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. 2013 Mar;15(3):315–327. doi: 10.1593/neo.121392

Removal of the BH4 Domain from Bcl-2 Protein Triggers an Autophagic Process that Impairs Tumor Growth1,2

Daniela Trisciuoglio *, Teresa De Luca *, Marianna Desideri *, Daniela Passeri †, Chiara Gabellini *, Stefania Scarpino ‡, Chengyu Liang §, Augusto Orlandi †, Donatella Del Bufalo *
PMCID: PMC3593154  PMID: 23479509

Abstract

Here, we show that forced expression of a B-cell lymphoma 2 (bcl-2) protein lacking residues 1 to 36 at the N-terminal, including the entire Bcl-2 homology 4 (BH4) domain, determines reduction of in vitro and in vivo human melanoma growth. Noteworthy, melanoma cells in vivo exhibit markedly increased autophagy, as response to expression of bcl-2 protein deleted of its BH4 domain. This observation led to the identification of a novel gain of function for bcl-2 protein lacking the BH4 domain. In particular, upon different autophagic stimuli in vitro, overexpression of bcl-2 protein deleted of BH4 domain induces autophagosome accumulation, conversion of microtubule-associated protein 1 light chain 3B-II, reduced expression of p62/SQSTM1 protein, and thereby enhanced autophagic flux. The relevance of Beclin-1 is evidenced by the fact that 1) the autophagy-promoting and growth-inhibiting properties are partially rescued by Beclin-1 knockdown in cells expressing bcl-2 protein lacking the BH4 domain, 2) Beclin-1 only interacts with wild-type but not with deleted bcl-2, and 3) BH4 domain removal from bcl-2 protein does not influence in vitro and in vivo growth of tumor cells expressing low levels of endogenous Beclin-1. These results provide new insight into molecular mechanism of bcl-2 functions and represent a rationale for the development of agents interfering with the BH4 domain of bcl-2 protein.

Introduction

Even though the B-cell lymphoma 2 (bcl-2) family proteins are critical regulator of apoptosis, they have also multiple apoptosis-independent functions that are involved in several phenomena including cell proliferation [1–3], tumor metastatization [4,5], angiogenesis [6–9], and autophagy [10].

Autophagy can be induced by different conditions, such as nutrient deprivation, chemotherapy, and hypoxia. It is controlled by conjugation of the ubiquitin-like protein microtubule-associated protein 1 light chain 3 (LC3) with phosphatidylethanolamine through an enzymatic cascade catalyzed by autophagy-related proteins. Phosphatidylethanolamine-conjugated LC3, known as LC3II, serves as a recognition site for LC3-binding chaperones, such as p62/SQSTM1, that deliver their cargo to autophagosomes [11,12].

Alterations in the pathways regulating autophagy may result in cancer development and progression [13]. A growing body of evidence indicates that antiapoptotic bcl-2 family members (bcl-2, bcl-xL, and mcl-1) inhibit induction of autophagy triggered by several stimuli through binding and blocking Beclin-1, a Bcl-2 homology 3 (BH3)-only protein with autophagy-promoting function [10]. However, bcl-2 expression silencing has been shown to induce autophagy [14], while bcl-2 inhibitors are able to trigger both Beclin-1.dependent and Beclin-1-independent autophagy [15].

Bcl-2 family members are characterized by the presence of four domains, BH1, BH2, BH3, and BH4: while some proteins possess up to four BH domains, other have either three (BH1, BH2, and BH3) or only one domain (BH3). The ability of many bcl-2 family members to form homodimers as well as heterodimers could be important for both activation and neutralization of specific functions. Further, bcl-2 protein has been identified as a caspase substrate, and its cleavage at the N-terminal appears to inactivate bcl-2 function in the apoptotic pathway [16]. Recently, we also demonstrated the mechanism by which bcl-2 through its BH4 domain regulates HIF-1-dependent angiogenesis that is independent of antiapoptotic and prosurvival function of bcl-2 [17].

By using in vitro human melanoma, lung, and colon carcinoma cell cultures and in vivo mouse xenograft models expressing bcl-2 protein wild type (wt) or deleted of BH4 domain, in this study we evaluated the role of BH4 deletion on in vitro and in vivo autophagy and tumor growth.

Materials and Methods

Cell Cultures

Human M14, A375SM-SC1, and JR8 melanoma, HT29 colon, and H1299 lung carcinoma cell lines were transfected and cultured as previously reported [17,18]. Control, bcl-2 wt, and bcl-2 deleted of its BH4 domain (bcl-2/BH4del) overexpressing clones derived from the M14, A375SM-SC1, H1299, JR8, and HT29 cells after stable transfection were cultured in the presence of puromycin (puro, 1 µg/ml; Sigma-Aldrich, St Louis, MO). For enhanced green fluorescent protein (EGFP). LC3B [19], mRFP-EGFP-LC3B (ptfLC3, Addgene, plasmid 21074), luciferase (pGL4.50-Luc2; Promega, Madison, WI), and shBeclin-1 subclones, a polyclonal population of stably transfected cells were cultured in the presence of geneticin (800 µg/ml; Sigma-Aldrich).

Treatments

Autophagy was induced by serum starvation, exposure to hypoxia (1% oxygen), or rapamycin (1 µM; Sigma-Aldrich). For some experiments, cells were treated with bafilomycin A1 (2.5 nM; Santa Cruz Biotechnology, Santa Cruz, CA), chloroquine (25 µM), hydrogen peroxide (H2O2, 50 µM, 24 hours), acridine orange (Sigma-Aldrich), and cisplatin (CDDP, 20 µM, 48 hours; Pfizer, New York, NY). Experiments by using z-VAD-fmk (50 µM; BD Pharmingen International, San Diego, CA) were performed, adding the inhibitor to the cells 1 hour before treatment and maintained in the medium thereafter. Cell proliferation, cell colony-forming ability, apoptosis, and progression of cells through cell cycle phases were analyzed as previously described [20,21]. Oligodeoxynucleotide treatment was performed as previously reported [21].

