Mar Lorente Guillermo Velasco

María Sofía Torres

María Salazar

Arkaitz Carracedo

Sonia Hernández-Tiedra

Fátima Rodríguez-Fornés

Elena García-Taboada

Bárbara Meléndez

Manuela Mollejo

Mar Lorente

Yolanda Campos-Martín

Sergey A Lakatosh

Juan Barcia

Manuel Guzmán

STIMULATION OF THE MIDKINE/ALK AXIS RENDERS GLIOMA CELLS RESISTANT TO CANNABINOID ANTI-TUMORAL ACTION

Keywords: Midkine, ALK, autophagy, DNA arrays, tumor xenografts

published or not. The documents may come

Introduction

Glioblastoma multiforme (GBM), or grade IV astrocytoma, is the most frequent class of malignant primary brain tumor and one of the most aggressive forms of cancer. As a consequence, median survival after diagnosis is usually just 12-15 months [START_REF] Nieder | Therapeutic options for recurrent highgrade glioma in adult patients: recent advances[END_REF][START_REF] Wong | Targeting malignant glioma survival signalling to improve clinical outcomes[END_REF][START_REF] Louis | The 2007 WHO classification of tumours of the central nervous system[END_REF]. This dramatic behavior is mainly due to the high invasiveness and proliferation rate of GBM.

In addition, GBM exhibits a high resistance to standard chemotherapy and radiotherapy.

Current strategies for the treatment of GBM are only palliative, and include surgical resection (which is frequently incomplete due to the proximity of the tumor to vital brain structures) and focal radiotherapy. A large number of chemotherapeutic agents (e.g. alkylating agents such as temozolomide and nitrosureas such as carmustine) have also been tested, but they display limited efficacy [START_REF] Nieder | Therapeutic options for recurrent highgrade glioma in adult patients: recent advances[END_REF][START_REF] Wong | Targeting malignant glioma survival signalling to improve clinical outcomes[END_REF]. It is therefore essential to develop new therapeutic strategies for the management of GBM. Recently, much effort has been made to understand GBM in terms of its underlying genetic lesions [START_REF] Verhaak | Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1[END_REF][START_REF] Purow | Advances in the genetics of glioblastoma: are we reaching critical mass?[END_REF]. This approach, together with the use of gene expression profile studies, may help to precisely characterize these tumors, thus allowing the identification of factors for resistance to different anti-glioma therapies and the development of specific targeted therapies [START_REF] Huse | Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma[END_REF].

The hemp plant Cannabis sativa produces approximately 70 unique compounds known as cannabinoids, of which Δ 9 -tetrahydrocannabinol (THC) is the best characterized owing to its high potency and abundance [START_REF] Gaoni | Isolation Structure + Partial Synthesis of Active Constituent of Hashish[END_REF]. THC exerts a wide variety of biological effects by mimicking endogenous substances -the endocannabinoids anandamide and 2-arachidonoylglycerol -that bind to and activate specific cannabinoid receptors [START_REF] Howlett | International Union of Pharmacology. XXVII. Classification of cannabinoid receptors[END_REF]. So far, two G protein-coupled cannabinoid-specific receptors have been cloned and characterized from mammalian tissues: CB 1 , abundantly expressed in the brain and at many peripheral sites, and CB 2 expressed in the immune system and also present in microglia, some neuron subpopulations and glioma cells [START_REF] Fernandez-Ruiz | Cannabinoid CB2 receptor: a new target for controlling neural cell survival?[END_REF].

One of the most active areas of research in the cannabinoid field is the study of the potential application of cannabinoids as anti-tumoral agents [START_REF] Guzman | Cannabinoids: potential anticancer agents[END_REF]. Thus, cannabinoid administration has been shown to curb the growth of several models of tumor xenografts in rats and mice [START_REF] Guzman | Cannabinoids: potential anticancer agents[END_REF], including gliomas [START_REF] Carracedo | The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells[END_REF][START_REF] Galve-Roperh | Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation[END_REF][START_REF] Velasco | Cannabinoids and gliomas[END_REF]. Based on this preclinical evidence, a pilot clinical study has been conducted to investigate the effect of THC on recurrent GBM [START_REF] Guzman | A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme[END_REF]. The mechanism of cannabinoid anti-tumoral action relies on the ability of these agents to inhibit tumor angiogenesis, inhibit cell cycle progression and stimulate cancer cell death [START_REF] Guzman | Cannabinoids: potential anticancer agents[END_REF][START_REF] Sarfaraz | Cannabinoids for cancer treatment: progress and promise[END_REF]. We have recently found that cannabinoids activate an ER stress-related signaling route that leads to the up-regulation of the transcriptional co-activator p8 and its target the pseudo-kinase tribbles homolog 3 (TRB3) [START_REF] Carracedo | The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells[END_REF][START_REF] Carracedo | Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related genes[END_REF].

The stimulation of this pathway promotes autophagy via TRB3-mediated inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis and is indispensable for the pro-apoptotic and anti-tumoral action of THC [START_REF] Salazar | Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells[END_REF].

The present work was undertaken to identify the molecular factors responsible for the resistance of glioma cells to cannabinoid antitumoral activity. By using DNA microarrays we have identified a gene expression profile characteristic of THC-resistant glioma cells. We also show that one of the genes up-regulated in cannabinoid-resistant glioma cells, the growth factor Mdk, acting through the anaplastic lymphoma kinase receptor (ALK), plays a pivotal role in enhancing the resistance to cannabinoid-evoked autophagy-mediated cell death. Our results support that targeting of the Mdk/ALK axis could help to improve the efficacy of anti-tumoral therapies for gliomas.

Results

Identification of the gene expression profile characteristic of THC-resistant glioma cells

To gain insight into the molecular features associated with the resistance of glioma cells to cannabinoid action, we initially characterized 10 human glioma cell lines on the basis of their different sensitivity to THC-induced cell death (Figure 1a). Thus, cell lines were classified as cannabinoid-sensitive (IC 50 for THC lower than 2.5 μM) and cannabinoidresistant (IC 50 for THC higher than 2.5 μM). To note, we did not find any correlation between the sensitivity of these cell lines to THC and to other two chemotherapeutic agents (carmustine and temozolomide) frequently used in glioma treatment (Supplementary Figure 1a, 1b and 1c), which indicates that the resistance to THC action is determined by specific mechanisms distinct from those promoting resistance to other drugs. In addition, differences in the levels of CB 1 or CB 2 receptors of glioma cells did not correlate with a higher or lower sensitivity to THC-induced cell death (Figure 1b), and pharmacological blockade of CB 1 receptors with the selective antagonist SR141716 or CB 2 receptors with the selective antagonist SR144528 abrogated THC-induced death in both sensitive and resistant cell lines (Figure 1c and Supplementary Figure 1d). This supports that mechanisms other than changes in the expression or the functionality of CB receptors are responsible for the differences in THC sensitivity of glioma cells. Analysis of some of the genes most frequently altered in gliomas (p16, PTEN, p53 and EGFR) did not reveal association between the presence of mutations, amplifications or deletions on those genes and sensitivity to cannabinoid treatment (data not shown).

Therefore, in order to identify the molecular factors that determine the different sensitivity of glioma cells to THC, we analyzed by DNA microarrays the gene expression profile of the 10 glioma cells lines. After selection of those genes whose expression was significantly changed (FDR<0.2) at least 2.5 fold in more than 3 sensitive or resistant cell lines with respect to their sensitive or resistant counterparts (Figure 2a, Supplementary Figure 2a and Supplementary Tables 1 and2) and real-time quantitative PCR validation of the array data (Figure 2b, Supplementary Figure 2b and Supplementary Tables 3 and5), we selected 20 genes as potential candidates to predict the resistance of glioma cells to cannabinoid action. Next, we analyzed the expression of these genes in primary cultures of glioma cells obtained from tumor samples of GBM patients (in which we confirmed that THC promoted cell death via stimulation of cannabinoid receptors; Supplementary Figure 3d). Data revealed that 8 of the 20 initially-selected genes were differentially up-regulated in the cultures that were more resistant to THC-induced cell death (Figure 2c and 2d, Supplementary Figure 3a and Supplementary Table 4). These observations support that this set of genes is strongly associated with a higher resistance of glioma cells to THC-induced death.

The growth factor midkine confers resistance to THC

One of the genes whose up-regulation was more strongly associated with the resistance of glioma cells to cannabinoid treatment was the growth factor Mdk; Mdk mRNA levels were 30-80-fold higher in the resistant than in the sensitive cell lines (Figure 2b and2d and Supplementary Figure 4a and4c). Mdk is a member of the pleiotrophin (PTN)/Mdk family and was originally described as a developmentally-regulated, heparin-bound, neurotrophic factor [START_REF] Kadomatsu | The midkine family in cancer, inflammation and neural development[END_REF][START_REF] Kadomatsu | cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis[END_REF]. Mdk has been proposed to modulate the proliferation and migration of various types of cancer cells and is often overexpressed in malignant tumors [START_REF] Chen | Midkine and LDL-receptor-related protein 1 contribute to the anchorage-independent cell growth of cancer cells[END_REF][START_REF] Ohuchida | Midkine protects hepatocellular carcinoma cells against TRAIL-mediated apoptosis through down-regulation of caspase-3 activity[END_REF][START_REF] Mirkin | Identification of midkine as a mediator for intercellular transfer of drug resistance[END_REF]. Of interest, analysis of samples from 201 glioma patients (including 115 GBMs) revealed that Mdk expression is associated to a lower overall survival of glioma, high-grade glioma and GBM patients (Figure 3a and Supplementary Figure 5). These analyses were validated with external data obtained from The Cancer Genome Atlas (4) (Supplementary Figure 5e and Supplementary Table 6).

We found that the levels of Mdk are increased in the supernatants of the cannabinoid-resistant glioma cell lines (Figure 3b). We therefore investigated whether this growth factor participates in the mechanism of resistance to cannabinoid antitumoral action of gliomas. In agreement with this possibility, Mdk silencing sensitized two cannabinoid-resistant cell lines (Figure 3c and Supplementary Figure 6a) as well as a primary culture of glioma cells (Figure 3d) to THC-induced cell death. Moreover, incubation with conditioned medium derived from the cannabinoid resistant cell line T98 (which was enriched in Mdk) (Figure 3e) or with exogenous Mdk (Figure 3f) rendered the cannabinoid-sensitive line U87 resistant to THC treatment, an effect that was abrogated by Mdk neutralization with an anti-Mdk antibody (Figure 3e and Supplementary Figure 6b). These observations strongly support that Mdk is a factor for resistance to THC-induced glioma cell death.

