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. 2012 Jul 26;120(4):858-67.
doi: 10.1182/blood-2012-02-407999. Epub 2012 Jun 8.

Cytotoxic effects of bortezomib in myelodysplastic syndrome/acute myeloid leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1

Jing Fang  1 , Garrett Rhyasen, Lyndsey Bolanos, Christopher Rasch, Melinda Varney, Mark Wunderlich, Susumu Goyama, Gerrit Jansen, Jacqueline Cloos, Carmela Rigolino, Agostino Cortelezzi, James C Mulloy, Esther N Oliva, Maria Cuzzola, Daniel T Starczynowski
Affiliations

Cytotoxic effects of bortezomib in myelodysplastic syndrome/acute myeloid leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1

Jing Fang et al. Blood. .

Erratum in

  • Blood. 2014 Jun 5;123(23):3682

Abstract

Bortezomib (Velcade) is used widely for the treatment of various human cancers; however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that bortezomib induces proteasome-independent degradation of the TRAF6 protein, but not mRNA, in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA _targeting TRAF6 were resensitized to the cytotoxic effects of bortezomib due to down-regulation of the proteasomal subunit α-1 (PSMA1). To determine the molecular consequences of loss of TRAF6 in MDS/AML cells, in the present study, we applied gene-expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe herein novel mechanisms by which TRAF6 is regulated through bortezomib/autophagy-mediated degradation and by which it alters MDS/AML sensitivity to bortezomib by controlling PSMA1 expression.

