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Suppression of programmed cell death 4 (PDCD4) protein expression by BCR-ABL-regulated engagement of the mTOR/p70 S6 kinase pathway - PubMed

️Tue Jan 01 2008

Suppression of programmed cell death 4 (PDCD4) protein expression by BCR-ABL-regulated engagement of the mTOR/p70 S6 kinase pathway

Nathalie Carayol et al. J Biol Chem. 2008.

Abstract

There is accumulating evidence that mammalian target of rapamycin (mTOR)-activated pathways play important roles in cell growth and survival of BCR-ABL-transformed cells. We have previously shown that the mTOR/p70 S6 kinase (p70 S6K) pathway is constitutively activated in BCR-ABL transformed cells and that inhibition of BCR-ABL kinase activity by imatinib mesylate abrogates such activation. We now provide evidence for the existence of a novel regulatory mechanism by which BCR-ABL promotes cell proliferation, involving p70 S6K-mediated suppression of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein that acts as an inhibitor of cap-dependent translation by blocking the translation initiation factor eIF4A. Our data also establish that second generation BCR-ABL kinase inhibitors block activation of p70 S6K and downstream engagement of the S6 ribosomal protein in BCR-ABL transformed cells. Moreover, PDCD4 protein expression is up-regulated by inhibition of the BCR-ABL kinase in K562 cells and BaF3/BCR-ABL transfectants, suggesting a mechanism for the generation of the proapoptotic effects of such inhibitors. Knockdown of PDCD4 expression results in reversal of the suppressive effects of nilotinib and imatinib mesylate on leukemic progenitor colony formation, suggesting an important role for this protein in the generation of antileukemic responses. Altogether, our studies identify a novel mechanism by which BCR-ABL may promote leukemic cell growth, involving sequential engagement of the mTOR/p70 S6K pathway and downstream suppression of PDCD4 expression.

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Figures

**FIGURE 1.**
**Effects of imatinib mesylate on the phosphorylation/activation status of elements of the mTOR pathway in Ba/F3 cells expressing BCR-ABL-resistant mutants.** A (*top*), Ba/F3 cells stably transfected with empty vector, wild type (native) BCR-ABL, or the Y253F, E255K, H396P, or T315I mutants were incubated with imatinib mesylate (STI571) (1 μ
m
) for 2 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of the p70 S6 kinase on Thr-389 or against GAPDH, as indicated. *Bottom*, the signals were quantitated by densitometry. Data are expressed ratios of phosphorylated p70 S6 kinase on Thr-389 to GAPDH levels and represent means ± S.E. of three independent experiments. Paired t test analysis for the phosphorylation of p70 S6 kinase on Thr-389 in wild type BCR-ABL-transfected cells treated with imatinib mesylate *versus* control untreated cells showed a p value of 0.023. Similar analysis for Y253F and H396P transfected cells showed p = 0.475 and p = 0.107, respectively. B (*top*), Ba/F3 cells stably transfected with empty vector, wild type (native) BCR-ABL, or the Y253F, E255K, H396P, or T315I mutants were incubated with imatinib mesylate (STI571) (1 μ
m
) for 2 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of the S6 ribosomal protein on Ser-240/244 or against GAPDH, as indicated. The signals were quantitated by densitometry. Data are expressed ratios of phosphorylated S6 ribosomal protein on Ser-240/244 to GAPDH and represent means ± S.E. of three independent experiments. Paired t test analysis for the phosphorylation of Ser-240/244 in wild type BCR-ABL-transfected cells treated with imatinib mesylate *versus* control untreated cells showed a p value of 0.029. Similar analysis for Y253F and H396P transfected cells showed p = 0.113 and p = 0.160, respectively. C, Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or E255K mutant were incubated with imatinib mesylate (STI571) (1 μ
m
) in the presence or absence of rapamycin (20 n
m
) for 30, 60, and 90 min, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236 or Ser 240/244 or against the phosphorylated form of the p70 S6 kinase on Thr-389 or with an antibody against GAPDH, as indicated.

**FIGURE 2.**
**Effects of nilotinib on the phosphorylation/activation status of elements of the mTOR pathway in Ba/F3 cells expressing BCR-ABL-resistant mutants.** A, growth-inhibitory effects of nilotinib (AMN107) in Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the E255K mutant. Cells were treated for 7 days with solvent control (Me₂SO) or with the indicated concentrations of nilotinib (AMN-107). Results are expressed as percentage of control (Me₂SO-treated) cells. B, Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the Y253F, E255K, H396P, or T315I BCR-ABL mutants were incubated with nilotinib (AMN107) (1 μ
m
) for 2 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236 or Ser-240/244 or against the phosphorylated form of the p70 S6 kinase on Thr-389 or antibodies against the S6 ribosomal protein or the p70 S6K, as indicated. C, Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the E255K BCR-ABL mutant were incubated with nilotinib (AMN107) (1 μ
m
) for 30, 60, and 90 min as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236 or Ser-240/244 or against the phosphorylated form of the p70 S6 kinase on Thr-389 or against GAPDH, as indicated. D, Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the E255K BCR-ABL mutant were incubated with nilotinib (AMN107) (1 μ
m
), in the presence or absence of rapamycin (20 n
m
) for 30, 60, and 90 min, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236 or Ser-240/244 or against the phosphorylated form of the p70 S6 kinase on Thr-389 or an antibody against GAPDH, as indicated.