Of note, the cannabinoid-resistant pancreatic cancer cell line Panc1 exhibited much higher levels of Mdk than the cannabinoid-sensitive pancreatic cancer cell line MiaPaca2 (Supplementary Figure 7a and7b). Furthermore, Mdk knock-down rendered Panc1 cells sensitive to THC, suggesting that a similar mechanism of resistance to cannabinoid-induced cell death also operates in pancreatic cancer cells (Supplementary Figure 7c).

Mdk promotes resistance to THC through stimulation of the ALK receptor

The mechanism by which Mdk mediates its different biological actions has not been completely clarified [START_REF] Kadomatsu | The midkine family in cancer, inflammation and neural development[END_REF] and may involve the stimulation of several receptors including ALK, protein-tyrosine phosphatase, receptor-type, zeta-1 (RPTPZ), and low density lipoprotein receptor-related protein 1 (LRP1) [START_REF] Kadomatsu | The midkine family in cancer, inflammation and neural development[END_REF]. It has been proposed that the prosurvival and pro-angiogenic effect of Mdk on tumor cells relies on the stimulation of the ALK receptor [START_REF] Stoica | Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types[END_REF]. ALK was expressed in all the cell lines and primary cultures of human glioma cells used in this study (Supplementary Figure 8 a, b andc), and therefore, we asked whether Mdk binding to ALK plays a role in the resistance of glioma cells to THC-induced cell death. In agreement with this idea: (i) ALK knockdown sensitized T98 and HG2 glioma cells to THC-induced cell death (Figure 4a and Supplementary Figure 9), whereas silencing of RPTPZ and LRP1 only modestly enhanced the response to THC treatment of T98 (Supplementary Figure 9c) and HG2 (Supplementary Figure 9d) cells, respectively; (ii) Incubation with T98 cell conditioned medium or addition of exogenous Mdk increased ALK phosphorylation of U87 cells (Supplementary Figure 10c and10d); (iii) silencing of ALK -but not of RPTPZ or LRP1 -abrogated the resistance to THC-induced cell death exerted by the addition of exogenous Mdk to U87 cells (Figure 4b and Supplementary Figure 10a); (iv) knockdown of ALK abolished the protective action exerted by T98-conditioned medium on U87 cells (Supplementary Figure 10b); and (v) incubation with the ALK selective inhibitor NVP-TAE-684 reduced ALK phosphorylation (Figure 4c) and enhanced THC action in T98 cells (Figure 4d). Altogether, these observations strongly support that Mdk promotes resistance to THC-induced cell death via ALK stimulation.

Mdk inhibits the activation of the cannabinoid-evoked autophagy-mediated cell death pathway

We have recently found that THC stimulates autophagy in glioma cells and that the activation of this cellular process is required for the pro-apoptotic and anti-tumoral action of cannabinoids [START_REF] Salazar | Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells[END_REF]. We therefore asked whether the mechanism by which Mdk promotes resistance to THC-induced cell death could be based on the modulation of the signalling route that regulates autophagy in response to cannabinoid treatment. In line with this hypothesis, knock-down of Mdk or ALK in T98 or HG2 cells enhanced THCinduced autophagy, as determined by the presence of cells with LC3 dots as well as by the accumulation of the lipidated form of LC3 (LC3-II) [two hallmarks of autophagy [START_REF] Rubinsztein | Potential therapeutic applications of autophagy[END_REF]] (Figure 5a and 5b and Supplementary Figure 11a), as well as THC-induced apoptosis (Figure 5c and Supplementary Figure 11b). Likewise, pharmacological inhibition of ALK enhanced THC-induced autophagy and apoptosis of T98 cells (Supplementary Figure 12). Moreover, incubation of U87 cells with T98 cell conditioned medium or with exogenous Mdk decreased THC-induced autophagy (Figure 5d) and apoptosis (Figure 5e) in response to THC. These data strongly support that the Mdk/ALK axis modulates THC-induced autophagy.

Induction of autophagy in many cellular settings relies on the inhibition of the mammalian target of rapamycin complex 1 (mTORC1), which in turn regulates the activity of several autophagy proteins involved in the early triggering of this cellular process [START_REF] Rubinsztein | Potential therapeutic applications of autophagy[END_REF]. Accordingly, THC stimulates an ER stress-related pathway that leads to the up-regulation of the transcription factor p8 and its downstream target TRB3. This latter protein triggers the inhibition of the Akt/mTORC1 axis, thus leading to the stimulation of autophagy-mediated cell death [START_REF] Salazar | Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells[END_REF]. On the other hand, ALK stimulation has been shown to activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway [START_REF] Stoica | Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types[END_REF].

Therefore, we investigated whether the resistance to THC-induced autophagy exerted by Mdk relied on the modulation of p8/TRB3 expression and/or on the activation of the Akt/mTORC1 axis. In line with this idea, THC-triggered p8 and TRB3 induction in T98 and HG2 cells was strongly enhanced by Mdk or ALK knock-down (Figure 6a and Supplementary Figure 11c). Furthermore, a decrease in the phosphorylation of Akt and S6 (a well-established readout of mTORC1 activity) was observed upon THC treatment of Mdk-or ALK-silenced T98 and HG2 cells (Figure 6b and Supplementary Figure 11d). On the other hand, incubation of U87 cells with T98 cell conditioned medium or with exogenous Mdk decreased THC-induced p8 and TRB3 up-regulation (Figure 6c) and attenuated THC inhibition of Akt and S6 phosphorylation (Figure 6d). Taken together, these observations strongly support that Mdk (via ALK) modulates p8/TRB3 expression as well as the activity of the Akt/mTORC1 axis to prevent the stimulation of autophagy-mediated cell death by cannabinoids.

In vivo silencing of midkine sensitizes cannabinoid-resistant tumors to THC anticancer action

To analyze the in vivo relevance of our findings, we generated tumor xenografts by subcutaneous injection of T98 or U87 cells in nude mice. In agreement with our in vitro data, T98 cell-derived tumors exhibited higher levels of Mdk and were more resistant to THC treatment than the ones generated with U87 cells (Supplementary Figure 13a, 13b and13c). To investigate whether Mdk is responsible for the resistance to THC antitumoral action we analyzed the effect of THC on the growth of T98 tumor xenografts in which Mdk expression had been knocked-down (Figure 7b and Supplementary Figure 13d). As shown in Figure 7a and in Supplementary Figure 13e, in vivo silencing of Mdk rendered established T98 cell-derived tumors sensitive to THC treatment, although it did not affect tumor growth by itself. Subsequent analysis of tumor samples revealed that Mdk silencing correlated with a decrease in ALK phosphorylation (Figure 7c).

Discussion

GBM is one of the most aggressive forms of cancer in part owing to its poor response to standard radiotherapy and chemotherapy. Consequently, it is currently believed that the design of successful therapeutic strategies to manage these tumors should be based on their detailed molecular characterization, which would allow the development of more specific and personalized treatments [START_REF] Verhaak | Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1[END_REF][START_REF] Purow | Advances in the genetics of glioblastoma: are we reaching critical mass?[END_REF][START_REF] Huse | Targeting brain cancer: advances in the molecular pathology of malignant glioma and medulloblastoma[END_REF]. In line with this line of reasoning, in this study we aimed to identify the molecular features associated with the resistance of gliomas to cannabinoid anti-tumoral action. Our data show that the level of expression of CB 1 or CB 2 receptors does not determine the sensitivity of glioma cells to THCinduced cell death. This is in line with previous observations by our group and others showing that activation of either CB 1 [START_REF] Carracedo | The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells[END_REF][START_REF] Galve-Roperh | Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation[END_REF][START_REF] Salazar | Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells[END_REF] or CB 2 [START_REF] Blazquez | Cannabinoid receptors as novel targets for the treatment of melanoma[END_REF][START_REF] Caffarel | Delta9tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through Cdc2 regulation[END_REF][START_REF] Herrera | p38 MAPK is involved in CB2 receptor-induced apoptosis of human leukaemia cells[END_REF] receptors leads to tumor cell death, thus suggesting that additional factors are responsible for the differences in cannabinoid sensitivity. Detailed analysis of the gene expression profile of a series of cell lines and primary cultures of human glioma cells has led us to find a panel of genes whose up-regulation is associated with differences in sensitivity to THCinduced cell death. On the one hand, this information will allow setting the basis for predicting which specific GBM patients could be more susceptible of being treated with cannabinoids. On the other hand, the genes that are selectively up-regulated in the cannabinoid-resistant glioma cells become potential candidates to play a direct role in the resistance to THC. Several observations strongly support that at least one of these genes, the growth factor Mdk, is directly responsible for the resistance to cannabinoid anti-tumoral action: (i) Mdk levels are much higher in cannabinoid-resistant than in cannabinoid-sensitive glioma cells, (ii) incubation with Mdk-enriched conditioned medium or with exogenous Mdk renders cannabinoid-sensitive glioma cells resistant to THC-induced cell death, (iii) knock-down of Mdk sensitizes cannabinoid-resistant glioma cells to THC killing action, and (iv) in vivo silencing of Mdk renders cannabinoid-resistant tumors sensitive to THC anti-tumoral action.

Mdk is frequently overexpressed in malignant tumors and has been proposed to act as a protective factor against cell death [START_REF] Tong | Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas[END_REF][START_REF] Xu | Midkine positively regulates the proliferation of human gastric cancer cells[END_REF], although the precise mechanism by which Mdk mediates this effect has not been clarified. At least four receptors, namely ALK, N-syndecan, RPTPz and LRP1, have been proposed to mediate the biological activities of Mdk [START_REF] Kadomatsu | The midkine family in cancer, inflammation and neural development[END_REF]. Although controversial data have been published with respect to the notion that midkine acts as a ligand of the ALK receptor [START_REF] Stoica | Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types[END_REF][START_REF] Kuo | Recruitment of insulin receptor substrate-1 and activation of NF-kappaB essential for midkine growth signaling through anaplastic lymphoma kinase[END_REF][START_REF] Moog-Lutz | Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin[END_REF][START_REF] Mourali | Anaplastic lymphoma kinase is a dependence receptor whose proapoptotic functions are activated by caspase cleavage[END_REF][START_REF] Dirks | Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines[END_REF], data presented here demonstrate that the mechanism by which Mdk promotes resistance to THCinduced cell death relies on ALK. Thus, we observed that ALK silencing abolishes the protective effect exerted by a MDK-enriched conditioned medium or by the addition of exogenous Mdk on glioma cells. Likewise, genetic or pharmacological inhibition of ALK enhances THC-induced cell death in cannabinoid-resistant cells. Moreover, treatment with the ALK-selective inhibitor NVP-TAE684 sensitizes cannabinoid resistant tumors to THC anticancer action.

