Cytokine 73 (2015) 219–224
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Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas } c, Anna Sebestyén a,b,⇑, Ágnes Márk a, Melinda Hajdu a, Noémi Nagy a, Anna Molnár a, Gyula Végso Gábor Barna a, László Kopper a a
}i út 26., Hungary Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest 1085, Üllo }i út 26, Hungary Tumor Progression Research Group of Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University, Budapest 1085, Üllo c Semmelweis University, Department of Transplantation and Surgery, Budapest 1082, Baross u. 23, Hungary b
a r t i c l e
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Article history: Received 4 November 2014 Received in revised form 24 January 2015 Accepted 3 February 2015
Keywords: Rapamycin TGF-b mTOR Apoptosis Lymphoma
a b s t r a c t TGF-b1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-b1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-b signaling, and through these pathways it is able to enhance TGF-b induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-b1 combination on cell growth and on TGF-b and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-b1 induced apoptosis and restored TGF-b1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-b sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-b1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-b1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-b resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-b1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Apoptosis is the default program for B-cells in the absence of survival stimuli. Therefore, the balance between pro- and antiapoptotic signals are essential for normal B-cell development and function [1]. Early stages of tumorigenesis share similarities with the suppression of apoptotic pathways leading to continuous survival. ⇑ Corresponding author at: Semmelweis University, 1st Department of Pathology } i út 26, Hungary. Tel.: +36 1 and Experimental Cancer Research, Budapest 1085, Üllo 2661638/54447; fax: +36 1 3171074. E-mail addresses: [email protected] (A. Sebestyén), [email protected] (Á. Márk), [email protected] (M. Hajdu), [email protected] (N. Nagy), [email protected] (A. Molnár), [email protected]. }), [email protected] (G. Barna), [email protected] (L. Kopper). hu (G. Végso
http://dx.doi.org/10.1016/j.cyto.2015.02.024 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Mammalian target of rapamycin complex 1 (mTORC1) lies at the crossroads of signalling networks [2,3], controlled by a wide variety of factors in favour of cell survival. The activation of mTORC1 is an early and frequent event in several tumors [4–6]. Rapamycin (an inhibitor of mTORC1 and a known immunosuppressive agent) and its analogues (rapalogues) inhibit the activity of mTORC1 and the phosphorylation of its downstream targets, p70 ribosomal S6 kinase (p70S6K), ribosomal S6 protein and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) [7]. The inhibition of mTORC1 yielded therapeutical benefits – such as inhibition of tumor progression and extended overall survival – in recent clinical trials for acute myeloid leukemia (AML), mantle cell lymphoma (MCL), other high grade lymphomas and in non-lymphoid malignancies as well [8–10]. TGF-b1 is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal
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lymphoid cells [11]. As expected, TGF-b1 acts as a potent tumour suppressor during the early stages of lymphomagenesis; however, it can promote progression at later stages [12]. The pro-oncogenic function of TGF-b1 signalling may be associated with the loss of TGF-b1 pathway activity and the engagement of other signalling mechanisms, which act in concert during the process of carcinogenesis [13]. While malignant lymphoid cells often lose their sensitivity to proapoptotic/antiproliferative regulators, the failure of the ’’classical’’ (Smad-dependent) TGF-b1 pathway is uncommon in lymphoid tumours [14], which raises the importance of ’’nonclassical’’ (Smad-independent) TGF-b1 signalling as well as the modulatory role of various interacting proteins [15,16]. We have previously shown that exogenous TGF-b1 can induce apoptosis in high grade lymphoma cells in a Smad4-independent and PP2A-dependent manner, primarily through the mitochondrial apoptotic pathway (caspase 9 and 3 activation mediated by FAS, TRAIL and TNF-a receptor independent mitochondrial depolarization) [17,18]. It has been suggested that TGF-b1 resistance could be reversed by lowering the ’’survival threshold’’ of cells in certain cases [17]. The interaction between TGF-b1 and mTORC1 signalling and their simultaneous targeting (by TGF-b1 plus rapamycin treatment) has been explored in some cell types, yielding conflicting results [19,20]. It is suggested that rapamycin can modulate TGF-b1 responses by effectively binding to FKBP-12, an inhibitor of TGF-b receptors. The sequestration of FKBP-12 may facilitate TGF-b responses; however, other mechanisms should also be considered. In the present work we found that inhibition of mTORC1 by rapamycin was able to restore the effectiveness of TGF-b1 to induce apoptosis in high grade B-cell non-Hodgkin lymphomas in vitro. 2. Materials and methods 2.1. Cell culture Experiments were performed on the following human B-cell non-Hodgkin lymphoma (B-NHL) cell lines: HT58 [21] and HT58r (EBV negative cell lines established in our laboratory; HT58r is a subclone of HT58 with low TGF-b1 sensitivity); BL41, BL41/95 (EBV-transfected variant of BL41); Ramos and U266. Cells were cultured in RPMI-1640 (Sigma, St. Louis, MO, USA) with 10% fetal bovine serum (GIBCO-BRL, Grand Island, NY, USA), 0.03% glutamine (, Sigma, St. Louis, MO, USA) and penicillin-streptomycin (100 U/ mL–100 lg/mL, Sigma, St. Louis, MO, USA), at 37 °C in 5% CO2 atmosphere. Cells in the exponential growth phase were used for all experiments. 2.2. Special treatments Cells (at a density of 1–2 105/ml) were treated with 1 ng/mL TGF-b1 (reconstituted with 4 mM HCl in 0.1% BSA, aliquoted and stored at 80 °C; R&D Systems, Minneapolis, MN, USA), 50 ng/ml rapamycin (also referred to as low dose – except where indicated otherwise; Sigma, St. Louis, MO, USA), SB431542 TGF-b RI/ALK1 (TGF-b receptor I/activin receptor like kinase-1) 5 lM inhibitor (Sigma, St. Louis, MO, USA), 100 nM okadaic acid (Sigma, St. Louis, MO, USA), LY294002 (5 lM, Calbiochem), Z-VAD-fmk (Sigma, St. Louis, MO, USA, 50 lM) for 0–72 h in 24-well plates or 25 cm2 flasks. Okadaic acid was used only in the first 4 h of the treatment (short pretreatment), cells were then washed and replated in rapamycin supplemented medium for 72 h. Rapamycin and TGF-b1 were added at the same time in our experiments, as 1–6–12 h pretreatment with either agent showed no
