The sodium/myo-inositol co-transporter SLC5A3 promotes non-small cell lung cancer cell growth

Identification of novel molecular signaling targets for non-small cell lung cancer (NSCLC) is important. The present study examined expression, functions and possible underlying mechanisms of the sodium/myo-inositol co-transporter SLC5A3 in NSCLC. The Cancer Genome Atlas (TCGA) database and local NSCLC tissue results demonstrated that SLC5A3 expression in NSCLC tissues (including patient-derived primary NSCLC cells) was significantly higher than that in normal lung tissues and lung epithelial cells. In primary NSCLC cells and immortalized lines, SLC5A3 depletion, using small hairpin RNA (shRNA) and CRSIRP/Cas9 methods, robustly impeded cell proliferation and migration, simultaneously provoking cell cycle arrest and apoptosis. Conversely, ectopic overexpression of SLC5A3 further enhanced proliferation and migration in primary NSCLC cells. The intracellular myo-inositol contents and Akt-mTOR activation were largely inhibited by SLC5A3 silencing or knockout (KO), but were augmented following SLC5A3 overexpression in primary NSCLC cells. Significantly, SLC5A3 KO-induced anti-NSCLC cell activity was largely ameliorated by exogenously adding myo-inositol or by a constitutively-active Akt construct. By employing the patient-derived xenograft (PDX) model, we found that the growth of subcutaneous NSCLC xenografts in nude mice was largely inhibited by intratumoral injection SLC5A3 shRNA adeno-associated virus (AAV). SLC5A3 silencing, myo-inositol depletion, Akt-mTOR inactivation and apoptosis induction were detected in SLC5A3 shRNA virus-injected NSCLC xenograft tissues. Together, elevated SLC5A3 promotes NSCLC cell growth possibly by maintaining myo-inositol contents and promoting Akt-mTOR activation.Subject terms: Non-small-cell lung cancer, Targeted therapies     

AMPK activation by ASP4132 inhibits non-small cell lung cancer cell growth

Activation of adenosine monophosphate-activated protein kinase (AMPK) is able to produce significant anti-non-small cell lung cancer (NSCLC) cell activity. ASP4132 is an orally active and highly effective AMPK activator. The current study tested its activity against NSCLC cells. In primary NSCLC cells and established cell lines (A549 and NCI-H1944) ASP4132 potently inhibited cell growth, proliferation and cell cycle progression as well as cell migration and invasion. Robust apoptosis activation was detected in ASP4132-treated NSCLC cells. Furthermore, ASP4132 treatment in NSCLC cells induced programmed necrosis, causing mitochondrial p53-cyclophilin D (CyPD)-adenine nucleotide translocase 1 (ANT1) association, mitochondrial depolarization and medium lactate dehydrogenase release. In NSCLC cells ASP4132 activated AMPK signaling, induced AMPKα1-ACC phosphorylation and increased AMPK activity. Furthermore, AMPK downstream events, including mTORC1 inhibition, receptor tyrosine kinases (PDGFRα and EGFR) degradation, Akt inhibition and autophagy induction, were detected in ASP4132-treated NSCLC cells. Importantly, AMPK inactivation by AMPKα1 shRNA, knockout (using CRISPR/Cas9 strategy) or dominant negative mutation (T172A) almost reversed ASP4132-induced anti-NSCLC cell activity. Conversely, a constitutively active AMPKα1 (T172D) mimicked and abolished ASP4132-induced actions in NSCLC cells. In vivo, oral administration of a single dose of ASP4132 largely inhibited NSCLC xenograft growth in SCID mice. AMPK activation, mTORC1 inhibition and EGFR-PDGFRα degradation as well as Akt inhibition and autophagy induction were detected in ASP4132-treated NSCLC xenograft tumor tissues. Together, activation of AMPK by ASP4132 potently inhibits NSCLC cell growth in vitro and in vivo.Subject terms: Targeted therapies, Non-small-cell lung cancer     