Of interest, alterations in ALK have been implicated in cancer progression. In particular, ALK fusion proteins associated to chromosome translocations are present in most anaplastic large cell lymphomas, a subset of non-Hodgkin lymphomas, as well as in several solid tumors including non-small cell lung cancer [START_REF] Palmer | Anaplastic lymphoma kinase: signalling in development and disease[END_REF][START_REF] Mosse | Inhibition of ALK signaling for cancer therapy[END_REF]. In addition, gainof-function mutations, mostly harbored in the kinase domain of ALK, have been associated to both familiar and sporadic cases of neuroblastoma [START_REF] Chen | Oncogenic mutations of ALK kinase in neuroblastoma[END_REF][START_REF] Mosse | Identification of ALK as a major familial neuroblastoma predisposition gene[END_REF][START_REF] George | Activating mutations in ALK provide a therapeutic target in neuroblastoma[END_REF][START_REF] Janoueix-Lerosey | Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma[END_REF]. By contrast, to the best of our knowledge no studies have been published reporting the presence of these alterations in glioma patients. Moreover, we did not find any mutation in the ALK kinase domain of the glioma cells used in this study (Supplementary Table 7). Taken together, these observations support that increased ligand production rather than mutations or amplifications of the ALK gene are responsible for the stimulation of this receptor in glioma cells. To note, no correlation between pleiotrophin (PTN, a Mdkrelated protein with high homology with Mdk) expression and resistance to THCinduced cell death was found in this study (Supplementary Figure 4b), which further supports that the mechanism of resistance to THC-induced cell death in glioma cells specifically relies on the increased expression of Mdk. Our data are therefore in agreement -at least in part -with the proposal that ALK is a dependence receptor [START_REF] Mourali | Anaplastic lymphoma kinase is a dependence receptor whose proapoptotic functions are activated by caspase cleavage[END_REF][START_REF] Allouche | ALK is a novel dependence receptor: potential implications in development and cancer[END_REF] that plays an anti-apoptotic role only when is activated/stimulated by its ligand. Of potential therapeutic relevance, we found that increased expression of Mdk is associated with decreased survival of GBM patients, which further supports that this ligand may play an important role in promoting resistance to cancer therapies.

We also show that Mdk, through stimulation of ALK, promotes resistance to THC-induced cell death by interfering with the induction of autophagy by cannabinoids.

The mechanism by which Mdk inhibits THC-induced autophagy relies on the modulation of the Akt/mTORC1 axis. ALK, as other tyrosine kinase receptors, is coupled to PI3K/Akt [START_REF] Powers | Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth[END_REF]. Accordingly, we found that Mdk stimulates Akt in human glioma cell lines. However, our data also show that, in the absence of THC, silencing of ALK or Mdk decreases Akt phosphorylation in cannabinoid-resistant glioma cells without leading to either inhibition of the mTORC1 complex or autophagy. These observations suggest that additional signals -as those triggered by THC -are necessary for the stimulation of autophagy-mediated cell death in glioma cells. Interestingly, we also found that p8 and TRB3 only become up-regulated in response to THC treatment in cannabinoid-resistant cells when Mdk or ALK have been silenced. Taken together, these results support that Mdk and ALK promote resistance to THC-induced autophagy via stimulation of Akt and modulation of the expression of the pro-autophagic genes p8 and TRB3.

In summary, we have found that increased expression of the growth factor Mdk and subsequent activation of ALK are critically involved in the promotion of glioma resistance to cannabinoid anti-tumoral action. Furthermore, experiments with tumor xenografts demonstrate that inhibition of the Mdk/ALK axis sensitizes cannabinoidresistant tumors to THC treatment, which correlates with an enhanced activation of autophagy and apoptosis in these tumors. Anti-tumoral therapies based on the selective targeting of tyrosine kinase receptors are currently being used for the management of different types of cancer [START_REF] Okamoto | Epidermal growth factor receptor in relation to tumor development: EGFR-targeted anticancer therapy[END_REF][START_REF] Ivy | An overview of small-molecule inhibitors of VEGFR signaling[END_REF][START_REF] De Bono | Translating cancer research into targeted therapeutics[END_REF]. Moreover, resistance to different chemoterapeutic agents frequently relies on a gain of function of the signaling routes activated by tyrosine kinase receptors [START_REF] Laurent-Puig | Mutations and response to epidermal growth factor receptor inhibitors[END_REF][START_REF] Dempke | Resistance to EGF-R (erbB-1) and VEGF-R modulating agents[END_REF][START_REF] Lorente | Amphiregulin is a factor for resistance of glioma cells to cannabinoid-induced apoptosis[END_REF]. In line with these observations, and with the growing evidence for the participation of ALK in the origin and progression of different types of tumors [START_REF] Mosse | Inhibition of ALK signaling for cancer therapy[END_REF][START_REF] Mano | Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung cancer[END_REF], our findings support that selective targeting of Mdk or ALK could be used to enhance the therapeutic efficacy of anti-tumoral therapies in gliomas.

Materials and methods

Reagents

THC was kindly provided by GW Pharmaceuticals (Salisbury, United Kingdom). The cannabinoid antagonists SR141716 (SR1) and SR144528 (SR2) were kindly donated by Sanofi-Aventis (Montpellier, France). Recombinant human Midkine was from Sigma (St. Louis, MO, USA). Double-stranded RNA duplexes corresponding to human Mdk, human ALK (smart-pool) and control siRNA were synthesized by Dharmacon (Lafayette, CO). The RNA sequence targeting human Mdk is described in [START_REF] Takei | Combinational antitumor effect of siRNA against midkine and paclitaxel on growth of human prostate cancer xenografts[END_REF]. The ALK inhibitor NVP-TAE 684 was synthesized by Sergey A. Lakatosh at the Gause Institute of New Antibiotics (Moscow, Russia). BCNU was purchased from Sigma and TMZ was kindly provided by Schering-Plough (Whitehouse Station, NJ, USA).

Cell culture and viability

The human glioma cell lines GOS3, U87MG (U87), A172, SW1783, U118 MG (U118), Human glioma or pancreatic cancer cell lines were cultured in DMEM containing 10% FBS. The preparation of adherent primary culture of brain tumor cells was as follows: tumor samples were homogenized, digested with type Ia collagenase (Sigma) for 90 min at 37ºC and incubated on ice for 10 min. The supernatant was collected and, after centrifugation to discard the remaining death-floating cells, resuspended in DMEM containing 15% FBS. Finally, cells were seeded at a density of 400,000 cells/cm 2 and kept in culture for 2 weeks in DMEM containing 15% FBS and 1% glutamine. Cells were transferred to a serum-free medium (human glioma cell lines), 0.5 % FBS medium (primary cultures of glioma cells) or conditioned medium 18 h before performing the different treatments. When indicated, cells were pre-incubated with exogenous Mdk for 2 h or with NVP-TAE-684, SR1 or SR2 for 1 h before treatment with THC. Cell viability was determined after 72 h of THC treatment by using the MTT test (Promega, Madison, WI) according to manufacturer's instructions.

THC stock solution was prepared in dimethylsulfoxide. Control incubations contained the same amount of dimethylsulfoxide and no significant effect was observed in any of the parameters determined throughout this study at the final concentration used (0.1-0.2%, v/v).

Analysis of gene expression by cDNA microarrays.

Total RNA was isolated from human glioma cells lines with RNeasy Mini Kit (Qiagen, Hilden, Germany). Double-stranded cDNA was amplified with Superscript Choice System and T7-(deoxythymidine)24 oligo-primers (Life Technologies, Inc., Gaithersburg, MD, USA) and in vitro transcription was carried out with Megascript TM T7 (Ambion, Austin, TX, USA). Samples were labelled with Cy3-dUTP (Amersham, Uppsala, Sweden) and Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) with Cy5-dUTP (Amersham) and hybridized on an advanced version of the Spanish National Cancer Research Centre (CNIO) Oncochip (http://bioinfo.cnio.es/data/oncochip) as described in the MIAME. Slides were scanned in a Scanarray 5000 XL scanner (GSI Lumonics Kanata, Ontario, Canada) and the data matrices were processed with GenePix 6.0 software (Axon, Instruments Inc., Union City, CA, USA).

Human Mdk ELISA bioassay

Soluble Mdk levels were determined by using the Human Midkine ELISA (Biovendor;

Heidelberg, Germany).

Conditioned medium experiments

U87 or T98 cells were seeded in complete medium at a density of 30.000 cells/cm 2 and, after 8 h, transferred to a serum-free medium for 18 h. Media were subsequently recovered, centrifuged and incubated alone or with an anti-Mdk antibody (20 μg/ml;

Ref: sc-20715, Sta. Cruz Biotechnology Inc, Sta. Cruz, CA, USA) or with an isotype antibody (rabbit IgG; 20 μg/ml; Amersham Biosciences Corp., Piscataway, NJ, USA)

for 1 h at 37ºC. To study the influence of the conditioned media (obtained as described above) on cell viability, U87 cells were seeded in complete medium at a density of 5000 cells/cm 2 and, after 8 h, transferred to the corresponding conditioned medium 18 h before performing the different treatments. Finally, cell viability was determined as described above.

Transfections

Seventy five percent-confluent T98, LN405, HG2, or Panc1 cells were transfected with control (siC), human Mdk (siMdk) or human ALK (siALK) siRNAs using the X-tremeGENE siRNA Transfection Reagent (Roche, Basel, Switzerland) according to manufacturer's instructions. Twenty four hours after transfection, cells were trypsinized and seeded in complete medium at a density of 5000 cells/cm 2 . After 8 h, cells were transferred to a serum-free (cell lines) or 0.5% FBS (primary culture) medium for 18 h and the different treatments were performed. Transfection efficiency was monitored using a control fluorescent siRNA (Qiagen, Hilden, Germany). The efficacy of transfection was higher than 80 % in all the experiments.

Reverse transcription and Real-time quantitive PCR analysis

RNA was isolated using the RNeasy Protect kit (Qiagen) including a DNase digestion step using the RNase-free DNase kit (Qiagen). cDNA was subsequently obtained using

Transcriptor Reverse Transcriptase (Roche). PCR reactions were performed using the following parameters: 95ºC for 5 min, 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min followed by a final extension step of 72°C for 5 min. The number of cycles [START_REF] Stoica | Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types[END_REF][START_REF] Rubinsztein | Potential therapeutic applications of autophagy[END_REF][START_REF] Blazquez | Cannabinoid receptors as novel targets for the treatment of melanoma[END_REF] cycles for human Mdk and 22-25 cycles for GAPDH) was adjusted to allow detection in the linear range. Finally, PCR products were separated on 1.5 % agarose gels. Real-time quantitive PCR assays were performed using the FastStart Master Mix with Rox (Roche) and probes were obtained from the Universal Probe Library Set (Roche).

Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems). Each value was adjusted using 18S RNA levels as reference. Primers sequences and probes used in this study can be found in Supplementary Table 8.

Tumor xenografts

Tumors were induced by subcutaneous injection in nude mice of 10 x 10 6 U87 or T98 cells in PBS supplemented with 0.1% glucose. When tumors had reached an average size of 250 mm 3 (an average of 2 weeks for U87 tumors and 14 weeks for T98 tumors), animals were assigned randomly to various groups and injected peritumorally for 15 days with the corresponding treatment [THC (15 mg/Kg/day), NVP-TAE-684 (1 mg/kg/day) or THC (15 mg/Kg/day) + NVP-TAE-684 (1 mg/kg/day)] or vehicle in 100 μl of PBS supplemented with 5 mg/ml defatted and dialyzed BSA. Tumors were measured with external caliper, and volume was calculated as (4π/3) x (width/2) 2 x (length/2). At the end of the treatment, animals were sacrified, all tumors were excised and their weights were determined.

In vivo silencing

In vivo Mdk silencing was performed by using a 1:1 mixture of AteloGeneTM (Koken Co. Ltd., Tokyo, Japan) and 10 μM HPLC-purified double-stranded siRNA (synthesized by Dharmacon), according to manufacturer's instructions. Briefly, the mix of AteloGene and siRNA (1 nmol of control or Mdk-selective siRNA per injection) was administered in each tumor at days 1 and 7 of vehicle or THC treatment. RNA sequence targeting human Mdk was 5'-GUUUGGAGCCGACUGCAAGdTdT-3' [START_REF] Takei | Combinational antitumor effect of siRNA against midkine and paclitaxel on growth of human prostate cancer xenografts[END_REF].

Western blot analysis

Western blot analysis was performed following standard methods. The antibodies used were: anti-phospho-S6 ribosomal protein Ser 235/236 and anti-total-Akt, anti-phospho-Akt Ser473 (1:1000; Cell Signaling Technology, Inc., Lake Placid, NY), anti-α-tubulin

Imunofluorescence from cell cultures

Cell cultures grown on 12 mm-coverslips were washed in PBS, fixed with 4% paraformaldehyde (20 min at room temperature), permeabilized and blocked to avoid non-specific binding with 10% goat antiserum and 0.25% Triton X-100 in PBS for 45 min, and subsequently incubated with the following primary antibodies (for 1 h at room temperature): rabbit polyclonal anti-CB 1 antibody and anti-CB

TUNEL

Tumor samples were fixed, blocked and permeabilized and TUNEL was performed as described [START_REF] Carracedo | The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells[END_REF].

Tissue microarray immunohistochemistry

Tissue microarrays (TMAs) were constructed as described previously (50) using formalin-fixed, paraffin-embedded archival tissue blocks from five non-tumoral tissues For immunohistochemical staining, 4-μm-thick paraffin-embedded tissue sections were subsequently dewaxed, rehydrated and subjected to antigen retrieval using EnVision FLEX Target Retrieval solution at pH10 and heated at 95ºC. The slides were cooled and treated with the EnVision FLEX Peroxidase-Blocking Reagent solution (Dako, Glostrup, Denmark) for 5 min. Sections were then immunostained with Mdk mouse monoclonal antibody A-9 (1:50; Santa Cruz Biotechnology INC, Santa Cruz, CA) using the Dako EnVision FLEX /HRP Technique (Dako), counterstained with hematoxylin and mounted. Mdk expression was considered positive when presence of cytoplasmic staining was observed.

Survival analyses

Survival curves were plotted according to the Kaplan-Meier method, and the log-rank test was used to establish the statistical significance of the differences between curves.

Differences of overall survival based on MDK positive or negative protein expression were analyzed. Survival analysis of the MDK microarray expression TCGA (The Cancer Genome Atlas) data reported by Verhaak et al. ( 4) was performed by categorizing patients into two groups according to the median MDK expression (high expression ≥ 0.12; low expression < 0.12). The association between continuous MDK expression levels and overall survival was determined by a Cox regression univariate model. The threshold for statistical significance in all cases was taken to be P < 0.05.

Statistical analyses were carried out with SPSS v18.0 (SPSS, Chicago, IL).

Statistics

  Moreover, THC treatment increased TRB3 expression (Figure 7d, upper panels), decreased S6 phosphorylation (Figure 7d, middle panels), enhanced autophagy [as determined by LC3 immunostaining (Figure 7d, lower panels)] and induced apoptosis [as determined by active caspase-3 immunostaining (Figure 7e)] only in those tumors in which Mdk expression had been knocked-down. Finally, we investigated the participation of ALK on the resistance of T98 tumors to THC anti-tumoral action. To this aim we analyzed the effect of the co-administration of THC and NVP-TAE-684 on the growth of T98 tumor xenografts. Similar to what we observed upon Mdk silencing, administration of NVP-TAE-684 decreased ALK phosphorylation and sensitized established T98 cell-derived tumors to THC treatment, but did not affect tumor growth by itself (Figure 8a and 8b). Furthermore, THC treatment increased TRB3 expression (Figure 8c, upper panels), decreased S6 phosphorylation (Figure 8c, middle panels), enhanced autophagy (Figure 8c, lower panels) and induced apoptosis (Figure 8d) only in those tumors in which ALK had been inhibited. Altogether, these results strongly support that the Mdk/ALK axis promotes resistance to THC anti-tumoral action in vivo by modulating the activation of the autophagy mediated cell-death pathway.

  U373MG (U373), T98G (T98), SW1088, CCF-STTG1 (CCF) and LN405 were from ATCC (Middlesex. UK), DSMZ (Braunschweig, Germany) or were kindly provided by Dr. Javier Castresana (Universidad Pública de Navarra, Pamplona, Spain). Primary cultures of brain tumor cells were obtained from biopsies donated by the Tumor Bank Network coordinated by the Spanish Cancer Research Centre and from the Hospital Clínico Universitario (Madrid, Spain). Human pancreatic cancer cell lines MiaPaca2 and Panc1 were kindly provided by Dr. J.L. Iovanna (Marseille, France).

( 1 :

 1 4000; Sigma), anti-LC3 (1:1000; polyclonal, MBL, Naka-Ku Nagoya, Japan, ref PM036), anti-CB 1 and anti-CB 2 (1:1000; Affinity Bioreagents, Golden, CO); anti-Mdk (1:200; Sta. Cruz Biotechnology Inc, Sta. Cruz, CA, USA), anti total ALK (1:250; rabbit polyclonal ab75953; Abcam) and anti-phospho ALK (Tyrosine 1604) (1:500; rabbit monoclonal [EP661-Y] ab51030, Abcam).

( 4

 4 normal brains and 1 tonsil) and 201 glioma samples (115 glioblastomas WHO grade IV, GBs; 14 anaplastic astrocytomas WHO grade III, AAs; 12 diffuse astrocytomas WHO grade II, DAs; 12 pilocytic astrocytomas WHO grade I, PAs; 4 anaplastic oligoastrocytomas WHO grade III, AOAs; 8 oligoastrocytomas WHO grade II, OAs; 19 anaplastic oligodendrogliomas WHO grade III, AOGs; and 17 oligodendrogliomas WHO grade II, OGs). All samples were reviewed by means of full tissue sections stained with hematoxylin and eosin (H&E) to verify tumor viability, select the representative areas of each tumor and confirm the diagnosis according to WHO guidelines (2).

-Figure 1 .

 1 Figure 1. Classification of human glioma cell lines according to their sensitivity to

Figure 2 .

 2 Figure 2. Identification of the gene expression profile associated with resistance to

Figure 3 .

 3 Figure 3. Increased Mdk expression confers human glioma cells resistance to THC

Figure 4 .

 4 Figure 4. Mdk promotes resistance to THC-induced cell death via ALK

Figure 5 .

 5 Figure 5. Mdk promotes resistance to THC-induced autophagy and apoptosis

Figure 6 .

 6 Figure 6. Mdk promotes resistance to THC-induced autophagy by preventing p8

Figure 7 .

 7 Figure 7. In vivo silencing of Mdk sensitizes T98 cell-derived tumors to THC anti-

Acknowledgments

This work was supported by grants from Spanish Ministry of Science and Innovation (MICINN) (PS09/01401; HF2005/0021, FR2009-0052 and IT2009-0053 to GV; SAF2006/00918 to MG), Santander-Complutense (PR34/07-15856 to GV), Comunidad de Madrid (S-SAL/0261/2006 to MG). ML was sequentially recipient of a "Juan de la Cierva" contract, a postdoctoral contract from Spanish Ministry of Education and Science (MEC) and a postdoctoral contract from Comunidad de Madrid. ST was recipient of a research formation contract from Comunidad de Madrid, MS was recipient of a fellowship from MEC and of a research formation contract from Comunidad de Madrid, AC was recipient of fellowships from Gobierno Vasco, FEBS and EMBO. SH has a technician contract from MICINN and Fondo Social Europeo. We thank Horacio Zimman and Carmen Moreno from Hospital Clínico San Carlos as well as Leyre Urigüen from Universidad del Pais Vasco for their kind collaboration in the processing and delivery of glioma samples; Miguel Ángel Piris, Raquel Villuendas, Paloma Cueva and Rosa Pérez for technical advice in the gene expression experiments and other members of our lab for their continuous support.

Conflict of interest:

The authors declare no conflict of interest.


Daniela Trisciuoglio

Chiara Gabellini

Marianna Desideri

Ylenia Ragazzoni

Teresa De Luca

Elio Ziparo

Donatella Del Bufalo
email: delbufalo@ifo.it

Donatella Del

Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells

Keywords: BH domain, HIF-1, VEGF, cancer

In addition to act as an antiapoptotic protein, bcl-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia inducible factor 1 (HIF-1) -mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogates the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia.