Analysis of Autophagy

Evaluation of autophagosomal structures was obtained by fluorescence microscopy observing LC3B puncta in EGFP-LC3B or mRFPEGFP-LC3B expressing cells or through LC3B antibody (Cell Signaling Technology, Danvers, MA) staining. For some experiments, a primary antibody against p62/SQSTM1 (Santa Cruz Biotechnology) was also used. Preparation of microscopy slides was performed as previously described [22]. The images were scanned under a x63 oil immersion objective, and to avoid bleed-through effects, each dye was scanned independently by using a Leica DMIRE2 microscope equipped with a Leica DFC 350FX camera, elaborated by a Leica FW4000 deconvolution software (Leica, Solms, Germany) and processed using Adobe PhotoShop software. Acridine orange (1 µg/ml, 15 minutes) staining was used to detect acidic vesicular organelles (AVOs) in live cells. Orange-positive cells were quantified by flow cytometric analysis. Cells with more than 10 puncta were considered autophagy positive.

Immunoprecipitation, Western Blot, and Immunohistochemical Analyses

Immunoprecipitation, Western blot, and immunohistochemical analyses were performed as previously reported [22,23]. For Western blot analysis, 40 µg of total proteins was loaded unless otherwise indicated. Antibodies against bcl-2, p62/SQSTM1, HA epitope (Santa Cruz Biotechnology), Beclin-1 (Cell Signaling Technology), poly (ADP-ribose) polymerase (PARP; BD Pharmingen International), β-actin, LC3B, bromodeoxyuridine (BrdU; Sigma-Aldrich), Ki67 (Novus Biologicals, Littleton, CO), and HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA) were used.

For morphologic and immunohistochemical analyses, tumors obtained 40 days after intramuscular (i.m.) injection were placed in 10% buffered formalin or zinc fixative for 24 hours, dehydrated, and embedded in paraffin. Four-micrometer-thick serial sections were stained with hematoxylin and eosin for the evaluation of cellularity and mitotic index or used for immunohistochemistry. Semiquantitative evaluation of immunoreactions was performed as reported [24].

In Vivo Tumorigenicity

In vivo experiments were performed as previously reported [24], injecting i.m. 5 x 106 cells (analysis of tumor growth) or from 5 x 105 to 5 x 106 cells for tumor take. For some experiments, BrdU (100 mg/kg) was administrated intraperitoneally 2 hours before animal sacrifice. Tumor sections were stained with anti-BrdU monoclonal antibody. For in vivo bioluminescence experiments, animals were subcutaneously injected with 2 x 106 cells and luciferase activity was quantified by IVIS Imaging System 200 (Caliper Life Sciences, Hopkinton, MA). Mice were anesthetized with a combination (i.m., 2 mg/kg) of tiletamine-zolazepam (Telazol, Virbac, Carros, France) and xylazine (Xilazyne/Rompun, Bayer, Leverkusen, Germany) and then injected intraperitoneally with 150 mg/kg D-luciferin (Caliper Life Sciences) and imaged 10 to 15 minutes after injection. Data were acquired and analyzed using Living Image software version 3.0 (Caliper Life Sciences).

Statistical Analysis

Differences between groups were analyzed with a two-sided paired or unpaired t test by use of GraphPad Prism 3.00 (GraphPad Software, San Diego, CA). Results were considered to be statistically significant if P < .05 (*, #). Experiments were replicated three times unless otherwise indicated.

Results

Deletion of BH4 Domain Affects In Vitro Autophagy of M14 Melanoma Cells

Bcl-2 protein has been found to inhibit autophagy [10]. We first investigated whether knockdown of bcl-2 expression leads to autophagy induction in M14 melanoma cells. To this purpose, M14 cells stably expressing EGFP-LC3B fusion protein were treated with oligonucleotide antisense (AS bcl-2) or with a control oligonucleotide sequence for 6 hours and the presence of punctate structures was evaluated after 48 hours. Formation of punctate structures with EGFP-LC3B fusion protein is a well-characterized marker to visualize autophagosomes and represents the accumulation of a membrane-bound form of LC3B-II on autophagic vesicles [25]. As reported in Figure W1, treatment with AS bcl-2 resulted in an increase in the characteristic redistribution of EGFP-LC3B, from a diffused staining to punctate vesicular structures, concomitantly to a decreased expression of bcl-2 protein. These results suggest that silencing of bcl-2 induces autophagy in M14 cells and further support the observation that bcl-2 confers protection to cells against autophagy [14,26].

Next, we studied the involvement of BH4 domain of bcl-2 protein in autophagy. To this purpose, M14 melanoma clones stably overexpressing bcl-2 protein wt or deleted of residues 1 to 36 at the N-terminal (bcl-2/BH4del; Figure 1A), previously characterized for their response to apoptotic and angiogenic stimuli [17], were used. We evaluated the response of M14 and its derivatives to canonical inducers of autophagic process, such as serum starvation, rapamycin, and hypoxia (Figure 1, B–D). Under basal conditions, no significant differences were observed in the different clones in terms of AVO formation, a hallmark of autophagic cells (Figure 1B). After a 48-hour serum starvation, about 15% of control cells showed prominent red fluorescence, while a lower percentage of AVO-positive cells (about 8%) was observed after bcl-2 wt overexpression. However, approximately 35% of AVO-positive cells were evidenced when overexpressing bcl-2/BH4del protein. Time-dependent increase of AVO formation was also observed after exposure to 8- to 24-hour serum starvation (data not shown). Increased autophagy was also evidenced in bcl-2/BH4del expressing cells compared to control clones in response to rapamycin and hypoxia, while bcl-2 wt overexpression reduces the number of AVO-positive cells induced by both stimuli.

