Proteasome Inhibition Leads to Dephosphorylation and Downregulation of Protein Expression of Members of the Akt&Sol;Mtor

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Letters to the Editor 2442 Proteasome inhibition leads to dephosphorylation and downregulation of protein expression of members of the Akt/mTOR pathway in MCL

Leukemia (2012) 26, 2442–2444; doi:10.1038/leu.2012.118 of the PI3K complex, a main regulator of the PI3K/Akt pathway involved in the pathogenesis of MCL.4 Analyzing the impact of bortezomib on the PI3K/Akt pathway members, we could show that exposure to bortezomib results in dephosphorylation of Akt As previously shown bortezomib exhibits significant antilymphoma on Ser273 accompanied by dephosphorylation of glycogen activity not only in the well-characterized MCL (mantle cell synthase kinase 3a/b and downregulation of CyclinD1 (CCND1) lymphoma) cell lines, but also in the T-cell leukemia cell line protein (Figures 2a–d) in those cell lines with Akt protein Jurkat, and the follicular B-cell lymphoma cell line lymphoma downregulation. These findings are confirming previous studies Karpas 422. To identify common patterns of downstream events in that CCND1 is regulated by Akt via glycogen synthase kinase 3.4 MCL following bortezomib exposure, protein expression analysis CyclinD1 gene expression is activated by Wnt/b-catenin signaling, and mRNA expression analysis were performed and compared with glycogen synthase kinase 3 having a critical role. In fact with cell proliferation, cell viability and apoptosis. CCND1 degradation is influenced by glycogen synthase Investigating the mRNA expression regulated by bortezomib we kinase 3b.4 Several studies have shown that glycogen synthase identified downregulation of 68 and upregulation of 53 mRNAs, kinase 3b Ser9 is phosphorylated by Akt.5 In conclusion our results whereby one of the main hubs of the network was the PI3K suggest that downregulation of CCND1 after bortezomib exposure signaling pathway. Beside that, eight genes (downregulated- is mediated through regulation of Akt (Figure 1). PI3K3CG, GST4A; upregulated-DNAJB, GPX2, HMOX1; MAFG, VCP, Another downstream target of the PI3K/Akt pathway is SQSTM) were involved in nuclear factor (erythroid-derived 2)-like 2 mammalian target of rapamycin (mTOR). The mTOR signaling -mediated oxidative stress response. These findings are in pathway is activated and may contribute to cell cycle progression concordance with data showing that Bortezomib produces mild and tumor cell survival in MCL.4 mTOR is a serine–threonine kinase oxidative stress that activates nuclear factor (erythroid-derived 2)- downstream of Akt, which catalytic subunit consists of the two like 2-mediated gene expression1 by increased expression of multi-protein complexes mTORC1 (mTOR-Raptor) and mTORC2 genes involved in antioxidative defense like GSR, GSS, SOD2 and (mTOR-Rictor), which have distinct substrates and mechanisms of GPX2,2 but also with the correlation between high basal nuclear activation.4 Analogs of rapamycin, a mTOR inhibitor, which blocks levels of nuclear factor (erythroid-derived 2)-like 2 and reduced some of the mTORC1 functions but does not have an effect on sensitivity to bortezomib in acute myelogenous leukemia (AML).3 mTORC2, has already been shown highly active in clinical trials 4 Interestingly one of the upstream regulatory genes of this and has been recently registered for the treatment of relapsed pathway affected by bortezomib, PIK3CG (Figure 1), is a member MCL in Europe. In our study we could show that beside moderate

PI3K IRS-1 Bortezomib

× CIP2A × × PDK -P PP2A

× P-Ser2481 mTORC1 RAF/MAPK/ERK P-Ser473 mTOR P-Ser2448 Akt × -P P-Thr1135 Rictor × × × P-Ser21 mTORC1 GSK3α/β P-Ser9 P-Ser2448 mTOR P-Ser2481 × +P P-Ser792 Raptor MNK1 × NRF2 P × 4EBP1 × eIF4E P-Ser209 CCND1 p70S6 GPX2, MAFG

Oxidative Cell cycle Cell growth Cap-dependent stress translation initiation response (CCND1, c-myc ...) Figure 1. Impact of bortezomib on members of the PI3K/Akt pathway in MCL. Indicated members of the PI3K pathway were analyzed for RNA–(gray) and protein expression (orange) and also for their phosphorylatioon status (red). k, downregulation; m, upregulation; -P, dephosphorylation; þ P, phosphorylation; dashed lines and X- assumed to be interrupted interactions, continuous lines-supposed to be activated interactions. The color reproduction of this ﬁgure is available at the Leukemia journal online.

