ADRB3 induces mobilization and inhibits differentiation of both breast cancer cells and myeloid-derived suppressor cells.

Metastatic tumors are mainly composed of neoplastic cells escaping from the primary tumor and inflammatory cells egressing from bone marrow. Cancer cell and inflammatory cell are remained in the state of immaturity during migration to distant organs. Here, we show that ADRB3 is crucial in cell mobilization and differentiation. Immunohistochemistry revealed ADRB3 expression is significantly more frequent in breast cancer tissues than in adjacent noncancerous tissues (92.1% vs. 31.5%). Expression of ADRB3 correlated with malignant degree, TNM stage and poor prognosis. Moreover, ADRB3 expression was markedly high in activated disseminated tumor cells, myeloid-derived suppressor cells (MDSCs), lymphocytes and neutrophil extracellular traps of patients. Importantly, ADRB3 promoted the expansion of MDSC through stimulation of bone marrow mobilization and inhibiting of the differentiation of immature myeloid cells. Furthermore, ADRB3 promoted MCF-7 cells proliferation and inhibited transdifferentiation into adipocyte-like cell by activating mTOR pathway. Ultimately, the MDSC-deficient phenotype of ADRB3 -/- PyMT mice was associated with impairment of mammary tumorigenesis and reduction in pulmonary metastasis. Collectively, ADRB3 promotes metastasis by inducing mobilization and inhibiting differentiation of both breast cancer cells and MDSCs.

Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells.

METHODS: Hypoxia in hBMSCs was induced for 0, 4, and 12 hours, and cellular senescence was evaluated by senescence-associated ß-galactosidase (SA-ß-gal) staining. Tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of hypoxia in hBMSCs. Parallel reaction monitoring (PRM) analysis was used to validate the candidate proteins. Verifications of signaling pathways were evaluated by western blotting. Cell apoptosis was evaluated using Annexin V/7-AAD staining by flow cytometry. The production of reactive oxygen species (ROS) was detected by the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). RESULTS: Cell senescence detected by SA-ß-gal activity was higher in the 12-hour hypoxia-induced group. TMT analysis of 12-hour hypoxia-induced cells identified over 6000 proteins, including 686 differentially expressed proteins. Based on biological pathway analysis, we found that the senescence-associated proteins were predominantly enriched in the cancer pathways, PI3K-Akt pathway, and cellular senescence signaling pathways. CDK1, CDK2, and CCND1 were important nodes in PPI analyses. Moreover, the CCND1, UQCRH, and COX7C expressions were verified by PRM. Hypoxia induction for 12 hours in hBMSCs reduced CCND1 expression but promoted ROS production and cell apoptosis. Such effects were markedly reduced by the PI3K agonist, 740 Y-P, and attenuated by LY294002. CONCLUSIONS: Hypoxia of hBMSCs inhibited CCND1 expression but promoted ROS production and cell apoptosis through activating the PI3K-dependent signaling pathway. These findings provided a detailed characterization of the proteomic profiles related to hypoxia-induced senescence of hBMSCs and facilitated our understanding of the molecular mechanisms leading to stem cell senescence.

ADRB3 expression in tumor cells is a poor prognostic factor and promotes proliferation in non-small cell lung carcinoma.

The cross-talk between cancer cells and monocyte-derived alveolar macrophages (Mo-AMs) promotes non-small cell lung carcinoma (NSCLC) progression. In this study, we report that both cancer cells and Mo-AMs robustly express beta 3-adrenergic receptor (ADRB3) in NSCLC. ADRB3 supports lung cancer cells proliferation and promotes chronic inflammation. Genetic and pharmacologic inhibition of ADRB3 reverses tumor growth and inflammation in mouse. Furthermore, we demonstrate that M5D1, a novel anti-ADRB3 monoclonal antibody, inhibits human lung cancer cells proliferation and inflammation via affecting the intracellular mTOR pathway and activating p53. In NSCLC patients, we confirmed that upregulation of ADRB3 expression correlates with tumor progression and poor prognosis. Altogether, these results shed light on the role of ADRB3 in NSCLC and suggest that M5D1 could become powerful antitumor weapons.

