SMARCB1 (Snf5/Ini1/Baf47) is a potent tumor suppressor, the loss of which serves as the diagnostic feature in malignant rhabdoid tumors (MRT) and atypical teratoid/rhabdoid tumors (AT/RT), two highly aggressive forms of pediatric neoplasms. SMARCB1 is a core subunit of Swi/Snf chromatin remodeling complexes, and loss of SMARCB1 or other subunits of these complexes has been observed in a variety of tumor types. Here, we restore Smarcb1 expression in cells derived from Smarcb1-deficient tumors, which developed in Smarcb1 heterozygous p53−/− mice. We find that while re-introduction of Smarcb1 does not induce growth arrest, it restores sensitivity to programmed cell death and completely abolishes the ability of the tumor cells to grow as xenografts. We describe persistent activation of AKT signaling in Smarcb1-deficient cells, which stems from PI3K (phosphatidylinositol 3′-kinase)-mediated signaling and which contributes to the survival and proliferation of the tumor cells. We further demonstrate that inhibition of AKT is effective in preventing proliferation of Smarcb1-deficient cells in vitro and inhibits the development of xenografted tumors in vivo. Profiling Smarcb1-dependent gene expression, we find genes that require Smarcb1 and Swi/Snf for their expression to be enriched for extracellular matrix and cell adhesion functions. We find that Smarcb1 is required for transcriptional activation of Igfbp7, a member of the insulin-like growth factor-binding proteins family and a tumor suppressor in itself, and show that re-introduction of Igfbp7 alone can hinder tumor development. Our results define a novel mechanism for Smarcb1-mediated tumorigenesis and highlight potential therapeutic targets.
Mutation and inactivation of the tumor-suppressor gene SMARCB1 (Snf5/Ini1/Baf47) have been well established as the underlying mechanism leading to malignant rhabdoid tumors (MRT) and atypical teratoid/rhabdoid tumors (AT/RT), two highly aggressive forms of pediatric neoplasms.1, 2, 3 More recently, recurrent mutations in SMARCB1 in many other tumor types were also reported.4 SMARCB1 codes for BAF47, a core subunit of the Swi/Snf ATP-dependent chromatin remodeling complex that facilitates nucleosome re-positioning relative to the DNA sequence.5 Notably, mutations in SMARCB1 and in other subunits of the Swi/Snf complex, such as the ATPases BRG1 and BRM, have been identified in a wide range of cancers, implicating the Swi/Snf complexes as a whole in tumor suppression.6, 7
Swi/Snf complexes are generally regarded as transcriptional co-activators/repressors. Accordingly, transformation associated with SMARCB1 inactivation is considered to stem from transcriptional deregulation of target genes.4 Several mechanisms have been put forward when considering the tumor-suppressive properties of SMARCB1. SMARCB1 was shown to transcriptionally regulate p16INK4a and/or p21, repress cyclin D1 and generally lead to inhibition of E2F activity.8, 9, 10 Consistent with these effects, cyclin D1 deficiency inhibits tumor formation in Smarcb1 heterozygous mice.11 Moreover, gene expression profiling in SMARCB1-deficient MRT tumors and in various cell lines following Smarcb1 conditional inactivation have also implicated SMARCB1 in the inhibition of E2F-target genes.12, 13 Further evidence suggests SMARCB1 as necessary for p53- and possibly c-MYC-driven transcriptional activation and cell-cycle control.13, 14 Recently, aberrant activation of the Hedgehog pathway in SMARCB1-deficient cells has been demonstrated as mediating tumorigenesis.15
Further complicating our understanding of Smarcb1-associated transformation is the finding that conditional knockout of Smarcb1 in primary cells results in rapid growth arrest and in p53-mediated programmed cell death.16 This observation suggests that Smarcb1 transformation requires additional enabling conditions such as mutations in additional genes or a unique cellular context allowing for Smarcb1-associated transformation. Indeed, the precise cellular origin of MRTs and AT/RTs is unknown.