significant difference in apoptosis compared to combined treatment (data not shown). 2.3. Cell cycle analysis and apoptosis detection Apoptosis detection by flow cytometry was performed according to Mihalik et al. [22]. Briefly, cells were fixed in 70% ethanol (20 °C) followed by alkalic extraction (200 mM Na2HPO4, pH 7.4 and 100 lg/ml RNase, Sigma, St. Louis, MO, USA) and ethidium bromide staining (10 lg/ml, Sigma, St. Louis, MO, USA). For each sample, 10,000-20,000 events were acquired using a FACScan flow cytometer (Becton-Dickinson, BD Biosciences, San Diego, CA, USA). Data were analyzed with WinList software (Verity Software House, Topsman, ME, USA). Cell morphology was evaluated on methanol fixed and H&E stained cytospin preparations. 2.4. Knockdown of Smad4 by siRNA Synthetic Smad4 siRNA (s: r(CAU-CCU-AGU-AAA-UGU-GUUA) dTdT; as: r(UAA-CAC-AUU-UAC-UAG-GAUG)dAdG) (Qiagen GmbH, Hilden, Germany) was used to silence Smad4. A fluorescein labelled negative siRNA control was used as a transfection control. Cells (3 106) were transfected with 5–10 ll (20 lM) siRNA and 24 ll HiPerFect reagent (Qiagen, Hilden, Germany) in 4 ml medium. Transfection efficiency was determined by flow cytometry after 6–74 h in Smad4 siRNA and fluorescent control siRNA (1:1) co-treated samples and by screening for Smad4 expression and activity. Smad4 expression was detected by Smad4 RT-PCR and Western-blot analysis. Smad4 activity was determined by screening for TIEG (TGF-b induced early gene) mRNA expression in TGF-b1 treated (1–2 h) cultures by RT-PCR. TGF-b1 and rapamycin treatment was initiated 6 h after siRNA transfection. 2.5. RT-PCR Total RNA was isolated from cells (5–10 106) with Qiagen RNeasy kit (Qiagen, Hilden, Germany). RNA was reverse transcribed using MMLV Reverse Transcriptase and random primers (Invitrogen, Carlsbad, CA, USA). cDNA (100 ng) was used for PCR. Equal quantity of cDNA was confirmed by b-actin amplification in control and TGF-b1 treated samples by semiquantitative RTPCR. PCR conditions were as follows: 94 °C 1 min, 55 °C or 60 °C 30 s, 72 °C 45 s; 26–30 cycles using RedTaq polymerase (Sigma, St. Louis, MO, USA). PCR products were resolved by agarose gel (1.5%) electrophoresis, stained with ethidium bromide and analyzed with an Eagle Eye video densitometer (Stratagene, La Jolla, CA, USA). Primers: Smad4 (205 bp, 26–28 cycles) 50 GTG GAA TAG CTC CAG CTA TC30 , 50 CGG CAT GGT ATG AAG TAC TCC30 ; TIEG (229 bp, 28 cycles) 50 ACA GGA GAA AAG CCT TTC AGC30 , 50 TTT TAC ATC ACC ACT GGC TCC30 ; beta-actin (538 bp, 23-25 cycles) 50 GTG-GGG-CGC-CCC-AGG-CAC-CA30 , 50 CTC-CTT-AAT-GTC-ACGCAC-GAT-TTC30 . 2.6. Western-blotting Cells (2 106) were lysed on ice in sample-buffer (100 ll; containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, 10 lg/ml leupeptide and 10% glycerol – Sigma, St. Louis, MO, USA; Bio-Rad, Hercules, CA, USA). Lysates were kept on ice for 10 min and centrifuged at 15,000 g for 20 min to collect the supernatant. Protein concentration was measured with the Bradford assay. Equal amounts of protein were diluted with 2xSDS protein sample buffer (60 mM Tris–HCl, 2% SDS, 20% glycerol, 2% b-mercaptoethanol, bromophenolblue; Sigma, St. Louis, MO, USA; Bio-Rad, Hercules, CA, USA), separated on 12.5% SDS-PAGE gels and blotted onto PVDF
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membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with anti-Smad4/DPC4 (Santa-Cruz Biotech., Santa Cruz, CA, USA) anti-phospho-S6, anti-phospho-p70S6K (Cell Signaling Technology, Beverly, MA, USA), followed by the appropriate secondary antibodies (HRP conjugated anti-mouse or HRP conjugated anti-rabbit; Cell Signaling Technology, Beverly, MA, USA), and developed with ECL (Pierce, Rochford, IL, USA). Membranes were stained with PonceauS and also stripped and re-probed with anti-beta-actin antibody (both from Sigma, St. Louis, MO, USA) to confirm equal protein loading. 2.7. In vivo xenograft model Xenograft tumors were established as described previously [23]. HT58 cells (2 107) were injected subcutaneously in SCID mice. Treatment was initiated after palpable tumours (of 5 mm diameter) formed (15 days after xenograft injection). Rapamycin (Rapamune, Wyeth-Pfizer) was administered by gavage at 3 mg/ kg body weight 5 times a week (n = 10), according to previously described and tested doses. Body weight and tumour diameter were monitored. Tumour weight was measured in euthanized animals after 14 days of treatment at the end of experiments. All procedures were approved by the Institutional Review Board of our university. 2.8. Immunohistochemistry and immunocytochemistry Antigen retrieval was achieved by heat treatment in a microwave oven or pressure cooker for 10–30 min in citrate buffer (pH6) on sections from formalin fixed paraffin embedded tissues. Slides were incubated with primary antibodies (anti-phospho-S6, anti-phospho-p70S6K, anti-phospho-4EBP1, anti-phospho-Histone H3 and anti-cleaved caspase 3 – Cell Signaling Technology) at 4 °C overnight. Vectastain Elite Universal (Vector Laboratories, Burlingame, CA, USA) or Novolink (Novocastra, Wetzlar, Germany) systems were used for immunodetection, visualized by DAB and counterstained with haematoxylin. For quantitative evaluation of phopho-Histone H3 and cleaved caspase 3 positivity in lymphoma xenografts, cells were counted in untreated and treated tissue samples; the number of positive cells/500 cells in untreated tissues was considered 100%. 2.9. Statistics Statistics was calculated with paired Student’s t-test using Microsoft Excel. P < 0.05 was considered statistically significant. 3. Results 3.1. Rapamycin restores or increases the apoptotic effect of TGF-b1 in high grade lymphoma cells in vitro Flow cytometry showed that rapamycin inhibited cell proliferation and lead to G1 cell cycle arrest in high grade lymphomas, in vitro (Fig. 1a). Due to this cell cycle block, cell numbers were reduced by 50–70%. No significant increase was detected in apoptosis (8–15%), even after 72 h treatment with high dose rapamycin (1–10 lg/ml). The doses used here were higher than those used for immunosuppression in organ transplantation. However, rapamycin (even in doses as low as 50 ng/ml) was able to restore the apoptotic response to TGF-b1 in cell lines with low TGF-b1 sensitivity such as HT58r. Furthermore, rapamycin + TGF-b1 treatment increased apoptosis in the majority of lymphoma cell lines (HT58, BL41 and Ramos) (Fig. 1b). Apoptosis induced by rapamycin + TGF-b1 was time-dependent. This combination
Fig. 1. Rapamycin treatment enhances the apoptotic effect of TGF-b1 in lymphoma cells in vitro. (a) Rapamycin induces G1 cell cycle arrest in lymphoma cells. Representative flow cytometric diagram shows cell cycle distribution of HT58 cells treated with rapamycin (Rap) for 72 h (arrows show G1 peaks in control and rapamycin treated cells). (b) Rapamycin (Rap) combination significantly enhances the apoptotic effect of TGF-b1 (TGF) treatment after 72 h in the majority of the examined cell lines (⁄p < 0.05, flow cytometry).
overcame TGF-b1 resistance in cell lines with low TGF-b1 sensitivity (HT58r), which was comparable to TGF-b1 monotreatment in HT58 or Ramos. However, rapamycin had no such significant effect in U266 and BL41/95 cells. 3.2. Characterization of Rapamycin + TGF-b1 induced effects and their role in the altered signalling mechanisms in lymphoma cells We confirmed in in vitro experiments that the kinase activity of TGF-b receptor I (TGF-b RI/ALK1) is required for its activation and apoptosis induction both by TGF-b1 and by rapamycin + TGF-b1 treatments. Apoptosis induced by these treatments was completely abolished by a TGF-b RI/ALK1-inhibitor (Fig. 2a). We also found that the active, phosphorylated form of p70S6K (pp70S6K) and ribosomal S6 protein (p-S6) – targets of mTORC1 – was slightly decreased by TGF-b1, remarkably decreased by rapamycin and completely eliminated by rapamycin + TGF-b1 in HT58 lymphoma cells after 24 h treatment (Fig. 2b). The in vitro effect of the PI3K inhibitor LY294002 was also examined in HT58r cells. LY294002 was able to enhance the apoptotic effect of TGF-b1 in HT58r cells, similarly to rapamycin. Moreover, the addition of LY294002 to rapamycin + TGF-b1 (triple combination) further increased the rate of apoptosis (Fig. 2a). It was shown previously that TGF-b1 induced apoptosis in lymphoma cells was PP2A-dependent, but independent of Smad4 (12). Similarly, apoptosis induced by rapamycin + TGF-b1 required no functioning Smad4 (Fig. 3a–c). Knockdown of Smad4 by siRNA inhibited the expression of TGF-b1 induced early gene (TIEG), which is a well known target gene of the ’’classical’’ TGF-b-Smad signalling pathway. A short pretreatment with the PP2A inhibitor okadaic acid (used only in the first 4 h of the treatment) blocked the apoptotic effect of rapamycin + TGF-b1, which supports the importance of PP2A-dependent induction in certain lymphoma cells (Fig. 4a).