Figure 1.

Figure 1

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Deletion of BH4 domain from bcl-2 protein in M14 and H1299 cells increases autophagosome formation in response to autophagic stimuli. (A) Western blot analysis of bcl-2 protein expression in M14 control clone (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del). β-Actin is shown as loading and transferring control. Two bands, indicative of the higher endogenous full-length and shorter exogenous bcl-2 protein, were observed in cells overexpressing bcl-2 deleted of BH4 region, while a single high band was observed in control cells (puro, transfected with empty vector) and in cells overexpressing bcl-2 wt protein. Representative Western blot of two independent ones with similar results is shown. (B) AVOs in M14 control clone and its derivatives grown in complete media, under serum starvation or exposed to rapamycin or hypoxia for 48 hours. (C) Representative images of fluorescence microscopy and (D) quantification of cells positive for autophagosomal structures in M14 and H1299 control and derivative clones stably transfected with EGFP-LC3B vector grown as reported in B. (B, D) The results represent the average ± SEM of three independent experiments. *P < .05, comparison between control cells and bcl-2 wt or mutant clones; #P < .05, comparison between wt and mutant bcl-2.

As shown in Figure 1, C and D, under growth in complete media M14 control cells expressing EGFP-LC3B showed diffuse cytoplasmic distribution of green fluorescence similar to that of bcl-2 wt clone, whereas an increase in the characteristic redistribution of EGFP-LC3B, from a diffused staining to punctate vesicular structures, was observed in the clone expressing bcl-2/BH4del.

Likewise, upon starvation or exposure to rapamycin or hypoxia, a significant increase in punctate vesicular structures was observed in clone expressing bcl-2/BH4del when compared to control and bcl-2 wt overexpressing transfectants. Similar results were also observed in EGFP-LC3B expressing H1299 lung carcinoma cells transfected with expression vectors encoding bcl-2 wt or bcl-2/BH4del (Figure 1, C and D), thus indicating that BH4 domain deletion from bcl-2 protein can induce autophagosome accumulation in different tumor histotypes.

Concurrent with autophagosome maturation, cargo incorporation occurs, and it can be monitored by analyzing p62/SQSTM1 and LC3B protein colocalization. As reported in Figure 2A under growth in complete media, p62/SQSTM1 and EGFP-LC3B proteins are predominantly diffuse in M14 control and bcl-2 wt overexpressing transfectants, whereas punctate structures colocalized with p62/SQSTM1 bodies in cells expressing bcl-2/BH4del, indicating incorporation of specific p62/SQSTM1-labeled cargo into autophagosomes. Likewise, after starvation, these structures are more evident in cells expressing bcl-2/BH4del than in control and bcl-2 wt transfectants.

Figure 2.

Figure 2

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Deletion of BH4 domain from bcl-2 protein enhances autophagic flux. (A) Immunofluorescence staining of p62/SQSTM1 protein in M14 control clone (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del) stably transfected with EGFP-LC3B vector grown in complete media or under serum starvation (48 hours). Representative images are shown. Western blot analysis of LC3B-I/II (B) and p62/SQSTM1 (C) protein expression in M14/EGFP-LC3B derivatives grown in complete media or under serum starvation for 48 hours, in the presence or absence of chloroquine (25 µM). Western blots, representative of three independent experiments with similar results, are shown. β-Actin is shown as loading and transferring control. LC3B-II levels are quantified by densitometric analyses and the fold of increase relative to control is presented. (D) Representative images of fluorescence microscopy of H1299 control (puro) and derivative clones (Bcl-2wt and BH4del) stably transfected with mRFP-GFP-LC3B constructs grown under serum starvation (24 hours).

Next, to monitor the activation of autophagic flux, we measured the conversion of LC3B-I to LC3B-II and the level of p62/SQSTM1 protein by Western blot analysis. As shown in Figure 2B, the level of LC3B-II was increased upon starvation in both control (about two-fold) and bcl-2/BH4del expressing cells (about three-fold), being the induction more evident in the latter ones. On the contrary, the level of LC3B-II slightly changed in cells stably overexpressing bcl-2 wt exposed to serum starvation (about 1.4-fold). At the same time, a strong decrease of p62/SQSTM1 protein expression was observed in control cells and in cells expressing bcl-2/BH4del in response to starvation but not in cells stably overexpressing bcl-2 wt (Figure 2C).

To determine whether the increase of LC3B-II conversion observed in cells expressing bcl-2/BH4del was due to autophagy induction or to inhibition of autophagic process completion, we used chloroquine, which blocks late stage of autophagy. As reported in Figure 2B, chloroquine causes an increase in LC3B-II conversion in cells incubated in complete growth media regardless bcl-2 protein status, reflecting the basal level of autophagy of these cells. This effect is more evident when cells were exposed to serum starvation, confirming that starvation can induce a complete autophagic flux in these cells. Bafilomycin A1, another inhibitor of late-stage autophagy, produced superimposable data (data not shown). Similar results were also obtained by analyzing the percentage of cells with punctate vesicular structures in EGFP-LC3B expressing H1299 cells and its derivatives. The combination of chloroquine with serum starvation in cells expressing bcl-2/BH4del resulted in a much stronger accumulation of EGFP-LC3 puncta than chloroquine alone, compared to control and bcl-2 wt overexpressing cells (data not shown).

