferation, highlighting a novel molecular mechanism which could provide differential therapeutic approaches especially in non-responsive lung cancer patients.

Topics: A549 Cells; Adenocarcinoma of Lung; Animals; Humans; Lung Neoplasms; Mice; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine-1-Phosphate Receptors

The purpose of the present study was to investigate the effect and potential mechanism of knockdown of sphingosine kinase-1 (SPHK1) on the proliferation, cell cycle and apoptosis of non-small cell lung cancer (NSCLC) cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect SPHK1 mRNA expression in human healthy lung fibroblasts (MRC-5 cells) and four NSCLC cell lines. Then, A549 and H1299 cells were transfected with SPHK1-shRNA and corresponding negative control. CCK-8, Annexin V-FITC/PI dual staining and cell cycle assay were performed to evaluate cell proliferation, apoptosis and cell cycle distribution, respectively. JC-1 mitochondrial membrane potential measurement kit was adopted to measure mitochondrial membrane potential. Western blot was used to detect the protein expression levels of cell cycle and mitochondrial apoptotic pathway-related proteins, as well as MEK/ERK signaling pathway. The results showed that the mRNA expression of SPHK1 in NSCLC cells was higher than that in MRC-5 cells. SPHK1-shRNA significantly inhibited the proliferation of A549 and H1299 cells, blocked the cell cycle in G0/G1 phase, and promoted cell apoptosis through the mitochondrial pathway. Compared with the control group, the expression of p-MEK and p-ERK proteins in the SPHK1-shRNA group was significantly down-regulated. Moreover, MEK/ERK inhibitor could dramatically suppress cell proliferation and promote cell apoptosis. These results suggest that SPHK1 knockdown can inhibit the proliferation of NSCLC cells and might promote mitochondrial apoptotic pathway by inhibiting MEK/ERK signaling pathway.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Gene Knockdown Techniques; Humans; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor)

Sphingosine kinase1 (SphK1) is an oncogenic enzyme that regulates tumor cell apoptosis, proliferation and survival. SphK1 has been reported to promote the development of non‑small cell lung cancer (NSCLC), although the underlying mechanism remains to be determined. The aim of the present study was to examine the expression and function of SphK1 in NSCLC and to explore the underlying molecular mechanism. The results of the present study demonstrated that SphK1 expression was upregulated in NSCLC tissues and cell lines. Overexpression of SphK1 increased the proliferation and migration of NSCLC cells. Additionally, overexpression of SphK1 induced expression of antiapoptotic and migration‑associated genes, such as Bcl‑2, matrix metallopeptidase 2 and cyclin D1. Of note, signal transducer and activator of transcription 3 (STAT3) was also activated in the SphK1‑overexpressing cells. By treatment with a STAT3 inhibitor, it was demonstrated that the SphK1‑induced changes in expression of target genes, as well as the increase in proliferation and migration of NSCLC cells were mediated by STAT3. In conclusion, the effects of SphK1 overexpression on the development of NSCLC were demonstrated to be mediated by the activation of STAT3. These results suggested that inhibition of the SphK1‑STAT3 axis may be a potential strategy for the treatment of NSCLC.

Topics: A549 Cells; Adult; Aged; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Phosphotransferases (Alcohol Group Acceptor); STAT3 Transcription Factor

PSMG3-AS1 is a characterized oncogenic lncRNA in breast cancer, while its role in other cancers remains unclear. This study was to investigate the role and underlying mechansim of PSMG3-AS1 in non-small cell lung cancer (NSCLC). In this study, we found that PSMG3-AS1 could interact with miR-613. The expression of PSMG3-AS1 was upregulated in NSCLC, while the expression of miR-613 was downregulated in NSCLC. However, PSMG3-AS1 and miR-613 were not significantly correlated with each other. In NSCLC cells, PSMG3-AS1 and miR-613 overexpression failed to regulate the expression of each other. Interestingly, PSMG3-AS1 overexpression led to upregulated SphK1, a downstream target of miR-613. In addition, PSMG3-AS1 overexpression reduced the inhibitory effects of miR-613 on NSCLC cell proliferation. Therefore, PSMG3-AS1 may promote the proliferation of NSCLC cells by sponging miR-613 to upregulate SphK1.