Aspiring to further understand Smarcb1-associated transformation, we utilized our previously described MRT mouse model1 to establish two Smarcb1-deficient cell lines from tumors that developed spontaneously in Smarcb1 heterozygous, p53-deficient mice. Re-introduction of Smarcb1 to these murine tumor cells resulted in complete loss of tumorigenicity but had only a minor effect on cell proliferation in vitro, suggesting that Smarcb1-associated transformation may involve other mechanisms in addition to the regulation of cell-cycle progression. This possibility is further supported by a recent study that examined SMARCB1 mutations in human familial schwannomatosis and found novel, cancer-causing mutations, which retain the ability to suppress cyclin D1.17
Here we identify the AKT signaling pathway as a central axis in survival and proliferation of Smarcb1-deficient cells. We further identify Igfbp7 as a Swi/Snf-regulated gene, the loss of which contributes to the tumorigenic capacity of Smarcb1-deficient cells.
Smarcb1-associated transformation relies on pathways additional to cell-cycle regulation
We established two cell lines from tumors that developed in Smarcb1+/−, p53−/− mice.16 Both cell lines were Baf47 deficient due to spontaneous loss of heterozygosity of the Smarcb1 wildtype allele (Figures 1a and b). Stable re-introduction of Smarcb1 into the tumor cells induced a drastic change in cell size and morphology (Figure 1c), but unlike similar experiments in human tumor cells,12, 18, 19, 20 Smarcb1 restoration had only a minor effect on growth rate in normal (10% fetal calf serum) serum conditions (Figure 1d). Upon serum withdrawal (0.1% fetal calf serum), Smarcb1-proficient tumor cells stopped proliferating and exhibited fast and dramatic cell death, an effect much delayed in Smarcb1-deficient tumor cells (Figures 1e–g). Both Smarcb1-deficient and -proficient tumor cells failed to grow in soft agar (not shown), yet in vivo, when injected subcutaneously to NOD-SCID mice, tumors developed exclusively from Smarcb1-deficient cells (Figure 1h). The fact that following restoration of Smarcb1, murine MRT cells continue to proliferate but lose tumorigenic capabilities indicate that Smarcb1 promotes cell transformation not only through cell-cycle regulation but via additional mechanisms as well. Moreover, these results reaffirm Smarcb1 as a potent tumor suppressor and assert the validity of this specific murine cellular system in studying transformation mediated by Smarcb1 deficiency.
Smarcb1-deficient tumor cells exhibit persistent AKT activation and signaling
Seeking to explain the Smarcb1-dependent differences in response to serum withdrawal, and based on previous indications21, 22, 23 we examined the levels of phosphorylated AKT (pAKT) in the two tumor cell lines and found them to be significantly elevated in Smarcb1-deficient cells relative to their Smarcb1-proficient counterparts (Figure 2a). AKT phosphorylation levels depend on external signals from serum components, and accordingly, in Smarcb1-proficient cells lowering serum concentration abolished AKT phosphorylation. However, in Smarcb1-deficient cells, pAKT was evident even in low serum. This result indicates that Smarcb1-deficient tumor cells have an aberrant and persistent activation of AKT that is corrected when Smarcb1 is restored.
AKT phosphorylation and activation promotes cell growth and proliferation while inhibiting apoptosis, an effect which is mediated through phosphorylation of various downstream targets. One such target is the transcription factor FoxO1, whose phosphorylation prevents it from entering the nucleus and promoting expression of pro-apoptotic genes and cyclin-dependent kinase inhibitors.24, 25, 26, 27, 28 In both tumor cell lines, re-introduction of Smarcb1 resulted in reduction of AKT phosphorylation and its downstream signaling, as evident by the transcriptional induction of the cyclin-dependent kinase inhibitor p27 and the pro-apoptotic gene Bim, two transcriptional targets of FoxO1, and by the decreased phosphorylation of S6 ribosomal protein (Figures 2a and b). These results demonstrate that Smarcb1 deficiency in these cells leads to activation of AKT signaling and provide a mechanistic explanation to the increased cell proliferation and survival of Smarcb1-deficient cells, particularly in low serum, as observed in Figure 1.
