text: LMP1 Promotes Tumor Growth and Survival through Activation of Akt, NFkappaB, and Stat3 Pathways
  To explore which pathways were required for the enhanced growth and survival of LMP1-induced lymphomas, splenocytes from wild-type and LMP1 transgenic mice were cultured in the presence of inhibitors for Akt, NFkappaB, Stat3, mTOR, or MAPK and assayed for growth and survival by the MTS assay. As previously shown, wild-type lymphocytes were not viable in culture and could not be tested with the inhibitors. However, the enhanced viability of LMP1 transgenic lymphocytes was effectively blocked by treatment with triciribine, BAY11-7085, cucurbitacin I, and slightly with SB203580, but not by treatment with rapamycin, U0126, or AG490 (Figure 7). Triciribine inhibits the activation of Akt and at 20 muM has been shown to induce growth arrest in cancer cells with aberrant Akt activity [46]. The effects of triciribine on cell growth of the transgenic lymphocytes and lymphomas were apparent as low as 1 muM, suggesting that activation of Akt is required for the survival and growth of LMP1 transgenic lymphocytes and lymphoma cells (Figure 7). The effects of the inhibitors were assessed by identifying phosphorylated Akt, Stat3, and total levels of IkappaBalpha (Figure 8). Treatment with triciribine effectively blocked phosphorylation of Akt, and phosphorylated Akt was no longer detected past 5 muM. Phosphorylated Stat3 and IkappaBalpha were still present at 25 muM. These findings suggest that triciribine specifically targets Akt and that Akt activation is required for the enhanced viability of the transgenic lymphocytes and lymphoma cells. 
  Inhibition of NFkappaB signaling rapidly induces cell death of EBV-transformed lymphocytes [17,18]. BAY11-7085, an inhibitor of NFkappaB signaling, also greatly decreased the viability of the LMP1 transgenic lymphocytes and lymphoma cells at doses as low as 1 muM, and at 5 muM the cells were completely nonviable (Figure 7). This is well within the reported IC50 of 10 muM. Phosphorylated Akt, Stat3, and total IkappaBalpha were still present up to treatment with 15 muM and then were no longer detected (Figure 8). This finding suggests that inhibition of NFkappaB can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells without significant effects on activation of Akt or Stat3. 
  Cucurbitacin I inhibits activation of Stat3 by suppressing the activation of its kinase JAK2. It has been shown to selectively inhibit the growth of tumors with constitutively activated Stat3 [47]. Similarly, LMP1 transgenic lymphocytes and lymphoma cells were susceptible to cucurbitacin I treatment starting at 0.1 muM, a dose that corresponds closely to the reported IC50 of 500 nM (Figure 7) [47]. Phosphorylated Akt, Stat3, and total IkappaBalpha were not detectable past 1 muM and at higher doses all protein levels were greatly decreased, indicative of the total loss of viability (Figure 8). A second reported inhibitor of Stat3, AG490, had no effect on growth (Figure 7), but activation of Akt, Stat3, or levels of IkappaBalpha were also not affected (Figure 8). These findings suggest that inhibition of Stat3 can induce cell death in LMP1 transgenic lymphocytes and lymphoma cells, but Stat3 inhibition also has considerable crossover effects on Akt and NFkappaB signaling. 
  LMP1 has also been shown to activate JNK and p38 MAPK pathways [13,48], and LMP1 transgenic lymphocytes were mildly susceptible to growth inhibition by SB202190, an inhibitor of p38 MAPK, but not U0126, an inhibitor of MEK1/2 activity. However, effects of SB202190 were only apparent at high doses (>10 muM), much higher than the reported IC50 of 0.35 muM, suggesting that p38 MAPK does not significantly contribute to the enhanced viability in LMP1 transgenic lymphocytes or lymphoma cells (Figure 7). Interestingly, both wild-type and LMP1 transgenic lymphomas were similarly susceptible to triciribine, BAY11-7085, and cucurbitacin I treatments, but not SB202190, AG490, or U0126 treatment, suggesting that activation of Akt, NFkappaB, and Stat3 but not MAPK pathways are characteristics associated with malignant transformation (Figure 7). rapamycin, an inhibitor of mTOR, did not affect the viability of the transgenic lymphocytes or lymphoma cells, confirming that mTOR is not targeted by Akt activation in LMP1 transgenic lymphocytes or malignant lymphoma cells (Figure 7). 
  