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

The pro-tumorigenic activity of p38γ overexpression in nasopharyngeal carcinoma

It is urgent to identify and validate biomarkers for early diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC). Recent studies have proposed p38 gamma (p38γ) as a cyclin-dependent kinase (CDK)-like kinase that phosphorylates retinoblastoma (Rb) to promote cyclins expression and tumorigenesis. Here the Gene Expression Profiling Interactive Analysis (GEPIA) database and results from the local NPC tissues demonstrate that p38γ is significantly upregulated in NPC tissues, correlating with poor overall survival. Furthermore, p38γ mRNA and protein expression is elevated in established NPC cell lines (CNE-1 HONE-1 and CNE-2) and primary human NPC cells, but low expression detected in human nasal epithelial cells. In established and primary NPC cells, p38γ depletion, using the shRNA strategy or the CRISPR/Cas9 gene-editing method, largely inhibited cell growth, proliferation and migration, and induced significant apoptosis activation. Contrarily, ectopic p38γ overexpression exerted opposite activity and promoted NPC cell proliferation and migration. Retinoblastoma (Rb) phosphorylation and cyclin E1/A expression were decreased in NPC cells with p38γ silencing or knockout, but increased after p38γ overexpression. Moreover, mitochondrial subcellular p38γ localization was detected in NPC cells. Significantly, p38γ depletion disrupted mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production, oxidative injury and ATP depletion in NPC cells. In vivo, intratumoral injection of adeno-associated virus-packed p38γ shRNA potently inhibited primary human NPC xenograft growth in nude mice. In p38γ shRNA virus-injected NPC xenograft tissues, p38γ expression, Rb phosphorylation, cyclin E1/A expression and ATP levels were dramatically decreased. Taken together, we conclude that p38γ overexpression is required for NPC cell growth, acting as a promising therapeutic target of NPC.Subject terms: Targeted therapies, Oncogenes     

Elevated expression of nuclear receptor-binding SET domain 3 promotes pancreatic cancer cell growth

The nuclear receptor-binding SET domain 3 (NSD3) catalyzes methylation of histone H3 at lysine 36 (H3K36), and promotes malignant transformation and progression of human cancer. Its expression, potential functions and underlying mechanisms in pancreatic cancer are studied. Bioinformatics studies and results from local human tissues show that NSD3 is upregulated in human pancreatic cancer tissues, which is correlated with poor overall survival. In primary and established pancreatic cancer cells, NSD3 silencing (by shRNAs) or CRISPR/Cas9-induced NSD3 knockout potently inhibited cell proliferation, migration and invasion, while provoking cell cycle arrest and apoptosis. Conversely, ectopic expression of NSD3-T1232A mutation significantly accelerated proliferation, migration, and invasion of pancreatic cancer cells. H3K36 dimethylation, expression of NSD3-dependent genes (Prkaa2, Myc, Irgm1, Adam12, and Notch3), and mTOR activation (S6K1 phosphorylation) were largely inhibited by NSD3 silencing or knockout. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD3 shRNA potently inhibited pancreatic cancer xenograft growth in nude mice. These results suggest that elevated NSD3 could be an important driver for the malignant progression of pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

Vaccinia H1-related phosphatase is a phosphatase of ErbB receptors and is down-regulated in non-small cell lung cancer

Vaccinia H1-related phosphatase (VHR) is classified as a dual specificity phosphatase. Unlike typical dual specificity phosphatases, VHR lacks the MAPK-binding domain and shows poor activity against MAPKs. We found that EGF receptor (EGFR) was a direct substrate of VHR and that overexpression of VHR down-regulated EGFR phosphorylation, particularly at Tyr-992 residue. Expression of VHR inhibited the activation of phospholipase Cγ and protein kinase C, both downstream effectors of Tyr-992 phosphorylation of EGFR. Decreasing VHR expression by RNA interference caused higher EGFR phosphorylation at Tyr-992. In addition to EGFR, VHR also directly dephosphorylated ErbB2. Consistent with these results, suppression of VHR augmented the foci formation ability of H1299 non-small cell lung cancer (NSCLC) cells, whereas overexpression of VHR suppressed cell growth in both two- and three-dimensional cultures. Expression of VHR also suppressed tumor formation in a mouse xenograft model. Furthermore, VHR expression was significantly lower in NSCLC tissues in comparison to that in normal lung tissues. Collectively, this study shows that down-regulation of VHR expression enhances the signaling of ErbB receptors and may be involved in NSCLC pathogenesis.     

