LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
LMP1 transformation of rodent fibroblasts requires activation of PI3K and Akt [5]. Additionally, activated pAkt is frequently detected in NPC and the neoplastic Reed-Sternberg cells of classical HD [39,40]. To determine if Akt signaling is activated in LMP1 transgenic mice, pAkt and several of its targets were assessed by immunoblotting of splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had increased levels of pAkt compared to wild-type lymphocytes; however, progression to lymphoma in both LMP1-positive and -negative lymphoma cells did not further increase pAkt levels. The Akt target glycogen synthase kinase 3 (GSK3) is inactivated by phosphorylation; however, increased phosphorylated GSK3 was not detected in the transgenic lymphocytes and was almost absent in the lymphoma samples (Figure 6A). This finding indicates that GSK3 is not a target of activated Akt in the LMP1 transgenic lymphocytes and lymphoma cells. Similarly, activation of Akt without phosphorylation of GSK3 has been previously shown in EBV-positive HD [40]. In contrast, the wild-type lymphocytes lacked activated Akt but did have detectable phosphorylated GSK3. This further suggests that additional pathways are involved in the regulation of GSK3.
Figure 6  LMP1 Activates Akt Signaling and Deregulates the Rb Cell Cycle Pathway
(A and B) Immunoblot analysis of purified B cells (CD19+) from the spleens of WT and LMP1 transgenic mice for Akt signaling, probing for (A) activated pAkt and downstream targets, including inactivated pGSK3α/β, and (B) activated p-mTOR, and total levels of FoxO1. Arrows indicate the positions of α and β isoforms of GSK3. The white line indicates that intervening lanes have been spliced out.
(C) Immunoblot analysis for cell cycle proteins regulating the Rb pathway, probing for activated pRb, and total levels of Cdk2 and the Cdk inhibitor p27. Actin was used as a loading control.   To identify other potential Akt targets, immunoblot analysis for p-mTOR was performed. Activated p-mTOR was not increased in LMP1 transgenic lymphocytes or lymphoma cells, indicating that this pathway is not affected by LMP1-induced Akt activation and does not contribute to lymphoma development (Figure 6B). Akt is also known to phosphorylate and induce the degradation of the pro-apoptotic Forkhead family of transcription factors, leading to cell cycle progression and survival in some human tumors [41,42]. Immunoblot analysis of splenic B cells did not consistently detect p-FoxO1 levels, a signal that targets FoxO1 for degradation. Hence, degradation of FoxO1 was assessed by detection of total FoxO1 levels. Immunoblot analysis indicated that total FoxO1 levels were greatly decreased in wild-type and LMP1 transgenic lymphomas (Figure 6B), suggesting that inhibition of the Forkhead signaling pathway is an important target of Akt in lymphoma development. However, considering that Akt activation did not induce FoxO1 degradation in LMP1 transgenic B cells, Akt may not be the sole regulator of FoxO1, and it may be that progression to lymphoma requires modulation of multiple pathways.
The Forkhead family of transcription factors is known to induce the expression of the Cdk inhibitor p27 [43,44]. LMP1-transformed rodent fibroblasts have decreased expression of p27, upregulation of Cdk2, and subsequent phosphorylation and inactivation of the tumor suppressor gene Rb [45]. To investigate whether LMP1 affected cell cycle regulation through the Rb pathway in B cells, immunoblot analyses for pRb, Cdk2, and p27 were performed on splenic CD19+ MACS-purified B cells. LMP1 transgenic B cells had enhanced levels of pRb with concomitant stabilization of total Rb levels and Cdk2 compared to wild-type B lymphocytes (Figure 6C). Progression to lymphoma in both wild-type and LMP1 transgenic lymphoma cells led to increased levels of Rb, correspondingly high levels of Cdk2, and decreased levels of p27 (Figure 6C). These data indicate that the Rb pathway is deregulated in LMP1 transgenic lymphocytes and that lymphoma cells are distinguished by loss of FoxO1 and decreased p27.