Topics: Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Phosphotransferases (Alcohol Group Acceptor); RNA, Long Noncoding; Up-Regulation

Lung cancer remains the leading cause of cancer-associated mortality in China and the world. Increasing numbers of studies have reported that sphingosine kinase 1 (SPK1) is frequently highly expressed in tumors of various origins, including lung cancer, and its high expression contributes toward tumor progression. However, the clinical significance of SPK1 and its role in the growth and metastasis of non-small-cell lung cancer (NSCLC) remain unclear. In the present study, we found that SPK1 expression was expressed highly in NSCLC tissues and cell lines. Knockdown of SPK1 suppressed cell growth, proliferation, migration, and invasion and increased apoptosis. Moreover, knocking down SPK1 expression inhibited the growth of tumors in nude mice. Mechanistically, silencing the expression of SPK1 inhibited the expression of p-extracellular signal-regulated kinase (ERK). Moreover, the ERK-specific inhibitor U0126 suppressed the expression of the epithelial-mesenchymal transition of lung cancer cells. Together, our findings indicated that SPK1 enhanced tumor growth in lung cancer and induced metastasis by activating the ERK1/2 signaling pathway, indicating its potential application in NSCLC diagnosis and therapy.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Animals; Carrier Proteins; Cell Line, Tumor; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Nude; Microfilament Proteins; NF-kappa B; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

LncRNA HULC has been proved to have important functions in the pathogenesis of several types of cancers. While its involvement in non-small cell lung cancer (NSCLC), which is one of the most common malignancies, still hasn't been reported to date. Therefore, we aimed to investigate the role of HULC in NSCLC and to explore the possible mechanisms.. Tumor tissues and adjacent healthy tissues were collected from NSCLC patients, and blood samples were collected from both NSCLC patients and healthy controls. Expression of HULU in those tissues was detected by qRT-PCR. All patients were followed up for 5 years. Diagnostic and prognostic values of serum HULU for NSCLC were investigated by ROC curve analysis and survival curve analysis, respectively. HULC overexpression NSCLC cell lines were established and its effects on cell proliferation as well as apoptosis were investigated by CCK-8 assay and MTT assay, respectively. Effects of HULC overexpression on sphingosine kinase 1 (SPHK1) and its downstream PI3K/Akt pathway were investigated by Western blot.. HULC expression level was increased in tumor tissues compared with adjacent healthy tissues in most patients. Serum level of HULC was higher in cancer patients than that in healthy control. Serum level of HULC was increased with the increased stage of primary tumor (T stage). Serum HULC can be used to accurately predict NSCLC and its prognosis. HULC overexpression promoted tumor cell proliferation, but inhibited cell apoptosis. HULC overexpression also increased expression level of SPHK1 and phosphorylation level of Akt in NSCLC cell, but showed on significant effects on Akt expression. Treatment with SPHK1 inhibitor and Akt reduced the effects of HULC overexpression on proliferation and apoptosis of NSCLC cells. But the treatment showed no significant effects on HULC expression. SPHK1 inhibitor treatment inhibited phosphorylation of Akt, while Akt inhibitor treatment showed no significant effects on SPHK1 expression.. LncRNA HULC overexpression can promote NSCLC cell proliferation and inhibit cell apoptosis by up-regulating sphingosine kinase 1 (SPHK1) and further induce the activation of its downstream PI3K/Akt pathway.