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Fig. 2. TGF-b RI/ALK1 and PI3K inhibitors modify the apoptotic effect of rapamycin and rapamycin + TGF-b1. (a) The effect of TGF-b RI/ALK1 inhibitor (ALK1-I) and PI3K inhibitor (LY294002) on rapamycin + TGF-b1 (Rap + TGF) induced apoptosis in lymphoma cells with low TGF-b1 sensitivity (HT58r) after 72 h treatment (⁄p < 0.05, flow cytometry). (b) Low dose rapamycin monotreatment and rapamycin + TGF-b1 (Rap + TGF) decreases the amount of p-p70S6K and its target p-S6 protein after 24 h in HT58 lymphoma cells (Western-blot).
pathway. Inhibition of mTORC1 by rapamycin was able to restore the ability of TGF-b1 to induce apoptosis in certain lymphoma cell lines. We also found that TGF-b1 sensitivity could not be restored by rapamycin in U266 and BL41/95 cells. In these cell lines, the downregulation/loss of TGF-b RII expression may be responsible for TGF-b1 resistance. These results emphasize the requirement of active TGF-b receptors for effective apoptosis induction by TGF-b1 and rapamycin + TGF-b1, which is further supported by our experiments using a TGF-b RI/ALK1-inhibitor. Similarly to our results, TGF-b induced differentiation showed synergism with mTOR inhibition (by rapamycin) in regulatory Tcell development in different models [25]. Moreover, mTOR activity inhibited Smad3 phosphorylation in HCV related hepatocellular carcinoma as well [19]. However, it is also known that TGF-b1 induced epithelial mesenchymal transition and kidney fibrogenesis may depend on TGF-b induced mTOR activity. In light of all these results, we should keep in mind that signalling networks are complex and cell type dependent, and TGF-b and mTOR signalling have diverse, sometimes opposing effects [26–28]. There are several explanations for the effect of combined rapamycin + TGF-b1 treatment in B lymphoma cells. A plausible target would be FKBP12, a guardian of TGF-b R, negatively regulating the receptor – preventing the receptor from leaky signals at suboptimal ligand concentration. At the same time, FKBP12 is a known rapamycin-binding protein [29]. Therefore, it is possible
TGF-b1 induced apoptosis was caspase-dependent. However, apoptosis induced by rapamycin + TGF-b1 was not completely eliminated by Z-VAD-fmk, a caspase inhibitor (Fig. 4b), indicating that co-treatment with rapamycin engaged a caspase-independent mechanism as well. 3.3. Rapamycin treatment inhibits proliferation and induces apoptosis in vivo in lymphoma xenografts mTOR activity related phosphorylated proteins (p-mTOR, p-p70S6K, p-4EBP1 and p-S6) were detected in biopsies of Burkitt-lymphoma patients [24] and in HT58 lymphoma xenografts, which suggests that enhanced mTOR signalling activity is present in vivo (Supplemented Figure), which may serve as a potential therapeutic target. To prove the in vivo relevance of our observations, mice with HT58 lymphoma xenografts were treated with low dose Rapamune. Short term (2 weeks) treatment lead to remarkable and significant growth suppression of the tumours as indicated by tumour weight (3.98 ± 0.25 g vs. 8.9 ± 0.6 g in untreated vs. Rapamune-treated animals, respectively). Immunohistochemical staining of xenografts for phospho-Histone H3 (pHH3; a mitotic marker) revealed inhibition of proliferation: the number of pHH3-positive cells was reduced to 27% in Rapamune-treated xenografts, compared to untreated tumours. In addition, Rapamune treatment resulted in an increase of apoptosis in vivo: the number of tumour cells positive for cleaved/activated caspase 3 (an apoptosis marker) showed a 6.64-fold increase (Supplemented Figure). 4. Discussion Our results suggest that elevated mTORC1 activity is likely to contribute to TGF-b1-resistance in several high grade B lymphoma cell lines, even in the presence of a theoretically functional TGF-b-
Fig. 3. The effect of rapamycin and TGF-b1 combination is Smad4-independent in lymphoma cells. (a) Percentage of apoptotic cells after TGF-b1 and rapamycin treatment in control and Smad4 siRNA transfected cells; 72 h treatment (flow cytometry). (b) Smad4 protein expression after siRNA transfection in control, rapamycin and TGF-b1 treated cells; 2 h treatment (Western-blot). (c) TIEG mRNA expression in siRNA transfected cells after rapamycin and TGF-b1 treatment; 2 h treatment (RT-PCR). (Co: control; TGF-b1: TGF; rapamycin: Rap; TGF-b1 and rapamycin treatment was initiated 6 h after siRNA transfection).
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Fig. 4. The apoptotic effect of rapamycin + TGF-b1 combination requires early PP2A-activity and is partially caspase dependent. (a) The PP2A inhibitor okadaic acid abolishes TGF-b1-induced and rapamycin + TGF-b1-induced apoptosis in HT58 lymphoma cells (flow cytometry). (b) Z-VAD-fmk caspase inhibitor completely abolishes TGF-b1-induced apoptosis but only partially inhibits rapamycin + TGF-b1 induced apoptosis in lymphoma cells with low TGF-b1 sensitivity (HT58r). (72 h treatment; co: control; TGF-b1: TGF; Rap: rapamycin; oka: okadaic acid; no inh: without PP2A inhibitor treatment; apoptosis was detected by flow cytometry.).