To confirm the induction of a complete autophagic flux, we used a pH-sensitive, double-tagged mRFP-GFP-LC3B reporter to determine the autophagosome maturation and autolysosome formation in H1299. As GFP but not mRFP fluorescence is lost in acidic compartments, mRFP-GFP-LC3B labels nonacidic autophagosomes as yellow fluorescence (positive for both green and red) but acidic autophagolysosomes as red fluorescence only [27]. The increase of red fluorescence observed under serum starvation indicates that autolysosome maturation proceeds normally; moreover, such red fluorescence was higher in control cells and in cells that express bcl-2/BH4del protein than in bcl-2 wt transfectants, confirming that autophagic flux is inhibited in bcl-2 wt overexpressing cells (Figure 2D). All together these data indicate that deletion of bcl-2 protein induces a significant level of autophagy rather than blocking the basal level of autophagosome degradation in M14 cells.

Because bcl-2 protein devoid of its BH4 domain can be generated by different agents [16,28], several conditions have been used to generate cleavage of endogenous bcl-2 protein in M14 cells. As reported by Western blot analysis in Figure W2A, in addition to the expected 26-kDa band corresponding to the bcl-2 protein, a bcl-2 cleavage product of about 23 kDa was evident in floating cells after exposure to H2O2 or CDDP or when cells were left to die 6 days after seeding in high cell density condition. Under these conditions, bcl-2 cleavage product was absent or barely detectable in adherent cells in both untreated cells and cells exposed to stress condition. As expected, bcl-2 cleavage occurred concomitantly with caspase activation as monitored by PARP cleavage. Notably, a concomitant increased expression of LC3B-II and decreased expression of p62/SQSTM1 proteins reflecting an autophagy induction was observed (Figure W2A). Importantly, PARP cleavage, increase of LC3B-II, and decreased expression of p62/SQSTM1 protein were completely inhibited by pretreatment of M14 cells with a pan-caspase inhibitor z-VAD-fmk before CDDP treatment (Figure W2B). Notably, in the presence of CDDP and pan-caspase inhibitor, the number of floating cells was irrelevant and no generation of the bcl-2 fragment was observed in the cell extracts from pooled adherent and floating cells (data not shown). These data demonstrated that induction of apoptosis in M14 cells is paralleled by caspase-dependent cleavage of bcl-2 and induction of autophagy.

Deletion of BH4 Domain Affects In Vitro Cell Proliferation and In Vivo Tumorigenicity

We next evaluated the effect of bcl-2/BH4del expression on in vitro and in vivo M14 cell growth. As reported in Figure 3A, starting from day 4 of growth two clones expressing bcl-2/BH4del showed a significant reduction in cell proliferation, when compared to both control and bcl-2 wt overexpressing clones (P < .05). We next performed a BrdU pulse/chase experiment to follow the kinetic of cell cycle progression (Figure W3A). The exit of cells out from S phase is quite similar for all the clones analyzed. However, the reappearance of BrdU-positive cells into the S phase began at about 12 hours for control and cells expressing bcl-2 wt, while cells expressing bcl-2/BH4del reentered into the S phase about 18 hours after BrdU pulse, suggesting a cell cycle delay in latter cells. No significant apoptosis was observed up to day 8 of cell growth regardless bcl-2 status (Figure W3B).

Figure 3.

Figure 3

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Deletion of BH4 domain from bcl-2 protein in M14 cells reduces tumorigenicity. In vitro (A) cell proliferation and (B) clonogenic ability and (C) in vivo tumor growth of M14 control (puro) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del). (A) The results represent the average ± SD of three independent experiments. (B) Three representative dishes of six are shown. (C) The results represent the average ± SD of one representative of three experiments with similar results. (D) Bioluminescence imaging of tumor-bearing mice 6 days after injection of luciferase expressing M14 control (puro) and its derivative clones. *P < .05, relative to control cells.

We also assessed the ability of M14 cells and its derivatives to form colonies (Figure 3B). While an increased number of colonies was observed after bcl-2 overexpression, a dramatic reduction in colony-forming potential was evidenced in cells expressing bcl-2/BH4del as compared with control transfectants.

To evaluate the effect of ectopic expression of bcl-2/BH4del in in vivo M14 tumorigenicity, parental cells and their derivatives were injected into nude mice. After i.m. injection of 5 x 106 M14 cells, all animals developed tumors and the analysis of the median time of tumor appearance revealed that mice injected with control and bcl-2 wt overexpressing cells began to develop tumors about 10 days after cell implant, similarly to mice injected with cells expressing bcl-2/BH4del. As Figure 3C illustrates, only about 37 days after cell implant, tumors derived from cells overexpressing bcl-2 wt had a significant growth advantage when compared with tumors derived from control cells (P < .05), whereas tumors derived from cells expressing bcl-2/BH4del exhibited tumor growth rates lower than tumors derived from both control cells and cells expressing bcl-2 wt, already evident at early stage of growth (P < .05, starting from days 26 and 29 for BH4del6 and BH4del5 clones, respectively).

The tumor-suppressor activity of bcl-2/BH4del protein is further illustrated by tumor incidence when mice were injected with a lower number of cells. In particular, 8 weeks after injection of 5 x 105 cells, only about 20% of mice injected with bcl-2/BH4del expressing cells developed tumors, whereas the percentage reached 100% in the mice injected with control or bcl-2 wt overexpressing cells. Moreover, the analysis of the median time of tumor appearance revealed that mice injected with 5 x 105 bcl-2/BH4del expressing cells began to develop tumors about 63 days after injection, whereas the same number of control and bcl-2 wt overexpressing cells resulted in tumors after about 48 days.

When mice were subcutaneously injected with 2 x 106 M14 cells stably expressing both luciferase and wt or deleted bcl-2 protein, imaging analysis evidenced that the luminescent signal was stronger in control and bcl-2 wt xenografts than in the tumors derived from cells expressing bcl-2/BH4del (Figure 3D). Removal of BH4 domain from bcl-2 protein in A375SM-SC1, another human melanoma cell line, also reduced in vitro and in vivo tumorigenicity (Figure 4, A–E).

