Rab8 GTPase regulates Klotho-mediated inhibition of cell growth and progression by directly modulating its surface expression in human non-small cell lung cancer.

BACKGROUND: The klotho (KL) gene is an anti-aging gene that has recently been shown to also function as a general tumor suppressor. However, there is currently only limited information regarding the potential molecular signals for regulation of Klotho without identifying precise molecular mechanisms or interactions. METHODS: We performed a mass spectrometry (MS) assay to screen candidate proteins complexed with Klotho derived from immunoprecipitation in human non-small cell lung cancer (NSCLC) cells, and identified Rab8 to be the protein that most prominently interacts with Klotho. We further investigated whether Rab8 can regulate trafficking of Klotho and which process it would modulate using surface biotinylation assay, immunofluorescence and fluorescence ratio microscopy. Furthermore, we explored whether Rab8 is involved in Klotho-mediated function in NSCLC, and verified the results which we found in vivo using xenograft mouse model. FINDINGS: We report discovery of Rab8 as a Klotho-interacting protein that acts as a critical modulator of Klotho surface expression in human NSCLC. In particular, we report that Rab8 is co-localized and associated with Klotho, and Klotho trafficking is regulated by Rab8. Moreover, we found that Rab8 modulates surface levels of Klotho via a post-biosynthetic pathway, as opposed to an endocytic pathway. Furthermore, we demonstrate that Rab8 is involved in Klotho-mediated regulation of cell proliferation, migration, invasiveness, epithelial-mesenchymal transition (EMT), and Wnt-ß-catenin signaling in NSCLC. Additionally, Rab8 overexpression was also found to increase Klotho-mediated inhibition of NSCLC tumorigenesis in vivo. INTERPRETATION: Overall, our findings suggest that Rab8 GTPase can regulate Klotho-mediated inhibition of Wnt signaling activity by modulating translocation of Klotho onto the cell surface, which in turn affects Klotho-mediated inhibition of cell proliferation, migration and invasiveness in NSCLC. These results have important implications for the development of new therapeutic targets, Klotho-related research in the context of NSCLC as well as other areas, and provide a working model for Rab8 function in the context of cancer and cancer biology.

Overexpression of Klotho Inhibits HELF Fibroblasts SASP-related Protumoral Effects on Non-small Cell Lung Cancer Cells.

Lung cancer (LC) is the most common cause of death from cancer worldwide, and it is also a closely aging-related disease. Klotho, a new anti-aging gene, has been proven to play a critical role in regulating aging and the development of age-related diseases including LC. However, whether Klotho is a key link between aging and LC is still unknown. Here we report that Klotho can indirectly inhibit LC growth and development through regulating senescence-associated secretory phenotype (SASP). We found that senescent lung fibroblasts (SLF) can promote production of IL-6 and IL-8, which can be effectively inhibited by overexpressing Klotho. Using conditioned medium (CM) derived from SLF to culture LC cells, the LC cells show obvious increase of viability and migration rates, significant increase expression of p-STAT3 and α-SMA, and decrease expression of P53 and E-cadherin. However, using CM derived from SLF overexpressed Klotho to culture LC cells, all above results are nearly completely reversed. Thus, these results suggest that Klotho can regulate SLF extracellular release of IL-6 and IL-8, which can influence STAT3 activation, P53 expression and epithelial-mesenchymal transition (EMT) of LC cells, finally inhibiting LC cells growth and migration indirectly.

DNA damage induced by human CD40 ligand mutant promotes senescence and induces demethylation of GATA4 in lung cancer.

The ligand of CD40, known as CD154 or CD40L, is the key to immunostimulatory and anticancer activity, but how CD40L affects cellular senescence is unclear. Thus, we studied a membrane­stable mutant form CD40L (CD40L­M) to explore tumor growth and cellular senescence in CD40­positive NSCLC cells. We found that CD40L­M­expressing cells had senescent characteristics, including reduced cell proliferation and enlargement, increased SA­ß­gal staining activity, and overexpression of several cell cycle regulators p53 and p21. In addition, expression of GATA4 was restored, and the NF­κB signaling pathway was activated in the CD40L­M­induced senescent cells. Mechanistic analyses revealed that CD40L­M expression triggered the ATM/Chk2 DNA damage response, which mediated cell senescence and GATA4 activation. Knockdown of GATA4 reversed CD40L­M­induced senescence and decreased NF­κB activity. Thus, CD40L­M contributes to induction of cell senescence in CD40­positive NSCLC cells, and GATA4 is a switch to activate the NF­κB pathway, which is positively regulated by DNA damage response (DDR) signaling kinases. Collectively, CD40L­M­induced senescence may be a barrier to the growth of lung cancer cells.