that rapamycin neutralizes available FKBP12 molecules, thereby allowing the activation of TGF-b R for apoptosis induction during rapamycin + TGF-b1 treatment. However, it is very likely that FKBP12 is not the sole mechanism of action for rapamycin, as mTORC1 signalling can promote survival in several ways. The role of mTORC1 activity in TGF-b1 resistance has already been observed in non lymphoid cell types, e.g. epithelial cell lines. IL-4 prevented TGF-b1 induced apoptosis through the activation of AKT and p70S6K in human hepatocellular and breast carcinoma cells [30]. It was also suggested that mTORC1 can activate SGK1 (serum- and glucocorticoid-inducible kinase 1) to regulate p27kip phosphorylation, leading to TGF-b1 resistance in mammalian carcinoma cells [31]. Our results are the first to demonstrate the contribution of mTORC1 activity to developing resistance against TGFb1 induced apoptosis in selected lymphoma cells. We found previously that the apoptotic effect of TGF-b1 was independent of Smad4, i.e. the ’’classical’’ TGF-b pathway in lymphoma cell lines [14]. Using Smad4 siRNA transfection, apoptosis induced by rapamycin + TGF-b1 proved to be also Smad4-independent in our present experiments. Our previous and current results suggest that the TGF-b1/TGF-bR/PP2A pathway has an important role in growth inhibition and apoptosis in lymphoma cells with inhibited mTORC1 activity. mTORC1 is an important element in the cellular signalling network, and a powerful supporter of cell survival. Its activity is able to block apoptosis (e.g. induced by TGF-b1) in lymphoma cells. Using a PI3K inhibitor in addition to rapamycin and TGF-b1, apoptosis was further increased, compared to the TGF-b + rapamycin combination. Similar observations were made in different tumours and hematological malignancies, which suggest that PI3K
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inhibitors and rapamycin can potentiate each other’s effect; many clinical studies have recently been initiated based on this, both in lymphoid and solid malignancies [32]. We previously found that caspase inhibitors blocked TGF-b1 induced apoptosis in lymphoma cells [17]. Interestingly, a portion (about a third) of rapamycin + TGF-b1 induced apoptosis was caspase independent. This pan-caspase inhibitor resistant cell death can be explained by different mechanisms, e.g. by a crosstalk between ’’self-eating’’/autophagy and ’’self-killing’’/apoptosis. The role of mTOR inhibitors in autophagy induction is well known, and TGF-b1 has also been reported to activate autophagy in kidney epithelial and hepatocellular carcinoma cells [33–35]. Caspase independent mitochondrial endonuclease G release was observed in HT58 lymphoma cells after HRP4/ATRA treatment [36], which can also contribute to caspase independent DNA fragmentation. Taken together: (a) PI3K/mTORC1 activity contributes to resistance against TGF-b1-induced apoptosis in high grade lymphoma cells; (b) rapamycin enhances the growth inhibitory effect of TGF-b1 and switches on the TGF-b1 dependent apoptotic program in B lymphoma cells; (c) rapamycin + TGF-b1 induced apoptosis is Smad4–independent and PP2A dependent in these lymphoma cells (Fig. 5). We confirmed the growth inhibitory effect of low dose Rapamune treatment in our in vivo xenograft model. We detected not only a reduction in cell proliferation but also apoptosis induction in tissue specimens after monotreatment, which is in contrast to our in vitro experiments, where rapamycin by itself did not increase apoptosis in the examined cell lines. These results underline the potential role of mTORC1 inhibition in switching on the apoptotic effect of pro-apoptotic regulators (such as TGF-b1) in the examined lymphomas. We focused on TGF-b1 in our study; however, there are many other negative regulators (e.g. cytokines) in tumour cells and also in their environment, which may be suppressed by mTORC1 activation – this may also explain the fact that rapamycin effectively induced apoptosis in vivo, but not in vitro. Our results suggest that high mTOR activity contributes to TGFb resistance and that lowering mTORC1 activity may provide a tool in high grade B-cell lymphoma therapy. We presume that mTOR inhibition may promote therapeutic success in lymphomas and
Fig. 5. PI3K/mTOR activity contribute to the survival and proliferation of TGF-b1 resistant lymphoma cells. The internal apoptotic program can be switched on by TGF-b1 in the examined B lymphoma cells. This mechanism is PP2A dependent and it is independent of ’’classical’’ Smad4/TGF-b signalling (as shown by siRNA knockdown). PI3K/mTOR activity can support survival and TGF-b1 resistance in lymphoma cells. Rapamycin and PI3K inhibitors can reverse this effect in TGF-b1 resistant lymphoma cells. Moreover, blocking PI3K activity can further increase the effect of rapamycin and TGF-b1 treatment. (inh: inhibitor).
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other tumours by increasing or restoring sensitivity to currently available treatment modalities [32,37–39] and to endogenous regulators such as TGF-b1. However, the complex in vivo effects of mTOR inhibition, the availability of endogenous negative regulators (including TGF-b1) and the net effect of these factors need to be clarified in further studies. Further data is required to elucidate the impact of mTOR inhibition on the microenvironment – such as tumour related inflammation in the case of non-lymphoid tumours – or on the pleiotropic effect of negative regulatory cytokines in tumor matrix elements. Studies on different lymphomas showed that rapamycin treatment could be beneficial in high grade lymphomas and refractory Hodgkin lymphomas as well. However, the role of potentially available in vivo negative regulators and their release under inhibition were not examined in these cases. The activity of mTOR is often elevated in a variety of tumours, which can be easily detected in biopsy materials. Increased mTOR activity very likely leads to resistance not only to endogenous negative regulators but also to therapeutic drugs. In these cases, drugs targeting PI3K or mTOR may be added to the regimen, in order to reverse therapeutic resistance, and they may also potentiate each other. As a result, a decrease in mTOR activity (after a shorter time period) may be followed by a beneficial therapeutic effect and a reduction in tumour burden after a longer time. Pharmacological inhibition of the PI3K-AKT-mTOR pathway is a puzzling but promising approach in the development of novel anticancer therapies [37], especially in the light of the emerging problem of drug resistance in targeted therapy. Data about elevated mTORC1-activation in numerous solid tumours and lymphomas are increasing. Our experimental and in vivo xenograft results suggest that the addition of rapalogues to treatment protocols could be beneficial not only in mantle cell lymphomas (MCLs) but also in other lymphomas with high mTORC1 activity – such as Hodgkin lymphomas, non-germinal center type diffuse large B-cell lymphomas and acute lymphoblastic leukemias [24,37–40]. Acknowledgements We would like to express our thanks to Gézáné Csorba, Lilla Kis, Viktória Varga, Titanilla Dankó and András Sztodola for their technical help. This work was supported by the Hungarian Scientific Research Found [grant numbers: OTKA F048380, T81624, T84262]. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.02.024. References [1] Buhl AM, Nemazee D, Cambier JC, Rickert R, Hertz M. B-cell antigen receptor competence regulate selection and survival. Immunol Rev 2000;176:154–70. [2] Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 2014;15:155–62. [3] Laplante M, Sabatini DM. MTOR signaling in growth control and disease. Cell 2012;149:274–93. [4] Ghayad SE, Cohen PA. Inhibitors of the PI3K/Akt/mTOR pathway: new hope for breast cancer patients. Recent Pat Anticancer Drug Discov 2010;5:29–57. [5] Wysocki PJ. MTOR in renal cell cancer: modulator of tumor biology and therapeutic target. Expert Rev Mol Diagn 2009;9:231–41. [6] Younes A, Samad N. Utility of mTOR inhibition in hematologic malignancies. Oncologist 2011;16:730–1. [7] Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N. Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta 2010;1804: 433–9. [8] Rivera-Rodriguez N, Cabanillas F. Recent advances in the management of mantle cell lymphoma. Curr Opin Oncol 2013;25:716–21. [9] Arita A, McFarland DC, Myklebust JH, Parekh S, Petersen B, Gabrilove J, et al. Signaling pathways in lymphoma: pathogenesis and therapeutic targets. Future Oncol 2013;9:1549–71.