AKT inhibition hinders tumor cell proliferation
To further validate the role of AKT signaling in survival and proliferation of Smarcb1-deficient cells and explore the possible therapeutic potential in targeting this pathway, we assayed the sensitivity of the tumor cells to an AKT inhibitor. Inhibition of AKT activity with an AKT1/2-specific inhibitor reduced proliferation rates of Smarcb1-deficient cells to a level comparable to that of their Smarcb1-proficient counterparts, while having little, if any, effect on Smarcb1-proficient cells (Figures 2c and d). To assess the contribution of AKT activation to survival of Smarcb1-deficient tumor cells in vivo, we treated mice carrying xenografted Smarcb1-deficient tumors with the AKT inhibitor Triciribine and found it to inhibit tumor growth (Figures 2e and f). Overall, these results indicate that the AKT signaling pathway contributes to survival of Smarcb1-deficient tumor cells and suggest that inhibition of AKT may be a beneficial approach for suppression of Smarcb1-deficient tumors, such as MRT and AT/RT.
Inhibition of phosphatidylinositol 3′-kinase (PI3K) abolishes aberrant AKT activation in Smarcb1-deficient tumor cells
Signaling leading to AKT phosphorylation is often mediated by PI3K.27 To determine whether the elevated level of pAKT observed in Smarcb1-deficient cells depends on PI3K activity, we used the PI3K inhibitor LY294002. As before, reducing serum concentration resulted in reduced AKT phosphorylation in both Smarcb1-deficient and -proficient cells. However, while in Smarcb1-expressing cells AKT phosphorylation was completely abolished in low serum, it persisted in Smarcb1-deficient cells (Figures 2a and 3a). This persistent, Smarcb1-dependent AKT phosphorylation was suppressed following treatment with LY294002, suggesting that it is mediated by PI3K activation (Figure 3a).
PI3K-mediated AKT phosphorylation and activation is stimulated upon diverse signals, including growth factor receptors, insulin, integrins, cytokines and so on.27, 29 Indeed, a western blot for phospho-Tyrosine (pTyr) in Smarcb1-deficient and -proficient cells reveals multiple differentially phosphorylated proteins (Figure 3b), suggesting that several players may contribute to AKT activation.
Transcriptional response to Smarcb1 restoration
Our findings thus far provide evidence for aberrant activation of AKT in Smarcb1-deficient tumor cells, which is mediated by PI3K and contributes to transformation. However, it remained unclear how deficiency in Smarcb1, a member of Swi/Snf chromatin remodeling complexes, can cause such an effect. Swi/Snf complexes are recognized mostly as transcriptional co-activators and co-repressors, and previous findings show that Smarcb1-dependent transformation is mediated by its transcriptional targets and not by promoting mutagenesis.11, 30 We therefore used DNA microarrays to characterize Smarcb1-dependent transcriptional changes in tumor cells. To enrich for transcriptional targets that are directly regulated by Smarcb1, we profiled gene expression in both the tumor cell lines following Smarcb1 re-introduction, as soon as the BAF47 protein could be detected. Following Smarcb1 re-expression, 490 and 371 genes were upregulated at least two fold in cell lines 167 and 365, respectively, with an overlap of 170 genes (Supplementary Figure S1a). In all, 186 and 209 genes were downregulated at least two fold in cell lines 167 and 365, respectively, with only 21 genes in common (Supplementary Figure S1a).
