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The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration.

Mohr CF, Kalmer M, Gross C, Mann MC, Sterz KR, Kieser A, Fleckenstein B, Kress AK - Cell Commun. Signal (2014)

Bottom Line: Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase β (IKKβ) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression.Beyond that, chemical inhibition of IKKβ significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix.While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The actin-bundling protein Fascin (FSCN1) is a tumor marker that is highly expressed in numerous types of cancer including lymphomas and is important for migration and metastasis of tumor cells. Fascin has also been detected in B lymphocytes that are freshly-infected with Epstein-Barr virus (EBV), however, both the inducers and the mechanisms of Fascin upregulation are still unclear.

Results: Here we show that the EBV-encoded oncoprotein latent membrane protein 1 (LMP1), a potent regulator of cellular signaling and transformation, is sufficient to induce both Fascin mRNA and protein in lymphocytes. Fascin expression is mainly regulated by LMP1 via the C-terminal activation region 2 (CTAR2). Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase β (IKKβ) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression. Furthermore, chemical inhibition of IKKβ reduced both Fascin mRNA and protein levels in EBV-transformed lymphoblastoid cell lines, indicating that canonical NF-κB signaling is required for LMP1-mediated regulation of Fascin both in transfected and transformed lymphocytes. Beyond that, chemical inhibition of IKKβ significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix. Transient transfection experiments revealed that Fascin contributed to LMP1-mediated enhancement of invasive migration through extracellular matrix. While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells.

Conclusions: Thus, our data show that LMP1-mediated upregulation of Fascin depends on NF-κB and both NF-κB and Fascin contribute to invasive migration of LMP1-expressing lymphocytes.

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NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P < 0.05. (B) Quantitative PCR of Fascin transcripts normalized to ACTB in LCL-B upon ACHP-and SP600125-treatment for 48 h. The means of three independent experiments +/− SE were normalized to solvent-treated cells and compared using a paired t-test. **indicates P < 0.01. (C) Detection of Fascin, LMP1, p100 processing (p100/p52) and IκBα by immunoblot after treatment of LCL-B with DMSO, 2.5 and 5 μM ACHP for 48 h. ACTB served as loading control. Samples were loaded on two gels in parallel. (D) LCL-B cells were cultured in presence of ACHP (5 μM) or solvent (DMSO) for 48 h and cells were serum-starved (1% fetal calf serum (FCS)) for 4 h. Invasion assays were performed using trans-wells coated with extracellular matrix for 24 h. Values shown in the upper panel reflect the percentage of invaded cells (measured at OD 560 nm) that are attached to the bottom of the membrane. The lower bar graphs show the percentage of invaded cells that are non-attached and have migrated to the medium (20% FCS) of the lower compartment. Solvent-treated cells (DMSO) were set as 100%. Mean values and error bars of three independent experiments each performed in triplicates are shown. Values were compared using a paired t-test. *indicates P < 0.05. n.s., not significant.
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Figure 5: NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P < 0.05. (B) Quantitative PCR of Fascin transcripts normalized to ACTB in LCL-B upon ACHP-and SP600125-treatment for 48 h. The means of three independent experiments +/− SE were normalized to solvent-treated cells and compared using a paired t-test. **indicates P < 0.01. (C) Detection of Fascin, LMP1, p100 processing (p100/p52) and IκBα by immunoblot after treatment of LCL-B with DMSO, 2.5 and 5 μM ACHP for 48 h. ACTB served as loading control. Samples were loaded on two gels in parallel. (D) LCL-B cells were cultured in presence of ACHP (5 μM) or solvent (DMSO) for 48 h and cells were serum-starved (1% fetal calf serum (FCS)) for 4 h. Invasion assays were performed using trans-wells coated with extracellular matrix for 24 h. Values shown in the upper panel reflect the percentage of invaded cells (measured at OD 560 nm) that are attached to the bottom of the membrane. The lower bar graphs show the percentage of invaded cells that are non-attached and have migrated to the medium (20% FCS) of the lower compartment. Solvent-treated cells (DMSO) were set as 100%. Mean values and error bars of three independent experiments each performed in triplicates are shown. Values were compared using a paired t-test. *indicates P < 0.05. n.s., not significant.

