Oncogene (2009) 28, 721–733 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE small-1 modulates Akt/FoxO3A signaling and apoptosis through PP2A

C Bertoli1, T Copetti1, EW-F Lam2, F Demarchi1 and C Schneider1,3

1Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Trieste, Italy; 2Cancer Research-United Kingdom Laboratories, Department of Oncology, Imperial College London, Hammersmith Hospital, London, UK and 3Dipartimento di Scienze e Tecnologie Biomediche, Universita’ degli Studi di Udine, Udine, Italy

Here, we show that FoxO3A transcription factor is signaling (Goll et al., 2003). Its involvement in a upregulated upon calpain small-1 (CAPNS1) depletion multiplicity of complex pathologies such as muscular both in mouse embryonic fibroblasts (MEFs) and in the dystrophies, neurodegeneration, type II diabetes human mammary carcinoma cell line MCF-7. On and cancer has been well documented (Zatz and starvation, CAPNS1 depletion is associated with a higher Starling, 2005), although its role in the patho- rate of FoxO3A dephosphorylation and translocation to genesis and disease progression has not been completely the nucleus and to a sharper increase in the levels of clarified. Understanding the pathways modulated p27Kip1 and Bim, the products of two FoxO target . by calpain might be instrumental in exploiting Notably, FoxO3A depletion in CAPNS1À/À MEFs calpain inhibition as a therapeutic strategy against reduces both the induction of Bim and apoptosis. Both cancer. Besides the involvement of calpain in cell death okadaic acid treatment and silencing of the (Goll et al., 2003), we and others have observed a phosphatase 2A (PP2A) catalytic subunit can partially pro-survival role of this in tumor-derived cell reduce starvation-induced FoxO3A activation and apop- lines in response to chemotherapeutic drugs and to tosis in CAPNS1À/À fibroblasts. PP2A associates more starvation (Demarchi et al., 2005, 2006; Guan et al., tightly with Akt in CAPNS1 knockout cells, indicating 2006; Tan et al., 2006; Darnell et al., 2007), a condition that PP2A is involved in calpain-mediated FoxO regula- usually occurring in tumors. tion. Finally, we show that PP2A regulatory subunits B56 Most of the cellular calpain activity is due to the alpha and gamma are in vitro substrates of calpain, and ubiquitous micro- and milli-calpain, requiring micro- calpain regulates B56 alpha stability in vivo, suggesting a and millimolar calcium concentrations, respectively, for direct role of calpain in the regulation of PP2A function. its function. Ubiquitous are heterodimers In conclusion, for the first time we report that CAPNS1 consisting of an 80-kDa catalytic subunit and of a interferes with PP2A-Akt interaction consequently affect- common 28-kDa regulatory subunit, calpain small-1 ing FoxO3A-dependent cell death. Calpain inhibition (CAPNS1), encoded by CAPNS1 (Goll et al., might therefore be exploited as a tool to induce apoptosis 2003), required for function (Arthur et al., 2000). in tumors sensitive to FoxO activation. One of the key regulators of cell survival and Oncogene (2009) 28, 721–733; doi:10.1038/onc.2008.425; growth is the PI3K/Akt pathway. Its activation published online 24 November 2008 occurs through phosphorylation by phosphoinositide- dependent kinase-1 (PDK-1) at Thr 308, and by Keywords: CAPNS-1; FoxO3A; apoptosis; PP2A; B56; mTORC2 (mammalian target of rapamycin complex) Akt at Ser 473, allowing the activation of the mTORC1 complex, which sustains protein synthesis and consequently cell growth, and the inactivation of involved in growth control and apoptosis such as Bad and FoxO (Manning and Cantley, 2007). PTEN Introduction (phosphatase and tensin homolog deleted from chromo- some ten) antagonizes PI3K function, consequently The calpain family of calcium-dependent cytosolic inhibiting downstream signaling through Akt. regulates multiple cellular functions such as Moreover, Akt is directly inhibited by PHLPP (PH spreading and migration, cell death, transcription and domain leucine-rich repeat protein phosphatase), which dephosphorylates Ser 473 (Gao et al., 2005), and by protein phosphatase 2A (PP2A), which targets Thr Correspondence: Dr F Demarchi or Professor C Schneider, Labor- 308 and Ser 473 (Sato et al., 2000; Resjo et al., 2002; atorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Rocher et al., 2007). AREA Science Park, Padriciano 99, 34012 Trieste, Italy. Downstream molecules of Akt include the FoxO E-mails: [email protected] or [email protected] Received 30 April 2008; revised 16 October 2008; accepted 24 October family of transcription factors. In mammals, there are 2008; published online 24 November 2008 three FoxO genes with overlapping functions: FoxO1, CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 722 3A and 4, which regulate a number of target genes acids and serum (Earle’s balanced salt solution (EBSS)) involved in cell cycle arrest such as p21 and p27Kip1, at different points of time, then the lysates were collected apoptotic molecules such as Bim, Puma and TRAIL and analysed for the presence of the 3 active (TNF-related apoptosis-inducing ligand), and reactive fragment, a well-established apoptotic marker. As oxygen species-detoxifying genes such as catalase and shown in Figure 1a, caspase 3 active fragment is MnSOD (Greer and Brunet, 2005; van der Horst and detectable after 4 h treatment in CAPNS1 KO MEFs, Burgering, 2007). Simultaneous deletion of FoxO1, 3A whereas it is barely visible at 10 h of treatment in WT and 4 leads to a progressive cancer-predisposed condi- MEFs, confirming that CAPNS1 KO MEFs are more tion characterized by thymic lymphomas and heman- prone to apoptosis than WT counterparts. To verify giomas (Paik et al., 2007), demonstrating the importance whether CAPNS1 is responsible for the reported cell of FoxO as tumor suppressors. Akt phosphorylation death, we performed a similar experiment in CAPNS1 promotes FoxO binding to 14-3-3 and its retention in KO cells stably expressing rat CAPNS1 R, in which the cytoplasm. On Akt inactivation, FoxO is depho- calpain activity is partially restored (Dourdin et al., sphorylated and translocates to the nucleus activating 2001). CAPNS1 KO, WT and CAPNS1 R were transcription (Biggs et al., 1999; Brunet et al., 1999; incubated with EBSS for 16 h, then they were stained Kops and Burgering, 1999; Tang et al., 1999). with V-fluorescein isothiocyanate (FITC) Overactivation of the PI3K/Akt pathway has been and analysed by flow cytometry. The histogram reported in a number of tumors, which is due to both reported in Figure 1b confirms the increased sensitivity excess of stimulation and reduced phosphatase activity of CAPNS1 KO cells compared with WT MEFs; in (Altomare and Testa, 2005). PTEN is deleted in addition, CAPNS1 reintroduction diminishes the per- numerous tumors such as prostate and breast carcino- centage of apoptotic cells. The reduced sensitivity to mas (Li et al., 1997; Altomare and Testa, 2005); EBSS-induced apoptosis in CAPNS1 R MEFs com- moreover, FoxO has been implicated in growth factor pared with KO ones was further confirmed by the withdrawal-induced apoptosis (You et al., 2006; Yan detection of caspase 3 active fragment by western blot et al., 2008) and in amyloid-beta-induced cell death (Figure 1c). (Yin et al., 2006). PP2A positively regulates FoxO both indirectly, through Akt (Trotman et al., 2006; Yin et al., 2006), FoxO transcription factors are more active in CAPNS1 and directly, by dephosphorylation both of FoxO1 (Yan MEFs et al., 2008) and FoxO3A (Barreyro et al., 2007), leading We hypothesized that the increased sensitivity to cell to FoxO-dependent cell death (Yin et al., 2006; Barreyro death observed in CAPNS1 KO MEFs could be et al., 2007; Yan et al., 2008). PP2A is predominantly partially due to an increased activation of FoxO, as present as a heterotrimer composed of a scaffolding these cells are partially defective in Akt activation in subunit (A), a catalytic subunit (C) and a variable response to TNF-alpha and staurosporine (Tan et al., regulatory subunit (B). The regulatory subunits belong 2006). to four major unrelated families (B, B0,B00 and B000), and To evaluate whether FoxO transcription factors were are believed to confer substrate specificity to the enzyme more active in the absence of calpain activity, CAPNS1 (Janssens and Goris, 2001). PP2A is an important tumor KO and WT MEFs were transfected with a luciferase suppressor targeted by inactivating mutations (Sablina reporter construct bearing FoxO-binding sites in the et al., 2007) and by viral oncoproteins in many tumors promoter together with a Renilla-expressing plasmid for (Mumby, 2007). Inhibition of PP2A can lead to cell normalization. After 24 h, the cells were stimulated with survival or death depending on the cellular context and EBSS for 4 or 8 h, or left in complete medium, then the growth conditions, indeed polyoma small-T- luciferase activity was detected. The histogram shown in mediated PP2A inhibition can promote cell death in Figure 2a indicates that FoxO-dependent luciferase complete medium, whereas it allows survival upon activity is higher in CAPNS1 KO than in WT MEFs, serum withdrawal, in an Akt-dependent manner both in complete medium and in EBSS. To investigate (Andrabi et al., 2007). the involvement of FoxO3A in this response a domi- nant-negative FoxO3A (DN-FoxO3A) was co-ex- pressed. As shown in Figure 2a, DN-FoxO3A is able to reduce FoxO luciferase activity in CAPNS1 KO Results cells both in basal conditions and on EBSS treatment, suggesting that FoxO3A is partially responsible for Starvation induces apoptosis in CAPNS1 knockout the observed transcriptional activation. A similar fibroblasts experiment was performed with ectopic FoxO1 and We have reported that the lack of calpain activity FoxO3A. The histogram reported in Figure 2b indicates sensitizes cells to death induced by a variety of stimuli, that both FoxO transcription factors appear more including growth factors and nutrient starvation (De- active in CAPNS1 KO cells in complete medium and marchi et al., 2005, 2006, 2007). To investigate the in EBSS. FoxO3A appears always more active involvement of caspase activation, CAPNS1 knockout than FoxO1 in CAPNS1 KO cells. To further verify (KO) and wild-type (WT) mouse embryonic fibroblasts whether CAPNS1 is responsible for the downregulation (MEFs) were treated with a medium lacking both amino of FoxO-responsive promoter a plasmid encoding