Gene set enrichment analysis (GSEA)31, 32 showed that genes that are upregulated following re-introduction of Smarcb1 are enriched for such gene ontology categories as cell adhesion, migration and extracellular components (Table 1, false discovery rate <0.001) and include central players such as fibronectin, paxilin, integrins and MMPs. These transcriptional changes offer several possible mechanistic explanations to the observed morphological changes in cell shape and size following Smarcb1 re-introduction. Consistent with similar studies in human MRT cells,13 genes downregulated following re-introduction of Smarcb1 in our experiment were found to be enriched for categories associated with DNA repair and replication and cell-cycle regulation (Table 1). As Swi/Snf complexes are known to be recruited by transcription factors, we used GSEA to search for enrichment in common DNA-binding motifs. Among the genes downregulated by Smarcb1, we found significant enrichment for targets of E2F1, Max and Elk1, while for genes upregulated by Smarcb1 we found a significant enrichment for targets of FoxO1, SRF and Smad4 (Table 2).
There is a significant correlation between the transcriptional changes observed in our study and those following re-expression of other subunits of the Swi/Snf complex in other experimental systems, specifically Smarca4 (BRG1) and Smarce1 (Baf57)33, 34 (Supplementary Figure S1b). The genes which contribute to this similarity (as defined by the GSEA ‘leading edge’ group) include extracellular matrix (ECM) components such as Fibronectin1 and Integrinb3 and other secreted molecules (Supplementary Table S1) and exhibit as a whole statistically significant enrichment for gene ontology terms related to ECM and adhesion (Supplementary Table S2). As Baf47 (Smarcb1) is a core component of Swi/Snf complexes, the similarity in transcriptional response to re-introduction of different complex subunits indicates that at least some of the transcriptional perturbations in Smarcb1-deficient cells reflect loss of Swi/Snf complex functionality in the absence of Baf47. However, it remains unknown to what degree these changes represent similarity in transformation mechanism.35
Inhibition of xenografted tumor growth following expression of Igfbp7 in Smarcb1-deficient tumor cells
Expression data also revealed several candidate genes whose low expression in Smarcb1-deficient cells may explain Smarcb1-associated transformation and the increased levels of AKT phosphorylation. We considered two such candidates which were among the most upregulated genes following Smarcb1 restoration: Caveolin1, which is involved in modulating epidermal growth factor receptor or transforming growth factor-β activity,36, 37 and IGFBP7, which is related to the insulin-like growth factor binding proteins family, whose members have been demonstrated to bind and sequester IGF-I and thus attenuate signaling through the IGF1 receptor.38, 39, 40, 41 Both genes were induced greatly following Smarcb1 re-expression, as validated by quantitative reverse transcriptase–PCR and by western blot (Figures 4a and b).
To test whether these proteins may contribute to AKT phosphorylation and to Smarcb1-dependant tumorigenicity, we expressed them in Smarcb1-deficient tumor cells (Supplementary Figure S2). In culture, re-expression of Igfbp7 or Cav1 did not lower the levels of pAKT or pS6 and did not confer any measurable effect on cell morphology or proliferation (data not shown). However, expression of Igfbp7 (but not Cav1) inhibited growth of xenografted tumor cells (Figures 4c and d). As expression of Igfbp7 in tumor cells inhibited tumor growth but did not prevent it completely, we could examine its effect on the AKT signaling pathway in vivo. In tumor cell line 167, we found that Igfbp7 expressing tumors had reduced levels of pAKT compared with control 167 cells (Figures 4e and f). This effect was not evident in line 365. However, in both the cell lines, expression of Igfbp7 resulted in significantly reduced S6 ribosomal protein phosphorylation (Figures 4e and f). Consistent with these results, we find a marked reduction in proliferating cell nuclear antigen (PCNA)-positive cells, indicating reduced in vivo proliferation of Igfbp7-expressing tumor cells (Figures 4e–g). These results indicate that the loss of Igfbp7 expression in Smarcb1-deficient tumor cells contributes to the transformed phenotype, yet it is still unclear whether Igfbp7 acts by interfering with IGFR signaling.