TAZ inhibition restores sensitivity of cisplatin via AKT/mTOR signaling in lung adenocarcinoma.

The cisplatin-based doublet remains the foundation of treatment for the majority of patients with advanced non-small cell lung cancer (NSCLC), however, many patients exhibit acquired drug resistance. Transcriptional co-activator with PDZ binding motif (TAZ) is an oncogene in lung cancer, important for lung tumorigenesis and metastasis. Recently, it was revealed that TAZ upregulation confers resistance against a diverse range of cytotoxic agents. The present study aimed to investigate whether TAZ is involved in cisplatin sensitivity in lung adenocarcinoma. In the present study, we investigated TAZ expression in lung adenocarcinoma tissues and cell lines with western blot and RT-PCR analyses. By knocking down TAZ using short hairpin RNAs in cisplatin-resistant A549 and H460 cells, cell proliferation and apoptosis were assessed by BrdUrd labeling and flow cytometric analysis, respectively. The IC50 value of cisplatin was also assessed with CCK-8 assays. The western blotting for AKT, p-AKT, S6K and p-S6K were performed in cells with TAZ knockdown or overexpression. When the AKT/mTOR pathway was blocked in A549 and H460 with TAZ overexpression, cisplatin sensitivity was assessed by IC50 value. High expression of TAZ was found in lung adenocarcinoma tissues and cell lines, which were associated with cisplatin resistance. Knockdown of TAZ using shRNAs confered decreasing proliferation, increasing apoptosis and enhanced cisplatin sensitivity in cisplatin­resistant cells. Additionally, TAZ knockdown decreased the AKT/mTOR pathway expression. Overexpression of TAZ increased p-AKT and p-S6K, which was inhibited by siAKT. Furthermore, we found that the inhibition of the AKT/mTOR pathway rescued the cells from cisplatin resistance caused by TAZ overexpression. Our data revealed that TAZ inhibition restores sensitivity of cisplatin in lung adenocarcinoma, which was, at least in part, AKT/mTOR signaling pathway-dependent. TAZ may be a potent therapeutic target for NSCLC in combination with conventional chemotherapy.

Depleted aldehyde dehydrogenase 1A1 (ALDH1A1) reverses cisplatin resistance of human lung adenocarcinoma cell A549/DDP.

BACKGROUND: Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible. METHODS: Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay. RESULTS: ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity. CONCLUSION: These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin.

Near-Infrared Light-Responsive Poly(N-isopropylacrylamide)/Graphene Oxide Nanocomposite Hydrogels with Ultrahigh Tensibility.

Novel near-infrared (NIR) light-responsive poly(N-isopropylacrylamide)/graphene oxide (PNIPAM-GO) nanocomposite hydrogels with ultrahigh tensibility are prepared by incorporating sparse chemical cross-linking of small molecules with physical cross-linking of graphene oxide (GO) nanosheets. Combination of the GO nanosheets and thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) polymeric networks provides the hydrogels with an excellent NIR light-responsive property. The ultrahigh tensibility of PNIPAM-GO nanocomposite hydrogels is achieved by simply using a very low concentration of N,N'-methylenebis(acrylamide) (BIS) molecules as chemical cross-linkers to generate a relatively homogeneous structure with flexible long polymer chains and rare chemically cross-linked dense clusters. Moreover, the oxidized groups of GO nanosheets enable the formation of a hydrogen bond interaction with the amide groups of PNIPAM chains, which could physically cross-link the PNIPAM chains to increase the toughness of the hydrogel networks. The prepared PNIPAM-GO nanocomposite hydrogels with ultrahigh tensibility exhibit rapid, reversible, and repeatable NIR light-responsive properties, which are highly promising for fabricating remote light-controlled devices, smart actuators, artificial muscles, and so on.

Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib.