HBO1 overexpression is important for hepatocellular carcinoma cell growth

Hepatocellular carcinoma (HCC) is a common primary liver malignancy lacking effective molecularly-targeted therapies. HBO1 (lysine acetyltransferase 7/KAT7) is a member of MYST histone acetyltransferase family. Its expression and potential function in HCC are studied. We show that HBO1 mRNA and protein expression is elevated in human HCC tissues and HCC cells. HBO1 expression is however low in cancer-surrounding normal liver tissues and hepatocytes. In HepG2 and primary human HCC cells, shRNA-induced HBO1 silencing or CRISPR/Cas9-induced HBO1 knockout potently inhibited cell viability, proliferation, migration, and invasion, while provoking mitochondrial depolarization and apoptosis induction. Conversely, ectopic overexpression of HBO1 by a lentiviral construct augmented HCC cell proliferation, migration and invasion. In vivo, xenografts-bearing HBO1-KO HCC cells grew significantly slower than xenografts with control HCC cells in severe combined immunodeficient mice. These results suggest HBO1 overexpression is important for HCC cell progression.Subject terms: Targeted therapies, Oncogenes     

Lentiviral delivery of a shRNA sequence analogous to miR-4319/miR-125-5p induces apoptosis in NSCLC cells by arresting G2/M phase

In this study, we explored the therapeutic potential of microRNA (miR) analogs against non–small-cell lung cancer (NSCLC) using lentiviral delivery of short hairpin RNA (shRNA). By using A549 as a model cell line, we used analogs and mimics of miR-4319/miR-125-5p to target the tumorigenic RAF1 gene. Lentiviral vectors carrying shRNA of a highly efficient miRNA analog of miR-4319/miR-125-5p, Analog2, were constructed to infect A549 cells. Our results showed that, compared with the noncancerous bronchial epithelial cell line 16HBE, lentivirus delivering Analog2 shRNA induced significant G2/M arrest and subsequent apoptosis in A549 cells, but not in 16HBE cells. Western blot analysis revealed that key factors regulating cell cycle were downregulated following RAF1 inhibition. In vivo xenograft experiments showed that lentivirus carrying Analog2 shRNA markedly decreased tumor size. Therefore, lentiviral delivery of Analog2 shRNA is a valid RNA interference-based treatment against NSCLC with high potency and specificity.     

AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR-96-5p-AIMP3-p53 axis

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a tumour suppressor, however, the roles of AIMP3 in non-small cell lung cancer (NSCLC) are not explored yet. Here, we reported that AIMP3 significantly inhibited the cell growth and metastasis of NSCLC (lung adenocarcinoma) in vitro and in vivo. We have firstly identified that AIMP3 was down-regulated in human NSCLC tissues compared with adjacent normal lung tissues using immunohistochemistry and western blot assays. Overexpression of AIMP3 markedly suppressed the proliferation and migration of cancer cells in a p53-dependent manner. Furthermore, we observed that AIMP3 significantly suppressed tumour growth and metastasis of A549 cells in xenograft nude mice. Mechanically, we identified that AIMP3 was a direct target of miR-96-5p, and we also observed that there was a negative correlation between AIMP3 and miR-96-5p expression in paired NSCLC clinic samples. Ectopic miR-96-5p expression promoted the proliferation and migration of cancer cells in vitro and tumour growth and metastasis in vivo which partially depended on AIMP3. Taken together, our results demonstrated that the axis of miR-96-5p-AIMP3-p53 played an important role in lung adenocarcinoma, which may provide a new strategy for the diagnosis and treatment of NSCLC.     

Angio-associated migratory cell protein interacts with epidermal growth factor receptor and enhances proliferation and drug resistance in human non-small cell lung cancer cells

Angio-associated migratory cell protein (AAMP) is expressed in some human cancer cells. Previous studies have shown AAMP high expression predicted poor prognosis. But its biological role in non-small cell lung cancer (NSCLC) cells is still unknown. In our present study, we attempted to explore the functions of AAMP in NSCLC cells. According to our findings, AAMP knockdown inhibited lung cancer cell proliferation and inhibited lung cancer cell tumorigenesis in the mouse xenograft model. Epidermal growth factor receptor (EGFR) is a primary receptor tyrosine kinase (RTK) that promotes proliferation and plays an important role in cancer pathology. We found AAMP interacted with EGFR and enhanced its dimerization and phosphorylation at tyrosine 1173 which activated ERK1/2 in NSCLC cells. In addition, we showed AAMP conferred the lung cancer cells resistance to chemotherapeutic agents such as icotinib and doxorubicin. Taken together, our data indicate that loss of AAMP from NSCLC inhibits tumor growth and elevates drug sensitivity, and these findings have clinical implications to treat NSCLC cancers.     