Igfbp7 has been studied as a modulator of angiogenesis and was found to inhibit tumor angiogenesis in several contexts and proposed to block vascular endothelial growth factor receptor.42, 43, 44, 45 We used the endothelial marker CD31 to examine the vasculature of Igfbp7-expressing xenograft tumors and control counterparts. Consistent with previous publications, expression of Igfbp7 in tumor cells had a marked effect on distribution and size of blood vessels (Supplementary Figure S3). It is therefore possible that the growth-suppressive effect of Igfbp7, observed only in vivo, stems from an effect on angiogenesis. Although expression of Igfbp7 alone did reduce tumor growth, it did not completely abolish tumor development and did not correct the persistent activation of AKT. We therefore conclude that transformation caused by loss of Smarcb1 involves components additional to Igfbp7 transcriptional downregulation.
In this study, we took advantage of a previously described mouse model for Smarcb1/Snf5/Ini1/Baf47-deficient tumors.16 Focusing on the contribution of Smarcb1 deficiency to the etiology of these tumors, we study phenotypic and molecular consequences of Smarcb1 re-introduction into tumor cells. As opposed to previous studies in human cells,12, 18, 19, 20 re-introduction of Smarcb1 in this system did not result in growth arrest (Figure 1d) but nevertheless abolished the tumorigenic capacity of the cells (Figure 1h). This outcome may reflect a difference between human and mouse Smarcb1-deficient tumors; however, p53 deficiency may also contribute to the escape from growth arrest following Smarcb1 re-expression. Even though the tumor cells are p53 deficient, they completely lose tumorigenic capability following re-introduction of Smarcb1. Here we focus on understanding this critical Smarcb1-dependent component of transformation.
Persistent AKT activation in Smarcb1-deficient tumor cells
Smarcb1-deficient tumor cells exhibit resistance to apoptosis and increased proliferation, particularly in low serum conditions, as compared with Smarcb1-expressing cells (Figure 1). At the molecular level, we find persistent, PI3K-dependent, activation of AKT in serum-deprived Smarcb1-deficient cells (Figure 2) that is abolished upon re-expression of Smarcb1. Accordingly, in Smarcb1-deficient cells we identify key downstream effects of AKT activation, including increased S6 phosphorylation and reduced p27 and Bim expression, which are known to promote proliferation and prevent apoptosis (Figures 2a and b). The dependence of tumor cells on AKT activity for survival and proliferation is also evident from the effect of AKT inhibition on cell proliferation, which is specific to Smarcb1-deficient cells (Figure 2d). The ability of an AKT inhibitor to attenuate growth of xenografted Smarcb1-deficient tumor cells further demonstrates the relevance of AKT signaling in the progression of these tumors (Figure 2f).
Smarcb1-dependent transcription in tumor cells
To further investigate the molecular basis of cellular phenotypes brought about by Smarcb1 deficiency, we compared gene expression profiles of Smarcb1-proficient and -deficient tumor cells. The similarity between Smarcb1-dependent transcription and BRG1- or BAF57-dependent transcription indicates that at least part of the transcriptional alterations in Smarcb1-deficient cells are the result of loss of Swi/Snf complex functionality in the absence of Smarcb1. However, it remains unknown to what degree these changes reflect a similar mechanism of transformation.35
Our analysis identifies candidate transcription factors that may recruit Swi/Snf complexes as co-activators/repressors in tumor cells. We find that Smarcb1 responsive genes are enriched in binding sites for E2F1, Elk1, SRF and Smad4, which have been reported previously to interact with Swi/Snf complex members for their transcriptional activity.46, 47, 48 The GSEA analysis also defines the list of putative target genes, which have the relevant binding site for these transcription factors, and which also change in expression soon after BAF47 re-expression. It remains to be determined which of the responsive genes are indeed directly regulated by Swi/Snf complexes.