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 723 CAPNS1 WT CAPNS1 KO Actin

Cleaved Caspase 3

- 4h 6h 8h 10h - 4h 6h 8h 10h EBSS EBSS

60 EBSS #* 50 Control * 40

30 # 20

10 % of Annexin V-positive cellse 0 CAPNS1 WT CAPNS1 KO CAPNS1 R

CAPNS1 WT CAPNS1 KO CAPNS1 R

Cleaved Caspase 3

0.1 0.7 1.2 1.2 0.1 0.1 0.6 0.8 0.6

Actin

EBSS 2h 4h 6h 8h 2h 4h 6h 8h - 2h 4h 6h 8h Figure 1 Calpain small-1 (CAPNS1) knockout (KO) fibroblasts are more sensitive to starvation-induced apoptosis. (a) CAPNS1 KO and wild-type (WT) mouse embryonic fibroblasts (MEFs) were treated for the indicated time points with Earle’s balanced salt solution (EBSS), or left in complete medium, then lysates were collected, separated by SDS–polyacrylamide gel electrophoresis (PAGE) and the western blots were performed with anti-caspase 3-cleaved fragment, or anti-actin as a loading control. (b) CAPNS1 KO, R and WT MEFs were treated with EBSS or complete medium for 16 h, then stained with Annexin V-fluorescein isothiocyanate (FITC) and analysed by fluorescence-activated cell sorting (FACS). The histogram represents the average of three independent experiments *Po0.05; #Po0.001. (c) CAPNS1 KO MEFs and a CAPNS1 KO cell line stably expressing CAPNS1 R were treated with EBSS for the indicated time points and analysed for caspase 3-cleaved fragment or actin by western blot. The numbers are the ratios between caspase 3 and actin for each lane quantified with the ImageJ program.

