Synergistic growth inhibition of anaplastic large cell lymphoma cells by combining cellular ALK gene silencing and a low dose of the kinase inhibitor U0126

Abstract

Abnormal expression of anaplastic lymphoma kinase (ALK) gene is an important pathogenic factor for anaplastic large cell lymphoma (ALCL). To study the function of ALK, an inducible short hairpin RNA (shRNA) system was stably introduced into cultured human ALCL cells. Inducing shRNA expression in the generated cells resulted in cellular ALK gene silencing and led to inactivation of multiple signaling pathways and growth arrest. Interestingly, a combination of ALK gene silencing with U0126, a kinase inhibitor specific for the extracellular signal-regulated kinases 1/2 pathway, resulted in an augmented reduction in cellular JunB expression. Functional studies indicated that combining ALK gene silencing with U0126 treatment provided a synergistic growth inhibition, which occurred faster and was more profound than with either treatment alone. This synergistic effect was also observed when measuring cell proliferation, apoptosis, and in vitro cell colony formation. Importantly, the combination of ALK gene silencing and U0126 had a prolonged inhibitory effect, preventing recovery of ALCL cell growth even after treatments were removed. Moreover, this synergistic inhibitory effect was confirmed in vivo using a mouse model with xenografted ALCL tumors. Our findings indicate that combining cellular ALK gene silencing with a low dose of U0126 may prove to be an effective and more specific therapeutic approach to treating ALCL.

Figure 1. Establishing a stable ALCL cell model that contains an inducible shRNA construct for specifically silencing the ALK gene. A, Overview of cell model

First, ‘conditioned cells’ are generated by stable introduction of pLenti/CMV/TetR/Blasticidin vector into cultured human ALCL cells (SUDHL-1) to constitutively express Tetracycline Repressor (TetR) under CMV promoter control. Subsequentially, the pLenti/H1/TO/shRNA-ALK/Zeocin construct is introduced to express shRNA-ALK that specifically targets the ALK portion of the NPM-ALK gene. The H1/TO promoter of shRNA-ALK construct is normally suppressed by the presence of TetR. As desired, addition of tetracycline (T) into cell media will lead to competitive binding of tetracycline to TetR, resulting in activation of H1/TO promoter. As a gene silencing control, pLenti/H1/TO/shRNA-Lamin vector is stably introduced into ‘conditioned cells’ to generate control cells that carry an inducible shRNA-Lamin construct. B, Stable TetR protein expression in ‘conditioned cells’. Western blot analysis confirmed stable protein expression of TetR (24-kDa band) in ‘conditioned cells’ (lane 2). Parental ALCL cells are shown in lane 1 as a negative control. C, DNA inserts encoding shRNAs and TetR gene. Upper panel: PCR analysis of DNA inserts encoding shRNAs. Lane 1: molecular weight markers; Lane 2: plasmid DNA control of pLenti/H1/TO/shRNA-ALK/Zeocin with a 213-bp band; Lane 3: parental ALCL cells with no amplified DNA products; Lane 4: ‘conditioned cells’ with no amplified DNA products; Lane 5: generated cells containing shRNA-ALK construct with a 213-bp band identical to that seen in plasmid DNA control (lane 2); and Lane 6: control cells with a 201-bp band corresponding to the DNA insert encoding shRNA-Lamin. Lower panel: PCR analysis of cellular TetR gene. Lane 1: molecular weight markers.; Lane 2: control plasmid of pLenti6/CMV/TetR/Blasticidin with a 973-bp band; Lane 3: parental SUDHL-1 cells with no amplified DNA products; Lane 4: ‘conditioned cells’ containing TetR gene with a 973-bp band identical to that seen in plasmid DNA control (lane 2); Lane 5: generated cells containing both shRNA-ALK and TetR gene with a 973-bp band identical to that seen in plasmid DNA control (lane 2); and Lane 6: control cells with a 973-bp band identical to that seen in plasmid DNA control (lane 2). D, Inducing shRNA-ALK suppresses NPM-ALK protein expression. Cells were treated with 3 µg/ml tetracycline for 6 days and ALK gene silencing was monitored by detecting cellular NPM-ALK protein expression using Western blotting. Lanes 1 and 2: generated cells containing an inducible shRNA-ALK. Lanes 3 and 4: control cells carrying an inducible shRNA-Lamin. Expression of cellular β-actin was used as an internal control for equivalent cellular protein loading. Experiments were repeated three times with similar results.

Figure 2. ALK gene silencing by inducing shRNA-ALK results in cell growth arrest, apoptosis, and death. A, Dose-dependent effect of tetracycline treatment on NPM-ALK expression

Cells were treated with the indicated concentration of tetracycline for 6 days, and NPM-ALK expression was detected by Western blot (upper panel). β-actin expression was used as internal control for equivalent cellular protein loading (lower panel). B, Corresponding effect on cell growth. Treated cell growth was monitored using trypan blue staining. C, Time course study of ALK gene silencing. Cells were treated with 3 µg/ml tetracycline for up to 8 days and NPM-ALK expression was monitored by Western blot. D, Corresponding effect on cell growth. Cell growth was examined by counting viable cells. E, Cell apoptosis. Cells were treated with 3 µg/ml tetracycline (solid bars) for 4 days, and apoptotic cells were detected by flow cytometry. Apoptosis rates (%) among cells containing shRNA-ALK (left) and control cells carrying an inducible shRNA-Lamin (right) are displayed. Experiments were repeated three times with similar results. Student’s t-test: ** p < .01 versus control.