Topics: Adult; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Chromones; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Morpholines; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Up-Regulation

Statins are the most effective drugs used in the reduction of intracellular synthesis of cholesterol. Numerous studies have confirmed that statins reduce the risk of multiple types of cancers. Statin use in cancer patients is associated with reduced cancer-related mortality. Epithelial-to-mesenchymal transition (EMT), a complicated process programmed by multiple genes, is an important mechanism of cancer metastasis. We explored the effect and mechanism of atorvastatin on the EMT process in A549 cells by establishing an EMT model in vitro induced by TGF-β1, and evaluated the effects of atorvastatin on the lower signaling pathway of TGF-β1 stimulation. Our results showed that atorvastatin partially inhibited the EMT process, and inhibited cell migration and actin filament remodeling. Transcriptional upregulation of ZEB1 and protein sphingosine kinase 1 (SphK1) induced by TGF-β1 was also suppressed. SphK1 plasmid transient transfection strengthened the EMT process induced by TGF-β1 in the presence of atorvastatin. Our experiments confirmed that atorvastatin can partially inhibit the EMT process of non-small cell lung cancer cells induced by TGF-β1 by attenuating the upregulation of SphK1.

Topics: A549 Cells; Atorvastatin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta1; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.

Topics: Adenocarcinoma; Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Binding Sites; Cell Line; Cell Line, Tumor; Enzyme Inhibitors; Female; HEK293 Cells; Humans; Lung Neoplasms; Male; MCF-7 Cells; Mice; Mice, Transgenic; Molecular Conformation; Mutagenesis; Mutation; Neoplasm Transplantation; Neovascularization, Pathologic; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Sphingolipids

Evidences suggest that tumor microenvironment may play an important role in cancer drug resistance. Sphingosine kinase 2 (SphK2) is proposed to be the key regulator of sphingolipid signaling. This study is aimed to investigate whether the combination of molecular targeting therapy using a specific inhibitor of SphK2 (ABC294640), with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can enhance the apoptosis of non-small cell lung cancer (NSCLC) cells. Our results revealed that NSCLC cells' sensitivity to TRAIL is correlated with the level of SphK2. Compared with TRAIL alone, the combination therapy enhanced the apoptosis induced by TRAIL, and knockdown of SphK2 by siRNA presented a similar effect. Combination therapy with ABC294640 increased the activity of caspase-3/8 and up-regulated the expression of death receptors (DR). Additional investigations revealed that translocation of DR4/5 to the cell membrane surface was promoted by adding ABC294640. However, expression of anti-apoptosis proteins such as Bcl(-)2 and IAPs was not significantly modified by this SphK2 inhibitor. Overall, this work demonstrates that SphK2 may contribute to the apoptosis resistance in NSCLC, thus indicating a new therapeutic target for resistant NSCLC cells.

Topics: Adamantane; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Molecular Targeted Therapy; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Protein Transport; Pyridines; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand

Sphingosine kinase 2 (SphK2) as a conserved lipid kinase has not been thoroughly elucidated in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate the expression of SphK2 in NSCLC tissues and to determine its correlation with clinicopathologic characteristics and its impact on patient prognosis. We assessed the expression of SphK2 and proliferating cell nuclear antigen (PCNA) (as a proliferative index) by immunohistochemistry in 180 NSCLC patient's formalin-fixed paraffin-embedded tissue blocks. Relationship between the expression of SphK2 and PCNA and various clinicopathological features in these patients was evaluated. We detected that expression of SphK2 was gradually upregulated from normal, metaplasia/dysplasia tissues to NSCLC tissues. At the same time, PCNA expression followed a similar pattern. Statistical analysis showed that expression of SphK2 in NSCLC tissues was strongly associated with PCNA expression, histology grade, live vaccine strain invasion, lymph node status, clinical stage, tumors size, and histology type. Patients with SphK2 overexpression in their tissues had lower overall survival (OS) and disease-free survival (DFS) rates than those with low SphK2 expression. Using uni- and multivariate analysis, we found that SphK2 overexpression was an independent prognostic factor for both OS and DFS. The expression of SphK2 parallels the progression of NSCLC, and SphK2 overexpression may represent a novel and potentially independent biomarker for the prognosis of patients with NSCLC.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Female; Gene Expression; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Proliferating Cell Nuclear Antigen; Risk Factors; Tumor Burden