CAPNS1 or a control vector were transfected in The kinetics of FoxO3A dephosphorylation and nuclear KO cells along with FoxO3A. The histogram in translocation is faster in CAPNS1 KO cells Figure 2c indicates that the presence of CAPNS1 As FoxO activation is often associated with a decrease reduces EBSS-induced FoxO3A activation. Conversely, in phosphorylation followed by nuclear translocation, transfection of calpastatin, calpain endogenous we evaluated the phosphorylation level of Thr 32, a site inhibitor, along with FoxO3A in WT cells increases phosphorylated by Akt, resulting in FoxO inhibition FoxO3A transcriptional activity in EBSS medium (Brunet et al., 1999). CAPNS1 KO and WT cells were (Figure 2d). treated with EBSS for 5, 10 or 20 min or left in complete Furthermore, both p27Kip1 and Bim, two FoxO medium, and the phosphorylation of Thr 32 at every endogenous target genes, are more efficiently induced by time point was evaluated by western blot (Figure 3a). EBSS in CAPNS1 KO than in WT MEFs (Figure 2e), Thr 32 basal phosphorylation is slightly reduced in although the basal protein level of p27Kip1 is higher in CAPNS1 KO cells compared with WT ones, and on WT cells. The experiment was performed three times, EBSS treatment it disappears more promptly than in analysed statistically, and the difference in Bim expres- WT cells; in parallel also Akt appears to be less sion relative to actin between CAPNS1 and WT cells is phosphorylated both at Thr 308 and Ser 473 in reported in Supplementary Figure 1a. In CAPNS1 KO CAPNS1 KO MEFs, and in response to EBSS it is cells, the expression of Bim is significantly more induced dephosphorylated more quickly. than in WT cells, further supporting a role of calpain in We further evaluated FoxO nuclear translocation by FoxO regulation. immunofluorescence. CAPNS1 KO and WT MEFs were

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 724 transfected with FoxO3A, 24 h later the cells were in complete medium and on EBSS treatment, consistent treated with EBSS for 15 min or left in complete with the lower level of Thr 32 phosphorylation medium, and the number of cells bearing nuclear, observed. An analogous experiment with FoxO1 also cytoplasmic or whole cell FoxO distribution was indicates that in this case the number of cells with a analysed. The data reported in Figure 3b indicate that nuclear localization is higher in CAPNS1 KO cells in CAPNS1 KO fibroblasts FoxO has a prominent compared with WT cells (Supplementary Figure 1b). A nuclear distribution in a higher percentage of cells both time course experiment of FoxO3A and FoxO1 dis-

4.5 7 * Control 4 EBSS 6 * 3.5 3 5 2.5 4 2 * 3 1.5 * * ** 2 1 ** 1 Luciferase arbitrary units 0.5 Luciferase arbitrary units * 0 0 Vector DN-FoxO3A Vector DN-FoxO3A FoxO3A FoxO1 FoxO3A FoxO1 CAPNS1 WT CAPNS1 KO CAPNS1 WT CAPNS1 KO FoxO3A-HA FoxO1 Actin

CAPNS1 KO MEFs CAPNS1 WT MEFs 20 8 18 7 * 16 6 14 5 12 4 10 3 8 * * 6 2 2 4 * * 2 1 Luciferase arbitrary units Luciferase arbitrary unitsf 0 0 EBSS 0 4h 8h 0 4h 8h EBSS 0 4h 8h 0 4h 8h Vector Calpain Small-1 Vector Calpastatin

FoxO3A-HA Calpastatin

Flag-Calpain small-1 FoxO3A-HA Actin Actin

CAPNS1 WT CAPNS1 KO p27Kip1 0.8 0.80.6 0.7 0.7 0.8 0.4 0.4 0.7 1.3 1.2 0.4 Actin - 2h4h 6h 8h - - 2h4h 6h 8h - EBSS EBSS BimEL

BimL

0.40.20.4 0.6 0.6 0.3 0.6 0.6 0.8 0.9 1.1 0.6 Actin - 2h4h 6h 8h - - 2h4h 6h 8h - EBSS EBSS

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 725 EBSS EBSS tribution in CAPNS1 KO and WT cells following EBSS - 5′ 10′ 20 - 5′ 10′ 20 treatment further indicates that the kinetics of FoxO Phospho-Thr 32 activation is accelerated in KO compared with WT

1 0.78 0.73 0.61 0.89 0.61 0.49 0.34 MEFs (Supplementary Figures 1c and 1d). * Altogether, the above data suggest that the lack of ] FoxO3A CAPNS1 is linked to an increase in FoxO activation both in basal conditions and following starvation. To further P-Akt Ser 473 verify such increase in FoxO activity, electrophoretic mobility shift assay assays were performed with nuclear P-Akt Thr 308 extracts prepared from CAPNS1 KO and WT cells using an oligonucleotide bearing FoxO-binding sites derived Akt from Fas promoter as a probe (Brunet et al., 1999; Yin et al., 2006). The autoradiography reported in Supple- P-PDK1 Ser 241 mentary Figure 2a shows that CAPNS1 KO MEFs have a higher amount of FoxO-binding activity compared with Actin the WT counterpart. An excess of specific cold compe- CAPNS1 WT CAPNS1 KO titor oligonucleotide clearly competes for binding and * Nonspecific band dramatically reduces the retarded bands, whereas a 120 mutated oligonucleotide leaves it unaffected.

100 Starvation-induced apoptosis of CAPNS1 KO fibroblasts 80 is associated with increased FoxO activity 60 To investigate whether FoxO activation is responsible for the increased apoptosis observed in CAPNS1 KO cells, 40 CAPNS1 KO fibroblasts were infected with pRetroSU-