Figure 4.

Figure 4

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Deletion of BH4 domain from bcl-2 protein in A375SM-SC1 cells reduces tumorigenicity. (A) Western blot analysis of bcl-2 protein expression in human A375SM-SC1 melanoma cells stably transfected with empty vector (puro) or with vectors encoding bcl-2 wt protein (Bcl-2wt) or bcl-2 protein deleted of BH4 domain (BH4del). (B) In vitro cell proliferation, (C) clonogenic ability, (D) in vivo tumor growth, and (E) tumor incidence of A375SM-SC1 cells and its derivatives. (B) The results represent the average ± SD of three independent experiments. (C) Three representative dishes of six are shown. (D, E) Representative experiments of two independent ones with similar results are shown. *P < .05, relative to control cells.

M14 Xenografts Carrying Bcl-2/BH4del Protein Show Decreased Proliferation and Increased Autophagy

To determine whether reduced in vivo tumor growth was paralleled by reduced expression of the proliferation marker Ki67, we performed immunohistochemical analyses of tumor sections derived from M14 cells and its derivatives. As reported in Figure 5, A and B, while tumors overexpressing bcl-2 wt protein showed a significantly higher number of Ki67-positive cells than those found after implantation of control cells, tumors carrying bcl-2/BH4del protein showed a significantly reduced number of cells expressing Ki67 when compared to both control and bcl-2 wt overexpressing xenografts. A decreased number of mitotic figures (Figure 5C) and a reduced in vivo BrdU (Figure 5D) incorporation were also found in tumors carrying bcl-2/BH4del protein when compared to both control and bcl-2 wt overexpressing xenografts.

Figure 5.

Figure 5

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M14 xenografts carrying bcl-2 protein lacking the BH4 domain show decreased proliferation and increased autophagy. (A) Representative images and (B) quantification of Ki67 immunostaining in tumor xenografts from M14 control (puro) and its derivatives overexpressing bcl-2 wt (Bcl-wt) or deleted of BH4 domain (BH4del). Original magnification, x200. Histochemical analysis of (C) mitosis (hematoxylin and eosin) and (D) BrdU staining in xenografts from M14 control (puro) and its derivatives. (B–D) The results represent the average ± SD of three independent experiments. (E) Western blot analyses of bcl-2 and LC3B-I/II protein expression in tumor xenografts obtained 40 days after i.m. implantation of M14 parental (puro) and its derivatives. β-Actin is shown as loading and transferring control. A representative Western blot of two independent ones with similar results is shown. (F) Quantification of LC3B-II/LC3B-I protein ratio by densitometric evaluation of Western blot analysis presented in E. *P < .05, relative to control cells.

We next examined the conversion of LC3B-I to LC3B-II protein in M14 tumor xenografts (Figure 5, E and F) by Western blot analysis. Notably, increased LC3B-II/LC3B-I ratio was observed in tumors derived from cells expressing bcl-2/BH4del when compared to both control and bcl-2 wt overexpressing xenografts. Moreover, all bcl-2-overexpressing tumors maintained a high level of wt or deleted bcl-2 protein, confirming the stability of ectopically expressed bcl-2 in vivo (Figure 5E).

Deletion of α1 Helix of Bcl-2 Does Not Affect In Vivo Tumor Growth of M14 Cells

BH4 domain of bcl-2 protein is characterized by the presence of a secondary α helix structure (α1 helix). To ascertain whether the deletion of the entire BH4 domain (residues 1–36) or of just the α1 helix (residues 1–22) of bcl-2 was necessary to delay tumor growth, we assessed the effect of the overexpression of a bcl-2 mutant deleted of residues 1 to 22 including the α1 helix of BH4 domain (bcl-2 Δα1) on in vivo tumor growth (Figure 6, B–D). This deletion mutant has been demonstrated to be defective in apoptosis inhibition but competent for autophagy suppression, and it has been described to promote cell growth as efficiently as bcl-2 wt in MCF7 breast cancer [29]. Mixed population of M14 human melanoma cells stably transfected with expression vectors encoding human wt bcl-2, bcl-2 Δα1, or bcl-2/BH4del were obtained. Upon starvation, an increase of AVO-positive cells was observed only in control (about 17%) and in bcl-2/BH4del mutant (about 49%) compared to the cells expressing bcl-2 wt protein, while as expected and previously reported [29], bcl-2 Δα1 expressing cells inhibited starvation-induced AVOs as effectively as the cells expressing bcl-2 wt protein (about 4%; Figure 6A), thus confirming that bcl-2 Δα1 protein retains the antiautophagic properties of wt bcl-2.

Figure 6.

Figure 6

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Deletion of α1 helix of bcl-2 does not affect in vivo tumor growth of M14 cells. (A) AVOs in cells grown in completemedia or under serum starvation (hours) and (B) in vivo tumor growth of M14 control (empty) and its derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of amino acid residues 1 to 36 (BH4del) or 1 to 22 (α1del). (A) The percentage of cells with prominent red fluorescence is shown. (A, B) Representative experiments of two independent ones with similar results are shown. (C) Immunohistochemical analysis and (D) semiquantitative evaluation of p62/SQSTM1 protein in M14 derivative xenografts 40 days after tumor implant. Original magnification, x400. Results are reported as means ± SEM. Representative images are shown. *P < .05, relative to control cells.

As shown in Figure 6B, we found that after i.m. injection of 5 x 106 cells, tumors derived from control cells and cells expressing bcl-2 wt and bcl-2 Δα1 protein cells do not show significant differences in growth rate until day 30. However, tumors derived from cells expressing bcl-2/BH4del, consistent with the findings reported in Figure 3, C and D, exhibited lower tumor growth rates when compared to control cells. Tumor tissue sections were also examined for p62/SQSTM1 expression by immunohistochemistry. As shown in Figure 6, C and D, bcl-2/BH4del tumor xenografts showed a weakly positive p62/SQSTM1 protein expression, whereas a moderate to strong immunoreactivity for p62/SQSTM1 was observed in bcl-2 Δα1 and bcl-2 wt overexpressing xenografts as documented by semiquantitative evaluation (Figure 6D).