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Figures

Figure 1
Figure 1
Inhibitory effect of bortezomib on MDS and AML cell viability and progenitor function. (A) THP1 cells were treated with 10nM bortezomib or DMSO for the indicated times. Protein lysates were immunoblotted for ubiquitin-conjugated proteins and GAPDH (left panel). BM cells from MDS (MDS-01) and AML (AML-03) patients were treated with 10nM bortezomib or DMSO for 24 hours and evaluated by immunoblotting for ubiquitin and GAPDH (right panels). (B) The indicated cell lines and primary patient samples were treated with bor-tezomib for 24 hours and analyzed for cell survival by staining for annexin V+ cells. (C) Hematopoietic stem/progenitor cell colony-forming potential of THP1 and MDSL cell lines treated with bortezomib (10nM) was determined in methylcellulose. (D) Hematopoietic stem/progenitor cell colony-forming potential of control CD34+ cells and BM cells from MDS (MDS-01 and MDS-02) and AML (AML-03) patients treated with bortezomib (10nM) was determined in methylcellulose. Colonies were evaluated after 10 days. *P < .05; #P < .1.
Figure 2
Figure 2
Reduced TRAF6 protein expression in bortezomib-treated cells coincides with autophagy activation. (A) TRAF6 mRNA was measured by quantitative PCR in the indicated cell lines and CD34+ cells treated with increasing concentrations of bortezomib for 24 hours. (B) TRAF6 protein was measured by immunoblotting of the indicated cell lines treated with increasing concentrations of bortezomib for 24 hours. (C) HL60 and TF-1 cell lines cultured with bortezomib for 24 hours were evaluated by immunoblotting for LC3-I/II, p62, and GAPDH. (D) TF-1 cells were treated with the proteasome inhibitor, MG-132 (0, 1, and 10μM) for 24 hours and evaluated by immunoblotting for TRAF6, LC3-I/II, p62, and GAPDH. (E) TF-1 cells were treated with an autophagy inducer (100nM rapamycin) and an ER stress inducer (100nM thapsigargin). (F) Cell viability of THP-1 cells that have acquired resistance to bortezomib (Bort-R) and that are sensitive (Bort-S) was determined after treatment with increasing concentrations of bortezomib for 24 hours. (G) TRAF6 protein was measured by immunoblotting Bort-S and Bort-R cells treated with increasing concentrations of bortezomib for 24 hours. Values below the gel represent the intensity of TRAF6 relative to ACTIN.
Figure 3
Figure 3
TRAF6 is degraded by the autophagy lysosome pathway. (A) The indicated cell lines were cultured with 10nM bortezomib and cotreated with 5mM 3-MA for 24 hours. TRAF6 and GAPDH protein was determined by immunoblot analysis. (B) In the absence of proteasome inhibition, TF-1 cells were treated with 5mM 3-MA for 24 hours, and TRAF6 protein expression was measured by immunoblot analysis. (C) cDNA from TF-1 cells treated with 3-MA (5mM) or 3-MA and bortezomib (10nM) was evaluated for TRAF6 mRNA expression. Data are relative to GAPDH and normalized to cells treated with DMSO. (D) Cell viability of THP1 and TF-1 cells was determined after treatment with DMSO, 10nM bortezomib, or bortezomib and 5mM 3-MA for 24 hours. *P < .05.
Figure 4
Figure 4
Levels of TRAF6 expression affect the cytotoxic effects of bortezomib. (A) THP1 cells were transduced with a bicistronic lentiviral vector encoding TRAF6 and green fluorescent protein (GFP) or an empty vector expressing only GFP. Transduced cells were sorted for GFP expression, cultured with bortezomib (10nM) for 24 hours, and protein lysates were evaluated for TRAF6 expression. (B) Cell viability of bortezomib-treated THP1 cells transduced with vector or TRAF6 was measured by annexin V/propidium iodide staining. (C) Summary of replicate experiments from panel B. (D) Bort-S and Bort-R THP1 cells were transduced with shRNA lentiviral vectors _targeting TRAF6 or shCTL. Knockdown of the TRAF6 protein was confirmed by immunoblot analysis. (E-F) Cell viability of Bort-S (E) and Bort-R (F) cells transduced with shTRAF6 or control vector was determined by MTT assay after treatment with increasing concentrations of bortezomib for 24 hours (P < .05). (G-H) Proteasome inhibition in bortezomib-treated Bort-S (G) and Bort-R (H) cells transduced with shTRAF6 or control vector was determined by immunoblotting for ubiquitinated proteins (Ub) on total cell lysates. #P < .1; *P < .05.
Figure 5
Figure 5
Depletion of TRF6 sensitizes cells to bortezomib by regulating PSMA1. (A) Microarray analysis was performed on TF-1 cells after knockdown of TRAF6. Shown are differential gene-expression data between shTRAF6 and vector-transduced cells for all proteasome subunit genes (see supplemental Table 1). Expression of PSMA1 (highlighted in red) was most significantly down-regulated in cells with reduced TRAF6 expression (P < .05). (B) TF-1, Bort-S THP-1, Bort-R THP-1, and MDS BM cells (MDS-02) were transduced with shRNA lentiviral vectors _targeting TRAF6 or a non_targeting control (shCTL). Down-regulation of PSMA1 in cells with reduced TRAF6 expression was confirmed by quantitative PCR. (C) Bort-R cells were transduced with 2 independent shRNA lentiviral vectors (sh#70 and sh#72) _targeting PSMA1 or shCTL. Knockdown of PSMA1 was confirmed by quantitative PCR (sh#70, 89.6% knockdown; sh#72, 85.6% knockdown). Cell viability of Bort-R cells transduced with shPSMA1 or control vector was determined by MTT assay after treatment with 10nM bortezomib for 24 hours. (D) Proteasome inhibition in bortezomib-treated Bort-R cells transduced with 2 independent shPSMA1 or control vector was determined by immunoblotting for ubiquitinated proteins on total cell lysates. *P < .05.
Figure 6
Figure 6
Depletion of TRAF6 impairs MDS and AML cell viability. (A) Microarray analysis was performed on TF-1 cells after knockdown of TRAF6 (as in Figure 6A). Gene set enrichment analysis was performed, and the profile of the P53 signaling gene set is shown. (B) Knockdown of TRAF6 mRNA and protein by shTRAF6 was confirmed by quantitative PCR (supplemental Figure 5) and immunoblot analysis, respectively. (C) The indicated cell lines transduced with shCTL or shTRAF6 were evaluated for cell viability using the MTT assay. Cell viability is shown at the indicated days after transduction. (D) Cell viability of transduced HL60 and AML-03 patient cells was measured by annexin V/propidium iodide staining. Knockdown of TRAF6 mRNA in AML-03 was confirmed by quantitative PCR and showed > 50% reduced expression (not shown). (E) Hematopoietic stem/progenitor cell colony-forming potential of the indicated cell lines and MDS/AML patient BM cells after knockdown of TRAF6 was determined in methylcellulose. Colonies were evaluated after 10 days. *P < .05.
Figure 7
Figure 7
Model of TRAF6 degradation by bortezomib-induced autophagy and its role in cell leukemia cell survival. (A) Under normal conditions, TRAF6 induces expression of survival genes. In addition, TRAF6 mediates the expression of PSMA1, an α-subunit of the proteasome. (B) Proteasome inhibition with bortezomib coincides with induction of autophagy, potentially due to ER stress of accumulated nondegraded proteins. TRAF6 is degraded by autophagic lysosomes, which can be blocked with the autophagy inhibitor 3-MA. Depletion of TRAF6 results in apoptotic cell death, in part by down-regulation of survival genes. In addition, loss of TRAF6 results in reduced PSMA1 expression and increased bortezomib sensitivity. α indicates the α-subunit; and β, the β-subunit.
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