Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

<div><p>Background</p><p>Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.</p> <p>Material and Methods</p><p>Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.</p> <p>Results</p><p>Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.</p> <p>Conclusion</p><p>Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.</p> </div

NPC cells stimulated with fibulin-5 protein encourages cell growth and motility.

<p>(A) Hone 1 cells were treated with indicated concentrations of fibulin-5, and cell growth was analyzed on days 0-3 by MTT assay. Data were normalized against the OD₅₇₀ value on day 1 of each treatment. The results represent the mean ± SD of 3 independent experiments. (B) Migration and invasion of Hone1 cells (200×). For the migration assays, Hone1 cells stimulated with indicate concentrations of fiblin-5 protein were seeded into the top of a Transwell insert. After 24 hour, the cells on top were scraped, and the cells that had migrated to the bottom were fixed and stained with Giemsa. The relative-fold migration values for the clones were normalized against the DMSO and are represented diagrammatically.</p

Immuno-histochemical staining, and disease-specific survival of fibulin-5 in patients with NPC.

<p>(A) The intensity of fibulin-5 expression in various tissues by immunohistochemical staining were evaluated in (a) normal tissue and tumor tissues of NPC patients with stage I (b), II (c), III (d), IV (e), lymph node-negative (f), and lymph node-positive (g) samples. Original magnification, 200×. (B) Survival of patients with high fibulin-5 expression (solid line) was significantly abbreviated in comparison to those with low expression (dashed line), with a statistically significant difference in survival (<i>p</i> = 0.003) by log-rank test.</p

The activity of AKT induced cell motility is regulated by FLJ10540 in NPC cells.

<p>(A) Hone1-expressing FLJ10540 cells were serum-starved for 24 hour and treated with or without AKT inhibitor. The Total cell lysates of vehicle-Hone and FLJ10540-Hone1, transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (B) A negative control and FLJ10540 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B). (C) Vehicle-Hone1 and FLJ10540-Hone1 transfected cells were serum-starved and treated with the AKT inhibitor for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described.</p

Exogenous fibulin-5 promotes cell growth, migration, and invasion in NPC cells.

<p>(A) DDK-tagged fibulin-5 was stably transfected into Hone1 cells, 2 clones were chosen, and DDK-fibulin-5 expression was determined by western blotting with anti-DDK and anti-β-actin antibodies. (B) Cell viability of Hone1 stable cell lines was measured by MTT assay. The cells were cultured for 0-3 days followed by MTT assay (OD₅₇₀) to quantitate the cell growth. Data were normalized against the OD₅₇₀ value on day 0 of each treatment. The results represent the mean ± SD of 3 independent experiments. (C) Wound healing assays with cells expressing empty vector or fibulin-5-Hone1. Representative images captured with a 10× objective at the time of wounding or 24 hour after. All experiments were repeated at least three times. The percentage of wound closure corresponds to the distance between wound edges in at least three randomly chosen regions relative to the distance at time 0 hour for each cell. (D) Migration and invasion of vehicle-Hone1 and fibulin-5-Hone1 stable cells (200×). For the migration assays, cells (vehicle-Hone1, fibulin-5-Hone1 stable clones) were seeded into the top of a Transwell insert. After 24 hour, the cells on top were scraped, and the cells that had migrated to the bottom were fixed and stained with Giemsa. The relative-fold migration values for the clones were normalized against the vehicle control and are represented diagrammatically. For the invasion assays, cells were seeded after the addition of Matrigel. The relative-fold invasion values for the stable clones were normalized against the vehicle cells and are represented diagrammatically.</p

Overexpressed fibulin-5 is up-regulation the expression of FLJ10540, which induced the cell motility in NPC cells.

<p>(A) The mRNA and protein expression levels of FLJ10540 were determined by Q-RT-PCR and western blotting in fibulin-5 transfectants. The result of mRNA was normalized against the expression level of GAPDH mRNA in each fibulin-5-stable clones. For protein analyses, the cell lysates (50 μg) of Hone1/fibulin-5 transfectants was subjected to immunoblot analysis with anti-FLJ10540 and β-actin antibodies. β-actin was used as a control. (B) Fibulin-5 siRNAs decreased the expression levels of FLJ10540 mRNA and protein in fibulin-5-NPC transfectants. The Q-RT-PCR and western blotting were performed as in (B). (C) The luciferase assays were done to detect promoter activities of FLJ10540 in cotransfected with in a dose-dependent manner of DDK-, DDK-fibulin-5-, negative control and sifibulin-5 in Hone1 cells. The luciferase activity in 1μg cell lysate was normalized to β-galactosidase activity. Data are representative of three independent experiments done in triplicates. The Western blotting of DDK-fibulin-5 in a dose-dependent manner was shown in the right side of the left panel. (D) ChIP analysis of endogenous FLJ10540 promoter in the presence and absence of fibulin-5 in Hone 1 cells. The protein-DNA complexes were immunoprecipitated with DDK and IgG antibodies, and FLJ10540 promoter element was detected by PCR. (E) Migration and invasion decreased in cells transfected with FLJ10540 siRNA in vehicle-Hone1 and fibulin-5-Hone1 transfectants. The relative-fold migration and invasion values for the stable clones were normalized against the vehicle cells and are represented diagrammatically. All data represent the mean ± SD of 3 independent experiments.</p

