Induction of autophagy does not alter the anti-tumor effects of HDAC inhibitors

Citation: Cell Death and Disease (2012) 3, e387; doi:10.1038/cddis.2012.128

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A Newbold1,3, SJ Vervoort1,3,4, BP Martin1, M Bots*,1,5 and RW Johnstone*,1,2,5

Cell Death and Disease (2012) 3, e387; doi:http://dx.doi.org/10.1038/cddis.2012.128

Web End =10.1038/cddis.2012.128 ; published online 6 September 2012 Subject Category: Cancer

Dear Editor,

HDAC inhibitors (HDACis) can suppress the growth and/or survival of tumor cells and thus far two HDACis, vorinostat and romidepsin, have been approved by the FDA for the treatment of hematological malignancies.1 Vorinostat is a potent HDACi that mediates tumor cell-selective apoptosis and we and others have previously shown that the induction of apoptosis correlates with therapeutic efcacy in mouse models of hematological cancer.25 Although apoptosis may be the preferred mode of HDACi-induced cell death, it is clear that in tumor cells with nonfunctional apoptotic cascades, HDACi are capable of inducing a caspase-independent form of cell death.57 We recently demonstrated that Em-myc lymphomas devoid of Apaf-1 displayed a delayed cell death in response to

HDACi treatment, and loss of Apaf-1 failed to affect therapeutic efcacy.5 The delayed HDACi-induced cell death was concomitant with biochemical and morphological changes characteristic of autophagy.5 Herein we utilized the genetically tractable Em-myc mouse model to address conicting reports in the eld regarding the importance of autophagy in regulating the anti-tumor responses of HDACi.68

To determine whether autophagy had any role in regulating the response of Em-myc/Apaf-1 / cells to HDACi, we knocked down the expression of two key autophagy proteins (Atg5 and Atg7) using constitutive short-hairpin RNAs (shRNAs). We developed two distinct shRNAs against Atg5 and one shRNA against Atg7 that efciently silenced the expression of Atg5 and Atg7, respectively (Figure 1a). To determine if knockdown of Atg5 or Atg7 impaired HDACi-mediated autophagy, we treated Atg5- and Atg7-shRNA-expressing Em-myc/Apaf-1 / lymphomas with vorinostat and determined the ratio of LC3-I/LC3-II by western blot. In

Em-myc/Apaf-1 / cells with decreased expression of Atg5 or Atg7, vorinostat-induced processing of LC3-I to LC3-II was greatly reduced (Figure 1a).

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Induction of autophagy does not alter the anti-tumor effects of HDAC inhibitors

We next evaluated the response of Atg5- and Atg7-shRNA-expressing Em-myc/Apaf-1 / lymphomas to increasing concentrations of vorinostat over time. Consistent with our previous results,5 a delay in the kinetics of cell death was observed in vorinostat-treated Em-myc/Apaf-1 / compared with Em-myc lymphomas (Figure 1b). However, depletion of

Atg5 or Atg7 did not affect vorinostat-mediated tumor cell death, indicating that inhibition of autophagy neither protected Em-myc/Apaf-1 / cells from HDACi-mediated cell death nor potentiated its anticancer effect. To evaluate the response of Atg5- and Atg7-shRNA-expressing Em-myc/Apaf-1 /

lymphomas to vorinostat in vivo, we transplanted lymphomas into recipient mice and treated tumor-bearing mice with vorinostat. Consistent with our in vitro data, Atg5- and Atg7-shRNA-expressing lymphomas were equally responsive to vorinostat and cleared with similar kinetics as control-shRNA-expressing lymphomas (Figure 1c). Finally, we determined whether inhibition of autophagy had any effect on the therapeutic efcacy of vorinostat in Em-myc/Apaf-1 /

lymphomas. We observed an enhanced survival in mice bearing Atg5-shRNA Em-myc/Apaf-1 / lymphomas after treatment with vorinostat (median survival vorinostat: 43.5 days, median survival vehicle: 25 days, Po0.0084), which was similar to the therapeutic response seen with control-shRNA-expressing lymphomas (median survival vorinostat: 48 days compared with vehicle: 23.5 days, Po0.0001). In conclusion, our data demonstrate that there is no evidence supporting an essential role for autophagy in regulating the response of apoptosis-decient tumors to HDACi. Recent data suggest that in tumor cells with apoptotic defects, inhibition of autophagy may potentiate the therapeutic response mediated by HDACi.7 In contrast, we provide evidence that combining autophagy inhibitors with HDACi may not be clinically benecial in lymphomas with apoptotic defects.

