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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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Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin (ACTB) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A), peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2B served as negative control.
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Figure 1: Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin (ACTB) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A), peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2B served as negative control.

Mentions: In search of the functional role of Fascin in EBV-transformed lymphocytes, we started to analyze the expression pattern of Fascin in a number of cell lines by quantitative PCR (qPCR; Figure 1A). Human T-lymphotropic virus type 1 (HTLV-1)-transformed MT-2 cells, which express high amounts of Fascin [[30]], served as a positive control. In contrast to Jurkat T-cells, which only expressed very low amounts of Fascin mRNA, EBV-transformed lymphoblastoid cell lines (LCLs) LCL-B and LCL-721 cells (latency type III) expressed high amounts of Fascin; in LCL-3 and LCL-4 (latency type III), expression of Fascin was enhanced as well, albeit to lower levels than in LCL-B and LCL 721 cells. Cell lines derived from Hodgkin lymphoma (HL; EBV-negative), including KM-H2, L428, and HDLM-2, expressed high amounts of Fascin. All cell lines derived from Burkitt lymphoma (BL; latency type I) did not express Fascin confirming earlier observations [[23]]. In B-cell lymphoma cell lines derived from Kaposi’s sarcoma herpes virus-associated malignancies like primary effusion lymphoma (PEL) including EBV-negative cell lines Bcbl-1 and BC-3, and EBV-positive JSC-1 cells (latency type II), Fascin was only detectable at low amounts in the PEL-cell line JSC-1. This cell line is known to express low amounts of LMP1, which can be detected by PCR, but not at the protein level [[32]]. Data obtained by qPCR (Figure 1A) were confirmed in immunoblots detecting Fascin protein (Figure 1B). Among all cell lines analyzed, LCL-B, LCL-721, LCL-3 and LCL-4 cells are also LMP1-positive (Figure 1B) [[33]]. Taken together, these results show that expression of Fascin is a specific feature of HL-derived cells [[24]], of LCLs, and of other LMP-1-expressing cell lines (JSC-1). To analyze the subcellular localization of Fascin in transformed, LMP-1 expressing B-cells, immunofluorescence analysis was performed in LCL-B cells (Figure 1C). Fascin was found in the cytoplasm and at the plasma membrane and colocalized with actin, suggesting that Fascin exerts its molecular function of stabilizing actin in EBV-transformed 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)

Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin (ACTB) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A), peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2B served as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin (ACTB) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A), peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2B served as negative control.
Mentions: In search of the functional role of Fascin in EBV-transformed lymphocytes, we started to analyze the expression pattern of Fascin in a number of cell lines by quantitative PCR (qPCR; Figure 1A). Human T-lymphotropic virus type 1 (HTLV-1)-transformed MT-2 cells, which express high amounts of Fascin [[30]], served as a positive control. In contrast to Jurkat T-cells, which only expressed very low amounts of Fascin mRNA, EBV-transformed lymphoblastoid cell lines (LCLs) LCL-B and LCL-721 cells (latency type III) expressed high amounts of Fascin; in LCL-3 and LCL-4 (latency type III), expression of Fascin was enhanced as well, albeit to lower levels than in LCL-B and LCL 721 cells. Cell lines derived from Hodgkin lymphoma (HL; EBV-negative), including KM-H2, L428, and HDLM-2, expressed high amounts of Fascin. All cell lines derived from Burkitt lymphoma (BL; latency type I) did not express Fascin confirming earlier observations [[23]]. In B-cell lymphoma cell lines derived from Kaposi’s sarcoma herpes virus-associated malignancies like primary effusion lymphoma (PEL) including EBV-negative cell lines Bcbl-1 and BC-3, and EBV-positive JSC-1 cells (latency type II), Fascin was only detectable at low amounts in the PEL-cell line JSC-1. This cell line is known to express low amounts of LMP1, which can be detected by PCR, but not at the protein level [[32]]. Data obtained by qPCR (Figure 1A) were confirmed in immunoblots detecting Fascin protein (Figure 1B). Among all cell lines analyzed, LCL-B, LCL-721, LCL-3 and LCL-4 cells are also LMP1-positive (Figure 1B) [[33]]. Taken together, these results show that expression of Fascin is a specific feature of HL-derived cells [[24]], of LCLs, and of other LMP-1-expressing cell lines (JSC-1). To analyze the subcellular localization of Fascin in transformed, LMP-1 expressing B-cells, immunofluorescence analysis was performed in LCL-B cells (Figure 1C). Fascin was found in the cytoplasm and at the plasma membrane and colocalized with actin, suggesting that Fascin exerts its molecular function of stabilizing actin in EBV-transformed 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