Integration of biochemical and topographic cues for the formation and spatial distribution of invadosomes in nasopharyngeal epithelial cells.

Invadosomes are invasive protrusions generated by cells which can secrete matrix metalloproteinases for focal digestion of extracellular matrix. They also aid invasive cancer cells in their transmigration through vascular endothelium. However, how the physical and chemical cues in a three-dimensional (3D) system signal the spatial localization of invadosomes remains largely unknown. Here we study the topographic guidance of invadosome formation in invasive nasopharyngeal cells under the stimulation of an inflammatory cytokine, TGF-ß1, using engineered gratings with different width and depth. We first report that TGF-ß1 can act as an external signal to upregulate the formation of invadosomes with a random distribution on a plane 2D surface. When the cells were seeded on parallel 3D gratings of 5 µm width and 1 µm depth, most of the invadosomes aligned to the edges of the gratings, indicating a topographic cue to the control of invadosome localization. While the number of invadosomes per cell were not upregulated when the cells were seeded on 3D topography, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges when the cells move at a lower speed and are guided along narrow gratings. The invadosomes forming at 3D edges also have a longer half-life than those forming on a plane surface. These data suggest that there are integrated biochemical and 3D geometric cues underlying the spatial regulation of invasive structures so as to elicit efficient invasion or metastasis of cells. STATEMENT OF SIGNIFICANCE: Nasopharyngeal cells were integrated with the biological cues and matrix topography to govern the activity and spatial distribution of invadosomes. The biochemical induction of invadosome formation by TGF-ß1 in nasopharyngeal cells was observed. When the cells were seeded on parallel 3D gratings, most of the invadosomes aligned to the edges of the gratings due to topographical induced invadosome localization. While the number of invadosomes per cell were not upregulated, guidance of invadosomes localization to edges is correlated with cell migration directionality on 1 µm deep gratings. Invadosomes preferentially form at edges with a higher stability when the cells are guided along narrow gratings. The integrated biochemical and 3D geometric cues could elicit efficient invasion or metastasis of cells.

Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.

Inhibition of c-Met downregulates TIGAR expression and reduces NADPH production leading to cell death.

c-Met represents an important emerging therapeutic target in cancer. In this study, we demonstrate the mechanism by which c-Met tyrosine kinase inhibition inhibits tumor growth in a highly invasive Asian-prevalent head and neck cancer, nasopharyngeal cancer (NPC). c-Met tyrosine kinase inhibitors (TKIs; AM7 and c-Met TKI tool compound SU11274) downregulated c-Met phosphorylation, resulting in marked inhibition of NPC cell growth and invasion. Strikingly, inhibition of c-Met resulted in significant downregulation of TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) and subsequent depletion of intracellular NADPH. Importantly, overexpression of TIGAR ameliorated the effects of c-Met kinase inhibition, confirming the importance of TIGAR downregulation in the growth inhibitory activity of c-Met TKI. The effects of c-Met inhibition on TIGAR and NADPH levels were observed with two different c-Met TKIs (AM7 and SU11274) and with multiple cell lines. As NADPH provides a crucial reducing power required for cell survival and proliferation, our findings reveal a novel mechanistic action of c-Met TKI, which may represent a key effect of c-Met kinase inhibition. Our data provide the first evidence linking c-Met, TIGAR and NADPH regulation in human cancer cells suggesting that inhibition of a tyrosine kinase/TIGAR/NADPH cascade may have therapeutic applicability in human cancers.

Impact of G2 checkpoint defect on centromeric instability.

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G(2) checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G(2) to M phase transition, impaired G(2) checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G(2) checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G(2) checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G(2) checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G(2) checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G(2) checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells.

FGF8b oncogene mediates proliferation and invasion of Epstein-Barr virus-associated nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b upregulation.

The fibroblast growth factor 8b (FGF8b) oncogene is known to be primarily involved in the tumorigenesis and progression of hormone-related cancers. Its role in other epithelial cancers has not been investigated, except for esophageal cancer, in which FGF8b overexpression was mainly found in tumor biopsies of male patients. These observations were consistent with previous findings in these cancer types that the male sex-hormone androgen is responsible for FGF8b expression. Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer of head and neck commonly found in Asia. It is etiologically associated with Epstein-Barr Virus (EBV) infection, inflammatory tumor microenvironment and relatively higher male predominance. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated non-hormone-related cancer of the head and neck, NPC. More importantly, overexpression of FGF8b mRNA and protein was detected in a large majority of NPC tumors from both male and female genders, in addition to multiple NPC cell lines. We hypothesized that FGF8b overexpression may contribute to NPC tumorigenesis. Using EBV-associated NPC cell lines, we demonstrated that specific knockdown of FGF8b by small interfering RNA inhibited cell proliferation, migration and invasion, whereas exogenous FGF8b stimulated these multiple phenotypes. Further mechanistic investigation revealed that in addition to NF-κB signaling (a major inflammatory signaling pathway known to be activated in NPC), an important EBV oncoprotein, the latent membrane protein 1 (LMP1), was found to be a direct inducer of FGF8b overexpression in NPC cells, whereas androgen (testosterone) has minimal effect on FGF8b expression in EBV-associated NPC cells. In summary, our study has identified LMP1 as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells. This novel mechanism of viral-mediated FGF8 upregulation may implicate a new role of oncoviruses in human carcinogenesis.

Molecular mechanisms for the release of chemokines from human leukemic mast cell line (HMC)-1 cells activated by SCF and TNF-alpha: roles of ERK, p38 MAPK, and NF-kappaB.

BACKGROUND: Mast cells play pivotal roles in IgE-mediated airway inflammation and other mast cell-mediated inflammation by activation and chemoattraction of inflammatory cells. OBJECTIVE: We investigated the intracellular signaling mechanisms regulating chemokine release from human mast cell line-1 (HMC-1) cells activated by stem cell factor (SCF) or tumor necrosis factor (TNF)-alpha. METHODS: Chemokine gene expressions were assessed by reverse transcription-polymerase chain reaction, while the releases of chemokines were determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA). To elucidate the intracellular signal transduction regulating the chemokine expression, phosphorylated-extracellular signal-regulated kinase (ERK), phosphorylated-p38 mitogen-activated protein kinase (MAPK) and nuclear translocated nuclear factor (NF)-kappaB-DNA binding were quantitatively assessed by ELISA. RESULTS: Either SCF or TNF-alpha could induce release from HMC-1 cells of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, regulated upon activation normal T-cell expressed and secreted (RANTES), and I-309, while SCF and TNF-alpha induced release of macrophage inflammatory protein (MIP)-1beta and interferon-gamma-inducible protein-10 (IP-10), respectively. Using various selective inhibitors for signaling molecules, we found that the inductions of IL-8, MCP-1, and I-309 were mediated by either SCF-activated ERK or TNF-alpha-activated p38 MAPK, while the induction of IP-10 by TNF-alpha was mediated by both activated p38 MAPK and NF-kappaB. The induction of RANTES by SCF or TNF-alpha was mediated by ERK and NF-kappaB, respectively, and SCF induced MIP-1beta release was mediated by ERK. CONCLUSION: The above results therefore elucidated the different intracellular signaling pathways regulating the release of different chemokines from SCF and TNF-alpha-activated mast cells, thereby shedding light for the immunopathological mechanisms of mast cell-mediated diseases.