Percentage of cells 20 PER encoding an shRNA targeting FoxO3A (Essafi et al., 2005) or empty vector, then they were treated with 0 C C+N N C C+N N C C+N N C C+N N EBSS. Lysates were collected at different time points and - EBSS 15′ - EBSS 15′ the expression of Bim and caspase 3 was analysed (Figure 4b). Figure 4a shows the reduction of FoxO3A CAPNS1 WT CAPNS1 KO expression obtained. As reported in Figure 4b, FoxO3A Figure 3 FoxO3A has an accelerated kinetics of dephosphoryla- silencing reduces EBSS-induced Bim upregulation and tion and nuclear translocation in calpain small-1 (CAPNS1) the associated cell death, as indicated by the reduction of knockout (KO) cells. (a) CAPNS1 KO and wild-type (WT) mouse caspase 3 active fragment protein levels accumulating embryonic fibroblasts (MEFs) were stimulated with Earle’s balanced salt solution (EBSS) for the indicated time points or left upon FoxO3A silencing. These data strongly argue for a untreated, then lysates were analysed by western blot with the major role of FoxO3A in the increased sensitivity to indicated antibodies. (b) CAPNS1 KO and WT MEFs were starvation of CAPNS1 KO fibroblasts. transfected with FoxO3A-HA, after 24 h the cells were treated with EBSS for 15 min or left in complete medium, an immunofluores- cence with anti-HA antibody was performed and the cells were CAPNS1 depletion increases FoxO3A activation scored for FoxO3A distribution. The histogram reports the average in MCF-7 cells of three independent experiments. N, staining mostly nuclear; C, staining mostly cytoplasmic; C þ N, staining equally distributed To determine whether calpain depletion could increase both in nucleus and in cytoplasm. FoxO activation in human cells also, mammary

Figure 2 FoxO transcription factors are more active in mouse embryonic fibroblasts (MEFs) lacking calpain activity. (a) Calpain small-1 (CAPNS1) knockout (KO) and wild-type (WT) MEFs were transfected with a FoxO-responsive luciferase vector and Renilla for normalization, 24 h later the cells were treated with Earle’s balanced salt solution (EBSS) for 8 h, the lysates were then analysed in a Dual-Luciferase Reporter assay. The histogram represents the average of a typical experiment performed in triplicate. The experiment was performed in the presence of DN-FoxO3A, or a control empty vector. Data are expressed as means±s.d. *Po0.05 versus the WT group. **Po0.05 versus the empty vector- transfected group. (b) CAPNS1 KO and WT MEFs were transfected with a FoxO-responsive luciferase vector and Renilla for normalization together with a vector expressing FoxO3A or FoxO1, 24 h later the cells were treated with EBSS for 6 h, the lysates were then analysed in a Dual-Luciferase Reporter assay. The histogram reports the average of three experiments. Data are expressed as means±s.d. *Po0.05 versus the WT group. (c) CAPNS1 KO MEFs were transfected with FoxO3A-HA, a FoxO-responsive luciferase vector and Renilla for normalization together with a vector encoding for human Flag-tagged CAPNS1, 24 h later the cells were treated with EBSS for 4 and 8 h, the lysates were then analysed in a Dual-Luciferase Reporter assay. The histogram reports the average of a typical experiment performed in triplicate. The expression of CAPNS1 and FoxO3A was checked by western blot. Data are expressed as means±s.d. *Po0.05 versus the empty vector-transfected group. (d) CAPNS1 WT MEFs were transfected with FoxO3A, a FoxO-responsive luciferase vector and Renilla for normalization together with a vector encoding for human calpastatin, 24 h later the cells were treated with EBSS for 4 and 8 h, the lysates were then analysed in a Dual- Luciferase Reporter assay. The histogram reports the average of a typical experiment performed in triplicate. The expression of calpastatin and FoxO3A was analysed by western blot with anti HA antibody. Data are expressed as means±s.d. *Po0.05 versus the empty vector-transfected group. (e) CAPNS1 KO and WT MEFs were treated with EBSS for the indicated time points, then lysates were collected, separated by SDS– polyacrylamide gel electrophoresis (PAGE) and western blot analysis was performed with anti-p27Kip1, Bim and actin antibody. The numbers represent the ratio between the above band and actin after ImageJ quantification.

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 726 carcinoma MCF-7cells were transfected with an siRNA As revealed in Figure 5c, the level of phosphorylation is targeting CAPNS1 or a control siRNA. After 72 h, a reduced in CAPNS1-depleted cells in both conditions, time course of EBSS treatment was performed and confirming the results obtained in the previous experi- lysates were analysed by western blot with an antibody ment. Similarly, also FoxO1 translocated more quickly against phospho-Thr 32 of FoxO3A (Figure 5a). In into the nucleus in CAPNS1-silenced cells, as shown in CAPNS1-depleted cells, FoxO3A dephosphorylation Supplementary Figure 2b. On the other hand, pharma- takes place at faster kinetics than in control cells. cological activation of calpain by thapsigargin treatment Consistently, Akt Ser 473 dephosphorylation occurred (Harriman et al., 2002) reduces FoxO3A dephospho- more quickly, whereas Thr 308 appeared less phos- rylation occurring on EBSS treatment (Supplementary phorylated in all the conditions and did not change Figure 3a). significantly. To further assess the increase in FoxO activation We further evaluated FoxO activation by fluorescence associated with calpain depletion, electrophoretic mobi- microscopy. CAPNS1-depleted MCF-7cells were trans- lity shift assay analysis was performed. MCF-7cells fected with GFP-FoxO3A and 24 h later they were were transfected with GFP-FoxO3A and incubated treated with EBSS or left in complete medium, then they either in EBSS for 1.5 h or in complete medium, in the were fixed and scored for FoxO3A distribution. The presence or absence of calpain inhibitor I. Nuclear histogram reported in Figure 5b shows that also in extracts were prepared and incubated with a radiola- MCF-7cells depletion of CAPNS1 leads to an increased beled FoxO consensus oligonucleotide derived from Fas nuclear relocation of FoxO in a higher percentage of promoter. The identity of the retarded FoxO3A contain- cells compared with control ones, in both the conditions ing band was assessed through the addition of anti-GFP tested. In parallel, GFP-FoxO3A was immunoprecipi- antibody, as indicated (Supplementary Figure 3b), and tated from CAPNS1-depleted and control MCF-7 by competing titration with either specific or mutated cells maintained in complete medium or in EBSS, and FoxO cold oligonucleotides (Supplementary Figure 3c). Thr 32 phosphorylation was evaluated by western blot. EBSS treatment efficiently induces FoxO binding. FoxO3A appears more active when calpain is chemically inhibited, particularly in complete medium, indeed the retarded band is more pronounced than in the untreated FoxO3A counterpart. Actin We then analysed the induction of two FoxO target genes (Figure 5d), and also in this case on EBSS

pRS pRS shFoxO3A stimulation, p27Kip1 and Bim protein levels appear to increase more in CAPNS1-depleted cells compared with EBSS 002h 4h 6h 8h 2h 4h 6h 8h the control ones. The experiment was repeated three BimEL times and the increase in Bim expression in CAPNS1- depleted cells compared with control ones was analysed 1.31.2 1.5 2.3 2.2 1.3 1.1 1.2 1.4 1.6 statistically, as represented in the histogram reported in Actin Supplementary Figure 3d. To evaluate the effect of calpain depletion on EBSS- Cleaved Caspase 3 induced death, MCF-7cells were transfected with