MicroRNA-758 inhibits cervical cancer cell proliferation and metastasis by targeting HMGB3 through the WNT/ß-catenin signaling pathway.

Cervical cancer (CC) remains a highly prevalent cancer and cause of mortality amongst women worldwide. miR-758 has been demonstrated to be associated with tumorigenesis by controlling the expression of oncogenic or tumor suppressor genes. However, the function and mechanisms of miR-758 in CC have not been well illustrated. The present study aimed to dissect the effect of miR-758 on the proliferation, migration and invasion of CC cells and determine the potential underlying molecular mechanism of these effects. qPCR results revealed that the expression of miR-758 was significantly decreased in CC tissues and cell lines compared with that in normal tissues and normal cells. Results of CCK-8, colony formation and Transwell assays revealed that miR-758 overexpression markedly decreased cell viability, proliferation, invasion and migration. However, miR-758 inhibitors significantly increased viability, proliferation, invasion and migration. In the mechanism study, we demonstrated that high mobility group box 3 (HMGB3) was a direct target of miR-758, and HMGB3 overexpression rescued the viability, proliferation, invasion and migration of HeLa cells reduced by an miR-758 mimic. It was demonstrated that HMGB3 regulated the WNT/ß-catenin signaling pathway under miR-758 regulation. In summary, these observations suggested that miR-758 is a tumor suppressor gene that can inhibit the metastatic phenotype of CC cells by negatively regulating HMGB3, which may present a path to novel therapeutic stratagems for CC therapy.

Fn14, a Downstream Target of the TGF-ß Signaling Pathway, Regulates Fibroblast Activation.

Fibrosis, the hallmark of human injuries and diseases such as serious burns, is characterized by excessive collagen synthesis and myofibroblast accumulation. Transforming growth factor-ß (TGF-ß), a potent inducer of collagen synthesis, has been implicated in fibrosis in animals. In addition to TGF-ß, fibroblast growth factor-inducible molecule 14 (Fn14) has been reported to play an important role in fibrotic diseases, such as cardiac fibrosis. However, the function and detailed regulatory mechanism of Fn14 in fibrosis are unclear. Here, we investigated the effect of Fn14 on the activation of human dermal fibroblasts. In normal dermal fibroblasts, TGF-ß signaling increased collagen production and Fn14 expression. Furthermore, Fn14 siRNA blocked extracellular matrix gene expression; even when TGF-ß signaling was activated by TGF-ß1, fibroblast activation remained blocked in the presence of Fn14 siRNA. Overexpressing Fn14 increased extracellular matrix gene expression. In determining the molecular regulatory mechanism, we discovered that SMAD4, an important TGF-ß signaling co-mediator, bound to the Fn14 promoter and activated Fn14 transcription. Taken together, these results indicate that the TGF-ß signaling pathway activates Fn14 expression through the transcription factor SMAD4 and that activated Fn14 expression increases extracellular matrix synthesis and fibroblast activation. Therefore, Fn14 may represent a promising approach to preventing the excessive accumulation of collagen or ECM in skin fibrosis.

Slc35c2 promotes Notch1 fucosylation and is required for optimal Notch signaling in mammalian cells.

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.

The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling.

Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-beta superfamily. Mouse embryos lacking either Cripto or Nodal have the same lethal phenotype at embryonic day 7.5. Previous studies suggest that O-fucosylation of the epidermal growth factor-like (EGF) repeat in Cripto is essential for the facilitation of Nodal signaling. Substitution of Ala for the Thr to which O-fucose is attached led to functional inactivation of both human and mouse Cripto. However, embryos null for protein O-fucosyltransferase 1, the enzyme that adds O-fucose to EGF repeats, do not exhibit a Cripto null phenotype and die at about embryonic day 9.5. This suggested that the loss of O-fucose from the EGF repeat may not have led to the inactivation of Cripto in previous studies. Here we investigate this hypothesis and show the following: 1) protein O-fucosyltransferase 1 is indeed the enzyme that adds O-fucose to Cripto; 2) Pofut1(-/-) embryonic stem cells behave the same as Pofut1(+/+) embryonic stem cells in a Nodal signaling assay; 3) Pofut1(-/-) and Pofut1(+/+) embryoid bodies are indistinguishable in their ability to differentiate into cardiomyocytes; and 4) none of 10 amino acid substitutions at Thr(72), including Ser which acquires O-fucose, rescues the activity of mouse Cripto in Nodal signaling assays. Therefore, the Thr to which O-fucose is linked in Cripto plays a key functional role, but O-fucose at Thr(72) is not required for Cripto to function in cell-based signaling assays or in vivo. By contrast, we show that O-fucose, and not the Thr to which it is attached, is required in the ligand-binding domain of Notch1 for Notch1 signaling.

Activation of bombesin receptor subtype-3 stimulates adhesion of lung cancer cells.

Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor having sequence homologies to gastrin-releasing peptide and neuromedin B receptors. [d-Phe6, beta-Ala11, Phe13, Nle14]bombesin(6-14) is known to act as a synthetic receptor agonist for BRS-3. To characterize BRS-3-mediated biological responses, we examined the effect of BRS-3 activation by [d-Phe6, beta-Ala11, Phe13, Nle14]Bn(6-14) on the adhesion of the small cell lung cancer NCI-N417 cells that express native BRS-3. We found that the BRS-3 agonist stimulated adhesion of NCI-N417 cells in laminin-coated culture wells. The adhesion of the cells to laminin induced by BRS-3 activation was accompanied by an increase in vinculin-like immunoreactivity and diminished in the presence of an anti-beta1 integrin antibody, suggesting that the receptor activation stimulates focal adhesion formation. We suggest that BRS-3 may be involved in invasion and metastasis of certain cancer cells, like small cell lung cancer cells, upon attachment to laminin.

Interactions between E2F1 and SirT1 regulate apoptotic response to DNA damage.

The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2 (silent information regulator 2) regulates gene silencing in yeast and promotes lifespan extension during caloric restriction. The mammalian homologue of Sir2 (SirT1) regulates p53, NF-kappaB and Forkhead transcription factors, and is implicated in stress response. This report shows that the cell-cycle and apoptosis regulator E2F1 induces SirT1 expression at the transcriptional level. Furthermore, SirT1 binds to E2F1 and inhibits E2F1 activities, forming a negative feedback loop. Knockdown of SirT1 by small interference RNA (siRNA) increases E2F1 transcriptional and apoptotic functions. DNA damage by etoposide causes E2F1-dependent induction of SirT1 expression and knockdown of SirT1 increases sensitivity to etoposide. These results reveal a mutual regulation between E2F1 and SirT1 that affects cellular sensitivity to DNA damage.

Activation of p27Kip1 Expression by E2F1. A negative feedback mechanism.

The E2F1 transcription factor is a critical regulator of cell cycle due to its ability to promote S phase entry. However, E2F1 overexpression also sensitizes cells to apoptosis and E2F1-null mice are predisposed to tumor development, suggesting that it also has properties of a growth suppressor. E2F1 transcription function is regulated by interaction with hypophosphorylated pRb. Cdk inhibitors such as p16INK4a and p27Kip1 inhibit pRb phosphorylation by the cyclin D/Cdk4 and cyclin E/Cdk2 complexes, thus keeping E2F1 in an inactive state. We found that E2F1 binds to the p27 promoter in vivo and activates p27 mRNA and protein expression. Depletion of endogenous E2F1 by siRNA causes a reduction in basal p27 expression level. Inhibition of endogenous p27 expression by siRNA increases E2F1 transcriptional activity and permits accelerated cell cycle progression by exogenous E2F1. These observations suggest that induction of p27 acts as a negative feedback mechanism for E2F1 and may also contribute to other functions of E2F1.