Figure 3. Combining ALK gene silencing and U0126 had a synergistic effect on cellular JunB protein expression and cell growth. A, Cooperative effect of ALK and EKR1/2 pathways on cellular JunB expression

Cells were treated with 3 µg/ml tetracycline alone for gene silencing (lanes 2 and 6), 10 µM U0126 alone for down-regulating the ERK1/2 pathway (lanes 3 and 7), or both (lanes 4 and 8) for 6 days, and changes in the activity of signaling pathway proteins were examined. NPM-ALK and JunB expression was detected by Western blot, and ERK1/2, Akt, and STAT3 activity was detected by evaluating their phosphorylated forms (p-ERK1/2, p-STAT3, and p-Akt). Lanes 1–4: cells containing shRNA-ALK. Lanes 5–8: control cells carrying shRNA-Lamin. NPM-ALK expression (top) confirms ALK gene silencing, while β-actin expression (bottom) verifies equivalent cellular protein loading. B, Synergistic effects of ALK gene silencing and U0126 on cell growth. Cells were treated with 3 µg/ml tetracycline for ALK gene silencing (solid bars) or without (open bars). In addition, cells were exposed to different concentrations of U0126. After treatment(s) for 6 days, viable cells in each condition were counted, and relative cell growth rates were calculated. Control cells carrying shRNA-Lamin were exposed to the same treatment(s). C, Time course study of synergistic effects of ALK gene silencing and low dose U0126 on cell growth. Cells were treated with 3 µg/ml tetracycline [Tet/(−)] and 0.5 µM U0126 [(−)/U0126], individually and in combination, for 8 days, and relative cell growth rates were determined. Arrows indicate synergistic inhibition of cell growth. Control cells carrying shRNA-Lamin were exposed to the same treatment(s) (right panel). Experiments were repeated three times with similar results. Student’s t-test: ** p < .01 versus control.

Figure 4. Synergistic inhibition of cell proliferation by combining ALK gene silencing and low dose U0126

Cells containing an inducible shRNA-ALK construct (A–D) were treated with 3 µg/ml tetracycline alone [Tet/(−)], different concentrations of U0126 alone [(−)/U0126], or both tetracycline and U0126 (Tet/U0126) for 4 days, and cell proliferation was measured using an MTT assay. Control cells carrying an inducible shRNA-Lamin (E–H) were exposed to the same treatments. Experiments were repeated three times with similar results. Student’s t-test: * p < .05 and ** p < .01 versus controls.

Figure 5. Synergistic effects of ALK gene silencing and low dose U0126 on cell apoptosis and in vitro cell colony formation. A, Cell apoptosis

Cells were treated with 3 µg/ml tetracycline (lighter gray bars), 0.5 µM U0126 (darker gray bars), or both (solid bars) for 4 days and stained with Annexin-V. Control cells carrying shRNA-Lamin exposed to the same treatments are shown in the right panel. B, Cell colony formation. Cells were cultured in semi-solid culture medium containing tetracycline (lighter gray bars), 0.5 µM U0126 (darker gray bars), or both (solid bars) for 7 days, and cell colonies were counted under a light microscope. The relative cell colony formation rate (%) is shown, with non-treated cells as a negative control (open bars). Control cells carrying shRNA-Lamin exposed to the same treatments are shown in the right panel. C, Cell colony size. The diameter of colonies (n =50) was measured under a light microscope equipped with ruler lens. Combination of ALK gene silencing with low dose U0126 (left panel) and control cells carrying shRNA-Lamin (right panel). Experiments were repeated three times with similar results. Student’s t-test: * p < .05 and ** p < .01 versus controls.

Figure 6. Prolonged inhibition of cell growth by combination of ALK gene silencing and low dose U0126. A, Overview of analysis

Cells were treated with 3 µg/ml tetracycline and/or 0.5 µM U0126 for 8, 10, and 12 days (solid arrows). Treatments were then removed by cell washing, and equal numbers of viable cells from each condition were cultured in fresh medium with no treatment added for 6 days (open arrows). B, Cell growth after 8-day treatment. Cells were treated for 8 days with tetracycline [Tet/(−)], 0.5 µM U0126 [(−)/U0126], or both (Tet/U0126), then washed, and cultured in fresh medium with no treatment. Cell growth post-treatment was monitored by counting viable cells every other day. C, Cell growth after 10-day treatment. Same as for panel B but cells were treated for 10 days. D, Cell growth after 12-day treatment. Same as for panel B but cells were treated for 12 days. E, Control cell growth after 12-day treatment. Control cells carrying an shRNA-Lamin were exposed to the same treatments as described above for 12 days. All experiments were repeated three times with similar results. Student’s t-test: ** p < .01 versus controls.

Figure 7. Synergistic inhibition of xenografted ALCL tumor growth by combination of ALK gene silencing and U0126 treatment

Mice bearing tumors derived from ALCL cells containing shRNA-ALK (A) and carrying shRNA-Lamin (B) were fed tetracycline [Tet/(−)], treated with U0126 [(−)/U0126], treated with both (Tet/U0126), or none [(−)/(−)]. Tumor mass was monitored by whole body bioluminescence scanning every 3 days, and the strength of bioluminescence signals from the xenografted tumors were measured as photons in a digital format (photons/second/cm²/steradian). n=5 mice per condition. Student’s t-test: * p < .05 versus control.