Finally we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

INTRODUCTION

Bcl-2 family proteins are defined by the sequence homology shared within four conserved bcl-2 homology (BH) domains, BH1, BH2, BH3 and BH4. Several antiapoptotic members, such as bcl-2 and bcl-xL, possess all four BH domains, whereas some proapoptotic members, such as bax and bak, conserve homology only in the BH1, BH2 and BH3 domains [START_REF] Cory | The Bcl2 family: regulators of the cellular life-or-death switch[END_REF] ; however a structure-based alignment of globular bcl-2 family proteins, recently revealed that a conserved BH4 motif is present in some proapoptotic proteins, as well as antiapoptotic proteins [START_REF] Kvansakul | Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands[END_REF] . Whereas the BH1 and BH2 domains of bcl-2 are required for dimerization with proapoptotic members, the BH3 domain is crucial for homo-and hetero-dimerization between the bcl-2 family members. The amino(N)terminal BH4 domain is critical for interaction of bcl-2 and bcl-xL with and/or regulation of several proteins involved in apoptosis [START_REF] Shibasaki | Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2[END_REF][START_REF] De Moissac | Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis[END_REF][START_REF] Ricca | bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells[END_REF][START_REF] Huang | The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4[END_REF] . All BH domains are necessary for the antiapoptotic function of bcl-2 and bcl-xL [START_REF] Hunter | Functional dissection of the human Bc12 protein: sequence requirements for inhibition of apoptosis[END_REF] . In addition to protecting from apoptosis and increasing survival, bcl-2 and bcl-xL are involved in several other important functions, including angiogenesis [START_REF] Trisciuoglio | Bcl-2 overexpression in melanoma cells increases tumor progression-associated properties and in vivo tumor growth[END_REF][START_REF] Iervolino | Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity[END_REF][START_REF] Giorgini | Modulation of bcl-xL in tumor cells regulates angiogenesis through CXCL8 expression[END_REF][START_REF] Biroccio | Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line[END_REF][START_REF] Karl | Bcl-2 acts in a proangiogenic signaling pathway through nuclear factor-kappaB and CXC chemokines[END_REF][START_REF] Diensthuber | Expression of bcl-2 is associated with microvessel density in olfactory neuroblastoma[END_REF][START_REF] Warner | Endothelial cells enhance tumor cell invasion through a crosstalk mediated by CXC chemokine signaling[END_REF] . In this context we have previously reported that bcl-2 expression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxiainducible factor-1 (HIF-1) 9;11 . Moreover, modulation of bcl-xL expression in glioblastoma cells regulates interleukin 8 protein expression through a nuclear factor-kappa B (NF-kB)-dependent mechanism 10;15 . In vitro and in vivo inhibition of bcl-2 functions has a strong effect on HIF-1α target genes that, in some cases, is functionally unrelated to the prosurvival effect of bcl-2 [START_REF] Bufalo | Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity[END_REF][START_REF] Zeitlin | Antiangiogenic effect of TW37, a small-molecule inhibitor of Bcl-2[END_REF][START_REF] Anai | Downregulation of BCL-2 induces downregulation of carbonic anhydrase IX, vascular endothelial growth factor, and pAkt and induces radiation sensitization[END_REF] .

These results, together with data demonstrating the ability of bcl-2 to orchestrate a contactindependent crosstalk between endothelial and tumor cells promoting tumor growth [START_REF] Kaneko | Bcl-2 orchestrates a cross-talk between endothelial and tumor cells that promotes tumor growth[END_REF] , identify a new function for bcl-2 and bcl-xL that goes beyond their role in cell survival. An important point, yet to be studied in more depth, is which domain(s) of bcl-2 account for the regulation of angiogenesis by bcl-2 through a VEGF-mediated HIF-1-dependent pathway. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor, composed of α and β subunits, that plays a key role in cellular adaptations to hypoxia by controlling the expression of a series of genes involved in angiogenesis (such as VEGF, VEGF receptor-1, Chemokine receptor CXCR4), glucose metabolism (such as glucose trasporter-1, hexokinase-2) and apoptosis (BNIP3, Wilms tumor suppressor). Whereas both α and β subunits are required for the formation of HIF-1 heterodimer, HIF-1α is the key regulatory subunit responsible for HIF transcriptional function [START_REF] Semenza | Targeting HIF-1 for cancer therapy[END_REF] .

Several major mechanisms underlying regulation of HIF-1α protein have been delineated, involving multiple pathways and regulatory factors, some of them still to be fully characterized, such as PHD1-3/VHL, HSP90/Receptor of Activated protein Kinase C (RACK1), and HSP70/CHIP (Carboxyl terminus of Hsp70-Interacting Protein) 21;22 . In addition to hypoxia, HIF-1α expression can be regulated by certain oncogenes, including bcl-2 9;11 . We recently demonstrated that bcl-2 induces HIF-1α protein stabilization through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90 [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] . In this work we investigated the possible role of the BH domains of bcl-2 in the ability of this protein to regulate HIF-1-mediated VEGF expression in cooperation with hypoxia. Our present study shows that bcl-2 is implicated in HIF-1α stabilization in hypoxic conditions, through a mechanism that involves the BH4 domain, but not the BH1 or BH2.

RESULTS

The BH4 domain, but not the BH1 or BH2, is necessary for VEGF induction by wild type bcl-

under hypoxia

Previously, we showed that bcl-2 expression in tumor cells exposed to hypoxia increases the expression of VEGF gene through the activation of HIF-1 9;11 . To address the relevance of the different domains of bcl-2 in its ability to increase VEGF expression under hypoxia, M14 human melanoma cells were transiently transfected with expression vectors encoding human wild type bcl-2, or different point-mutated or deleted forms of this protein (Figure 1A,B). As shown by ELISA (Figure 1C), while under normoxia no differences were observed between the effect of wild type or mutated bcl-2 forced expression on VEGF protein level, under hypoxia the VEGF protein level increased in all cells compared to normoxia. In addition, under hypoxia higher levels of VEGF protein were observed in cells overexpressing bcl-2 wild type or deleted in the BH1 (BH1 del) or BH2 (BH2 del) domains when compared with control cells, besides higher level of bcl-2 protein was reached in cells overexpressing the wild type compared to mutated bcl-2 protein (Figure 1B), thus indicating that those low levels of BH1/BH2 deleted proteins are sufficient to sustain HIF-1/VEGF pathway activation in hypoxia. On the contrary, the level of the VEGF protein was not increased in cells expressing bcl-2 deleted in the BH4 domain (BH4 del) relative to control cells. To better define the relevance of the BH domains in bcl-2-induced VEGF expression under hypoxia, we transiently transfected M14 cells with vectors encoding bcl-2 single-point mutants of highly conserved residues within the BH1 (G145E) or BH2 (W188A) domains, or dicodon mutants into the BH4 domain (G14-15, G18-19, G22-23, G6-7, G10-11, G16-17). As reported in Figure 1C all mutations in the residues of BH4 domain, abrogated the ability of the wild type form of bcl-2, to cooperate with hypoxia to induce VEGF expression. On the contrary, point-mutations within the BH1 or BH2 domains did not affect bcl-2-induced VEGF protein secretion. Thus, demonstrating that VEGF activation by bcl-2 under hypoxia involves its BH4 domain, but not BH1 or BH2. To confirm the results obtained from transient-transfection assay, we obtained stably transfected clones using expression vectors encoding wild type bcl-2 (Bcl-2/clone5 and Bcl-2/clone 37), bcl-2 deleted of BH4 (BH4 del/clone 8 and BH4 del/clone 42) or point-mutated within the BH1 [BH1(G145E)/clone 14 and BH1(G145E)/clone 25] or BH2 [BH2(W188A)/clone 6 and BH2 (W188A)/clone 16] domains (Figure 2A). The role of the different mutations on bcl-2-induced VEGF expression (Figure 2B) and VEGF transcriptional activity (Figure 2C) was evaluated under normoxia or hypoxia. Under normoxia, there were not differences in VEGF protein secretion in normoxia regardless bcl-2 status. By contrast, under hypoxia wild type bcl-2 overexpression significantly increased both the VEGF protein secretion (Figure 2B) and VEGF promoter activity (Figure 2C) when compared to control cells. However, the deletion of the BH4 domain abrogates the ability of bcl-2 to cooperate with hypoxia to induce VEGF expression and promoter activity: both parameters were similar in the control clone and in bcl-2 deleted of BH4 transfectants (Figure 2B,C). On the contrary, point-mutations at the BH1 or BH2 domains did not affect bcl-2-induced VEGF protein secretion and promoter activity under hypoxia. Altogether, these data demonstrate that the induction of VEGF expression by bcl-2 under hypoxia requires the BH4 domain, but not the BH1 or BH2.

The BH4 domain is necessary for VEGF hypoxic induction by bcl-2 in tumor cells of different origin

To extend the BH4-dependent role of bcl-2 in the regulation of hypoxia-induced VEGF expression to other melanoma cells and other tumor histotypes, we transiently overexpressed bcl-2 wild type or the same protein deleted of its BH4 domain, in different tumor cells (Figure 3). In normoxia, the levels of VEGF protein secreted were similar in all cells regardless of bcl-2 status and expression. By contrast, the hypoxic induction of the VEGF protein was increased after forced expression of wild type bcl-2 in PLF2 and JR1 melanoma cells when compared to control cells, while no differences were observed after forced expression of the bcl-2 protein deleted of the BH4 domain (Figure 3A). Enhanced VEGF expression after overexpression of wild type bcl-2, but not of bcl-2 deleted of its BH4 domain, was also shown in colon (HT29), ovarian (CaOV3) and lung (H1299) carcinoma cell lines (Figure 3B), thus confirming the capability of bcl-2 to modulate, through its BH4 domain, VEGF expression under hypoxia in tumor cells of different origin.

The increase of HIF-1α protein expression by bcl-2 is dependent on its BH4 domain

We have previously demonstrated that the modulation of bcl-2 expression in tumor cells regulates VEGF expression under hypoxia through a HIF-1-dependent pathway 9;11;23;24 . Thus, we investigated on the role of different bcl-2 mutations on the bcl-2 induced HIF-1 transcriptional activity and HIF-1 proteins expression in melanoma cells, stably or transiently transfected with wild type or different mutants bcl-2. HIF-1 transcriptional activity and the hypoxic induction of HIF-1α and HIF-1β proteins control cells (puro), cells stably overexpressing bcl-2 wild type (Bcl-2/clone5 and Bcl-2/clone 37) or deleted of BH4 domain (BH4 del/clone 8 and BH4 del/clone 42) are reported in Figure 4A and Figure 4B, respectively. Under hypoxia wild type bcl-2 clones showed about 2-fold increase in the activity of the HIF-1-dependent gene transcription when compared with the transfected control cells, whereas the clones overexpressing bcl-2 deleted of its BH4 domain showed HIF-1 transcriptional activity similar to that observed in the transfected control cells (Figure 4A). Transfectants expressing point-mutants within the BH1 or BH2 domains of bcl-2 showed HIF-1 transcriptional activity similar to that of the clones overexpressing the wild type bcl-2 protein (data not shown). As shown in Figure 4B, after exposure to hypoxia, the HIF-1α nuclear protein expression was detectable in control cells (puro), while it became more evident after forced expression of wild type bcl-2 (Bcl-2/clone5 and Bcl-2/clone 37). Similar to control cells, both clones carrying bcl-2 deleted of the BH4 domain (BH4 del/clone 8 and BH4 del/clone 42) showed a low HIF-1α protein expression. Under hypoxia, high levels of HIF-1α protein were also observed in the cells transiently transfected with wild type bcl-2 when compared with the cells transiently transfected with both empty vector and vector encoding bcl-2 deleted of its BH4 domain. On the other hand, similar levels of the HIF-1α protein were observed in cells transiently transfected with wild type bcl-2 and in those expressing bcl-2 point-mutants within the BH1 or BH2 domains (Figure 4C). Besides, cells expressing bcl-2 dicodon mutants in the BH4 domain showed levels of the HIF-1α protein comparable to those showed by cells transiently transfected with the empty vector (Figure 4D), independently from their response to apoptosis. Moreover, under hypoxia HIF-1β expression was not modulated in any of the cell lysates regardless of the bcl-2 status (Figure 4B-D).