Transcriptional downregulation of Igfbp7 in Smarcb1-deficient tumor cells contributes to their transformed phenotype
Although multiple mechanisms can lead to AKT activation, we found that inhibition of PI3K can attenuate the persistent phosphorylation of AKT in Smarcb1-deficient cells (Figure 3), implicating signaling upstream of AKT in its persistent activation. Activation of the IGF1R-AKT signaling pathway was previously observed in human MRT and AT/RT tumor sections, and studies in cell lines derived from human Rhabdoid tumors suggest aberrant activity of the IGF1-AKT signaling pathway and dependence on this pathway for tumor cell survival.21, 22, 23, 49, 50 Our results emphasize the central role this pathway has in survival and proliferation of Smarcb1-deficient tumor cells and demonstrate that AKT aberrant activation is dependent on absence of Smarcb1 (Figure 3).
One the most upregulated genes following Smarcb1 re-introduction to tumor cells was Igfbp7, which was recently highlighted as a potential tumor-suppressor gene in various solid tumors through a mechanism which remains to be elucidated.43, 44, 51, 52, 53 Other members of the IGFBP family were found to bind IGF and affect its bioavailability44, 54 but IGFBP7 is less related to this group and is thought to have only low affinity to IGF. Interestingly, IGFBP7 was shown to have a high affinity to insulin,55 which was suggested to act as an autocrine growth factor in Rhabdoid tumor cells.50
Re-expression of Igfbp7 alone in Smarcb1-deficient tumor cells inhibited xenograft growth in vivo and reduced AKT pathway activity, yet Igfbp7 deficiency does not fully account for Smarcb1-associated transformation: while re-introduction of Smarcb1 completely prevented the development of xenograft, Igfbp7 had only an inhibitory effect (Figures 1h and 4c). In addition, expression of IGFBP7 alone did not reduce AKT phosphorylation in cultured tumor cells. IGFBP7 is a secreted factor and is known to depend on ECM components for its activity.56 ECM and adhesion-associated functions are underexpressed in Smarcb1-deficient cells (including fibronectin, integrins and MMPs, which were shown to function in coordination with IGFBPs56, 57, 58). It is thus possible that the tumorigenic effect of Smarcb1 deficiency is the result of mis-regulation of multiple genes, which together lead to AKT phosphorylation and result in transformation. Further support to this possibility comes from the western blot for pTyr, where re-introduction of Smarcb1 appeared to attenuate the phosphorylation levels of multiple proteins (Figure 3b).
In conclusion, our results demonstrate that targeting AKT signaling pathway may prove effective in treatment of Smarcb1-deficient tumors. As more tumor types are found to harbor mutation in Smarcb1 or to lose its expression,59 our results may be applicable for such tumors as well and not only to MRT and AT/RT. Our results further show that expression of Igfbp7 is dependent on Smarcb1/Swi/Snf activity and that Igfbp7 has a tumor-suppressing effect in SMARCB1-deficient tumor cells. Igfbp7 was not studied in human rhabdoid tumors; however, comparison of Igfbp7 expression across a spectrum of human tumor types and tumor cell lines reveals that Igfbp7 expression levels are among the lowest in human MRT and AT/RT samples (Supplementary Figure S4). This suggests that although its mechanism of action remains to be explored, IGFBP7 repression may also have a role in promoting human tumorigenesis following loss of SMARCB1.
Materials and methods
Cell line establishment and culture
Spontaneous tumors developing in Scmarb1 heterozygote p53-deficient mice were removed, dissected and suspended in serum-free DMEM (Dulbecco’s modified Eagle’s medium) containing 0.75 g/ml collagenase (Sigma, Rehovot, Israel, No. c5138) for 2 h. Following over night incubation, cells were mechanically separated by pipetting and suspended in growth medium. All cells were grown in DMEM supplemented with 10% Hyclone fetal bovine serum, penicillin (50 mg/ml), streptomycin (50 mg/ml), 2 mM l-Glutamine, 0.1 nM non essential amino acids, 0.1 mM β-mercaptoethanol and 1 mM sodium pyruvate. For low serum conditions, cells were washed twice in phosphate-buffered saline (PBS) before being transferred to medium containing 0.1% fetal bovine serum. PI3K inhibitor LY20094 (Sigma), AKT inhibitor 1/2 (Calbiochem, Darmstadt, Germany, AKT inhibitor VIII No. 124018) were solubilized according to the manufacturer’s instructions before addition to medium in indicated concentrations.