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Smad4-independent, PP2Adependent apoptotic effect of exogenous transforming growth factor beta 1 in lymphoma cells. Exp Cell Res 2007;313:3167–74. [18] Tótth Á, Sebestyén A, Barna G, Nagy K, Göndör A, Bocsi J, et al. TGFb1 induces caspase-dependent but death-receptor independent apoptosis in lymphoid cells. Anticancer Res 2001;21:1207–12. [19] Song K, Wang H, Krebs TL, Danielpour D. Novel roles of Akt and mTOR in suppressing TGF-beta/ALK5-mediated Smad3 activation. EMBO J 2006;25: 58–69. [20] Lamouille S, Derynck R. Cell size and invasion in TGF-beta-induced epithelial to mesenchymal transition is regulated by activation of the mTOR pathway. J Cell Biol 2007;178:437–51. [21] Kopper L, Bánkfalvy A, Mihalik R, Nagy P, Fülöp Cs, Sármay G. New in vitro line from a human (B) non-Hodgkin lymphoma. Anticancer Res 1991;11:1645–50. [22] Mihalik R, Uher F, Pocsik É, Berczi L, Benczur M, Kopper L. Detection of druginduced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells. Pathol Oncol Res 1996;2:78–83. } Gy, Sebestyén A, Paku S, Barna G, Hajdu M, Tóth M, et al. [23] Végso Antiproliferative and apoptotic effects of mycophenolic acid in human B-cell non-Hodgkin lymphomas. Leuk Res 2007;31:1003–8. [24] Márk Á, Hajdu M, Váradi Zs, Sticz TB, Nagy N, Csomor J, et al. Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease–a combined tissue microarray, in vitro and in vivo study. BMC Cancer 2013;13:250. http://dx.doi.org/10.1186/1471-2407-13-25. [25] Jhunjhunwala S, Cen LC, Nichols EE, Thomson AW, Raimondi G, Little SR. Alltrans retinoic acid and rapamycin synergize with transforming growth factorb1 to induce regulatory T cells but confer different migratory capacities. J Leukoc Biol 2013. http://dx.doi.org/10.1189/jlb.0312167. [26] Zhang L, Zhou F, ten Dijke P. Signaling interplay between transforming growth factor-b receptor and PI3K/Akt pathways in cancer. Trends Biochem Sci 2013;38:612–20. [27] Rozen ZB, Hayashida T, Hubchak SC, Hanna C, Platanias LC, William Schnaper H. TGF-b/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1a expression. Am J Physiol Renal Physiol 2013;305. F485-94. [28] Chen CI, Tsukamoto H, Liu JC, Kashiwabare C, Feldman D, Sher L, et al. Reciprocal regulation by TLR4 and TGF-b in tumor infiltrating stem-like cells. J Clin Invest 2013;123:2832–49. [29] Wang T, Donahoe PK. The immunophilin FKBP12: a molecular guardian of the TGF-beta family type I receptors. Front Biosci 2004;9:619–31. [30] Lin SJ, Chang C, Ng AK, Wang SH, Li JJ, Hu CP. Prevention of TGF-beta-induced apoptosis by interleukin-4 through Akt activation and p70S6K survival signaling pathways. Apoptosis 2007;12:1659–70. [31] Hong F, Larrea MD, Doughty C, Kwiatkowski DJ, Squillace R, Slingerland JM. MTOR-raptor binds and activates SGK1 to regulate p27 phosphorylation. Mol Cell 2008;30:701–11. [32] Rodon J, Dienstmann R, Serra V, Tabernero J. Development of PI3K inhibitors: lessons from early clinical trials. Nat Rev Clin Oncol 2013;10:143–53. [33] Ding Y, Kim JK, Kim SI, Na HJ, Jun SY, Lee SJ, et al. TGF-beta1 protects against mesangial cell apoptosis via induction of autophagy. J Biol Chem 2010;285:37909–19. [34] Gajewska M, Gajkowska B, Motyl T. Apoptosis and autophagy induced by TGFb1 in bovine mammary epithelial BME-UV1 cells. J Physiol Pharmacol 2005;56:143–57. [35] Xu Y, Kim SO, Li Y, Han J. Autophagy contributes to caspase-independent macrophage cell death. J Biol Chem 2006;281:19179–87. [36] Barna G, Sebestyén A, Weischede S, Peták I, Mihalik R, Formelli F, et al. Different ways to induce apoptosis by fenretinide and all-trans-retinoic acid in human B lymphoma cells. Anticancer Res 2005;25:4179–85. [37] Fruman DA, Rommel C. 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Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas } c, Anna Sebestyén a,b,⇑, Ágnes Márk a, Melinda Hajdu a, Noémi Nagy a, Anna Molnár a, Gyula Végso Gábor Barna a, László Kopper a a
}i út 26., Hungary Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest 1085, Üllo }i út 26, Hungary Tumor Progression Research Group of Joint Research Organization of the Hungarian Academy of Sciences and Semmelweis University, Budapest 1085, Üllo c Semmelweis University, Department of Transplantation and Surgery, Budapest 1082, Baross u. 23, Hungary b
a r t i c l e
i n f o
Article history: Received 4 November 2014 Received in revised form 24 January 2015 Accepted 3 February 2015
Keywords: Rapamycin TGF-b mTOR Apoptosis Lymphoma
a b s t r a c t TGF-b1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-b1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-b signaling, and through these pathways it is able to enhance TGF-b induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-b1 combination on cell growth and on TGF-b and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-b1 induced apoptosis and restored TGF-b1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-b sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin + TGF-b1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin + TGF-b1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-b resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-b1. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Apoptosis is the default program for B-cells in the absence of survival stimuli. Therefore, the balance between pro- and antiapoptotic signals are essential for normal B-cell development and function [1]. Early stages of tumorigenesis share similarities with the suppression of apoptotic pathways leading to continuous survival. ⇑ Corresponding author at: Semmelweis University, 1st Department of Pathology } i út 26, Hungary. Tel.: +36 1 and Experimental Cancer Research, Budapest 1085, Üllo 2661638/54447; fax: +36 1 3171074. E-mail addresses: [email protected] (A. Sebestyén), [email protected] (Á. Márk), [email protected] (M. Hajdu), [email protected] (N. Nagy), [email protected] (A. Molnár), [email protected]. }), [email protected] (G. Barna), [email protected] (L. Kopper). hu (G. Végso
http://dx.doi.org/10.1016/j.cyto.2015.02.024 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
Mammalian target of rapamycin complex 1 (mTORC1) lies at the crossroads of signalling networks [2,3], controlled by a wide variety of factors in favour of cell survival. The activation of mTORC1 is an early and frequent event in several tumors [4–6]. Rapamycin (an inhibitor of mTORC1 and a known immunosuppressive agent) and its analogues (rapalogues) inhibit the activity of mTORC1 and the phosphorylation of its downstream targets, p70 ribosomal S6 kinase (p70S6K), ribosomal S6 protein and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) [7]. The inhibition of mTORC1 yielded therapeutical benefits – such as inhibition of tumor progression and extended overall survival – in recent clinical trials for acute myeloid leukemia (AML), mantle cell lymphoma (MCL), other high grade lymphomas and in non-lymphoid malignancies as well [8–10]. TGF-b1 is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal
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lymphoid cells [11]. As expected, TGF-b1 acts as a potent tumour suppressor during the early stages of lymphomagenesis; however, it can promote progression at later stages [12]. The pro-oncogenic function of TGF-b1 signalling may be associated with the loss of TGF-b1 pathway activity and the engagement of other signalling mechanisms, which act in concert during the process of carcinogenesis [13]. While malignant lymphoid cells often lose their sensitivity to proapoptotic/antiproliferative regulators, the failure of the ’’classical’’ (Smad-dependent) TGF-b1 pathway is uncommon in lymphoid tumours [14], which raises the importance of ’’nonclassical’’ (Smad-independent) TGF-b1 signalling as well as the modulatory role of various interacting proteins [15,16]. We have previously shown that exogenous TGF-b1 can induce apoptosis in high grade lymphoma cells in a Smad4-independent and PP2A-dependent manner, primarily through the mitochondrial apoptotic pathway (caspase 9 and 3 activation mediated by FAS, TRAIL and TNF-a receptor independent mitochondrial depolarization) [17,18]. It has been suggested that TGF-b1 resistance could be reversed by lowering the ’’survival threshold’’ of cells in certain cases [17]. The interaction between TGF-b1 and mTORC1 signalling and their simultaneous targeting (by TGF-b1 plus rapamycin treatment) has been explored in some cell types, yielding conflicting results [19,20]. It is suggested that rapamycin can modulate TGF-b1 responses by effectively binding to FKBP-12, an inhibitor of TGF-b receptors. The sequestration of FKBP-12 may facilitate TGF-b responses; however, other mechanisms should also be considered. In the present work we found that inhibition of mTORC1 by rapamycin was able to restore the effectiveness of TGF-b1 to induce apoptosis in high grade B-cell non-Hodgkin lymphomas in vitro. 