Lung cancer is the leading cause of cancer-related deaths worldwide. Approximately 15% of all histological types consist of small cell lung cancer (SCLC). Chemotherapy is one of the major treatment method. Though the current first-line standard chemotherapy regimen for SCLC is active in most SCLC cases, however the disease recurs shortly after the first successful treatment with multi-drug resistance (MDR) phenotype. Our previously study showed that SPHK1 was associated with MDR in SCLC. The aim of this study is to investigate the role of sphingosine kinase 1 (SPHK1) showed in small cell lung multi-drug resistance.. Firstly, the analysis of QRT-PCR and Western blot were used to study differential expression of SPHK1 from mRNA and protein levels in both the H69 and H69AR cell lines. Then, Downregulation of SPHK1 by transfection with siRNA in H69AR. Moreover, the sensitivities of cells to chemotherapy drugs such as ADM, DDP, VP-16 were detected by CCK8 assay. The change of cell cycle and apoptosis rate were detected by flow cytometry. Meanwhile, expression of SPHK1 in clinical specimens were detected by QT-PCR and immunohistochemistry. Relation of SPHK1 expression with clinicopathological features and prognosis of patients was studied.. The expression of SPHK1 was significantly decreased in H69AR cells that in the H69 cells. The sensitivities of H69AR cells to chemotherapy drugs were increased when up-regulated the expression of SPHK1, enforced SPHK1 expression increased cell apoptosis and the cell cycle arrest in G0/G1 phase in H69AR cells, the expression of SPHK1 was not associated with gender, age, but significantly correlated with clinical stage, chemosensitivity and overall survival (P<0.05).. Our results suggest that SPHK1 is involved in the regulation of small cell lung cancer multi-drug resistance, SPHK1 may be as potentialtarget gene to evaluate the chemosensitivity and clinical prognostic for SCLC.. 背景与目的 小细胞肺癌约占全部肺癌的15%，化疗是其主要的治疗方法之一，虽然早期对一线化疗方案敏感，但极易出现多药耐药而导致治疗失败。前期基因芯片发现SPHK1与小细胞肺癌的耐药性相关，本研究进一步探讨SPHK1在小细胞肺癌多药耐药中的作用。方法 首先通过QRT-PCR和Western blot从基因和蛋白水平检测化疗敏感细胞株H69及多药耐药细胞株H69AR中SPHK1的差异表达；转染siRNA下调H69AR细胞中的SPHK1的表达，通过CCK8检测细胞对各种化疗药物（ADM, DDP, VP-16）的敏感性变化，流式细胞仪检测细胞周期及凋亡的变化。同时收集小细胞肺癌化疗前组织和血液标本，将其分为化疗敏感组和耐药组，QRT-PCR检测小细胞肺癌患者血液标本中SPHK1的表达，免疫组化法检测小细胞肺癌患者组织标本中SPHK1的表达，分析SPHK1与小细胞肺癌患者预后相关性。结果 SPHK1在耐药细胞H69AR中的表达明显高于H69，下调H69AR中SPHK1的表达能够增加细胞对化疗药物的敏感性，促进细胞的凋亡，细胞周期发生G0/G1期阻滞，SPHK1在小细胞肺癌耐药患者中的表达较敏感患者明显增加，SPHK1的表达与患者的性别、年龄无关，与疾病的分期、对化疗的敏感性及生存时间密切相关，差异具有统计学意义（P<0.05）。结论 SPHK1参与调节小细胞肺癌多药耐药，SPHK1可作为评估小细胞肺癌化疗敏感性及临床预后的潜在靶基因。

Topics: Adult; Aged; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle Checkpoints; Cell Line, Tumor; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Phosphotransferases (Alcohol Group Acceptor); Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Small Cell Lung Carcinoma; Treatment Outcome

