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     

Identification of Gαi3 as a promising target for osteosarcoma treatment

Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression. In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression.     

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     

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     

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.     

shRNA‑mediated knockdown of KNTC1 inhibits non-small-cell lung cancer through regulating PSMB8

In view of the important roles played by Kinetochore proteins in mitosis, we believed that they may contribute to the development and progression of human cancers, which has been reported recently elsewhere. Kinetochore-associated 1 (KNTC1) participates in the segregation of sister chromatids during mitosis, the effects of which on non-small-cell lung cancer (NSCLC) remain unclear. Here, we sought to identify the biological significance of KNTC1 in NSCLC. KNTC1 protein expression in NSCLC tissues was investigated by immunohistochemistry. Lentivirus delivered short hairpin RNA (shRNA) was utilized to establish KNTC1 silence NSCLC cell lines. The effects of KNTC1 depletion on NSCLC cell proliferation, migration, apoptosis, and tumor formation were analyzed by MTT assay, wound-healing assay, transwell assay, flow cytometry assay, and in nude mouse models in vivo. After KNTC1 reduction, NSCLC cell viability, proliferation, migration, and invasion were restrained. A xenograft tumor model was also provided to demonstrate the inhibited tumorigenesis in NSCLC. In addition, the downstream mechanism analysis indicated that KNTC1 depletion was positively associated with PSMB8. The findings of the present study suggested that KNTC1 may have a pivotal role in mediating NSCLC progression and may act as a novel therapeutic target for NSCLC.Subject terms: Non-small-cell lung cancer, Cell migration     

The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer

Background

SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown.

Results

Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway.

Conclusions

For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.     

Downregulation of fibroblast growth factor 5 inhibits cell growth and invasion of human nonsmall-cell lung cancer cells

The morbidity and mortality rates of nonsmall-cell lung cancer (NSCLC) have increased in recent years. We aimed to explore the biological role of fibroblast growth factor 5 (FGF5) in NSCLC. We first established that the expression of FGF5 was increased in NSCLC tissues compared with the normal adjacent tissues. The expression of FGF5 was also increased in NSCLC cell lines. The effect of FGF5 silencing on cell proliferation, cell cycle, apoptosis, migration, and invasion of H661 and CALU1 cells was then examined. Downregulation of FGF5 significantly inhibited cell proliferation and induced G1 phase cell cycle arrest compared with the negative control small interfering (siNC) groups. Cell apoptosis was promoted by siFGF5 treatment. Cell migration and invasion of H661 and CALU1 cells with siFGF5 transfection were markedly diminished compared with the siNC groups. In addition, migration and invasion-associated proteins (E-cadherin, matrix metalloproteinase-2 [MMP-2], and MMP-9) and epithelial mesenchymal transition markers (N-cadherin, vimentin, snail, and slug) were also regulated by FGF5 siRNA treatment. Gene set enrichment analysis on The Cancer Genome Atlas dataset showed that the Kyoto Encyclopedia of Genes and Genomes (KEGG) cell cycle and vascular endothelial growth factor (VEGF) pathways were correlated with FGF5 expression, which was further confirmed in NSCLC cells by Western blot analysis. Our results indicated that FGF5 silencing suppressed cell growth and invasion via regulation of the cell cycle and VEGF pathways. Therefore, FGF5 may serve as a promising therapeutic strategy for NSCLC.     

MicroRNA-193b modulates proliferation, migration, and invasion of non-small cell lung cancer cells

MicroRNAs have been reported to be closely related to the development of human lung cancers. However, the functions of microRNAs in non-small cell lung cancer (NSCLC) remain largely undefined. Here, we investigated the role of microRNA-193b (miR-193b) in NSCLC. Our data showed that miR-193b was markedly down-regulated in NSCLC cancer tissues compared with adjacent normal tissues. The NSCLC cell line (A549) transfected with the miR-193b exhibited significantly decreased proliferation, migration, and invasion capacities when compared with the control cells. In contrast, inhibition of miR-193b increased the proliferation, migration, and invasion of A549 cells. Moreover, miR-193b repressed the expressions of cyclin D1 and urokinase-type plasminogen activator in A549 cells. These data suggest that miR-193b is a tumor suppressor in NSCLC.     

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     

Circ_0025039 acts an oncogenic role in the progression of non-small cell lung cancer through miR-636-dependent regulation of CORO1C

Non-small cell lung cancer remains the leading cause of cancer-related death worldwide. Circular RNA plays vital roles in NSCLC progression. This study is designed to reveal the role of circ_0025039 in NSCLC cell malignancy. The RNA expression of circ_0025039, microRNA-636 (miR-636), and coronin 1C was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot analysis or immunohistochemistry assay. Cell proliferation, migration, invasion, tube formation ability, sphere formation capacity, and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine, transwell assay, tube formation assay, sphere formation assay, and flow cytometry analysis, respectively. Mouse model assay was conducted to reveal the effect of circ_0025039 silencing on tumor formation in vivo. The interaction between miR-636 and circ_0025039 or CORO1C was identified through dual-luciferase reporter and RNA pull-down assays. The expression of circ_0025039 and CORO1C was significantly increased, while miR-636 was decreased in NSCLC tissues and cells compared with controls. Circ_0025039 depletion repressed NSCLC cell proliferation, migration, invasion, tube-forming capacity, and sphere formation ability, but induced cell apoptosis. The neoplasm formation was repressed after circ_0025039 silencing. Additionally, circ_0025039 acted as a sponge for miR-636, which was found to target CORO1C. Importantly, the contribution of circ_0025039 to NSCLC progression was mediated by miR-636/CORO1C axis. Circ_0025039 silencing repressed NSCLC malignant progression by reducing CORO1C expression through miR-636, showing the possibility of circ_0025039 as a therapeutic target for NSCLC.

     

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.     

LncRNA DGUOK-AS1 facilitates non-small cell lung cancer growth and metastasis through increasing TRPM7 stability via m6A modification

BackgroundN6-methyladenosine (m6A) modification plays key roles in tumor progression. LncRNA deoxyguanosine kinase antisense RNA 1 (DGUOK-AS1) has been reported as a promoter in tumors, but its role and mechanism in non-small cell lung cancer (NSCLC) development remain uncertain.MethodsCell proliferation, migration, invasion and angiogenesis were investigated via CCK-8, colony formation, transwell, and tube formation assays, respectively. The location of DGUOK-AS1 was detected via FISH assay. The interaction relationship among DGUOK-AS1, IGF2BP2 and TRPM7 was confirmed by RIP and MeRIP assays. The effects of DGUOK-AS1 on NSCLC growth and metastasis in vivo were investigated using xenograft and pulmonary metastatic models.ResultsDGUOK-AS1 was upregulated in NSCLC. DGUOK-AS1 silencing inhibited NSCLC cell proliferation, migration, invasion and angiogenesis. DGUOK-AS1 was mostly expressed in cytoplasm, and positively regulated IGF2BP2. METTL3/IGF2BP2 axis could increase TRPM7 mRNA stability in m6A-dependent manner. TRPM7 overexpression reversed the inhibitive function of DGUOK-AS1 silencing on NSCLC development. DGUOK-AS1 knockdown suppressed NSCLC cell growth and metastasis in nude mice.ConclusionDGUOK-AS1 silencing restrains NSCLC cell growth and metastasis through decreasing TRPM7 stability via regulation of the METTL3/IGF2BP2-mediated m6A modification.