Accepted article preview online 3 May 2012; advance online publication, 15 June 2012

Leukemia (2012) 2414 – 2444 & 2012 Macmillan Publishers Limited Letters to the Editor 2443 MCL cell lines Control cell lines Jeko-1 Rec-1 Hbl-2 Granta NCEB-1 Karpas Jurkat mTOR 0.97 0.6 0.03 0.3 0.6 0.25 0.24 Ratio to untreated 1.00 MCL cell lines Control cell lines phmTOR(Ser2481) 0.80 0.05 0.03 0.1 0.08 0.17 0.11 0.12 Ratio to untreated

0.60 4EB-P1 0.40

0.20 p70S6 Ratio to untreated 0.35 0.2 0.35 0.39 0.4 0.4 0.17 Ratio to untreated 0.00 Jeko Rec Hbl Granta Karpas Jurkat βActin -+-+ -+-+ - + -+ -+Bortezomib (25nM) phAkt Akt βActin MCL cell lines Control cell lines Jeko-1 Rec-1 Hbl-2 Granta NCEB-1 Karpas Jurkat phEIF4E 2.00 MCL cell lines Control cell lines 0.141.6 0.11 0.37 1.65 4.9 Ratio to untreated

1.50 EIF4E 0.46 0.76 0.59 1.06 0.82 1.16 Ratio to untreated 1.00 MNK 0.50 0.11 1.2 0.1 0.73 0.75 1.3 Ratio to untreated Ratio to untreated β 0.00 Actin JekoRec Hbl Granta NCEB Karpas Jurkat -+-+-+-+-+- +Bortezomib (25nM) phGSK3α GSK3α Hbl-2 Jeko-1 βActin ph-Rictor (Thr1135) 11.2 1.38 0.4 0.04 0.130.05 0.04 Ratio to untreated

Rictor 2.00 MCL cell lines Control cell lines 11.2 0.36 0.12 0.03 0.680.51 0.07 Ratio to untreated 1.50 ph-Raptor (Ser792) 1.00 1.2 0.92 1 1 0.36 1.10.27 0.33 Ratio to untreated 0.50 Raptor Ratio to untreated 0.00 0.89 0.87 0.58 0.1 0.23 0.7 0.86 0.64 Ratio to untreated JekoRec Hbl Granta NCEB Karpas Jurkat phGSK3β phmTOR(2448) β GSK3 1.14 1.55 1.0 1.37 0.24 0.48 0.46 0.04 Ratio to untreated βActin phmTOR(2481)

Jeko-1 Rec-1 Hbl-2 Granta519 NCEB-1 1.07 1.19 2.78 0.3 0.01 0.570.24 0.04 Ratio to untreated BCL-2 mTOR 1.2 1.6 1.7 1.16 Ratio to untreated CCND1 0.75 0.94 0.83 0.79 0.36 1.02 1.2 0.67 Ratio to untreated 0.06 0.09 0.2 1.2 0.5 Ratio to untreated PDK c-myc 0.75 0.94 0.83 0.79 0.36 1.04 2.1 0.29 1.08 0.9 Ratio to untreated βActin CIP2A -+ -+ -+ -+ -+Bortezomib (25nM) 1.00 1.11 0.49 0.41 0.21 0.831.16 0.74 Ratio to untreated βActin