In normoxia, levels of HIF-1α protein were undetectable in all cells regardless of bcl-2 status and expression (data not shown).

The increase of HIF-1α protein stability by bcl-2 is dependent on its BH4 domain

We previously reported that while bcl-2 overexpression under hypoxia did not alter HIF-1α mRNA levels [START_REF] Iervolino | Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity[END_REF] , it affects HIF-1α protein expression at the posttranscriptional level [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] . RT-PCR analysis (Figure 5A) showed no difference in HIF-1α mRNA levels between cells exposed to normoxia or hypoxia regardless of bcl-2 status, thus confirming that bcl-2 overexpression under hypoxia did not modulate HIF-1α gene expression at transcriptional level and excluding the involvement of BH4 domain in the regulation of HIF-1α mRNA levels. To elucidate the mechanism by which bcl-2 activates HIF-1, we examined the effects of ectopic expression of wild type bcl-2 or bcl-2 deleted in BH4 domain of bcl-2 on HIF-1α protein at the posttranscriptional level. To this purpose, we firstly performed experiments using the proteasome inhibitor MG132 (Figure 5B). Since the blockade of proteasome function inhibits HIF-1α proteasome-mediated degradation in normoxic conditions, the detected HIF-1α protein levels will predominantly reflect the translation rate of the HIF-1α protein.

The accumulation of HIF-1α protein after MG132 treatment was identical in all cells regardless bcl-2 status, indicating that neither wild type nor mutated from of bcl-2 does not affect HIF-1α protein synthesis (Figure 5B). To directly test whether bcl-2 status in hypoxic cells is associated with a modulation of HIF-1α protein stability, we measured HIF-1α protein half-life. As depicted in In particular, while HIF-1α protein was still well detectable after 60 minutes treatment with the CHX in wild type bcl-2 transfectants (Bcl-2/37), HIF-1α protein was strongly reduced already after 15 minutes from CHX adding and it was not detectable at 60 minutes of CHX treatment in both control cells (puro) and BH4 deleted bcl-2 overexpressing clone. Densitometric analysis revelead that, as previously reported [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] , bcl-2 overexpression increased the HIF-1α protein half-life from 15±5 min to 45±5 min under hypoxic conditions. By contrast, no significant difference in the HIF-1α protein half-life was observed between control cells and stable clones expressing bcl-2 deleted of its BH4 domain (BH4 del/42, HIF-1α protein half-life: 13±7 min). Similarly, while a reduced HIF-1α ubiquitination is observed in Bcl-2/37, transfectant expressing wild type bcl-2, when compared to control cells (puro), similar modulation of HIF-1α ubiquitination was observed in BH4 del/8, transfectant carrying the deletion of BH4 domain and control cells (puro) (Figure 5E). All together these results demonstrate that the degradation rate of HIF-1α protein is faster in the control and bcl-2 deleted of BH4 domain overexpressing cells than in wild type bcl-2 transfectants, thus indicating an effect of bcl-2 on HIF-1α protein accumulation through its BH4 domain.

The effect of bcl-2 mutations on HIF-1/VEGF signaling is independent of their relevance in apoptosis.

There are evidences that mutations or deletions of bcl-2 protein can affect bcl-2 ability to inhibit apoptosis 7;25;26 , transient overexpression of wild type bcl-2 may induce rather than protect cells from apoptosis [START_REF] Portier | Bcl-2 localized at the nuclear compartment induces apoptosis after transient overexpression[END_REF] and deletion of BH4 domain can convert bcl-2 into a proapototic protein [START_REF] Huang | The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4[END_REF] . To exclude the possibility that the effect of bcl-2 on HIF-1/VEGF expression and activation could be a consequence of a different modulation of the apoptotic pathway by ectopic expression of different forms of bcl-2, we evaluated the response of cells in terms of apoptotic cell death induced by campthotecin (CPT), a drug able to activate the apoptotic program in several experimental models [START_REF] Kawatani | Deletion of the BH1 domain of Bcl-2 accelerates apoptosis by acting in a dominant negative fashion[END_REF] . As reported in Figure 6A-B, Annexin V/Propidium Iodide staining analysis by flow cytometry shows that CPT treatment induced about 50% of apoptosis in untrasfected cells or cells transfected with empty vector, while it induced a very low percentage of apoptosis in cells overexpressing wild type bcl-2. CPT-induced apoptosis was also observed in cells transfected with expression vectors encoding BH1, BH2 and BH4 domain deletion mutants of bcl-2, encoding bcl-2 single-point mutants of highly conserved residues within the BH1 (G145E) or BH2 (W188A) domains, or dicodon mutants into the BH4 domain (G14-15, G18-19, G22-23). On the other hand, the rate of apoptotic events in the cells transfected with bcl-2 mutations into the residues 6-7, 10-11, and 16-17 of BH4 domain was quite similar to that observed following CPT treatment in cells overexpressing wild type bcl-2, confirming that these residues are not required for bcl-2 antiapoptotic activity 6;7;28 . These results obtained in cells carrying bcl-2 deleted of its BH4 domain and mutations into BH4 domain, were also confirmed by the activation of caspase-3(-like) proteases, as estimated by the cleavage of PARP after treatment with CPT (Figure 6C).

Overexpression of wild type bcl-2 but not of bcl-2 deleted in BH4 domain prevented PARP cleavage induced by CPT treatment, indicating the BH4 domain was required to inhibit apoptosis. Further, PARP cleavage was markedly reduced only in the cells overexpressing bcl-2 protein mutated into amino acids 6-7, 10-11 and 16-17 within BH4 domain. Next, we evaluated whether mutated bcl-2 protein induced cell death and ROS production under hypoxia. As depicted in Figure 6 A,B the exposure of cells to hypoxia for 24 h did not induce apoptosis either in control or in cells overexpressing wild type or mutated bcl-2. Less than 10% of apoptotic cells were observed regardless bcl-2 expression. According with these results, hypoxia was not found to induce the generation of reactive oxygen species (ROS) (Supplementary Figure 1). Less than 5% of ROS was observed after 24 h exposure to hypoxia, with no differences between control and cells overexpressing wild type or mutated bcl-2.

Since all mutations into BH4 domain of bcl-2 are relevant for HIF-1/VEGF induction by wild type form of bcl-2 under hypoxia, regardless their relevance in the ability of bcl-2 protein to block apoptosis, our data demonstrate that the proangiogenic function of bcl-2 is not related to its antiapoptotic role of bcl-2.

TAT-BH4 peptide alone is sufficient to enhance HIF-1/VEGF axis

The results described above indicate that the BH4 domain has a role in the hypoxic induction of HIF-1/VEGF by bcl-2. To confirm this hypothesis we determined whether the exogenous application of TAT-BH4 peptide, a cell-penetrating peptide of human immunodeficiency virus TAT fused to a synthetic peptide corresponding to the BH4 domain, is able to modulate VEGF protein expression and its promoter activity, HIF-1α protein stabilization and transcriptional activity in M14 cells. Firstly, to confirm the anti-apoptotic activity of TAT-BH4 peptide 29;30 , we evaluated by Annexin V/Propidium Iodide staining the response of M14 cells to CPT. Figure 7A showed that the addition of TAT-BH4 significantly decreased the percentage of apoptosis induced by CPT: approximately 30% and 11% early apoptotic cells were observed after treatment with CPT in presence of control (TAT-CTRL) or TAT-BH4 peptides, respectively. Further, we also tested the effect on VEGF/HIF-1 signaling of a mutant version of TAT-BH4, where the TAT peptide is fused to a synthetic peptide corresponding to a mutated version of BH4 domain into amino acids 6-7, mutations that did not abolish TAT-BH4 anti-apoptotic effect (Figure 7A). The percentage of early apoptotic events in the cells treated with CPT and exposed to the mutant version of TAT-BH4 was quite similar to that observed after treatment with CPT and TAT-BH4 peptide, confirming that mutant version of TAT-BH4 still retains the antiapoptotic activity showed by TAT-BH4. As shown by ELISA assay (Figure 7B), no modulation of VEGF protein secretion was induced by TAT-BH4 peptide treatment under normoxia. On the contrary, under hypoxia the exogenous application of TAT-BH4 peptide at the doses of 2μM induced approximate 2-fold increase in the level of secreted VEGF protein when compared to M14 untreated or TAT-CTRL treated cells. Moreover, TAT-BH4 peptide treatment significantly enhanced VEGF promoter activity (Figure 7C) and HIF-1α transcriptional activity (Figure 7D) under hypoxic conditions in a dose-dependent manner in the M14 line when compared to the untreated or TAT-CTRL treated cells. VEGF protein secretion, VEGF promoter and HIF-1 transcriptional activity were not increased after the treatment of M14 cells with mutant version of TAT-BH4 compared to control cells. As shown in Figure 7E, an increase in HIF-1α protein levels was also observed after exposure to TAT-BH4 peptide under hypoxia when compared to TAT-CTRL treated cells, while the levels of HIF-1α protein in cells after treatment with mutant version of TAT-BH4 was quite similar to TAT-CTRL treated cells. Similar results in terms of VEGF and HIF-1α protein induction were also obtained using a BH4 antiapoptotic peptide [START_REF] Hotchkiss | TAT-BH4 and TAT-Bcl-xL peptides protect against sepsis-induced lymphocyte apoptosis in vivo[END_REF] from bcl-xL (data not shown). Finally exogenous application of TAT-BH4 peptide increases HIF-1α protein half-life (Figure 7F) and reduces HIF-1α protein ubiquitination under hypoxic conditions (Figure 7G). Thus, demonstrating that the addition of TAT-BH4 alone managed the hypoxia-induced enhancement of VEGF protein secretion and promoter activity, HIF-1α expression and HIF-1 transcriptional activity. Taken together, these findings indicate that the BH4 domain of bcl-2 is able to induce HIF-1/VEGF signaling, independently from its ability to prevent apoptotic cell death. Since we have obtained evidence that the localization of bcl-2 protein is important for its effect on HIF-1/VEGF signaling [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] , we evaluated the presence of TAT-BH4 peptide in the cellular compartments where generally bcl-2 protein localizes [START_REF] Hockenbery | Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death[END_REF] , such as the outer mitochondrial membrane, the endoplasmatic reticulum and the nuclear envelope, and the possible effect of hypoxia on TAT-BH4 localization.