CAPNS1 KO CAPNS1 siRNA or with a control siRNA, after 72 h they were treated with EBSS for 20 h, stained with Figure 4 Starvation-induced apoptosis of calpain small-1 propidium iodide and Annexin V and analysed by flow (CAPNS1) knockout (KO) fibroblasts is associated with increased FoxO activity. (a) CAPNS1 KO mouse embryonic fibroblasts cytometry. The plots reported in Figure 5e indicate that (MEFs) were infected with pRetroSUPER shFoxO3A or empty MCF-7cells are more sensitive to starvation, when vector, then lysates were subjected to western blot with anti- CAPNS1 is depleted, confirming the results observed in FoxO3A and anti-actin antibody. (b) pRetroSUPER shFoxO3A or murine fibroblasts. Next, we tested whether other empty vector-infected CAPNS1 KO cells were treated with Earle’s balanced salt solution (EBSS) for the indicated time points, and the mammary carcinoma cell lines were also sensitive to lysates were analysed for Bim, caspase 3 active fragment and actin. calpain inhibition. In particular, T47D and MDA- Numbers represent the ratio between Bim and actin in each lane. MB468 cells were treated with calpain inhibitor I, and

Figure 5 Calpain small-1 (CAPNS1) depletion increases FoxO3A activation in MCF-7cells. ( a and d) MCF-7cells were transfected with siRNA targeting CAPNS1 or control siRNA, 72 h later they were treated with Earle’s balanced salt solution (EBSS) for the indicted time points or left untreated, then the lysates were analysed with the indicated antibodies. Quantification of the band normalized with the corresponding actin band. Quantification was performed with the ImageJ program and numbers are the ratio between the intensities of the above band and the corresponding actin. (b) CAPNS1 or control silenced MCF-7was transfected with GFP-FoxO3A, after 24 h the cells were fixed and the distribution of FoxO3A was analysed in 250 cells. The histogram reports the mean value of nuclear, cytoplasmic and whole cell distribution in a typical experiment. (c) CAPNS1 or control silenced MCF-7was transfected with GFP-FoxO3A, after 24 h they were treated with EBSS for 10 min or left untreated, then the lysates were immunoprecipitated with anti-GFP and analysed by western blot with anti-phospho-Thr 32 antibody or anti-FoxO3A antibody. (e) MCF-7cells were transfected with siRNA targeting CAPNS1 or control siRNA, 72h later they were treated with EBSS for 20 h or left in complete medium, then they were trypsinized, stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analysed by flow cytometry. The dot plots reported represent the results of typical experiments. The numbers are means with standard deviation. Po0.05 between the EBSS-treated samples.

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 727 Bim protein levels and the appearance of PARP-cleaved CAPNS1 modulation of FoxO involves PP2A fragment were analysed by western blot. As reported in The data reported above suggest that calpain could Supplementary Figure 3e, Bim protein levels increase modulate FoxO through Akt regulation, as Akt appears and PARP cleavage is induced following calpain to be less phosphorylated in the absence of calpain inhibition, strongly suggesting that calpain inhibition activity (Figures 3a and 5a). We therefore hypothesized is associated with apoptosis induction. that there could be a phosphatase activity more

EBSS 030′ 60′ 90′ 120′ 0 30′ 60′ 90′ 120′

P-Thr32 90 80 FoxO3A 70 60 P-Ser 473 50 P-Thr 308 40 30 Akt Percentage of cells 20 10 CAPNS1 0 C C+NN C C+N N C C+N N C C+N N Actin --EBSS 60’ EBSS 60’ siCONT siCAPNS1 siCONT siCAPNS1

EBSS EBSS - 2h 4h 6h 8h 9h - 2h 4h 6h 8h 9h

siCONT siCAPNS1 BimEL + + EBSS 1.0 1.2 1.5 1.2 1.2 1.3 1.3 1.5 1.6 2.0 1.7 1.9

Phospho-Thr 32 p27Kip1

0.60.5 0.5 0.6 0.5 0.6 0.5 0.4 0.6 0.6 0.8 0.7 GFP-FoxO3A CAPNS1 IP anti-GFP Actin

siCONT siCAPNS1

siCONT siCONT EBSS 4.8 ± 3.9 4.8 ± 0.7 8.5 ± 0.9 2.8 ± 0.2 104 104

103 103

2 13.4 2 9.4 FL-2H 10 FL-2H 10 ± 4.9 ± 0.1 1 4 1 10 10 R3 10 R3

100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H siCAPNS1 siCAPNS1 EBSS 3.7 ± 1.7 5.2 ±1.9 4.0 ± 0.9 8.2 ± 2.9 104 104 Propidium Iodide 103 103

2 2 FL-2H 9.2 FL-2H 10 10 23.9 ± 3.8 ± 3.6 101 101 R3 R3

100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H

Annexin V-FITC

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 728 pronounced in CAPNS1 KO MEFs compared with WT CAPNS1 KO MEFs FoxO3A transcriptional activity is cells, because we did not observe any differences in inhibited by OA, suggesting that PP2A could be PDK-1 phosphorylation between the two cell lines. To involved in such activation. We also analysed the evaluate the involvement of PP2A in the increased subcellular location of FoxO3A and found that OA FoxO3A activity observed in CAPNS1 KO cells, increased the percentage of cells with cytoplasmic okadaic acid (OA), a PP2A inhibitor (Millward et al., FoxO3A in EBSS both after 15 min treatment 1999), was employed. As a first approach, we performed (Figure 6b) and after 2 h (data not shown). After OA a FoxO reporter luciferase assay in CAPNS1 KO and treatment, the percentage of CAPNS1 KO cells with WT cells transfected with FoxO3A; 24 h after transfec- cytoplasmic FoxO3A in EBSS is similar to that observed tion, the cells were treated with EBSS or left in complete in WT cells in EBSS, suggesting that an increased medium in the presence of different concentrations of phosphatase activity sensitive to OA could be present in OA. The histogram shown in Figure 6a indicates that in the cells lacking CAPNS1.