Xenografts and mice handling
Experiments were approved by the Israeli Animal Care and Use Committee (NS-09-12222-3). Mice were kept in specific pathogen-free approved facility. For xenografting, weaned Nod-SCID males aged 4 weeks were sedated with Isoflurane. Using a 27-G needle one million cells suspended in a total volume of 100 μl growing media were injected subcutaneously to each flank. When treated with the AKT inhibitor Triciribine (Merck, Darmstadt, Germany, AKT inhibitor V No. 124012), 1 μg per gram body weight per day were injected intraperitoneally in a total volume of 100 μl PBS. Inhibitor was injected daily starting the day following injection of cells, for 10 days. Mice were weighed daily to monitor any changes in body mass, and their general health examined throughout the duration of the experiment. Tumors were measured using a caliper, and tumor volume was calculated using the modified ellipsoidal formula.60
Primers listed in Supplementary Information (Supplementary Table S3) were used to generate full-length Smarcb1 cDNA transcript by PCR amplification of cDNA generated from human ES cells as listed below. The resulting amplicon was cloned into the BglII and BamHI sites of the pMig retroviral vector (Addgene, Cambridge, MA, USA, No. 9044) and sequenced. Igfbp7 and Cav1 were PCR amplified from cDNA generated from primary murine embryonic fibroblasts. Resulting amplicons were digested with BamH1 and EcoR1/Sal1, respectively, and cloned into the pBabe-puro retroviral vector. For generation of viral vectors, plasmids were co-transfected with pCL-Eco61 into 293T cells using the calcium phosphate protocol. Infection was carried out for 2 sequential days with 8 μg/ml Polybrene followed by selection with 5 μg/ml Puromycin for the pBabe-puro vector.
Protein extraction and western blot analysis
Proteins were extracted using p300 lysis buffer (20 mM NaH2PO4, 250 mM NaCl, 30 mM Na4P2O7*10H2O, 0.1% NP-40 and 5 mM EDTA) supplemented with 1 μM dithiothreitol, 1 μM phenylmethanesulfonylfluoride, 1 μM pepstatin, 1 μg/ml Aprotenin, 0.5 μg/ml Leupeptin and 1 mM Na3VO4. Following 10 min on ice, the lysate was centrifuged at 12 000 r.p.m. and the pellet discarded. The rabbit polyclonal anti-SNF5 was described previously,62 anti-pAKT serine473 (Cell Signaling, Danvers, MA, USA, No. 4058), anti-t-AKT (Cell Signaling No. 11E7), anti-p27 (Santa Cruz, Dallas, TX, USA, sc-529), anti-β-Actin (Abcam, Cambridge, MA, USA, ab6276), anti-p6S (Cell Signaling No. 2215s), anti-Igfbp7 (Santa Cruz sc-13095), anti-Cav1 (Cell Signaling No. 3267x) and anti-pTyr (4G10 hybridoma) Secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
Growth curves, bromodeoxyuridine (BrdU) labeling and cell-cycle
A standard MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used for plotting growth curves: 1500 cells were plated in triplets per day in a 96-well plate. For each time point, cells were incubated for 1 h in 100 μl medium supplemented with a final concentration of 400 μM MTT followed by two washes in PBS and extraction in 100 μl dimethyl sulfoxide. Plate was read at 570 nm and the background absorbance at 690 nm subtracted. For BrdU labeling, cells were plated on coverslips, left to adhere over night and 10 μM BrdU was added for 30 min before fixation in ice-cold methanol for 10 min at room temperature. Fixed cells were treated with 2 M HCl for 30 min followed by two washes in NaBO4 0.1 M pH 8.5 and two washes in PBS. For immunostaining, anti-BrdU (cell proliferation kit, 1:300; GE Healthcare Life Sciences, Uppsala, Sweeden) was diluted in PBS supplemented with 0.5% BSA and 0.05% Tween for 45 min followed by secondary antibody and DAPI (4,6-diamidino-2-phenylindole) before mounting. Images were collected on a Nikon TE-2000 (Nikon, Melville, NY, USA) inverted microscope and processed using NIS-elements software (Nikon). Identical camera and microscope settings were employed to allow valid comparison between images of Smarcb1-deficient and -proficient cells. For cell-cycle analysis, cells were trypsinized, washed and re-suspended in 300 μl PBS. Five milliliter ice-cold methanol was slowly added for fixation followed by incubation in −20 °C for at least 1 h. Cells were subsequently washed, re-suspended in 1 ml PBS and incubated on ice for 30 min. Subsequently, cells were re-suspended in 1 ml propidium iodide solution (PBS supplemented with 50 μg/ml propidium iodide and 250 μg/ml RNase A ) and incubated in 37 °C for 30 min.