To this aim we performed laser scanning confocal microscopy experiments after treating cells with TAT-BH4 peptide labeled with 5(6)-carboxyfluorescein, in normoxic or hypoxic conditions for 24 h. Firstly, we confirmed by flow cytometric analysis the efficient incorporation of TAT-BH4 peptide into the cells: a dose-dependent increase of fluorescence intensity was observed after treatment with increasing doses of TAT-BH4 peptide (Figure 8A). As shown in Figure 8B, we confirmed by confocal microscopy that the peptides were internalized in cells, forming several aggregates. Using different organelle-specific labelings, we found that TAT-BH4 peptide did not reside in the classical compartments in which full length bcl-2 is generally found, such as nucleus, mitochondria or endoplasmic reticulum in normoxic conditions, and hypoxia did not alter this localization of TAT-BH4.

The effect of BH4 domain on HIF-1/VEGF pathway activation is independent from the presence of endogenous bcl-2 protein

Some authors have reported that the deletion of BH4 domain converts bcl-2 into a dominant negative inhibitor of bcl-2 itself [START_REF] Hunter | Functional dissection of the human Bc12 protein: sequence requirements for inhibition of apoptosis[END_REF] , while other groups have suggested that bcl-2 deleted of its BH4 domain functions, like bax protein, to promote rather than inhibit cell death [START_REF] Huang | The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4[END_REF] . To test whether in our model the overexpression of bcl-2 deleted of BH4 domain protein could suppress the activity of endogenous protein, we assessed the BH4-dependent role of bcl-2 in the regulation of hypoxiainduced VEGF expression in the colon carcinoma cell line HT29, which is deficient for bcl-2 expression [START_REF] O'connell | Interferongamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis[END_REF] . In particular, bcl-2 wild type or deleted of its BH4 domain was stably expressed in HT29 cells that do not express detectable levels of endogenous bcl-2, as confirmed by Western blot and RT-PCR analyses (Supplementary Figure 2). As shown in Figure 9 A,B wild type bcl-2overexpressing HT29 cells (Bcl-2/1 and Bcl-2/2 clones) exposed to hypoxia for 24 h secrete higher level of VEGF protein than the parental cells. Both HT29 clones carrying bcl-2 deleted of the BH4 domain (BH4 del/1 and BH4 del/2) expressed VEGF protein at a level that is comparable to that one of control cells. Next, we evaluated whether exogenous application of TAT-BH4 peptide is sufficient to enhance HIF-1/VEGF axis also in a context of bcl-2 deficient background. An increase in both VEGF (Figure 9 C,D) and HIF-1α (Figure 9D) protein levels in HT29 cells was observed after hypoxic exposure to TAT-BH4 peptide when compared to untreated cells. Both VEGF promoter and HIF-1-dependent transcriptional activity were also increased after the treatment of HT29 cells with TAT-BH4 peptide, under hypoxic conditions (data not shown). Thus, demonstrating that the effect of BH4 domain is sufficient to activate HIF-1/VEGF pathway even in absence of endogenous bcl-2 protein, and that bcl-2 deleted of its BH4 domain does not elicit a dominant-negative activity.

DISCUSSION

Recent studies, including ours, have demonstrated that bcl-2 not only functions as an antiapoptotic and a prosurvival molecule 1 , but it also promotes tumor progression and angiogenesis in different tumor histotypes 9;11;33 . By using antisense oligonucleotide or RNA interference directed to bcl-2 mRNA, or overexpressing bcl-2 protein, we have previously demonstrated the ability of bcl-2 protein to regulate the expression of VEGF gene through HIF-1 activation in melanoma [START_REF] Iervolino | Bcl-2 overexpression in human melanoma cells increases angiogenesis through VEGF mRNA stabilization and HIF-1-mediated transcriptional activity[END_REF] and breast carcinoma [START_REF] Biroccio | Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line[END_REF] histotypes, independently of bcl-2 effect on cell survival [START_REF] Bufalo | Treatment of melanoma cells with a bcl-2/bcl-xL antisense oligonucleotide induces antiangiogenic activity[END_REF] . More recently, we have also reported that bcl-2 induced HIF-1α stabilization in response to hypoxia through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90 [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] . In the present study, we investigated the possible role of the BH1, BH2 and BH4 domains of bcl-2 protein in the regulation of HIF-1-mediated VEGF expression under hypoxia. We demonstrated that the deletion of the BH4 domain abrogated the ability of bcl-2 to cooperate with hypoxia to induce VEGF protein expression in tumor cells with different origin. The relevance of BH4 domain on the regulation of HIF-1/VEGF axis under hypoxia was showed in a human melanoma model. In particular, deletion of or point mutations into the BH4 region resulted in impaired ability of wild type bcl-2 protein to induce VEGF promoter activity, HIF-1α protein stabilization and ubiquitination under hypoxia.

Even though a recent prominent report suggested that there is a BH4 consensus sequence in some proapoptotic members, such as bax and bak 2 , the BH4 domain is mainly found in the bcl-2 family members with death-repressing activity. BH4 is one of the principal domains for the full multifunctional ability of bcl-2, and it appears to mediate the antiapoptotic activity of bcl-2 independently from the interaction of this protein with other bcl-2 family members. The BH4 domain is necessary and sufficient for bcl-2 to prevent apoptosis, bind to bax, translocate into the nucleus, reduce cell proliferation, induce NF-kB activity and regulate DNA repair 4;5;34 . In addition, some authors have suggested that bcl-2 deleted of its BH4 domain functions like bax to promote, rather than inhibit, cell death 6 , while other groups have reported that BH4 deletion converts bcl-2 into a dominant negative inhibitor of bcl-2 [START_REF] Hunter | Functional dissection of the human Bc12 protein: sequence requirements for inhibition of apoptosis[END_REF] . In our experimental model the bcl-2 protein deleted of BH4 domain did not function as a dominant negative inhibitor towards the bcl-2 ability to protect from apoptosis. In fact, cells overexpressing bcl-2 protein, whose BH4 domain was deleted or presented point mutations, undergo to apoptosis after treatment with CPT, a drug able to induce apoptotic cell death in parental but not in bcl-2 wild type overexpressing cells.

The molecular mechanism by which bcl-2 increases HIF-1-mediated VEGF expression in melanoma cells under hypoxia might be found in the ability of BH4 domain to directly or indirectly interact with HIF-1α protein, or to modulate the activity of some proteins found to interact with the BH4 region. It has been documented that the BH4 domain of bcl-2 interacts with a number of different factors, such as Ras [START_REF] Denis | Bcl-2, via its BH4 domain, blocks apoptotic signaling mediated by mitochondrial Ras[END_REF] , Raf-1 [START_REF] Wang | Bcl-2 targets the protein kinase Raf-1 to mitochondria[END_REF] , c-Myc 37 , NF-KB 4;5 , FK-506 binding protein 38 (FKBP38) [START_REF] Kang | The flexible loop of Bcl-2 is required for molecular interaction with immunosuppressant FK-506 binding protein 38 (FKBP38)[END_REF] , and inositol 1,4,5-triphosphate receptor [START_REF] Barth | The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4hydroxylase PHD2 protein stability[END_REF] . Moreover, some bcl-2 protein interactors have been demonstrated to be involved in oxygen-dependent and independent regulation of the HIF-1α protein. In particular, the bcl-2 mitochondrial chaperone, FKBP38, the orphan nuclear receptor, Nur77, and the molecular chaperone, HSP90, have been found to be implicated in HIF-1α turnover 22;39;40 . Very recently, we have demonstrated in a melanoma model that bcl-2 protein under hypoxic conditions interacts with both HIF-1α and HSP90 proteins contributing to the enhancement of HIF-1α protein stability [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] . Besides, we cannot exclude that also other factors can be involved in bcl-2-induced HIF-1α/VEGF regulation.

We have also evaluated the effect of exogenous application of the cell-permeable TAT-BH4 peptide in melanoma cells exposed to hypoxia. This peptide acts like full length wild type bcl-2 mimicking bcl-2 functions in terms of HIF-1/VEGF regulation. In fact, we showed that the TAT-BH4 peptide had a significant effect on VEGF protein induction and promoter activity, and regulated HIF-1α protein expression increasing the half-life and decreasing the ubiquitination of the protein. Our results clearly indicate that the BH4 domain is sufficient to induce HIF-1-mediated VEGF protein expression under hypoxia. TAT-control as well as TAT-BH4 mutant peptides showed no such effects, confirming the specific effect of TAT-BH4 peptide. We have also evidence that TAT-BH4 peptide localization was not responsible of the effects exerted by the TAT-BH4 peptide under hypoxic condition, in fact similar localization in the cytosol was found regardless oxygen tension.

Our results also demonstrated that the overexpression of bcl-2 protein carrying point-mutations at or deletion of the BH1 or BH2 domains while required for the antiapoptotic activity of bcl-2, they are not indispensable for bcl-2-dependent VEGF/ HIF-1 induction under hypoxia. In fact, bcl-2 protein deleted in BH1 or BH2 domains is still able to cooperate with hypoxia to activate HIF-1/VEGF signaling, as observed for wild type bcl-2, despite the lower deleted protein levels showed by the cells overexpressing BH1 and BH2 mutants in comparison to wild type bcl-2 overexpressing cells.

So this finding indicates that those low levels of BH1/BH2 deleted proteins are sufficient to sustain HIF-1/VEGF pathway activation in hypoxia. The fact that the level of BH4 deleted protein obtained after overexpression is comparable with the ones of cells expressing BH1/BH2 deleted bcl-2 protein, is an indirect demonstration that the loss of bcl-2 activity showed after BH4 domain deletion is not due to the lower level of expression of bcl-2 mutated in comparison to wild type bcl-2 protein expression. Besides, when we performed transient overexpression of bcl-2 constructs we generally reached higher level of proteins in cells overexpressing the wild type bcl-2 protein compared to deleted forms of protein. This observation, as also demonstrated by other authors [START_REF] Huang | The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4[END_REF] , may be due to an higher stability of full length wild type bcl-2 protein or to a different percentage of transfected cells.