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Topics: Animals; Disease Models, Animal; Humans; Lung Neoplasms; Lysophospholipids; Male; Mice; Mice, Knockout; Neoplasm Metastasis; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Receptors, Lysosphingolipid; Repressor Proteins; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Urinary Bladder Neoplasms

The present study was to examine the effect of sphingosine kinase-1 (SPHK1) on chemotherapeutics-induced apoptosis in non-small cell lung cancer (NSCLC) cells, which is relatively insensitive to chemotherapy, and its clinical significance in NSCLC progression.. The correlation of SPHK1 expression and clinical features of NSCLC was analyzed in 218 paraffin-embedded archived NSCLC specimens by immunohistochemical analysis. The effect of SPHK1 on apoptosis induced by chemotherapeutics was examined both in vitro and in vivo, using Annexin V staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assays. Western blotting and luciferase analysis were performed to examine the impact of SPHK1 on the PI3K/Akt/NF-κB signaling.. The expression of SPHK1 was markedly increased in NSCLC and correlated with tumor progression and poor survival of patients with NSCLC. Upregulation of SPHK1 significantly inhibited doxorubicin- or docetaxel-induced apoptosis, associated with induction of antiapoptotic proteins Bcl-xl, c-IAP1, c-IAP2, and TRAF1. In contrast, silencing SPHK1 expression or inhibiting SPHK1 activity with specific inhibitor, SK1-I, significantly enhanced the sensitivity of NSCLC cells to apoptosis induced by chemotherapeutics both in vitro and in vivo. Moreover, we demonstrated that upregulation of SPHK1 activated the PI3K/Akt/NF-κB pathway, and that inhibition of the PI3K/Akt/NF-κB pathway abrogated the antiapoptotic effect of SPHK1 on NSCLC cells.. Our results suggest that SPHK1 is a potential pharmacologic target for the treatment of NSCLC and inhibition of SPHK1 expression or its kinase activity might represent a novel strategy to sensitize NSCLC to chemotherapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Docetaxel; Drug Resistance, Neoplasm; Drug Tolerance; Female; Genes, Reporter; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Luciferases, Renilla; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA Interference; Signal Transduction; Taxoids; Transplantation, Heterologous

Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P(1)/S1P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P(1)/S1P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P(1)/S1P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Hypoxia; Cell Line, Tumor; Culture Media; Drug Resistance, Neoplasm; Etoposide; Humans; Lung Neoplasms; Lysophospholipids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Small Interfering; Sphingosine; Transfection

Sphingosine kinase 1 (SK1) is a key enzyme critical to the sphingolipid metabolic pathway responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphate. SK1-mediated production of sphingosine-1-phosphate has been shown to stimulate such biological processes as cell growth, differentiation, migration, angiogenesis, and inhibition of apoptosis. In this study, cell type-specific immunolocalization of SK1 was examined in the bronchus/terminal bronchiole of the lung. Strong immunopositive staining was evident at the apical surface of pseudostratified epithelial cells of the bronchus and underlying smooth muscle cells, submucosal serous glands, immature chondrocytes, type II alveolar cells, foamy macrophages, endothelial cells of blood vessels, and neural bundles. Immunohistochemical screening for SK1 expression was performed in 25 samples of normal/tumor patient matched non-small-cell lung cancer tissue and found that 25 of 25 tumor samples (carcinoid [5 samples], squamous [10 samples], and adenocarcinoma tumors [10 samples]), exhibited overwhelmingly positive immunostaining for SK1 as compared with patient-matched normal tissue. In addition, an approximately 2-fold elevation of SK1 mRNA expression was observed in lung cancer tissue versus normal tissue, as well as in several other solid tumors. Taken together, these findings define the localization of SK1 in lung and provide clues as to how SK1 may play a role in normal lung physiology and the pathophysiology of lung cancer.

Topics: Adenocarcinoma; Animals; Antibody Specificity; Bronchi; Carcinoid Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Rabbits; RNA, Messenger