2. Materials and methods 2.1. Cell culture Experiments were performed on the following human B-cell non-Hodgkin lymphoma (B-NHL) cell lines: HT58 [21] and HT58r (EBV negative cell lines established in our laboratory; HT58r is a subclone of HT58 with low TGF-b1 sensitivity); BL41, BL41/95 (EBV-transfected variant of BL41); Ramos and U266. Cells were cultured in RPMI-1640 (Sigma, St. Louis, MO, USA) with 10% fetal bovine serum (GIBCO-BRL, Grand Island, NY, USA), 0.03% glutamine (, Sigma, St. Louis, MO, USA) and penicillin-streptomycin (100 U/ mL–100 lg/mL, Sigma, St. Louis, MO, USA), at 37 °C in 5% CO2 atmosphere. Cells in the exponential growth phase were used for all experiments. 2.2. Special treatments Cells (at a density of 1–2 105/ml) were treated with 1 ng/mL TGF-b1 (reconstituted with 4 mM HCl in 0.1% BSA, aliquoted and stored at 80 °C; R&D Systems, Minneapolis, MN, USA), 50 ng/ml rapamycin (also referred to as low dose – except where indicated otherwise; Sigma, St. Louis, MO, USA), SB431542 TGF-b RI/ALK1 (TGF-b receptor I/activin receptor like kinase-1) 5 lM inhibitor (Sigma, St. Louis, MO, USA), 100 nM okadaic acid (Sigma, St. Louis, MO, USA), LY294002 (5 lM, Calbiochem), Z-VAD-fmk (Sigma, St. Louis, MO, USA, 50 lM) for 0–72 h in 24-well plates or 25 cm2 flasks. Okadaic acid was used only in the first 4 h of the treatment (short pretreatment), cells were then washed and replated in rapamycin supplemented medium for 72 h. Rapamycin and TGF-b1 were added at the same time in our experiments, as 1–6–12 h pretreatment with either agent showed no
significant difference in apoptosis compared to combined treatment (data not shown). 2.3. Cell cycle analysis and apoptosis detection Apoptosis detection by flow cytometry was performed according to Mihalik et al. [22]. Briefly, cells were fixed in 70% ethanol (20 °C) followed by alkalic extraction (200 mM Na2HPO4, pH 7.4 and 100 lg/ml RNase, Sigma, St. Louis, MO, USA) and ethidium bromide staining (10 lg/ml, Sigma, St. Louis, MO, USA). For each sample, 10,000-20,000 events were acquired using a FACScan flow cytometer (Becton-Dickinson, BD Biosciences, San Diego, CA, USA). Data were analyzed with WinList software (Verity Software House, Topsman, ME, USA). Cell morphology was evaluated on methanol fixed and H&E stained cytospin preparations. 2.4. Knockdown of Smad4 by siRNA Synthetic Smad4 siRNA (s: r(CAU-CCU-AGU-AAA-UGU-GUUA) dTdT; as: r(UAA-CAC-AUU-UAC-UAG-GAUG)dAdG) (Qiagen GmbH, Hilden, Germany) was used to silence Smad4. A fluorescein labelled negative siRNA control was used as a transfection control. Cells (3 106) were transfected with 5–10 ll (20 lM) siRNA and 24 ll HiPerFect reagent (Qiagen, Hilden, Germany) in 4 ml medium. Transfection efficiency was determined by flow cytometry after 6–74 h in Smad4 siRNA and fluorescent control siRNA (1:1) co-treated samples and by screening for Smad4 expression and activity. Smad4 expression was detected by Smad4 RT-PCR and Western-blot analysis. Smad4 activity was determined by screening for TIEG (TGF-b induced early gene) mRNA expression in TGF-b1 treated (1–2 h) cultures by RT-PCR. TGF-b1 and rapamycin treatment was initiated 6 h after siRNA transfection. 2.5. RT-PCR Total RNA was isolated from cells (5–10 106) with Qiagen RNeasy kit (Qiagen, Hilden, Germany). RNA was reverse transcribed using MMLV Reverse Transcriptase and random primers (Invitrogen, Carlsbad, CA, USA). cDNA (100 ng) was used for PCR. Equal quantity of cDNA was confirmed by b-actin amplification in control and TGF-b1 treated samples by semiquantitative RTPCR. PCR conditions were as follows: 94 °C 1 min, 55 °C or 60 °C 30 s, 72 °C 45 s; 26–30 cycles using RedTaq polymerase (Sigma, St. Louis, MO, USA). PCR products were resolved by agarose gel (1.5%) electrophoresis, stained with ethidium bromide and analyzed with an Eagle Eye video densitometer (Stratagene, La Jolla, CA, USA). Primers: Smad4 (205 bp, 26–28 cycles) 50 GTG GAA TAG CTC CAG CTA TC30 , 50 CGG CAT GGT ATG AAG TAC TCC30 ; TIEG (229 bp, 28 cycles) 50 ACA GGA GAA AAG CCT TTC AGC30 , 50 TTT TAC ATC ACC ACT GGC TCC30 ; beta-actin (538 bp, 23-25 cycles) 50 GTG-GGG-CGC-CCC-AGG-CAC-CA30 , 50 CTC-CTT-AAT-GTC-ACGCAC-GAT-TTC30 . 2.6. Western-blotting Cells (2 106) were lysed on ice in sample-buffer (100 ll; containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium vanadate, 10 lg/ml leupeptide and 10% glycerol – Sigma, St. Louis, MO, USA; Bio-Rad, Hercules, CA, USA). Lysates were kept on ice for 10 min and centrifuged at 15,000 g for 20 min to collect the supernatant. Protein concentration was measured with the Bradford assay. Equal amounts of protein were diluted with 2xSDS protein sample buffer (60 mM Tris–HCl, 2% SDS, 20% glycerol, 2% b-mercaptoethanol, bromophenolblue; Sigma, St. Louis, MO, USA; Bio-Rad, Hercules, CA, USA), separated on 12.5% SDS-PAGE gels and blotted onto PVDF
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membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with anti-Smad4/DPC4 (Santa-Cruz Biotech., Santa Cruz, CA, USA) anti-phospho-S6, anti-phospho-p70S6K (Cell Signaling Technology, Beverly, MA, USA), followed by the appropriate secondary antibodies (HRP conjugated anti-mouse or HRP conjugated anti-rabbit; Cell Signaling Technology, Beverly, MA, USA), and developed with ECL (Pierce, Rochford, IL, USA). Membranes were stained with PonceauS and also stripped and re-probed with anti-beta-actin antibody (both from Sigma, St. Louis, MO, USA) to confirm equal protein loading. 2.7. In vivo xenograft model Xenograft tumors were established as described previously [23]. HT58 cells (2 107) were injected subcutaneously in SCID mice. Treatment was initiated after palpable tumours (of 5 mm diameter) formed (15 days after xenograft injection). Rapamycin (Rapamune, Wyeth-Pfizer) was administered by gavage at 3 mg/ kg body weight 5 times a week (n = 10), according to previously described and tested doses. Body weight and tumour diameter were monitored. Tumour weight was measured in euthanized animals after 14 days of treatment at the end of experiments. All procedures were approved by the Institutional Review Board of our university. 2.8. Immunohistochemistry and immunocytochemistry Antigen retrieval was achieved by heat treatment in a microwave oven or pressure cooker for 10–30 min in citrate buffer (pH6) on sections from formalin fixed paraffin embedded tissues. Slides were incubated with primary antibodies (anti-phospho-S6, anti-phospho-p70S6K, anti-phospho-4EBP1, anti-phospho-Histone H3 and anti-cleaved caspase 3 – Cell Signaling Technology) at 4 °C overnight. Vectastain Elite Universal (Vector Laboratories, Burlingame, CA, USA) or Novolink (Novocastra, Wetzlar, Germany) systems were used for immunodetection, visualized by DAB and counterstained with haematoxylin. For quantitative evaluation of phopho-Histone H3 and cleaved caspase 3 positivity in lymphoma xenografts, cells were counted in untreated and treated tissue samples; the number of positive cells/500 cells in untreated tissues was considered 100%. 2.9. Statistics Statistics was calculated with paired Student’s t-test using Microsoft Excel. P < 0.05 was considered statistically significant. 3. Results 3.1. Rapamycin restores or increases the apoptotic effect of TGF-b1 in high grade lymphoma cells in vitro Flow cytometry showed that rapamycin inhibited cell proliferation and lead to G1 cell cycle arrest in high grade lymphomas, in vitro (Fig. 1a). Due to this cell cycle block, cell numbers were reduced by 50–70%. No significant increase was detected in apoptosis (8–15%), even after 72 h treatment with high dose rapamycin (1–10 lg/ml). The doses used here were higher than those used for immunosuppression in organ transplantation. However, rapamycin (even in doses as low as 50 ng/ml) was able to restore the apoptotic response to TGF-b1 in cell lines with low TGF-b1 sensitivity such as HT58r. Furthermore, rapamycin + TGF-b1 treatment increased apoptosis in the majority of lymphoma cell lines (HT58, BL41 and Ramos) (Fig. 1b). Apoptosis induced by rapamycin + TGF-b1 was time-dependent. This combination
Fig. 1. Rapamycin treatment enhances the apoptotic effect of TGF-b1 in lymphoma cells in vitro. (a) Rapamycin induces G1 cell cycle arrest in lymphoma cells. Representative flow cytometric diagram shows cell cycle distribution of HT58 cells treated with rapamycin (Rap) for 72 h (arrows show G1 peaks in control and rapamycin treated cells). (b) Rapamycin (Rap) combination significantly enhances the apoptotic effect of TGF-b1 (TGF) treatment after 72 h in the majority of the examined cell lines (⁄p < 0.05, flow cytometry).