BACKGROUND: The T790M mutation of epithelial growth factor receptor (EGFR) is a major cause of the acquired resistance to EGFR tyrosine kinase inhibitor (EGFR-TKIs) treatment for lung cancer patients. The Hippo pathway effector, TAZ, has emerged as a key player in organ growth and tumorigenesis, including lung cancer. RESULTS: In this study, we have discovered high TAZ expression in non-small cell lung cancer (NSCLC) cells harboring dual mutation and TAZ depletion sensitized their response to EGFR-TKIs. Mechanistically, knockdown of TAZ in T790M-induced resistant cells leaded to reduced anchorage-independent growth in vitro, tumor formation and resistance to gefitinib in vivo, correlated with epithelial-mesenchymal transition (EMT) and suppressed migration and invasion. Furthermore, we confirmed CTGF and AXL, novel EMT markers and potential therapeutic targets for overcoming EGFR inhibitor resistance, as directly transcriptional targets of TAZ. CONCLUSIONS: Taken together, this study suggests that expression of TAZ is an intrinsic mechanism of T790M-induced resistance in response to EGFR-TKIs. Combinational targeting on both EGFR and TAZ may enhance the efficacy of EGFR-TKIs in acquired resistance of NSCLC.

Self-complementary adeno-associated virus 5-mediated gene transduction of a novel CD40L mutant confers direct antitumor effects in lung carcinoma.

CD40 ligand (CD40L) gene therapy offers a potentially useful option for lung cancer due to its multiple antitumor activities. However, membrane-bound CD40L may be proteolytically cleaved to form soluble CD40L (sCD40L), which results in adverse effects. In a previous study by our group, it was demonstrated that recombinant self-complementary adeno-associated virus 5 (scAAV5) efficiently delivered genes to lung cancer cells. In the present study, an scAAV5 expressing a non-cleavable human CD40L mutant (scAAV5-CD40L-M) was generated and its direct antitumor effects in lung cancer were evaluated. Transduction with scAAV5-CD40L-M resulted in effective expression of CD40L on the cell surface with low levels of cleaved sCD40L, which significantly reduced the percentage of viable cells and promoted caspase-3-dependent apoptosis of CD40-positive lung carcinoma A549 cells, compared with scAAV5-CD40L transduction (P<0.05). Furthermore, treatment with scAAV5-CD40L-M exerted a significant antitumor effect against CD40-positive A549 xenografts by inducing apoptosis (P<0.05) with few side effects. Gene therapy using an scAAV5 vector expressing non-cleavable human CD40L mutant may therefore have direct antitumor effects against CD40-positive lung cancers. These tumoricidal effects of scAAV5-CD40L-M treatment make it a promising therapeutic technique for the treatment of lung cancer.

In situ fabrication of a temperature- and ethanol-responsive smart membrane in a microchip.

Here we report a simple and versatile strategy for the in situ fabrication of nanogel-containing smart membranes in microchannels of microchips. The fabrication approach is demonstrated by the in situ formation of a chitosan membrane containing poly(N-isopropylacrylamide) (PNIPAM) nanogels in a microchannel of a microchip. The PNIPAM nanogels, that allow temperature- and ethanol-responsive swelling-shrinking volume transitions, serve as smart nanovalves for controlling the diffusional permeability of solutes across the membrane. Such self-regulation of the membrane permeability is investigated by using fluorescein isothiocyanate (FITC) as a tracer molecule. This approach provides a promising strategy for the in situ fabrication of versatile nanogel-containing smart membranes within microchips via simply changing the functional nanogels for developing micro-scale detectors, sensors, separators and controlled release systems.

[The expression and significance of stem cell transcription factor Sox2 in lung carcinoma].

BACKGROUND AND OBJECTIVE: Transcription factor Sox2 remains the pluripotency of stem cell, participates in self-renew of cancer stem cell and plays important role during the initiation and development of various cancers. This study intends to investigate the expression and significance of Sox2 and Sox2 autoantibodies (Sox2-Ab) in tissue and serum of patients with non-small cell lung cancer (NSCLC). METHODS: Expression of Sox2 gene and protein was tested in 58 cases of NSCLC, 16 patients with other tumors and 20 cases of normal lung tissue specimens by quantitative PCR and immunohistochemical assay, respectively. Serum Sox2-Ab level was detected in 30 cases of NSCLC patients and 30 healthy controls by ELISA method. Clinical and pathological data from patients were collected and analyzed retrospectively. RESULTS: Expression levels of Sox2 mRNA and protein were higher in patient with NSCLC than other groups, with statistically significant differences (P<0.01), respectively. Meanwhile, Sox2 mRNA expression increased in NSCLC patients associated with histological type and tumor size. No significant differences in Sox2-Ab serum levels were found between NSCLC patients and normal subjects. CONCLUSIONS: Sox2 in NSCLC have a higher expression, which is closely related to histological type and tumor size. Sox2 might use as a potential biomarker and therapeutic target for lung cancer.