RNA and DNA extraction, reverse transcription and real-time PCR
Total RNA was extracted using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) for purification of total RNA and in accordance with the manufacturer’s instructions. All other procedures were performed using standard techniques and kits as described in Barzily-Rokni et al.63
Expression array and data analysis
Expression profiling was carried out on Affymetrix Mouse Gene 1.0st platform (Affymetrix, Santa Clara, CA, USA). GeneChip Whole Transcript (WT) Sense Target Labeling Assay (Affymetrix) was employed followed by GeneChip Hybridization, Wash and Stain Kit (Affymetrix). Scanning was performed with GeneChip Scanner 3000 7G (Affymetrix controlled by GeneChip Operating Software (Affymetrix). Raw probe intensities were normalized by the robust multiarray average method using the Affymetrix expression console program (Affymetrix). Probes with raw intensity value <5 were discarded. We applied GSEA analysis using the MSigDB collections31 to identify gene sets that were significantly enriched in either cell line. In our analysis, we considered gene sets with false discovery rate <0.01 to be significant. Expression data are available with GEO accession GSE46017.
Tumors were harvested, fixed in 4% PFA-PBS and embedded in paraplast. Five micron sections were de-parafinized and rehydrated. Following antigen retrieval in 10 mM citrate buffer pH 6.0 and inhibition of endogenous peroxidases with 3% H2O2, sections were blocked for 30 min and then incubated with the primary antibody (anti-pAKT473 No. 736e11 from Cell Signaling diluted 1:50 in TNB buffer, anti-P6S S240/S244 No. 2215s from Cell Signaling diluted 1:50 in CAS-Block, anti-Igfbp7 sc-13095 from Santa Cruz diluted 1:50 in CAS-Block, anti-PCNA from BioLegend, San Diego, CA, USA, No. 307901 diluted 1:50 in CAS-Block) over night at 4 °C. Biotinylated secondary antibody diluted 1:200 was incubated for 30 min at room temperature. For pAKT staining, incubation with Streptavidin-peroxidase conjugate from the PerkinElmer TSA kit (Perkin-Elmer, Waltham, MA, USA) and the Biotynil Tyramide was carried out in accordance to the manufacturer’s instructions. Sections were then incubated with Avidin horseradish peroxidase (extraAvidin-Peroxidase E2886 Sigma) diluted 1:100 in 1% BSA-PBS for 15 min followed by incubation with the DAB substrate from the DAB chromogen KIT DB801R by Biocare Medical (Concord, CA, USA). Sections were stained with hematoxylin and dehydrated before mounting.
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We thank Dr Ittai Ben-Porath, Professor Eli Keshet and Professor Alex Levitzki for reagents and helpful discussions. This work was funded by the Israel Cancer Research Fund (ICRF, 2011-3094-PG) and by the Association for International Cancer Research (AICR 03-109).
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Darr, J., Klochendler, A., Isaac, S. et al. Loss of IGFBP7 expression and persistent AKT activation contribute to SMARCB1/Snf5-mediated tumorigenesis. Oncogene 33, 3024–3032 (2014). https://doi.org/10.1038/onc.2013.261
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