Overall, these results reveal that the deletion of different lengths of N-terminal from bcl-2 protein can differently affect tumor growth.

Bcl-2 Protein Lacking the BH4 Domain Strongly Impairs the Binding between Bcl-2 and Its Interacting Proteins

Bcl-2 protein modulates either apoptosis or autophagy through its interaction with proapoptotic and Beclin-1 proteins, respectively [10,29]. To investigate whether BH4 domain is required for bcl-2 interaction with Beclin-1, we tested the ability of bcl-2 protein deleted of BH4 domain to associate with this protein by immunoprecipitation experiments. As control, interaction with bax was also evaluated [30]. As reported in Figure 7, when immunoprecipitation of total extract from M14 cells and its derivatives was carried out using an antibody against Beclin-1 or bax proteins, and Western blot analysis was performed using an antibody against amino acids 41 to 54 of bcl-2 (100), that specifically recognizes both wt and deleted bcl-2 protein (Figure 7A), we found that, as expected, both bax and Beclin-1 proteins bind bcl-2 wt protein. On the contrary, deletion of BH4 domain strongly impairs the binding between bcl-2 and its interacting proteins (Figure 7B). Because bcl-2 protein can form both heterodimers with other proapoptotic members and homodimers [31], we determined whether bcl-2 protein lacking the BH4 domain might bind to endogenous bcl-2 to prevent it from binding to Beclin-1 or bax. To test this hypothesis, we performed immunoprecipitation experiments using an antibody that, recognizing an epitope of bcl-2 protein mapping at the amino-terminus domain (N19), only immunoprecipitates bcl-2 wt protein but not bcl-2/BH4del (Figure 7A). Using the two antibodies specific for the different forms of bcl-2 protein, we showed that bcl-2/BH4del protein was not present in a protein complex with the endogenous bcl-2 protein, demonstrating that full-length bcl-2 protein cannot bind bcl-2 deleted protein. Similar data were obtained when H1299 cells transiently expressing both EGFP-bcl-2 wt and bcl-2/BH4del were immunoprecipitated with an EGFP antibody and then analyzed by Western blot analysis using a bcl-2 antibody that specifically recognizes both wt and deleted bcl-2 proteins (data not shown). Overall, these data indicate that bcl-2 protein blocks apoptosis [17] (Figure W3B) and autophagy (Figure 1) through its BH4 domain, being this domain critical for the interaction of bcl-2 protein with both bax and Beclin-1 proteins.

Figure 7.

Figure 7

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Bcl-2 protein lacking the BH4 domain strongly impairs the binding between bcl-2 and bax or Beclin-1. (A) Western blot analysis of bcl-2, bax, and Beclin-1 proteins in total lysates of M14 control cells (puro) and its derivatives overexpressing bcl-2 wt (Bcl-wt) or deleted of BH4 domain (BH4del). (A) β-Actin is shown as loading and transferring control. (B) Immunoprecipitation (IP) of whole-cell lysates with the indicated antibodies against Beclin-1, bax, and bcl-2. (A, B) Western blot analysis of bcl-2 expression was performed using antibodies against amino acids 41 to 54 of bcl-2, which specifically recognizes both wt and deleted bcl-2 protein [bcl-2 (100)], or against an epitope of bcl-2 protein mapping at the amino-terminus domain that recognizes only the wt protein [bcl-2 (N19)].

Beclin-1 Knockdown Enhances Cell Proliferation in M14 Cells Expressing Bcl-2 Protein Lacking the BH4 Domain

To investigate whether Beclin-1 is necessary for tumor growth inhibition by bcl-2/BH4del protein in M14 cells, stable cell subclones expressing control short hairpin (sh) RNA (BH4del-shControl) or shRNA directed against the essential autophagy gene Beclin-1 (BH4del-shBeclin-1) were generated from cells expressing bcl-2/BH4del (Figure 7B). Upon starvation, as a consequence of Beclin-1 knockdown, a reduction of both LC3B-II/LC3B-I ratio (Figure 8A) and AVO-positive cells (Figure 8B) was observed in cells expressing bcl-2/BH4del when compared to bcl-2/BH4del cells expressing shControl, thus indicating an attenuation of autophagy activation. Knockdown of Beclin-1 also resulted in significant increase of in vitro proliferation (Figure 8C) and in long-term survival being the clonogenic efficiency of bcl-2/BH4del cells enhanced from about 40% to 70% after Beclin-1 knockdown. According to in vitro results, Beclin-1 down-regulation rescued BH4-dependent inhibition of in vivo tumor growth (Figure 8D), thus indicating that the effect of bcl-2/BH4del protein on cell growth is strongly correlated with Beclin-1 expression, being Beclin-1 required for the ability of bcl-2/BH4del to induce autophagy and to modulate cell growth. To study the extent of contribution of autophagy on cell proliferation reduction by bcl-2/BH4del protein, we evaluated the effect of chloroquine treatment on in vitro cell growth. As reported in Figure 8E, we found that chloroquine treatment of M14 control and bcl-2 wt overexpressing cells had minimal or no effects on cell proliferation and vitality, while it decreased proliferation and vitality of cells expressing bcl-2/BH4del. At day 7 of growth, a nonsignificant reduction of about 2% and 15% was observed in control and bcl-2 wt clones, respectively, while a significant reduction of about 30% (P < .05) was evidenced in bcl-2/BH4del overexpressing clones.

Figure 8.