70 Control 60 EBSS 4h 50 40 30 20 10 Luciferase arbitrary units 0 OA - ---

CAPNS1 WT CAPNS1 KO

120 C 100 C+N N 80 60 40 * 20 * 0 Okadaic acid --++ -- ++ 10% EBSS 15′ 10% EBSS 15′ CAPNS1 WT CAPNS1 KO

CAPNS1 KO CAPNS1 WT

BimEL

BimL 0.50.7 0.86 1 0.5 0.6 0.7 0.7 0.2 0.4 0.5 0.5 Cleaved Caspase 3

Actin

EBSS 2h4h 6h 8h 2h 4h 6h 8h 2h 4h 6h 8h OA 500 nM Figure 6 Okadaic acid (OA) is partially inhibiting the increased FoxO activity of calpain small-1 (CAPNS1) knockout (KO) mouse embryonic fibroblasts (MEFs). (a) CAPNS1 KO and wild-type (WT) MEFs were transfected with a FoxO-responsive luciferase vector and Renilla for normalization together with a vector expressing FoxO3A, 24 h later they were treated with Earle’s balanced salt solution (EBSS) for 4 in the presence or absence of OA, luciferase activity was then evaluated by Dual-Luciferase Reporter assay. The histogram reports the average of three experiments. (b) CAPNS1 KO and WT MEFs were transfected with FoxO3A-HA, after 24 h the cells were treated with EBSS for 15 min or left in complete medium in the presence or absence of OA, an immunofluorescence with anti- HA antibody was performed and the cells were scored for FoxO3A distribution. The histogram reports the average of three independent experiments. *Po0.05. (c) CAPNS1 KO and WT MEFs were incubated with EBSS in the presence or absence of OA, as indicated, at different points of time and western blot was performed with the reported antibodies. The numbers represent the ratio between the above band and actin after ImageJ quantification.

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 729 shCONT shPP2A shCONT shPP2A shCONT shPP2A EBSS 16h + + + + PP2AC Actin Cleaved Caspase 3 Actin 0.4 0.24 CAPNS1 WT CAPNS1 KO

shCONT shPP2AC EBSS - 2h4h 6h - 2h 4h 6h

p27Kip1 0.3 0.3 0.5 0.84 0.2 0.2 0.6 0.6

Actin

CAPNS1 KO

shCONT shPP2AC shCONT EBSS - 2h4h 6h - 2h 4h 6h - 2h 4h 6h BimEL

BimL 0.10.13 0.33 0.64 0.1 0.2 0.2 0.39 0.13 0.17 0.15 0.12 Actin

CAPNS1 KO CAPNS1 WT Figure 7 Calpain small-1 (CAPNS1) modulation of FoxO involves protein phosphatase 2A (PP2A). (a) Western blot of the PP2AC subunit in CAPNS1 knockout (KO) infected with a vector encoding shRNA targeting PP2AC or a control. (b–d) PP2A-depleted or control CAPNS1 KO cells were treated with Earle’s balanced salt solution (EBSS) for the indicated times, then lysates were collected and analysed with the indicated antibodies. The numbers represent the ratio between the above band and actin after ImageJ quantification.

To further investigate the ability of OA to inhibit the the lack of CAPNS1 affects PP2A-Akt interaction. Akt increased FoxO3A activation observed in CAPNS1 KO was immunoprecipitated from CAPNS1 KO and WT cells, Bim protein levels were analysed by western blot in cells and the presence of co-precipitated PP2A was a time course EBSS experiment. As observed in investigated by western blot (Figure 8a). Akt is highly Figure 6c, treatment with OA reduces the increase in associated with PP2A in CAPNS1 KO cells than in WT Bim protein level triggered by EBSS, and the associated ones, indicating that PP2A is indeed the phosphatase cell death, as demonstrated by the analysis of caspase 3 responsible for the reduced Akt activity and the active fragment. OA reduces the levels of Bim also in consequent FoxO activation observed in cells lacking WT cells, as expected (data not shown); however, the calpain activity. induction of Bim in these cells is far less evident than in As PP2A global activity was not affected by calpain CAPNS1 KO cells at these time points, as shown in deletion (data not shown), we hypothesized that some Figure 2e. regulatory subunits of the phosphatase with substrate To further assess the role of PP2A in FoxO specificity were modulated by calpain, namely the B hyperactivation associated with CAPNS1 depletion, we subunits, which belong to four families: the B (PR55), employed a construct encoding a short hairpin targeting the B0 (B56), the B00(PR72) and the most recently the PP2A catalytic subunit to specifically downregulate identified B000 family. Every family accounts for a PP2A activity (Figure 7a). PP2A catalytic subunit number of isoforms, in particular the B56 family depletion decreases CAPNS1 KO cell death in starva- involves the alpha, beta, gamma, delta and epsilon tion medium (Figure 7b), and in parallel it reduces the members, which share high homology. Interestingly, induction of p27Kip1 (Figure 7c) and Bim (Figure 7d), B56 has been implicated in the modulation of Akt confirming the role of PP2A in FoxO activation in phosphorylation (Van Kanegan et al., 2005; Yin et al., CAPNS1 KO cells. 2006; Rocher et al., 2007). As a first attempt to understand whether they could be regulated by calpain, we performed in vitro proteo- PP2A B56 subunits are calpain substrates lysis assays. Among the B56 family, we chose to analyse To gain insight into the molecular mechanism under- B56 alpha and gamma. The HA-tagged B56 alpha lying the calpain-PP2A-FoxO axis, we checked whether and gamma proteins were immunoprecipitated from