overcame TGF-b1 resistance in cell lines with low TGF-b1 sensitivity (HT58r), which was comparable to TGF-b1 monotreatment in HT58 or Ramos. However, rapamycin had no such significant effect in U266 and BL41/95 cells. 3.2. Characterization of Rapamycin + TGF-b1 induced effects and their role in the altered signalling mechanisms in lymphoma cells We confirmed in in vitro experiments that the kinase activity of TGF-b receptor I (TGF-b RI/ALK1) is required for its activation and apoptosis induction both by TGF-b1 and by rapamycin + TGF-b1 treatments. Apoptosis induced by these treatments was completely abolished by a TGF-b RI/ALK1-inhibitor (Fig. 2a). We also found that the active, phosphorylated form of p70S6K (pp70S6K) and ribosomal S6 protein (p-S6) – targets of mTORC1 – was slightly decreased by TGF-b1, remarkably decreased by rapamycin and completely eliminated by rapamycin + TGF-b1 in HT58 lymphoma cells after 24 h treatment (Fig. 2b). The in vitro effect of the PI3K inhibitor LY294002 was also examined in HT58r cells. LY294002 was able to enhance the apoptotic effect of TGF-b1 in HT58r cells, similarly to rapamycin. Moreover, the addition of LY294002 to rapamycin + TGF-b1 (triple combination) further increased the rate of apoptosis (Fig. 2a). It was shown previously that TGF-b1 induced apoptosis in lymphoma cells was PP2A-dependent, but independent of Smad4 (12). Similarly, apoptosis induced by rapamycin + TGF-b1 required no functioning Smad4 (Fig. 3a–c). Knockdown of Smad4 by siRNA inhibited the expression of TGF-b1 induced early gene (TIEG), which is a well known target gene of the ’’classical’’ TGF-b-Smad signalling pathway. A short pretreatment with the PP2A inhibitor okadaic acid (used only in the first 4 h of the treatment) blocked the apoptotic effect of rapamycin + TGF-b1, which supports the importance of PP2A-dependent induction in certain lymphoma cells (Fig. 4a).
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Fig. 2. TGF-b RI/ALK1 and PI3K inhibitors modify the apoptotic effect of rapamycin and rapamycin + TGF-b1. (a) The effect of TGF-b RI/ALK1 inhibitor (ALK1-I) and PI3K inhibitor (LY294002) on rapamycin + TGF-b1 (Rap + TGF) induced apoptosis in lymphoma cells with low TGF-b1 sensitivity (HT58r) after 72 h treatment (⁄p < 0.05, flow cytometry). (b) Low dose rapamycin monotreatment and rapamycin + TGF-b1 (Rap + TGF) decreases the amount of p-p70S6K and its target p-S6 protein after 24 h in HT58 lymphoma cells (Western-blot).
pathway. Inhibition of mTORC1 by rapamycin was able to restore the ability of TGF-b1 to induce apoptosis in certain lymphoma cell lines. We also found that TGF-b1 sensitivity could not be restored by rapamycin in U266 and BL41/95 cells. In these cell lines, the downregulation/loss of TGF-b RII expression may be responsible for TGF-b1 resistance. These results emphasize the requirement of active TGF-b receptors for effective apoptosis induction by TGF-b1 and rapamycin + TGF-b1, which is further supported by our experiments using a TGF-b RI/ALK1-inhibitor. Similarly to our results, TGF-b induced differentiation showed synergism with mTOR inhibition (by rapamycin) in regulatory Tcell development in different models [25]. Moreover, mTOR activity inhibited Smad3 phosphorylation in HCV related hepatocellular carcinoma as well [19]. However, it is also known that TGF-b1 induced epithelial mesenchymal transition and kidney fibrogenesis may depend on TGF-b induced mTOR activity. In light of all these results, we should keep in mind that signalling networks are complex and cell type dependent, and TGF-b and mTOR signalling have diverse, sometimes opposing effects [26–28]. There are several explanations for the effect of combined rapamycin + TGF-b1 treatment in B lymphoma cells. A plausible target would be FKBP12, a guardian of TGF-b R, negatively regulating the receptor – preventing the receptor from leaky signals at suboptimal ligand concentration. At the same time, FKBP12 is a known rapamycin-binding protein [29]. Therefore, it is possible
TGF-b1 induced apoptosis was caspase-dependent. However, apoptosis induced by rapamycin + TGF-b1 was not completely eliminated by Z-VAD-fmk, a caspase inhibitor (Fig. 4b), indicating that co-treatment with rapamycin engaged a caspase-independent mechanism as well. 3.3. Rapamycin treatment inhibits proliferation and induces apoptosis in vivo in lymphoma xenografts mTOR activity related phosphorylated proteins (p-mTOR, p-p70S6K, p-4EBP1 and p-S6) were detected in biopsies of Burkitt-lymphoma patients [24] and in HT58 lymphoma xenografts, which suggests that enhanced mTOR signalling activity is present in vivo (Supplemented Figure), which may serve as a potential therapeutic target. To prove the in vivo relevance of our observations, mice with HT58 lymphoma xenografts were treated with low dose Rapamune. Short term (2 weeks) treatment lead to remarkable and significant growth suppression of the tumours as indicated by tumour weight (3.98 ± 0.25 g vs. 8.9 ± 0.6 g in untreated vs. Rapamune-treated animals, respectively). Immunohistochemical staining of xenografts for phospho-Histone H3 (pHH3; a mitotic marker) revealed inhibition of proliferation: the number of pHH3-positive cells was reduced to 27% in Rapamune-treated xenografts, compared to untreated tumours. In addition, Rapamune treatment resulted in an increase of apoptosis in vivo: the number of tumour cells positive for cleaved/activated caspase 3 (an apoptosis marker) showed a 6.64-fold increase (Supplemented Figure). 4. Discussion Our results suggest that elevated mTORC1 activity is likely to contribute to TGF-b1-resistance in several high grade B lymphoma cell lines, even in the presence of a theoretically functional TGF-b-
Fig. 3. The effect of rapamycin and TGF-b1 combination is Smad4-independent in lymphoma cells. (a) Percentage of apoptotic cells after TGF-b1 and rapamycin treatment in control and Smad4 siRNA transfected cells; 72 h treatment (flow cytometry). (b) Smad4 protein expression after siRNA transfection in control, rapamycin and TGF-b1 treated cells; 2 h treatment (Western-blot). (c) TIEG mRNA expression in siRNA transfected cells after rapamycin and TGF-b1 treatment; 2 h treatment (RT-PCR). (Co: control; TGF-b1: TGF; rapamycin: Rap; TGF-b1 and rapamycin treatment was initiated 6 h after siRNA transfection).
