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. 2016 Dec 6;7(49):81474-81492.
doi: 10.18632/oncotarget.13034.

TWEAK activation of the non-canonical NF-κB signaling pathway differentially regulates melanoma and prostate cancer cell invasion

Cheryl L Armstrong  1   2   3 , Rebeca Galisteo  1   2   3 , Sharron A N Brown  1   2   3 , Jeffrey A Winkles  1   2   3
Affiliations

TWEAK activation of the non-canonical NF-κB signaling pathway differentially regulates melanoma and prostate cancer cell invasion

Cheryl L Armstrong et al. Oncotarget. .

Abstract

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that binds with high affinity to a plasma membrane-anchored receptor named Fn14. Both TWEAK and Fn14 expression has been detected in human cancer tissue, and studies have shown that TWEAK/Fn14 signaling can promote either "pro-cancer" or "anti-cancer" cellular effects in vitro, depending on the cancer cell line under investigation. In this study, we engineered murine B16 melanoma cells to secrete high levels of soluble TWEAK and examined their properties. TWEAK production by B16 cells preferentially activated the non-canonical NF-κB signaling pathway and increased the expression of several previously described TWEAK-inducible genes, including Fn14. TWEAK overexpression in B16 cells inhibited both cell growth and invasion in vitro. The TWEAK-mediated reduction in B16 cell invasive capacity was dependent on activation of the non-canonical NF-κB signaling pathway. Finally, we found that this same signaling pathway was also important for TWEAK-stimulated human DU145 prostate cancer cell invasion. Therefore, even though TWEAK:Fn14 binding activates non-canonical NF-κB signaling in both melanoma and prostate cancer cells, this shared cellular response can trigger a very different downstream outcome (inhibition or stimulation of cell invasiveness, respectively).