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 730 WTKO WT KO Akt

PP2A IP anti-Akt Input

Calpain 0′ 5′ 15′ 30′ Calpain buffer PR72 0 5′ 15′ 30′ 15′ 30′

B56 alpha PR55

κ B56 gamma NF- B p50

MCF-7 shCONT shCAPNS1 - Chx - Chx B56 alpha

0.43 0.2 0.6 0.6 Actin

Calpain small-1

Figure 8 Protein phosphatase 2A (PP2A) B56 subunits are calpain substrates. (a) Endogenous Akt was immunoprecipitated from calpain small-1 (CAPNS1) knockout (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs), and western blot with anti-PP2A antibody was performed. (b) 293 cells were transfected with HA-B56 alpha and gamma. Anti-HA immunoprecipitation was performed, and the immunoprecipitated products were incubated with recombinant calpain for the indicated time points, separated by SDS– polyacrylamide gel electrophoresis (PAGE) and analysed with anti-HA antibody. (c) Autoradiography of in vitro translated PR72, PR55 and p50 incubated with recombinant calpain for the indicated time points. (d) MCF-7stably expressing sh-CAPNS1 or sh- control untreated or treated with cycloheximide for 15 h were lysed and western blot was performed with the indicated antibodies.

previously transfected cells and incubated with or in Figure 8d, CAPNS1 depletion is associated with a without calpain at different points of time; EGTA was slight increase in B56 alpha protein level, which becomes included in a control sample. The western blot reported more consistent on cycloheximide addition. These in Figure 8b clearly indicates that both B56 alpha and results indicate that calpain may modulate B56 levels gamma are in vitro substrates of calpain and the in living MCF-7cells. cleavage can be specifically inhibited by EGTA, which The overexpression of B56 alpha and gamma inhibits calpain activity. We also analysed PR55 and enhances FoxO3A transcriptional activity more consis- PR72 to check whether there was any difference in their tently in CAPNS1 KO cells than in WT ones, as assessed regulation by calpain. In vitro translated radiolabeled in luciferase assay (Supplementary Figure 4), indicating PR55, PR72 and NF-kB p50, as positive control, were that these subunits are involved in the upregulation of incubated with calpain at different points of time, then FoxO3A upon calpain deletion. the reaction was stopped by the addition of sample buffer and the proteins were separated by SDS– polyacrylamide gel electrophoresis (PAGE). The auto- radiography reported in Figure 8c shows that calpain Discussion very efficiently degrades PR72, whereas PR55 is resistant to degradation, indicating that these subunits In this paper, we demonstrate that FoxO transcription are differently regulated by calpain. We therefore factors, in particular FoxO3A, are negatively regulated evaluated whether the stability of B56 subunits was by calpain, and can be therefore activated upon calpain affected by calpain also in vivo. MCF-7cells stably inhibition. Calpain inhibition-mediated FoxO activation expressing an shRNA targeting CAPNS1 or a control is particularly evident upon nutrient withdrawal, shRNA were treated with cycloheximide for 15 h, to a condition that mimics the core of neoplastic lesions, evaluate the stability of the protein, and then B56 alpha where blood supply is reduced or absent. The activation protein levels were analysed by western blot. As shown of FoxO has been demonstrated both in murine

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 731 fibroblasts and in human mammary carcinoma cell lines, serum in CAPNS1 KO cells, where it contributes to the a context where the inactivation of PTEN (Li et al., activation of FoxO. It is, however, possible that calpain 1997; Altomare and Testa, 2005) and FoxO has been affects FoxO also in a PP2A-Akt-independent way. recognized as a major tumor-promoting event (Reagan- Calpain is often hyperactivated in tumor cells Shaw and Ahmad, 2006). Overexpression of active (Carragher et al., 2004; Libertini et al., 2005), where it FoxO mutants in PTEN-deficient prostate cancer cells plays a major role in migration and metastasis due to its leads to apoptosis (Nakamura et al., 2000); moreover, ability to cleave cytoskeletal proteins. Interestingly, PI3K depletion in mammary carcinoma cell lines causes PP2A negatively regulates calpain in nicotine-stimulated cell death, which is inhibited by FoxO1 and FoxO3A cell migration (Xu and Deng, 2006), in line with its silencing (Reagan-Shaw and Ahmad, 2006). FoxO- tumor suppressor role. Our findings suggest that calpain dependent Bim activation is an apoptotic mechanism negatively affects some specific PP2A functions, parti- induced by chemotherapeutics targeting breast carcino- cularly those depending on B56 alpha and gamma. ma cells (Sunters et al., 2003) and leukemia cells (Essafi In conclusion, we have demonstrated the existence of et al., 2005). a pathway linking calpain, PP2A and FoxO, which The role of FoxO in apoptosis induction has been could be exploited to induce tumor cell apoptosis. demonstrated in a number of reports (Brunet et al., 1999; Nakamura et al., 2000; Dijkers et al., 2002; Sunters et al., 2003; Essafi et al., 2005; You et al., 2006; Materials and methods Barreyro et al., 2007), in line with its tumor suppressor role, and in many instances the induction of cell death Plasmid constructs correlated with the induction of the pro-apoptotic Foxo-binding site luciferase construct (6 Â ) (DBE) was kindly molecule Bim. In agreement with these reports, we gifted by Dr M Sandri (Padova, Italy) (Sandri et al., 2004), observed a FoxO3A-dependent increase in Bim expres- pECE FoxO3A-HA and pECE Dn-FoxO3A were kind gifts by sion that correlated with the induction of apoptosis in Dr ME Greenberg (Boston, MA, USA) (Brunet et al., 1999); CAPNS1 KO cells, further confirming the importance of pEGFP-FoxO3A was obtained by subcloning the PCR- FoxO3A and Bim in growth factor withdrawal-induced amplified FoxO3A. The pRetroSUPER-shFoxO3A was cell death. already described (Brummelkamp et al., 2002). pSIREN- CAPNS1 inhibition is associated with a decrease in shPP2Ac is a kind gift by Dr Lee JM (St Louis, MO, USA) (Yin et al., 2006). pEGFP-FoxO1 was donated by Akt phosphorylation, correlating with a reduced FoxO Dr Unterman TG (Chicago, IL, USA) (Zhang et al., 2002). phosphorylation. We demonstrate that the PP2A pCEP-B56 constructs (Seeling et al., 1999), pcDNA-PR55 and catalytic subunit is more tightly associated with Akt in pcDNA-PR72 were gifted by Dr D Pim (Trieste, Italy). Flag- calpain-deficient cells compared with WT ones, indicat- CAPNS1 was produced by subcloning human CAPNS1 in ing that this phosphatase plays a major role in the pcDNA3/Flag vector. HA-calpastatin was produced by sub- reduction of Akt activity. Moreover, we show that cloning human calpastatin into pcDNA3 vector. calpain efficiently cleaves B56 alpha and gamma subunits in vitro, and affects B56 alpha stability also in Cell culture vivo. Notably, B56 subunits have been demonstrated to WT and CAPNS1À/À MEFs (Dourdin et al., 2001) were a regulate Akt phosphorylation in many reports (Van kind gift of Dr Peter A Greer (Kingston, Ontario, Canada); Kanegan et al., 2005; Yin et al., 2006; Rocher et al., MCF-7cells and MEFs were grown in Dulbecco’s modified 2007). On the basis of our findings, we may speculate Eagle’s medium supplemented with 10% fetal calf serum. that calpain could modulate Akt through the regulation of B56 subunits, and hence PP2A recruitment. Transfection and reporter assay The importance of Akt regulatory mechanisms in MCF-7, CAPNS1À/À and control mouse fibroblasts at 60– tumor suppression has been demonstrated in particular 80% confluency were transiently transfected using Fugene in prostate and breast cancer, where PTEN is often (Roche Diagnostics GmbH, Mannheim, Germany) or Oligo- depleted and Akt is hyperactivated (Li et al., 1997; fectamine (Invitrogen, Carlsbad, CA, USA) according to the Trotman et al., 2003; Altomare and Testa, 2005). manufacturer’s instructions. Recently, Trotman et al. (2006) have highlighted the For reporter assays, cells were transfected with DBE importance of a PP2A-dependent Akt regulatory luciferase plasmid and Renilla, together with the indicated mechanism activating FoxO3A to inhibit cell prolifera- plasmids, after 24 h the cells were washed three times and stimulated with EBSS or Dulbecco’s modified Eagle’s medium tion and survival, in particular in conditions of PTEN supplemented with 10% fetal calf serum. Lysates were depletion. harvested in passive lysis buffer (Promega, Madison, WI, In this study, we show that in CAPNS1-deficient cells USA) at the indicated time points. Luciferase activity was starvation activates a pro-apoptotic pathway relying on detected with the Dual-Luciferase Reporter Assay System a PP2A-mediated FoxO activation, and we provide (Promega). evidence of a calpain-PP2A-Akt-FoxO regulatory path- way. Cell infections The role of PP2A in the regulation of cell death in the 293 Phoenix Ampho packaging cells were transfected with the absence of serum has been recently addressed (Andrabi calcium phosphate method with pRetroSUPER-shFoxO3A, et al., 2007), in line with this study our findings indicate pSIREN shPP2A or vector, after 72 h the supernatant was that PP2A has a pro-apoptotic role in the absence of harvested, filtered and added to CAPNS1 KO fibroblasts. The