Overexpression of Fibulin-5 in NPC specimens.

<p>Representative (A) RT-PCR and (B) Q-RT-PCR analyses of <i>fibulin-5</i> expression in six-NPC samples (T) compared to that in three-normal tissues (N). GAPDH was used as internal control. (C) Western blot analyses of fibulin-5 protein expression level in NPC samples (T) and normal tissues (N) (<i>p</i> < 0.001) Total proteins were extracted from respective tissues and probed with antibody against fibulin-5. β-actin was used as a control.</p

The distributions of fibulin-5 in NPC and other human cancer cell lines.

<p>(A) Immunoblot analysis of total protein from normal tissues, TW01, TW04, and Hone1 cells using monoclonal anti-fibulin-5 antibody. (B) Hone1, A-431 and U2-OS cells were grown on glass cover slips and cultured overnight. Indirect immunofluorescence of endogenous fibulin-5 protein in Hone1 cells was detected by using anti-fibulin-5 antibody. (Bottom panel) Hone1 cells were transfected with DDK-fused fibulin-5 by Plus/Lipofectamine reagents and cultured overnight. Indirect immunofluorescence of highly expressed fibulin-5 protein in Hone1 cells was detected with anti-DDK antibody. The cells were double-stained with DAPI to detect DNA. (C) (Upper panel) Western blot analysis for fibulin-5 of cytoplasm extracts (left) and nuclear extracts (right) of TW01 and Hone1 cells. β-actin and LaminA/C were used as control. (Bottom panel) Immunoprecipitation analysis for fibulin-5 of cell culture supernatants and intra-cellular total cell extracts of TW01 and Hone1 cells.</p

The proliferation, migratory, and invasive abilities of NPC cells are inhibited by <i>fibulin-5</i>-specific siRNA.

<p>(A) A negative control siRNA plus <i>fibulin-5</i> siRNA was transfected into Hone1 cells for 24 hour. After transfection, western blotting was performed with anti-fibulin-5 and β-actin antibodies. The mRNA expression level of endogenous fibulin-5 was also measured by Q-RT-PCR. (B) Using the same panel, the sifibulin-5 transfectants and negative control were seeded into 96-well plates with 5.0% FBS. The cells were cultured for 0-3 days followed by MTT assay to quantitate cell growth. The data were normalized against the value on day 0 of each treatment. The growth curves of Hone1 cells are shown as the mean ± SD of 3 independent experiments. (C and D) The wound healing, migration, and invasion results of negative control-Hone1 and sifibulin-5-Hone1 stable cells are shown (200×). The relative-fold migration and invasion of sifibulin-5-Hone1 cells were normalized against the values for the negative control cells and are represented diagrammatically. The results represent the mean ± SD of 3 independent experiments. </p

Fibulin-5 modulates cell migration and invasion through enhanced AKT signaling.

<p>(A) Vehicle-Hone1, fibulin-5-Hone1, vehicle-TW01, and fibulin-5-TW01 transfected cells were serum-starved and treated with the indicated inhibitors, SB202190, PD98059, and LY294002 or solvent for 24 hour. The migration and invasion ratios of vehicle-Hone1 and fibulin-5-Hone1 transfected cells were determined as previously described. (B) The fibulin-5 expressing cells of Hone1 and TW01 were serum-starved for 24 hour and treated with or without LY294002 at the final concentration of 10 μM. The Total cell lysates of vehicle-Hone1, fibulin-5-Hone1, vehicle-TW01, and fibulin-5-TW01 transfected cells were immunoblotted for the unphosphorylated and phosphorylated forms of AKT. β-actin was used as the internal loading control. (C) A negative control and fibulin-5 siRNAs were transfected into Hone1 cells for 24 hour and western blotting was performed as in (B).</p