EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration

Abstract

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-kappaB) signaling. Inhibitors of PI3K, Akt, and NF-kappaB signaling dramatically reduced these enhanced properties. An IkappaBalpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappaB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-kappaB-activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-kappaB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.

Figure 1

LMP1 down-regulates plakoglobin levels. (A) Stable expression of HA-tagged LMP1, CTAR1, CTAR2 or LMP2A from the pBabe vector in C666-1 cells was analyzed by immunoblot analysis. Arrows indicate the LMP1-specific band. (B) The effects of LMP1, CTAR1, CTAR2 and LMP2A expression on phosphorylated Akt (pAkt), phosphorylated GSK3αβ, IκBα and plakoglobin levels were analyzed by immunoblot analysis. Actin was used as a loading control. Densitometry with Image J software was performed to normalize plakoglobin levels to the corresponding actin levels. Fold change of the normalized plakoglobin levels relative to the pBabe control is indicated below the immunoblots.

Figure 2

Down-regulation of plakoglobin by LMP1 is dependent on the IκBα regulated canonical NFκB pathway. (A) Expression and the effects of the HA-tagged IκBα super-repressor (IκBα^SS32/36AA) from the pHSCG vector, on LMP1’s down-regulation of plakoglobin was analyzed by immunoblot analysis. Activation of Akt was detected by the phosphorylation of Akt (pAkt). (B) Expression and the effects of the constitutively active myristylated-Akt (myr-Akt) and LMP1 on plakoglobin levels was compared by immunoblot analysis. Actin was used as a loading control. Densitometry with Image J software was performed to normalize pAkt and plakoglobin levels to the corresponding actin levels. Fold change of the normalized pAkt and plakoglobin levels relative to the pBabe control is indicated below the immunoblots.

Figure 3

LMP1 induced growth requires both CTAR1 and 2 domains, and is dependent on NFκB activation. (A-C) MTS assays were performed to measure the metabolic activity of C666-1 cells stably expressing LMP1, CTAR1, CTAR2, LMP2A, the constitutively activated myristylated-Akt (myr-Akt), the pBabe vector control and the effects of the IκBα supper-repressor (IκBα^SS32/36AA) on the growth of LMP1-expressing cells.

Figure 4

LMP1 enhanced soft agar colony formation requires both CTAR1 and 2 domains and is dependent on PI3K and NFκB signaling. (A) C666-1 cells stably expressing LMP1, CTAR1, CTAR2, LMP2A or the pBabe vector control was assessed for anchorage-independent growth using the soft agar colony assay. (B) The signals required by LMP1 for enhanced soft agar colony formation was assessed by using inhibitors of PI3K (LY294002), Akt (Akt inhibitor I) and NFκB (BAY11-7085) signaling, compared to the DMSO control. (C) The requirement of canonical NFκB signaling for LMP1-induced soft agar colony formation was assessed using the IκBα super-repressor (IκBα^SS32/36AA), compared to the pHSCG vector control. (D) Expression of the constitutively active myristylated Akt (myr-Akt) was used to assess whether activation of Akt signaling is sufficient to enhance soft agar colony growth.

Figure 5

LMP1 induced migration requires both CTAR1 and 2 domains and is dependent on PI3K and NFκB signaling. (A) C666-1 cells stably expressing LMP1, CTAR1, CTAR2, LMP2A or the pBabe vector control was assessed for metastasis in the transwell migration assay. (B) The signals required by LMP1 for enhanced migration was assessed by using inhibitors of PI3K (LY294002), Akt (Akt inhibitor I) and NFκB (BAY11-7085) signaling at the indicated concentrations, compared to the DMSO control. (C) The ability of the PI3K inhibitor (LY294002), NFκB inhibitor (BAY11-7085) and Akt inhibitor (Akt inhibitor I) to block PI3K/Akt and IκBα-dependent NFκB signaling was assessed by immunoblot analysis after an overnight treatment, and blotted for activated phosphorylated Akt (pAkt), inactivated phosphorylated GSK3 (pGSK3αβ) and total IκBα levels. Actin was used as a loading control. Densitometry with Image J software was performed to normalize pAkt and IκBα levels to the corresponding actin levels. Fold change of the normalized pAkt and IκBα levels relative to the pBabe control is indicated below the immunoblots. White line indicates that intervening lanes have been spliced out. (D) LMP1-induced migration is dependent on IκBα-dependent NFκB signaling and activation of Akt is not sufficient to enhance migration. Expression of the constitutively active myristylated Akt (myr-Akt) was used to assess whether activation of Akt signaling is sufficient to enhance migration. The requirement of canonical NFκB signaling for LMP1-induced migration was assessed using the IκBα super-repressor (IκBα^SS32/36AA), compared to the pHSCG vector control.

Figure 6

LMP1 induced migration is blocked by restoring plakoglobin levels. (A) Stable expression of LMP1 in the pBabe vector and a myc-tagged plakoglobin (PG) or the DsRed vector control from C666-1 cells was analyzed by immunoblot analysis. Actin and GAPDH were used as loading controls. Densitometry with Image J software was performed to normalize plakoglobin levels to the corresponding GAPDH levels. Fold change of the normalized plakoglobin levels relative to the pBabe/DsRed control is indicated below the immunoblots. (B) The effects of restoring PG levels on the migration of LMP1-expressing C666-1 cells were assessed by the transwell migration assay. (C) Soft agar transformation and (D) MTS assays were performed to assess the effect of expressing a PG construct on the growth of pBabe and LMP1-expressing C666-1 cells, compared to the DsRed vector control.