Oncogene CAPNS1 regulates Akt/FoxO3A through PP2A C Bertoli et al 732 infected MEFs were selected with puromycin and after 14 days specific oligonucleotide (Brunet et al., 1999; Yin et al., 2006) FoxO3A or PP2A expression was checked by western blot. and analysed on native 5% gels. Supershift assay was performed by pre-incubating nuclear extracts with anti-GFP Western blot immunofluorescence and immunoprecipitation antibody for 15 min. After washing three times with phosphate-buffered saline, fibroblasts and MCF-7were incubated with EBSS or complete In vitro protease assay medium for the indicated times and then lysed. Western 35S-labeled in vitro translated protein (Promega) and immuno- blotting was performed according to standard protocols using precipitated products were incubated with 0.005 U/ml micro- the following antibodies: anti-FoxO3A, phospho-FoxO3A- calpain at room temperature in: 30 mM Tris, pH 7.5, 1.5 mM Thr 32 and PP2Ac (Upstate Cell Signaling, Lake Placid, dithiothreitol, 750 mM CaCl2 for micro-calpain and 40 mM NY, USA), Akt detection kit (Cell Signaling Technologies, Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM CaCl2 Danvers, MA, USA), actin and p27Kip1 (Sigma, for milli-calpain. Reactions were terminated at the indicated St Louis, MO, USA), Bim (Pro-Science, Woburn, MA, time points by adding SDS–PAGE loading buffer and USA), caspase 3 Asp 175 (Cell Signaling Technologies). analysed on a 12.5% SDS–PAGE. Immunofluorescence was performed by fixing the cells in 3% paraformaldehyde and staining with anti-HA antibody. Immunoprecipitation was performed with anti-GFP, or Akt Statistical analysis antibody according to standard protocols. Results are expressed as means±standard deviation of at least three independent experiments. Statistical analysis was per- Annexin V and propidium iodide staining and fluorescence- formed using Student’s t-test with the level of significance set activated cell sorting analysis at Po0.05. Translocation of phosphatidylserine to the cell surface was monitored by using an Annexin V-FITC apoptosis detection Acknowledgements kit (Sigma-Aldrich) according to the manufacturer’s instruc- tions. In total, 15 000 events were collected by FACScalibur We acknowledge Silvano Piazza for help in statistics. CB was a (BD Biosciences, San Jose, CA, USA) in list mode manner and recipient of an FIRC (Fondazione italiana per la ricerca sul analysed by the Cell Quest software. cancro) fellowship. This study was supported by PRIN (Programmi di Ricerca Scientifica di Rilevante Interesse Electrophoretic mobility shift assay Nazionale) 2006-prot. 2006053543_003, and TRANSFOG Nuclear extracts and oligonucleotide probes were prepared by (Translational and Functinal Onco-Genomics) EU Contract standard procedures. Here, 10 mg (MCF-7) or 20 mg (MEFs) of 503438, AIRC (Associazione italiana per la ricerca sul cancro) nuclear extracts was incubated with [g-32P]ATP (Amersham Grant proposal 2007Cod. 4752,and Progetto FAR CT cod. Biosciences Corp., Piscataway, NJ, USA)-labeled FoxO- MIUR prog. 10036.

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