Figure 8

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Beclin-1 down-regulation enhances cell proliferation of M14 cells expressing bcl-2 protein lacking the BH4 domain. (A) Western blot analysis of LC3B-I/II and Beclin-1 protein expression and (B) AVOs in cells grown in complete media or under serum starvation (48 hours), (C) in vitro cell proliferation, and (D) tumor volume calculated at day 24 after cell injection of M14 control (puro) and its derivative expressing bcl-2 deleted of BH4 domain (BH4del) stably expressing control shRNA (shControl) or an shRNA directed against Beclin-1 (shBeclin-1). (A, B) Representative experiments of three independent ones with similar results are shown. (A) β-Actin is shown as loading and transferring control. (B) The percentage of the cells with prominent red fluorescence is shown. (D) A representative experiment ± SD of two independent ones with similar results is shown. (E) Growth curves of M14 cells and its derivatives untreated or treated with chloroquine (25 µM). (C, E) The results represent the average ± SD of three independent experiments. *P < .05, relative to control cells (C, D), or untreated cells (E).

Finally, we evaluated whether endogenous Beclin-1 is required for the ability of bcl-2/BH4del to induce autophagy and to modulate cell growth. To address this issue, JR8 human melanoma and HT29 human colon carcinoma cells, both characterized by undetectable or very low levels of endogenous bcl-2 [17] and Beclin-1 proteins (Figure 9A), were stably transfected with expression vectors for bcl-2 wt or bcl-2/BH4del. As reported in Figure 9B, upon starvation HT29 cells were reluctant to induce AVO formation and localization of LC3B-II to autophagosome membrane regardless bcl-2 protein status. Moreover, removal of BH4 domain from bcl-2 protein in HT29 did not affect cell proliferation, being in vitro (Figure 9C) and in vivo (Figure 9D) growth rate of control and bcl-2 wt overexpressing cells superimposable to that of cells expressing bcl-2/BH4del. Similar results in terms of response to autophagic stimuli (data not shown) and tumor growth (Figure 9, E and F) were also observed in melanoma line JR8 stably expressing bcl-2 wt or bcl-2/BH4del. Overall, these results indicate that bcl-2 protein deleted of its BH4 domain does not play a role on in vitro and in vivo growth of tumor cells with low level of bcl-2 and Beclin-1 proteins.

Figure 9.

Figure 9

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Deletion of BH4 domain from bcl-2 protein in HT29 colon carcinoma and JR8 melanoma cells does not affect tumorigenicity. (A) Western blot analysis of bcl-2 and Beclin-1 proteins, (C, E) in vitro cell proliferation, and (D, F) in vivo tumor growth of HT29 colon carcinoma and JR8 melanoma cells stably transfected with empty vector (puro) or with vectors encoding wt (Bcl-2wt) or mutated bcl-2 protein (BH4del). (C, E) The results represent the average ± SD of three independent experiments. (A, D, F) Representative experiments of two independent ones with similar results are shown. (B) AVOs and immunofluorescence staining of LC3B expression (red) in HT29 control (puro) and their derivatives overexpressing bcl-2 wt (Bcl-2wt) or deleted of BH4 domain (BH4del) grown for 48 hours in complete medium or under serum starvation. Representative pictures are shown.

Discussion

Bcl-2 protein plays an important role in several cell functions, including autophagy. Unlikely, the relevance of various BH domains of bcl-2 protein in autophagy has been poorly investigated. By using oligonucleotides antisense against bcl-2 mRNA, we have found that downregulation of bcl-2 was paralleled by activation of autophagic program. More importantly, by using melanoma cells overexpressing bcl-2 protein wt or deleted of its BH4 domain, previously characterized for their effect on tumor angiogenesis and apoptosis [17], we have demonstrated that forced expression of bcl-2/BH4del protein leads to a restricted tumorigenicity. In particular, cells expressing bcl-2/BH4del showed a reduction of in vitro cell proliferation, colony formation, and in vivo tumor growth when compared to control and bcl-2-transfected cells. Consistent with these results, a reduction of mitotic and proliferating cells in bcl-2/BH4del-xenotransplanted tumors was observed. An increased time of tumor appearance, indicating an effect on the early stages of tumor growth, was also evidenced in bcl-2/BH4del-carrying animals and paralleled by a reduced tumor incidence.

Interestingly, increased autophagy was found in vivo after forced expression of bcl-2/BH4del protein, and this process, concomitant with reduced proliferation, might explain the tumor growth delay observed in bcl-2/BH4del-xenotransplanted tumors. For the first time, our data show that BH4 domain of bcl-2 is involved in the autophagosome formation and that autophagic flux is increased by BH4 domain removal. Thus, it is plausible that in our experimental models at the early stages of tumorigenesis bcl-2 protein lacking BH4 domain regulates tumor development by suppressing tumor growth through regulating cell proliferation, whereas at later stages BH4 domain deletion may lead to cell death through autophagy induction.

The significance of our study is enhanced by the evidence that also cleavage of bcl-2 protein by apoptotic proteases, generated in the cells by stimuli such as H2O2 and CDDP, recapitulates some of the results obtained after ectopical introduction of bcl-2 devoid of its BH4 domain, such as up-regulation of autophagosome marker LC3B-II and down-regulation of autophagic substrate p62/SQSTM1. The use of the z-VAD-fmk pan-caspase inhibitor confirmed the caspase-dependent cleavage of bcl-2 at the N-terminal domain, as previously demonstrated [16,28,32], and supported the role of bcl-2 cleavage in autophagy induction. In fact, z-VAD-fmk blocked the cleavage of bcl-2 protein and the ability of CDDP to modulate the expression of autophagic proteins. We cannot exclude that besides bcl-2, other proteins can be cleaved and play a role in the activation of autophagic process.