**FIGURE 3.**
**Effects of nilotinib on the phosphorylation/activation status of elements of the mTOR pathway and PDCD4 expression in Ba/F3 cells expressing BCR-ABL-resistant mutants.**A, cell lysates from Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the Y253F or E255K or H396P or T315I BCR-ABL mutants were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. B, cell lysates from Ba/F3 cells stably transfected with MIGR1 empty vector, MIGRI wild type BCR-ABL, or MIG P210 KI mutant were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. C, Ba/F3 cells stably transfected with empty vector or wild type BCR-ABL were incubated with nilotinib (AMN107) (1 μ
m
) for 12 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236, against the phosphorylated form of the p70 S6 kinase on Thr-389, against PDCD4, or against GAPDH, as indicated. D, basal expression of mRNA for the *pdcd4* gene was evaluated by quantitative RT-PCR (TaqMan) in Ba/F3 cells stably transfected with empty vector, wild type BCR-ABL, or the Y253F or E255K or H396P or T315I BCR-ABL mutants. GAPDH was used for normalization. Data are expressed as -fold change over Ba/F3 cells stably transfected with empty vector and represent means ± S.E. of three independent experiments. Two-tailed paired t test analysis for the -fold changes of the various transfectants showed the indicated p values. E, Ba/F3 cells stably transfected with empty vector or wild type BCR-ABL were incubated for 12 h at 37 °C in the absence or presence of AMN107 (100 n
m
). Expression of mRNA for the *pdcd4* gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over AMN107-untreated samples and represent means ± S.E. of two independent experiments. Paired two-tailed t test analysis showed a two-tailed p = 0.045 for AMN107-treated *versus* untreated wild type BCR-ABL-expressing cells.

**FIGURE 4.**
**Up-regulation of PDCD4 protein expression by BCR-ABL kinase inhibitors.** A, K562 cells were incubated with nilotinib (AMN107) (100 n
m
) for 12 h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of the S6 ribosomal protein on Ser-235/236 or the phosphorylated form of the p70 S6 kinase on Thr-389 or PDCD4 or GAPDH, as indicated. B, K562 cells were incubated with nilotinib (AMN107 100 n
m
) for 1, 2, 4, 6, 12, and 24, h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. C, K562 cells were incubated with nilotinib (100 n
m
), STI 571 (1 μ
m
), or rapamycin (20 n
m
) for 12 h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or the GAPDH, as indicated. D, K562 cells were incubated for 12 h at 37 °C in the absence or presence of AMN107 (100 n
m
). Expression of mRNA for the *pdcd4* gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over AMN107-untreated samples and represent means ± S.E. of two experiments. Paired two-tailed t test analysis showed a two-tailed p = 0.022.

**FIGURE 5.**
**Effect of cycloheximide and actinomycin D on up-regulation of PDCD4 protein expression by AMN107.** A, Ba/F3-BCR-ABL cells were incubated with nilotinib (AMN107) (100 n
m
) in the presence or absence of cycloheximide (20 μ
m
) for 12 h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. B, the signals were quantitated by densitometry, and the ratios of PDCD4 over GAPDH were calculated. Data are expressed as -fold induction for PDCD4 levels normalized to GAPDH, over untreated samples in response to the indicated treatments and represent means ± S.E. of three independent experiments. C, Ba/F3-BCR-ABL cells were incubated with nilotinib (AMN107) (100 n
m
) in the presence or absence of actinomycin D (2 μg/ml) for 12 h as indicated. Expression of mRNA for the *pdcd4* gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over AMN107-untreated samples and represent means ± S.E. of two experiments. D, Ba/F3-BCR-ABL cells were incubated with nilotinib (AMN107) (100 n
m
) in the presence or absence of actinomycin D (2 μg/ml) for 12 h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. E, the signals for the PDCD4 protein were quantitated by densitometry, and the ratios of PDCD4 over GAPDH were calculated. Data are expressed as -fold induction for PDCD4 levels normalized to GAPDH over untreated samples in response to the indicated treatments and represent means ± S.E. of three independent experiments.