K 0.25 2.5 6.25 12.5 25 K 6.25 12.5 25 nM bortezomib

Figure 2. Protein levels of total and phosphorylated proteins of the PI3K/AKT/mTOR pathway after 24 h bortezomib (25 nM) treatment as determined by western blot analyses of three independent experiments. (a–c) Diagram of the protein- and phosphorylation levels of Akt and GSK3a/b after treatment with bortezomib executed as the ratio to untreated after normalization to b-actin together with western blot images (left-untreated; right-treated). (d–g) Western blot images together with the quantiﬁed protein– and phosphorylation levels as a ratio to untreated after normalization to bactin of downstream (d–f) and upstream targets (g) as well as members of the mTOR complexes (e, g). downregulation and dephosphorylation of mTOR bortezomib activating Akt via mTORC2 due to inhibition of mTORC1, downregulated and dephosphorylated Raptor (Ser792), as well as described for rapamycin and analogs,4 will be inhibited (Figure 1). Rictor (Thr1135) (Figures 2e and f). This data indicate that Bortezomib also induced dephosphorylation and downregula- bortezomib has an effect on both mTOR complexes, mTORC1 tion of substrates of TORC1, 4E-BP1 and p70S6. It is known that and mTORC2 (Figure 1 and Figures 2e and f). Accordingly phosphorylation of 4E-BP1 is a crucial step in the oncogenic bortezomib not only induced cell cycle arrest, as do rapamycin pathway downstream of Akt/mTOR and results in increased analogs,4 but also apoptosis. Thus, bortezomib acts on both translation of eIF-4E–dependent proteins, like cyclinD1, BCL2 and complexes simultaneously and the negative feedback loop c-myc.4 Therefore dephosphorylation of 4E-BP1 should lead to

& 2012 Macmillan Publishers Limited Leukemia (2012) 2414 – 2444 Letters to the Editor 2444 decrease of translation efficiency of the target proteins (Figure 1). development of future combined approaches in mantle cell However, only downregulation of CCND1 was detected, while Bcl2 lymphoma, based on the identified molecular mechanisms of and c-myc were not significantly influenced except in one cell line, proteasome inhibitors and other small molecules targeting the HBL-2, with c-myc downregulation (Figure 2d). Thus, an alternative PI3K/mTOR–or related (B-ceIl receptor) pathways. mechanism of translation initiation could use an internal ribosomal entry site (IRES).6 In fact c-myc,7 Bcl2(ref. 8) and CCND1(ref. 9) mRNAs contain an internal ribosomal entry site in the CONFLICT OF INTEREST 50untranslated regions that maintain protein expression after a The coauthor M Dreyling is a member of the scientific advisory board of Janssen and number of pathophysiological signals including apoptosis, received speakers honoraria and support of investigator-initiated trials by Janssen. genotoxic stress and viral infection.9 Importantly, the internal ribosomal entry site can stimulate translation initiation during the G Hutter1,2, Y Zimmermann1,2, M Rieken1,2, E Hartmann3, G2/M phase whereas cap-dependent protein synthesis is A Rosenwald3, W Hiddemann1,2 and M Dreyling1,2 inhibited.10 These data may explain that in our study Bcl2 and 1Department of Medicine III, University Hospital Grosshadern/LMU, c-myc proteins were not downregulated despite the observed Munich, Germany; cell apoptosis. Nevertheless the downregulation of CCND1 by 2CCG Leukemia, Helmholtz Zentrum, Munich, Germany and bortezomib correlated with that of eIF-4E expression, a trans- 3Institute of Pathology, University of Wuerzburg, lation initiation factor of cap-dependent translation initiation. Wuerzburg, Germany Interestingly eIF-4E phosphorylation was downregulated in Correspondence: E-mail: [email protected] concordance with the downregulation of MAP kinase interacting kinase 1, which phosphorylates EIF-4E11 (Figure 2g). The mitogen- activated protein kinase (MAPK) signaling is another downstream REFERENCES target of Akt and is linked to the mTOR signaling through MAP 1 Du ZX, Zhang HY, Meng X, Guan Y, Wang HQ. Role of oxidative stress and kinase interacting kinase. In the present study protein expression intracellular glutathione in the sensitivity to apoptosis induced by proteasome of other downstream targets of this signal pathway, namely the inhibitor in thyroid cancer cells. 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Supplementary Informationaccompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia (2012) 2414 – 2444 & 2012 Macmillan Publishers Limited