CXCR4/MIF axis amplifies tumor growth and epithelial-mesenchymal interaction in non-small cell lung cancer

Overexpression of C-X-C chemokine receptor type 4 (CXCR4) has been shown in several cancers, including non-small cell lung cancer (NSCLC) and is linked to early metastasis and worse prognosis. The crosstalk between cancer cells and tumor stroma promotes the growth and metastasis and CXCR4 signaling is a key element of this crosstalk.To test the effects of CXCR4 overexpression (CXCR4-OE), we transduced the human NSCLC cell line A549 by using a lentiviral vector. A 3D cell culture model showed generations of tumorspheres and the effects derived by the co-culturing of lung fibroblasts. Using a xenograft mouse model, we also studied the effects of CXCR4-OE in pulmonary cell engraftment and tumor burden in vivo.Our data indicate that CXCR4-OE leads to increased tumorsphere formation and epithelial-mesenchymal transition (EMT). CXCR4-OE by A549 cells resulted in a significant increase in the production of the CXCR4-ligand macrophage migration inhibitory factor (MIF) compared to those transduced with an empty vector (EV) or in which the CXCR4 expression was deleted (KO). In our in vitro system, we did not detect any production of the canonical CXCR4 ligand CXCL12. Autocrine MIF production and CXCR4 signaling are part of a self-perpetuating loop that amplifies tumor growth and EMT. Co-culture with lung fibroblasts further increased tumorsphere formation, partially driven by an increase in IL-6 production. When A549 cells were injected into murine lungs, we observed more abundant and significantly larger tumor lesions in recipients of CXCR4-OE A549 cells compared to those receiving EV or KO cells, consistent with our in vitro findings. Treatment of mice with the MIF antagonist ISO-1 resulted in significantly less tumor burden.In conclusion, our data highlight the role of the CXCR4-OE/MIF/IL-6 axis in epithelial mesenchymal crosstalk and NSCLC progression.     

CXCL12 induces lung cancer cell migration by polarized mtDNA redistribution

Instability of mitochondrial DNA (mtDNA) has been associated with the initiation and development of cancer, but the specific role of mtDNA in the invasiveness and migration of cancer cells remains unclear. In this study, we investigated whether the chemokine CXCL12 causes intact mitochondria to redistribute in cancer cells and, in this way, to increase cell invasiveness and migration. A549 lung cancer cells with intact mtDNA (mtDNA+) and ρ⁰A549 cells depleted of mtDNA (mtDNA?) by long-term ethidium bromide incubation were examined for their responses to CXCL12 in a transwell migration assay and for mitochondrial distribution by fluorescence microscopy. Intact A549 cells showed significantly increased migration and increased polar distribution of mitochondria (asymmetry) in response to CXCL12. However, ρ⁰A549 cells showed no changes in mitochondrial distribution in response to CXCL12, and only a few ρ⁰A549 cells migrated across the transwell membrane after CXCL12 treatment. These results demonstrate that, in A549 lung cancer cells, intact mitochondrial DNA is necessary for mitochondrial redistribution and a chemotactic response to CXCL12.     

Long non‐coding RNA SNHG15 promotes CDK14 expression via miR‐486 to accelerate non‐small cell lung cancer cells progression and metastasis

Long non‐coding RNAs (lncRNAs) have been validated to play important role in multiple cancers, including non‐small cell lung cancer (NSCLC). In present study, our team investigate the biologic role of SNHG15 in the NSCLC tumorigenesis. LncRNA SNHG15 was significantly upregulated in NSCLC tissue samples and cells, and its overexpression was associated with poor prognosis of NSCLC patients. In vitro, loss‐of‐functional cellular experiments showed that SNHG15 silencing significantly inhibited the proliferation, promoted the apoptosis, and induced the cycle arrest at G0//G1 phase. In vivo, xenograft assay showed that SNHG15 silencing suppressed tumor growth of NSCLC cells. Besides, SNHG15 silencing decreased CDK14 protein expression both in vivo and vitro. Bioinformatics tools and luciferase reporter assay confirmed that miR‐486 both targeted the 3′‐UTR of SNHG15 and CDK14 and was negatively correlated with their expression levels. In summary, our study conclude that the ectopic overexpression of SNHG15 contribute to the NSCLC tumorigenesis by regulating CDK14 protein via sponging miR‐486, providing a novel insight for NSCLC pathogenesis and potential therapeutic strategy for NSCLC patients.