Keywords: Fn14; NF-κB; TWEAK; invasion; melanoma.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Human sTWEAK overexpression in murine B16 melanoma cells increases Fn14 expression
A. Vector control (V1, V2) and TWEAK-myc plasmid-transfected (T1, T2) B16 clonal cell lines were harvested and cell lysate and conditioned media samples were prepared. TWEAK and GAPDH levels were analyzed by Western blotting using anti-myc tag and anti-GAPDH antibodies, respectively. This Western blot was done three times. B. Human sTWEAK levels in conditioned media samples collected from the four B16 cell lines were determined by ELISA. The results shown are the combination of 2 independent experiments for V1/T1 and 3 independent experiments for V2/T2. C. The four B16 cell lines were harvested and Fn14 and GAPDH levels were evaluated by Western blot analysis. This Western blot was done three times.
Figure 2
Figure 2. TWEAK overexpression in B16 cells reduces growth in vitro but not in vivo
A. The four B16 cell lines were cultured for 72 hr in low serum media (0.5% FBS) and cell growth was assayed using WST-1 reagent. The values shown are mean ± SEM of four replicates per cell line. Significance was measured by Student's t-test. This growth assay was performed four times and the results of one representative experiment are shown here. B. The four B16 cell lines were cultured for 72 hr in normal growth media (10% FBS) and cell growth was assayed as above. The values shown are mean ± SEM of four replicates per cell line. Significance was measured by Student's t-test. This growth assay was performed four times and the results of one representative experiment are shown here. C. The four B16 cell lines were injected subcutaneously into syngeneic C57BL/6 mice (n = 7 animals per cell line) and tumor growth was monitored over time. The values shown are the mean ± SEM. Significance was measured by Student's t-test (ns = not significant; p > 0.05). This in vivo experiment was conducted twice and the results of one representative experiment are shown here.
Figure 3
Figure 3. TWEAK overexpression does not alter B16 cell migration
A. The four B16 cell lines were grown to confluence and a scratch wound was made using a pipette tip. Wound closure was monitored by light microscopy and representative photographs (x10 magnification) of the V1 and T1 cell lines at 0 and 24 hr after wounding are shown. B. Wound width was calculated at 0 and 24 hr for all four B16 cell lines and the difference plotted as percentage wound closure. The values shown are the mean ± SEM for three replicates per cell line. Significance was measured by Student's t-test (ns = not significant; p > 0.05). The migration assay was performed three times and the results of one representative experiment are shown here.
Figure 4
Figure 4. Both TWEAK overexpression in B16 cells and exogenous TWEAK treatment of B16 cells inhibits cell invasion
A. The four B16 cell lines were evaluated for invasive capacity using the Matrigel invasion assay. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per cell line. Significance was measured by Student's t-test. B. V1 cells were either left untreated or treated with 200 ng/ml of TWEAK (TWK) for 24 hr prior to plating in Matrigel invasion chambers. T1 cell invasion was also evaluated. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test. C. V2 cells were either left untreated or treated with 200 ng/ml of TWEAK (TWK) for 24 hr prior to plating in Matrigel invasion chambers. T2 cell invasion was also evaluated. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test. All invasion assays were performed at least two times and the results of one representative experiment are shown here.
Figure 5
Figure 5. TWEAK overexpression in B16 cells activates the non-canonical NF-κB pathway and Fn14 depletion attenuates this effect
A. Human T98G cells (positive control) and the four engineered B16 cell lines were harvested and phospho-IκBα, IκBα, and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice. B. The four B16 cell lines were harvested and phospho-p100, NF-κB2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done three times. C. V1 and T1 cells were either left untreated or T1 cells were transiently transfected with control (CTL) siRNA, Fn14 siRNA #1 or Fn14 siRNA #4 for 48 hr. Cells were harvested and Fn14, NF-κB2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done once. D. V2 and T2 cells were either left untreated or T2 cells were transiently transfected with control (CTL) siRNA, Fn14 siRNA #1 or Fn14 siRNA #4 for 48 hr. Cells were harvested and Fn14, NF-κB2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done once.
Figure 6
Figure 6. NF-κB2 p100 depletion in B16 T2 cells increases cell invasion
A. V2 and T2 cells were either left untreated or T2 cells were transiently transfected with control (CTL) siRNA, p100 siRNA #3 or p100 siRNA #4 for 48 hr. Cells were harvested and NF-κB2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice. B. V2 and T2 cells were either left untreated or T2 cells were transiently transfected with control (CTL) siRNA, p100 siRNA #3 or p100 siRNA #4 for 24 hr prior to cell harvest and plating in Matrigel invasion chambers. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per condition, with each chamber done independently. Significance was measured by Student's t-test (ns = not significant; p > 0.05).
Figure 7
Figure 7. TWEAK treatment of human A375 melanoma cells decreases invasion and activates both the canonical and non-canonical NF-κB pathways
A. A375 cells were either left untreated or treated with 200 ng/ml of TWEAK for 24 hr prior to harvesting and plating in Matrigel invasion chambers. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test. The invasion assay was done twice and the results of one representative experiment are shown here. B. A375 cells were either left untreated or treated with TWEAK for 24 hr. Cells were harvested and NF-κB2 (p100/52), IκBα and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice.
Figure 8
Figure 8. TWEAK treatment of human PC-3 and DU145 prostate cancer cells increases invasion and activates the non-canonical NF-κB pathway
PC-3 A. and DU145 B. cells were either left untreated or treated with 200 ng/ml of TWEAK for 24 hr prior to harvesting and plating in Matrigel invasion chambers. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test. The invasion assay was repeated one more time using one chamber per condition with similar results. C. PC-3 and DU145 cells were either left untreated or treated with TWEAK for 24 hr. Cells were harvested and NF-κB2 (p100/52), IκBα and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice.
Figure 9
Figure 9. NF-κB2 p100 depletion attenuates TWEAK-stimulated DU145 cell invasion
A. DU145 cells were either left untreated (NT, no treatment) or transiently transfected with control (CTL) siRNA or p100 siRNA #4, treated with 200 ng/ml TWEAK (TWK), and harvested 24 hr post-transfection. Lysates were prepared and NF-κB2 (p100/p52) and GAPDH levels were evaluated by Western blot analysis. This Western blot was done twice. B. DU145 cells were either left untreated or transiently transfected with CTL siRNA or p100 siRNA #4, treated with 200 ng/ml TWEAK, harvested 24 hr post-transfection and plated in Matrigel invasion chambers. The number of invaded cells for each invasion chamber was determined from photographs of eight randomly chosen fields. The values shown are the mean ± SEM for three chambers per condition. Significance was measured by Student's t-test (ns = not significant; p > 0.05). The invasion assay was done twice and the results of one representative experiment are shown here.
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