To the best of our knowledge, this is the first demonstration that deletion of 36 residues at the N-terminus of bcl-2 protein does reduce tumor growth. In agreement with the results demonstrating that targeted down-regulation of bcl-2 results in cancer cell death [14,21,26], our findings evidenced a novel mechanism of tumor growth inhibition induced by affecting bcl-2 functions. By immunoprecipitation experiments, we have demonstrated that BH4 domain is critical for the interaction of bcl-2 protein with both bax and Beclin-1. We can speculate that the removal of the entire BH4 region from bcl-2 protein could alter the conformation of the hydrophobic cleft composed by the BH1, BH2, and BH3 domains [30], thus interfering with the functions of proteins involved in cell death, such as the proapoptotic protein bax and the BH3-only protein Beclin-1, a key player in autophagy process. Beclin-1 under normal steady-state growth conditions is bound to various antiapoptotic members of the bcl-2 family, including bcl-2. Besides, disruption or reduction of the interaction of the BH3 domain of Beclin-1 with the binding hydrophobic pocket of bcl-2 protein formed by the BH1.3 domain, promotes autophagy [25,33]. Moreover, BH3-mimetic compounds (ABT-737), “BH3-only” proteins (i-e., BNIP3, Bad), c-Jun N-terminal kinases (JNK)-mediated bcl-2 phosphorylation, and death-associated protein kinase-induced phosphorylation of Beclin-1 have been demonstrated to induce autophagy through disruption of such interaction [34–37].

The fact that 1) bcl-2 wt but not deleted of BH4 domain interacts with Beclin-1, 2) down-regulation of Beclin-1 in M14 cells expressing bcl-2/BH4del partially rescues cell proliferation and colony-forming ability, and 3) overexpression of bcl-2 wt or truncated in the BH4 domain did not result in any in vitro and in vivo growth retardation of the JR8 melanoma and HT29 colon carcinoma lines, both expressing very low levels of Beclin-1, indicates an involvement of Beclin-1 in the inhibitory effect of bcl-2/BH4del on in vivo tumor growth. Our findings also evidenced that the full-length endogenous bcl-2 protein fails to bind bcl-2 deleted protein, ruling out the hypothesis that bcl-2/BH4del prevents bcl-2 binding to Beclin-1. However, other bcl-2-interacting proteins might be also involved in BH4 domain function.

Noteworthy, when the bcl-2 Δα1 protein, lacking 22 residues at the N-terminus and retaining the ability to bind Beclin-1 and to protect from autophagic stimuli and losing the antiapoptotic properties of bcl-2 [29], was expressed in M14 cells, tumor growth rate was superimposable to that of control xenografts, thus indicating that removal of different lengths of N-terminus from bcl-2 protein can differently affect in vitro and in vivo cell death and consequently tumor growth.

Because the two mutants we used differ for residues 23 to 36, belonging to part of BH4 domain and a short part of the loop region, we can speculate that the gain of function phenotype that we have observed in bcl-2/BH4del may be the result of an interplay of these residues with other functional domains of bcl-2 or with other proteins. However, we cannot exclude that posttranslational modifications, such as phosphorylation of serine/tyrosine residues within 23 to 36 amino acid sequence of bcl-2, could be responsible of the difference observed between bcl-2 Δα1 and bcl-2/BH4del expressing cells in terms of cell death and tumor growth. Bcl-2 protein contains a number of possible serine phosphorylation sites that play a regulatory role in its functions, including serine 24 [38], serine 70 [39], and serine 87 [40], and these posttranslational modifications could directly or indirectly affect the interaction of bcl-2 with molecules involved in activation of cell death. It is possible that the lacking of the potential phosphorylation site in serine 24 in bcl-2/BH4del expressing cells could be responsible of the novel proautophagic and tumor suppressor functions of bcl-2/BH4del protein.

In conclusion, careful dissection of the biologic and biochemical effects of bcl-2 wt or lacking different amino acidic regions in different cell types may contribute to the understanding of the function of this protein and the relevance of the various domains in its functions. Our findings provide new insight into molecular mechanism of bcl-2 functions and an opportunity to develop new agents to promote tumor cell death by interfering with BH4 domain functions. The targeting of autophagic/apoptotic pathways by gene therapeutic or pharmacologic strategies could represent a very promising option for the treatment of cancer. Agents interfering with the critical residues of the BH4 domain or expression of a bcl-2.modified protein in cancer cells may provide a new strategy in cancer therapy by impairing bcl-2 function and inducing autophagy.

Supplementary Material

Supplementary Figures and Tables
neo1503_0315SD1.pdf (323KB, pdf)

Acknowledgments

We are grateful to Ameeta Kelekar and Jonathan Howard for providing shBeclin-1 and EGFP-LC3B plasmids, respectively. We thank Giuseppe Starace for cell sorting of EGFP-LC3B.positive cells, Adele Petricca for secretarial assistance, and Simone Bonacelli for English revision.

Abbreviations

Bcl-2

B-cell lymphoma 2

BH

Bcl-2 homology

LC3B

microtubuleassociated protein 1 light chain 3B

EGFP

enhanced green fluorescent protein

sh

short hairpin

wt

wild type

AVOs

acidic vesicular organelles

BrdU

bromodeoxyuridine

i.m.

intramuscular

PARP

poly(ADP-ribose) polymerase

CDDP

cisplatin

Footnotes

1

This work was supported by grants from the Italian Association for Cancer Research (IG 10239). Teresa De Luca is a recipient of a fellowship from the Italian Foundation for Cancer Research. The authors declare no conflict of interest.

2

This article refers to supplementary materials, which are designated by Figures W1 to W3 and are available online at www.neoplasia.com.

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Supplementary Materials

Supplementary Figures and Tables
neo1503_0315SD1.pdf (323KB, pdf)

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