The role of the BH3-driven protein-protein interaction on bcl-2-dependent regulation of HIF-1/VEGF was excluded by using HA14-1, a BH3 mimetic antagonist of bcl-2 (Supplementary Figure 3). In fact, exposure of bcl-2 overexpressing cells to HA14-1 did not affect the increased expression of VEGF observed after hypoxic exposure of bcl-2 transfectants, thus indicating that bcl-2-induced VEGF/HIF1 expression and activity is not related to the ability of bcl-2 to interact with the BH3 domain of proapoptotic proteins. By other hand, mutations of bcl-2 protein into BH1 (G145E), BH2 (W188A) and BH4 (G14-15) domains, disrupting the heterodimerization of bcl-2 with BH3-containing pro-apoptotic proteins, confirm these results. In fact, even though all these mutations damp bcl-2 antiapoptotic activity, only BH4 (G14-15) mutant affect bcl-2-induced VEGF/HIF1 expression and activity. Also the evidence that mutations on all the analyzed residues into BH4 abrogated bcl-2 ability to induce HIF-1/VEGF signaling under hypoxia, whereas they differently affect bcl-2 ability to prevent apoptosis, sustained our hypothesis that the antiapoptotic function of bcl-2 is not required for its effect on angiogenesis. These results were also corroborated by the evidence that TAT-BH4 mutated peptide, that still retains anti-apoptotic effect, it is not able to activate HIF-1/VEGF signaling. We further exclude the possibility that the effect of bcl-2 on the VEGF/HIF-1 pathway was attributed to the activity of bcl-2 as a prosurvival and antiapoptotic protein, since transient transfection of wild type or mutated bcl-2 proteins did not induce "per se" cell death in our experimental model, and less than 10% of apoptotic cells were observed in all the cell lines tested at the end of hypoxia exposure, regardless of bcl-2 status expression.

By using the HT29 colon carcinoma cell line which is deficient for bcl-2 expression [START_REF] O'connell | Interferongamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis[END_REF] , we have also showed that bcl-2 deleted of its BH4 domain is not a dominant-negative inhibitor of endogenous bcl-2 protein in terms of VEGF expression. In fact, wild type bcl-2-overexpressing HT29 cells exposed to hypoxia secrete higher level of VEGF protein than the parental cells and, similarly to control cells, HT29 clones carrying bcl-2 deleted of the BH4 domain showed a low level of VEGF protein. In the same context of bcl-2 deficient background the application of TAT-BH4 peptide is sufficient to enhance HIF-1/VEGF axis. Thus, demonstrating that the effect of BH4 domain is not mediated by the presence of endogenous bcl-2 protein.

In summary, our results demonstrated that the ability of wild type bcl-2 to enhance angiogenesis through the BH4 domain is not related its ability to prevent apoptosis, and indicate that the development of inhibitors based on the BH4 domain of bcl-2 can be useful to better define the ml). Following centrifugation, the supernatant was precleared with protein A/G agarose beads coupled with rabbit IgG for >2 h and then 1 µg of the HIF-1α antibody (clone H-206, Santa Cruz Biotechnology, Santa Cruz, CA) was added, and incubated overnight at 4°C with protein A/Gagarose beads (Amersham Biosciences Europe, Milan, Italy). Immunoprecipitates were washed four times in the lysis buffer before Western blot analysis. For some immunoprecipitation experiments we used ExactaCruz™ reagents (Santa Cruz Biotecnology) to detect the bcl-2 protein without detection of the light chain of the immunoprecipitation antibody. Immunoprecipitation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (100, Santa Cruz Biotecnology) and HIF-1α (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein.

Antibodies against HIF-1α (clone 54, BD Pharmingen, San Jose, CA), HIF-1β (clone 29, BD Pharmingen), PARP (clone 4C10-5, BD Pharmingen), bcl-2 (clones N-19 and C-2, Santa Cruz Biotechnology; clone 124, Dako, Milan, Italia), ubiquitin (clone P4G7, Santa Cruz Biotechnology), β-actin (clone AC-15, Sigma) and HSP72/73 (clone W27, Calbiochem, EMD Biosciences, La Jolla, CA) were used.

Reverse Transcriptase-Polymerase Chain reaction (RT-PCR).

Total cellular RNA was isolated using Trizol reagent (Invitrogen). cDNA equivalent to 1µg of total RNA was generated by reverse transcription reaction using (oligo dT) 18 primer (Fermentas, Burlington, Ontario, Canada) according to the manufacturer's instruction and then 2µl of obtained cDNA were added to PCR reaction mix (Fermentas) containing exon-specific primers targeting HIF-1α (sense CCCAGATTCAGGATCAGACA, antisense CCATCATGTTCCATTTTTCGC) and β-actin (sense, AAGAGAGGCATCCTCACCCT, antisense ATCTCTTGCTCGAAGTCCAG) for 25 cycles with an annealing temperature of 56°C. PCR products were separated by 1.5% agarose gel electrophoresis and gel was scanned with Gel Doc XR (Biorad). The intensity of the bands was quantified by densitometry using Molecular Analyst Software (Biorad) and the optical density of each HIF-1α amplified band was normalized to the intensity of each β-actin band.

Confocal analysis

Cells were treated with 0.5µM TAT-BH4 peptide modificated at N-terminal with 5(6)carboxyfluorescein and then exposed to normoxia or hypoxia. After 24h, cells were fixed in 3.7% formaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature and then incubated with anti-calreticulin (Affinity Bioreagents, Golden CO) or anti-HSP60 (BD Transduction Laboratories™, Franklin Lakes, NJ) to localize endoplasmic reticulum and mitochondria, respectively. The cells were incubated with TRITC conjugated Goat anti-Rabbit or anti-Mouse (Jackson Lab, West Grove, PA). Nuclei were visualized using TO-PRO3® (Invitrogen). The images were scanned under a ×40 oil immersion objective and to avoid bleed-through effects, each dye was scanned independently by a Leica confocal microscope (laserscanning TCS SP2) equipped with Ar/ArKr and HeNe lasers. The images were acquired and electronically merged utilizing the Leica confocal software (Leica Microsystems Heidelberg GmbH, Mannheim, Germany). Figures were processed using Adobe PhotoShop software.

Statistical Analysis. Differences between groups were analyzed with a two-sided paired or unpaired t test by use of GraphPad Prism 3.00 (GraphPadSoftware, San Diego, CA). Results were considered to be statistically significant if p<0.05. Experiments were replicated three times unless otherwise indicated. Whole cell lysates were immunoprecipitated (IP) with anti-HIF-1α antibody and then Western blot analysis was performed using anti-Ubiquitin antibody.

TITLES AND LEGENDS TO FIGURES

Figure 5 C

 5 Figure 5 C,D a time-dependent decrease of HIF-1α protein level was observed in the cells after

Figure 1 .

 1 Figure 1. The BH4 domain, but not BH1 or BH2, is necessary for VEGF induction by wild

Figure 2 .

 2 Figure 2. The BH4 domain, but not the BH1 or BH2, is necessary for VEGF induction by wild

Figure 3 .

 3 Figure 3. Forced expression of wild type bcl-2, but not bcl-2 deleted of BH4 domain, increases

Figure 4 .

 4 Figure 4. The BH4 domain is necessary for HIF-1 protein expression and activity induced by

Figure 5 .

 5 Figure 5. The BH4 domain is necessary for HIF-1α protein stabilization by wild type bcl-2.

Figure 6 .

 6 Figure 6. The effect of bcl-2 mutations on HIF-1/VEGF signaling is independent of their

Figure 7 .

 7 Figure 7. VEGF protein expression is induced under hypoxia by exogenous application of the

Figure 8 .

 8 Figure 8. Cellular localization of TAT-BH4 peptide in M14 cells exposed to normoxia and

Figure 9 .

 9 Figure 9. bcl-2 deleted of its BH4 domain is not a dominant-negative inhibitor of the

ACKNOWLEDGMENTS

We are grateful, to David Huang and Tristram G Parslow for providing all the bcl-2 plasmids, to Adele Petricca for secretarial assistance and to Paula Frank for English revision of the manuscript.

We thank Stefania De Grossi and Carla Ramina for technical assistance in confocal microscopy analysis.

bcl-2, This work was supported by grants from Italian Association for Cancer Research (D.D.B.) and Italian Ministry of Health (D.D.B.). G.C. is a PhD student of Sapienza University. Y.R. is recipient of a fellowship from the Italian Foundation for Cancer Research.

functions of BH4 domain and for the treatment of tumors associated with increased expression of VEGF or HIF-1α.

MATERIALS AND METHODS

Cell cultures, reagents and transfections. Human M14, PLF2 and JR1 (melanoma), HT29 (colon carcinoma), CaOV3 (ovarian carcinoma), and H1299 (lung carcinoma) were cultured in 10% FBS RPMI medium (Invitrogen, Carlsbad, CA). The cells were stably or transiently transfected with the expression vectors encoding the human wild type bcl-2 or different bcl-2 mutants (Figure 1A).

Transfections of expression vectors and hypoxia treatment were performed as previously reported [START_REF] Biroccio | Bcl-2 overexpression and hypoxia synergistically act to modulate vascular endothelial growth factor expression and in vivo angiogenesis in a breast carcinoma line[END_REF] .

Two wild type bcl-2 overexpressing (Bcl-2/clone 5 and Bcl-2/clone 37), two bcl-2 mutants at the were obtained from PRIMM (Milan, Italy) ). For some experiments, TAT-BH4 peptide was labeled at amino-terminal with 5(6)-carboxyfluorescein. Camptothecin (CPT), Cyclohexamide (CHX), Zleu-leu-leu-CHO (MG132) were purchased from Sigma-Aldrich.

ELISA, Western blot and Flow cytometric analyses and Promoter activity. ELISA, Flow

cytometric analysis and evaluation of promoter activity were done as previously reported 9;24 .

Nuclear and cytoplasmic fractions were prepared as previously reported [START_REF] Trisciuoglio | Bcl-2 regulates HIF-1alpha protein stabilization in hypoxic melanoma cells via the molecular chaperone HSP90[END_REF] . For immunoprecipitation assays and Western blot analysis, the cells were lysed in an 0.3% CHAPS buffer (40 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na 3 VO 4 , 0.3% CHAPS, and one tablet EDTA-free protease inhibitors [Roche] per 50