**FIGURE 6.**
**Effects of dasatinib on PDCD4 expression in BCR-ABL-expressing cells.** A, K562 cells were incubated with nilotinib (AMN107) (100 n
m
), STI 571 (1 μ
m
), or dasatinib (10 n
m
) for 12 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or Ser-240/244 rpS6 or a cleaved form of poly(ADP-ribose) polymerase (Asp-214) or GAPDH, as indicated. B, the signals for PDCD4 were quantitated by densitometry. Data are expressed ratios of PDCD4 protein to GAPDH levels and represent means ± S.E. of two independent experiments. C, K562 cells were incubated with dasatinib (10 n
m
) for 6, 12, and 24 h as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated forms of the S6 ribosomal protein on Ser-235/236 or Ser-240/244 or GAPDH, as indicated. D, K562 cells were incubated with dasatinib (10 n
m
) for 6, 12, and 24 h, as indicated. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against the phosphorylated form of the p70 S6 kinase on Thr-389 or GAPDH, as indicated.

**FIGURE 7.**
**Effects of PDCD4 knockdown on nilotinib-mediated suppression of leukemic progenitor (CFU-L).** A, Ba/F3-BCR-ABL cells were transfected with control siRNA or PDCD4 siRNA (targeting mouse PDCD4) and were incubated for 12 h, in the presence of nilotinib. Expression of mRNA for the *pdcd4* gene was evaluated by quantitative RT-PCR (TaqMan), using *Gapdh* for normalization. Data are expressed as percent control siRNA-transfected samples and represent means ± S.E. of two independent experiments. B, Ba/F3-BCR-ABL cells were transfected with control siRNA or PDCD4 siRNA and subsequently incubated in methylcellulose, in the presence or absence of nilotinib (100 n
m
), and leukemic CFU-L colony formation was assessed. Data are expressed as percentage of control colony formation of untreated samples for each condition and represent means ± S.E. of five independent experiments. Paired t test analysis comparing the effects of nilotinib in the presence of PDCD4 siRNA *versus* control siRNA showed a two-tailed p = 0.034. C, Ba/F3-BCR-ABL cells were transfected with control siRNA or PDCD4 siRNA and subsequently incubated in methylcellulose, in the presence or absence of imatinib mesylate (STI571) (1 μ
m
), and leukemic CFU-L colony formation was assessed. Data are expressed as percentage of control colony formation of untreated samples for each condition and represent means ± S.E. of five independent experiments. Paired t test analysis comparing the effects of imatinib mesylate in the presence of PDCD4 siRNA *versus* control siRNA showed a two-tailed p = 0.03.

**FIGURE 8.**
**Reversal of nilotinib-mediated suppression of leukemic progenitor (CFU-L) growth by PDCD4 knockdown and negative regulatory effects of PDCD4 on BCR-ABL-expressing leukemic progenitor growth.** A, Ba/F3-BCR-ABL cells were transfected with control siRNA or mouse PDCD4 siRNA and were or were not concomitantly transfected with a construct for human PDCD4 cDNA, as indicated. After nilotinib treatment, total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against PDCD4 or GAPDH, as indicated. B, Ba/F3-BCR-ABL cells were transfected with control siRNA or PDCD4 siRNA (targeting mouse PDCD4) and were or were not concomitantly transfected with a construct for human PDCD4 cDNA, as indicated. The cells were subsequently plated in methylcellulose, in the presence or absence of nilotinib (100 n
m
), and leukemic CFU-L colony formation was assessed. Data are expressed as percentage of control colony formation of untreated samples for each condition and represent means ± S.E. of five independent experiments. Paired t test analysis comparing the effects of nilotinib in the presence of PDCD4 siRNA *versus* control siRNA showed a p value of 0.0074 and a p value of 0.0067 for the effects of nilotinib in the presence of PDCD4 siRNA and human PDCD4 cDNA *versus* PDCD4 siRNA alone. C, Ba/F3-BCR-ABL cells were transfected with PCDNA3 vector control or PDCD4 S67A/S71A mutant. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against HA tag and GAPDH as indicated. D, Ba/F3-BCR-ABL cells were transfected with control empty vector (pcDNA3) or a pcDNA3-PDCD4-S67A/S71A construct. The cells were subsequently plated in methylcellulose, and leukemic CFU-L colony formation was assessed. Data are expressed as colony numbers for each condition and represent means ± S.E. of five independent experiments. Paired t test analysis comparing the effects of PDCD4 overexpression *versus* control empty vector showed a p value of 0.027.

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Suppression of programmed cell death 4 (PDCD4) protein expression by BCR-ABL-regulated engagement of the mTOR/p70 S6 kinase pathway - PubMed