Mentions: To analyse whether canonical NF-κB signals are also required for Fascin expression in EBV transformed LMP1-expressing B-cells, LCL-B cells were incubated with increasing amounts of the IKKβ-inhibitor ACHP (Figure 5A-C). Treatment of cells with a selective inhibitor of the JNK pathway (SP600125; 10 μM) served as specificity control [[38]]. After 48 h, viability of cells was determined by flow cytometry and RNA was extracted. Forward-side-scatter (FSC/SSC) analysis revealed that low concentrations of ACHP (1, 2.5, 5 μM) only slightly affected viability of the LCL-B culture compared to the solvent control DMSO (p > 0.05; paired t-test; Figure 5A). However, high concentrations of ACHP (10, 25 μM) reduced viability of LCL by 50-75% (p < 0.05; paired t-test) confirming earlier observations [[39]]. Quantitation of Fascin copy numbers by qPCR showed that even at low concentrations of ACHP (2.5, 5 μM), Fascin copy numbers were significantly and dose-dependently reduced (Figure 5B; p < 0.01; paired t-test), while inhibition of JNK signaling with SP600125 did not affect Fascin expression. To ensure specificity of the IKKβ-inhibitor ACHP in LCLs, transcripts of the NF-κB-dependent LMP1-target gene 4-1BB were measured (Additional file 2) [[37]]. Already at low concentrations of ACHP (1μM), expression of 4-1BB was diminished significantly (p < 0.01). While Fascin was only affected by treatment with ACHP, 4-1BB was also diminished upon treatment with the JNK-inhibitor SP600125, which confirms earlier findings showing a role of both NF-κB and JNK signaling in 4-1BB regulation [[40]]. To further address the influence of NF-κB signals on Fascin protein, Western blot analysis was performed upon treatment of LCLs with low doses of ACHP (2.5 μM; 5 μM). These data revealed that also Fascin protein is reduced upon treatment of LCLs with ACHP, despite the presence of LMP1 (Figure 5C). Beyond that, treatment of LCLs with ACHP led to less production of p100, a classical target of canonical NF-κB signaling, while processing of p100 to p52 was not affected. Finally, we observed an accumulation of IκBα, suggesting that (1) IκBα gets less degraded in presence of ACHP, and that (2) canonical NF-κB signals are blocked. In summary, these data show that Fascin is regulated by canonical NF-κB signals not only in LMP1-transfected cells, but also in LMP1-expressing, EBV-transformed lymphoblastoid B-cells.


The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration.

Mohr CF, Kalmer M, Gross C, Mann MC, Sterz KR, Kieser A, Fleckenstein B, Kress AK - Cell Commun. Signal (2014)

NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P < 0.05. (B) Quantitative PCR of Fascin transcripts normalized to ACTB in LCL-B upon ACHP-and SP600125-treatment for 48 h. The means of three independent experiments +/− SE were normalized to solvent-treated cells and compared using a paired t-test. **indicates P < 0.01. (C) Detection of Fascin, LMP1, p100 processing (p100/p52) and IκBα by immunoblot after treatment of LCL-B with DMSO, 2.5 and 5 μM ACHP for 48 h. ACTB served as loading control. Samples were loaded on two gels in parallel. (D) LCL-B cells were cultured in presence of ACHP (5 μM) or solvent (DMSO) for 48 h and cells were serum-starved (1% fetal calf serum (FCS)) for 4 h. Invasion assays were performed using trans-wells coated with extracellular matrix for 24 h. Values shown in the upper panel reflect the percentage of invaded cells (measured at OD 560 nm) that are attached to the bottom of the membrane. The lower bar graphs show the percentage of invaded cells that are non-attached and have migrated to the medium (20% FCS) of the lower compartment. Solvent-treated cells (DMSO) were set as 100%. Mean values and error bars of three independent experiments each performed in triplicates are shown. Values were compared using a paired t-test. *indicates P < 0.05. n.s., not significant.
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Figure 5: NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P < 0.05. (B) Quantitative PCR of Fascin transcripts normalized to ACTB in LCL-B upon ACHP-and SP600125-treatment for 48 h. The means of three independent experiments +/− SE were normalized to solvent-treated cells and compared using a paired t-test. **indicates P < 0.01. (C) Detection of Fascin, LMP1, p100 processing (p100/p52) and IκBα by immunoblot after treatment of LCL-B with DMSO, 2.5 and 5 μM ACHP for 48 h. ACTB served as loading control. Samples were loaded on two gels in parallel. (D) LCL-B cells were cultured in presence of ACHP (5 μM) or solvent (DMSO) for 48 h and cells were serum-starved (1% fetal calf serum (FCS)) for 4 h. Invasion assays were performed using trans-wells coated with extracellular matrix for 24 h. Values shown in the upper panel reflect the percentage of invaded cells (measured at OD 560 nm) that are attached to the bottom of the membrane. The lower bar graphs show the percentage of invaded cells that are non-attached and have migrated to the medium (20% FCS) of the lower compartment. Solvent-treated cells (DMSO) were set as 100%. Mean values and error bars of three independent experiments each performed in triplicates are shown. Values were compared using a paired t-test. *indicates P < 0.05. n.s., not significant.
Mentions: To analyse whether canonical NF-κB signals are also required for Fascin expression in EBV transformed LMP1-expressing B-cells, LCL-B cells were incubated with increasing amounts of the IKKβ-inhibitor ACHP (Figure 5A-C). Treatment of cells with a selective inhibitor of the JNK pathway (SP600125; 10 μM) served as specificity control [[38]]. After 48 h, viability of cells was determined by flow cytometry and RNA was extracted. Forward-side-scatter (FSC/SSC) analysis revealed that low concentrations of ACHP (1, 2.5, 5 μM) only slightly affected viability of the LCL-B culture compared to the solvent control DMSO (p > 0.05; paired t-test; Figure 5A). However, high concentrations of ACHP (10, 25 μM) reduced viability of LCL by 50-75% (p < 0.05; paired t-test) confirming earlier observations [[39]]. Quantitation of Fascin copy numbers by qPCR showed that even at low concentrations of ACHP (2.5, 5 μM), Fascin copy numbers were significantly and dose-dependently reduced (Figure 5B; p < 0.01; paired t-test), while inhibition of JNK signaling with SP600125 did not affect Fascin expression. To ensure specificity of the IKKβ-inhibitor ACHP in LCLs, transcripts of the NF-κB-dependent LMP1-target gene 4-1BB were measured (Additional file 2) [[37]]. Already at low concentrations of ACHP (1μM), expression of 4-1BB was diminished significantly (p < 0.01). While Fascin was only affected by treatment with ACHP, 4-1BB was also diminished upon treatment with the JNK-inhibitor SP600125, which confirms earlier findings showing a role of both NF-κB and JNK signaling in 4-1BB regulation [[40]]. To further address the influence of NF-κB signals on Fascin protein, Western blot analysis was performed upon treatment of LCLs with low doses of ACHP (2.5 μM; 5 μM). These data revealed that also Fascin protein is reduced upon treatment of LCLs with ACHP, despite the presence of LMP1 (Figure 5C). Beyond that, treatment of LCLs with ACHP led to less production of p100, a classical target of canonical NF-κB signaling, while processing of p100 to p52 was not affected. Finally, we observed an accumulation of IκBα, suggesting that (1) IκBα gets less degraded in presence of ACHP, and that (2) canonical NF-κB signals are blocked. In summary, these data show that Fascin is regulated by canonical NF-κB signals not only in LMP1-transfected cells, but also in LMP1-expressing, EBV-transformed lymphoblastoid B-cells.

Bottom Line: Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase β (IKKβ) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression.Beyond that, chemical inhibition of IKKβ significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix.While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The actin-bundling protein Fascin (FSCN1) is a tumor marker that is highly expressed in numerous types of cancer including lymphomas and is important for migration and metastasis of tumor cells. Fascin has also been detected in B lymphocytes that are freshly-infected with Epstein-Barr virus (EBV), however, both the inducers and the mechanisms of Fascin upregulation are still unclear.

Results: Here we show that the EBV-encoded oncoprotein latent membrane protein 1 (LMP1), a potent regulator of cellular signaling and transformation, is sufficient to induce both Fascin mRNA and protein in lymphocytes. Fascin expression is mainly regulated by LMP1 via the C-terminal activation region 2 (CTAR2). Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase β (IKKβ) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression. Furthermore, chemical inhibition of IKKβ reduced both Fascin mRNA and protein levels in EBV-transformed lymphoblastoid cell lines, indicating that canonical NF-κB signaling is required for LMP1-mediated regulation of Fascin both in transfected and transformed lymphocytes. Beyond that, chemical inhibition of IKKβ significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix. Transient transfection experiments revealed that Fascin contributed to LMP1-mediated enhancement of invasive migration through extracellular matrix. While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells.

Conclusions: Thus, our data show that LMP1-mediated upregulation of Fascin depends on NF-κB and both NF-κB and Fascin contribute to invasive migration of LMP1-expressing lymphocytes.

Show MeSH
Related in: MedlinePlus