1Cancer Therapeutics Program, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne 3002, Victoria, Australia and 2Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville 3010, Victoria, Australia*Corresponding authors: M Bots. Current address: Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected] or RW Johnstone, Cancer Therapeutics Program, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne 3002, Victoria, Australia. Tel: [phone]; Fax: [phone]; E-mail: mailto:[email protected]

Web End [email protected]

3Co-rst authors.

4Current address: Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands.

5Co-senior authors.

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2

shATG5 (1472)

shATG5 (1389)

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[afii9825]-[afii9826]-Actin

1:1 1:5 1:2 1:38 1:1 1:17 1:1 1:2

LC3I

LC3II

Loading controls (Erk, Actin)

(LC3I: LC3II)

shATG7

shATG7 (698)

shSCR

shATG7 (698)

shSCR

D

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shATG7 (698)

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24 hours

80

100

0 0.0 0.5 1.0 1.5 2.0

48 hours

% Annexin/PI positive

cellss

% Annexin/PI positive

cells

V

100 EmycEmyc/Apaf1-/-

Emyc/Apaf1-/-shSCR

Emyc Emyc/Apaf1-/-

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Emyc/Apaf1-/-shATG7

80

60

60

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40

40

20

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vorinostat ([afii9839]M)

vorinostat ([afii9839]M)

Figure 1 Vorinostat does not require a functional autophagic pathway to induce tumor cell death in Em-myc/Apaf-1 / lymphomas. (a) Em-myc/Apaf-1 / lymphomas were retrovirally transduced with constructs expressing shRNAs targeting Atg5 (clone 1472 and 1389, respectively), Atg7 (clone 698) or a scrambled control. Knockdown efciency was tested via western blot using antibodies specic for Atg5 and Atg7 (Cell Signaling Technologies, Inc., Danvers, MA, USA, no. 2630 and 2631). Anti-b-actin was used as a loading control. Results are a representative of at least three separate westerns. Atg5-shRNA-expressing Em-myc/Apaf-1 / (1389 and 1472), Atg7-shRNA-expressing Em-myc/Apaf-1 / (698) and control-shRNA-expressing Em-myc/Apaf-1 / lymphomas were treated with 1 mM vorinostat (V) or DMSO (D) for 24 h and LC3 processing was determined by western blot using antibody specic for LC3 (NanoTools Antikoerpertechnik GmbH & Co. KG., Teningen, Germany, no. 0260-100/LC3-2G6).

Anti-p44/42 ERK (Cell Signaling Technology, Inc., Danvers, MA, USA, no. 9107) or anti-b-actin were used as loading controls. Quantitative western analysis was performed using ImageJ software (public domain software by Wayne Rasband), National Institute of Health, USA. Results are representative of at least three separate westerns.

(b) Em-myc, Em-myc/Apaf-1 / and Em-myc/Apaf-1 / lymphomas expressing the various shRNA constructs were treated with increasing concentrations of vorinostat for 24 and 48 h. Cell viability was assessed by annexin V/propidium iodide staining and error bars indicate S.E.M. of at least three independent experiments. (c) C57Bl/6 mice bearing Atg5-shRNA-expressing Em-myc/Apaf-1 / (1389), Atg7-shRNA-expressing Em-myc/Apaf-1 / (698) or control-shRNA-expressing Em-myc/Apaf-1 /

lymphomas were treated with one dose of vorinostat (200 mg/kg). Spleen and lymph nodes were harvested at the indicated time points (h) following HDACi treatment and the percentage of lymphoma cells in lymph nodes was determined using ow cytometry in the presence of propidium iodide. Each time point is representative of 23 mice

Cell Death and Disease

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3. Lindemann RK et al. Proc Natl Acad Sci USA 2007; 104: 80718076.4. Newbold A et al. Mol Cancer Ther 2008; 7: 10661079.5. Ellis L et al. Blood 2009; 114: 380393.6. Shao Y et al. Proc Natl Acad Sci USA 2004; 101: 1803018035.7. Gammoh N et al. Proc Natl Acad Sci USA 2012; 109: 65616565.8. Liu YL et al. Autophagy 2010; 6: 10571065.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. MB was supported by the Dutch Cancer Society (AMC2009-4457). RWJ is a Principal Research Fellow of the National Health and Medical Research Council of Australia (NHMRC) and supported by NHMRC Program and Project Grants, the Susan G Komen Breast Cancer Foundation, Cancer Council Victoria, The Victorian Cancer Agency, The Leukemia Foundation of Australia and the Victorian Breast Cancer Research Consortium.

1. Bolden JE, Peart MJ, Johnstone RW. Nat Rev Drug Discov 2006; 5: 769784.2. Nebbioso A et al. Nat Med 2005; 11: 7784.

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