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Fig. 4. The apoptotic effect of rapamycin + TGF-b1 combination requires early PP2A-activity and is partially caspase dependent. (a) The PP2A inhibitor okadaic acid abolishes TGF-b1-induced and rapamycin + TGF-b1-induced apoptosis in HT58 lymphoma cells (flow cytometry). (b) Z-VAD-fmk caspase inhibitor completely abolishes TGF-b1-induced apoptosis but only partially inhibits rapamycin + TGF-b1 induced apoptosis in lymphoma cells with low TGF-b1 sensitivity (HT58r). (72 h treatment; co: control; TGF-b1: TGF; Rap: rapamycin; oka: okadaic acid; no inh: without PP2A inhibitor treatment; apoptosis was detected by flow cytometry.).
that rapamycin neutralizes available FKBP12 molecules, thereby allowing the activation of TGF-b R for apoptosis induction during rapamycin + TGF-b1 treatment. However, it is very likely that FKBP12 is not the sole mechanism of action for rapamycin, as mTORC1 signalling can promote survival in several ways. The role of mTORC1 activity in TGF-b1 resistance has already been observed in non lymphoid cell types, e.g. epithelial cell lines. IL-4 prevented TGF-b1 induced apoptosis through the activation of AKT and p70S6K in human hepatocellular and breast carcinoma cells [30]. It was also suggested that mTORC1 can activate SGK1 (serum- and glucocorticoid-inducible kinase 1) to regulate p27kip phosphorylation, leading to TGF-b1 resistance in mammalian carcinoma cells [31]. Our results are the first to demonstrate the contribution of mTORC1 activity to developing resistance against TGFb1 induced apoptosis in selected lymphoma cells. We found previously that the apoptotic effect of TGF-b1 was independent of Smad4, i.e. the ’’classical’’ TGF-b pathway in lymphoma cell lines [14]. Using Smad4 siRNA transfection, apoptosis induced by rapamycin + TGF-b1 proved to be also Smad4-independent in our present experiments. Our previous and current results suggest that the TGF-b1/TGF-bR/PP2A pathway has an important role in growth inhibition and apoptosis in lymphoma cells with inhibited mTORC1 activity. mTORC1 is an important element in the cellular signalling network, and a powerful supporter of cell survival. Its activity is able to block apoptosis (e.g. induced by TGF-b1) in lymphoma cells. Using a PI3K inhibitor in addition to rapamycin and TGF-b1, apoptosis was further increased, compared to the TGF-b + rapamycin combination. Similar observations were made in different tumours and hematological malignancies, which suggest that PI3K
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inhibitors and rapamycin can potentiate each other’s effect; many clinical studies have recently been initiated based on this, both in lymphoid and solid malignancies [32]. We previously found that caspase inhibitors blocked TGF-b1 induced apoptosis in lymphoma cells [17]. Interestingly, a portion (about a third) of rapamycin + TGF-b1 induced apoptosis was caspase independent. This pan-caspase inhibitor resistant cell death can be explained by different mechanisms, e.g. by a crosstalk between ’’self-eating’’/autophagy and ’’self-killing’’/apoptosis. The role of mTOR inhibitors in autophagy induction is well known, and TGF-b1 has also been reported to activate autophagy in kidney epithelial and hepatocellular carcinoma cells [33–35]. Caspase independent mitochondrial endonuclease G release was observed in HT58 lymphoma cells after HRP4/ATRA treatment [36], which can also contribute to caspase independent DNA fragmentation. Taken together: (a) PI3K/mTORC1 activity contributes to resistance against TGF-b1-induced apoptosis in high grade lymphoma cells; (b) rapamycin enhances the growth inhibitory effect of TGF-b1 and switches on the TGF-b1 dependent apoptotic program in B lymphoma cells; (c) rapamycin + TGF-b1 induced apoptosis is Smad4–independent and PP2A dependent in these lymphoma cells (Fig. 5). We confirmed the growth inhibitory effect of low dose Rapamune treatment in our in vivo xenograft model. We detected not only a reduction in cell proliferation but also apoptosis induction in tissue specimens after monotreatment, which is in contrast to our in vitro experiments, where rapamycin by itself did not increase apoptosis in the examined cell lines. These results underline the potential role of mTORC1 inhibition in switching on the apoptotic effect of pro-apoptotic regulators (such as TGF-b1) in the examined lymphomas. We focused on TGF-b1 in our study; however, there are many other negative regulators (e.g. cytokines) in tumour cells and also in their environment, which may be suppressed by mTORC1 activation – this may also explain the fact that rapamycin effectively induced apoptosis in vivo, but not in vitro. Our results suggest that high mTOR activity contributes to TGFb resistance and that lowering mTORC1 activity may provide a tool in high grade B-cell lymphoma therapy. We presume that mTOR inhibition may promote therapeutic success in lymphomas and
Fig. 5. PI3K/mTOR activity contribute to the survival and proliferation of TGF-b1 resistant lymphoma cells. The internal apoptotic program can be switched on by TGF-b1 in the examined B lymphoma cells. This mechanism is PP2A dependent and it is independent of ’’classical’’ Smad4/TGF-b signalling (as shown by siRNA knockdown). PI3K/mTOR activity can support survival and TGF-b1 resistance in lymphoma cells. Rapamycin and PI3K inhibitors can reverse this effect in TGF-b1 resistant lymphoma cells. Moreover, blocking PI3K activity can further increase the effect of rapamycin and TGF-b1 treatment. (inh: inhibitor).
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other tumours by increasing or restoring sensitivity to currently available treatment modalities [32,37–39] and to endogenous regulators such as TGF-b1. However, the complex in vivo effects of mTOR inhibition, the availability of endogenous negative regulators (including TGF-b1) and the net effect of these factors need to be clarified in further studies. Further data is required to elucidate the impact of mTOR inhibition on the microenvironment – such as tumour related inflammation in the case of non-lymphoid tumours – or on the pleiotropic effect of negative regulatory cytokines in tumor matrix elements. Studies on different lymphomas showed that rapamycin treatment could be beneficial in high grade lymphomas and refractory Hodgkin lymphomas as well. However, the role of potentially available in vivo negative regulators and their release under inhibition were not examined in these cases. The activity of mTOR is often elevated in a variety of tumours, which can be easily detected in biopsy materials. Increased mTOR activity very likely leads to resistance not only to endogenous negative regulators but also to therapeutic drugs. In these cases, drugs targeting PI3K or mTOR may be added to the regimen, in order to reverse therapeutic resistance, and they may also potentiate each other. As a result, a decrease in mTOR activity (after a shorter time period) may be followed by a beneficial therapeutic effect and a reduction in tumour burden after a longer time. Pharmacological inhibition of the PI3K-AKT-mTOR pathway is a puzzling but promising approach in the development of novel anticancer therapies [37], especially in the light of the emerging problem of drug resistance in targeted therapy. Data about elevated mTORC1-activation in numerous solid tumours and lymphomas are increasing. Our experimental and in vivo xenograft results suggest that the addition of rapalogues to treatment protocols could be beneficial not only in mantle cell lymphomas (MCLs) but also in other lymphomas with high mTORC1 activity – such as Hodgkin lymphomas, non-germinal center type diffuse large B-cell lymphomas and acute lymphoblastic leukemias [24,37–40]. Acknowledgements We would like to express our thanks to Gézáné Csorba, Lilla Kis, Viktória Varga, Titanilla Dankó and András Sztodola for their technical help. This work was supported by the Hungarian Scientific Research Found [grant numbers: OTKA F048380, T81624, T84262]. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.02.024. References [1] Buhl AM, Nemazee D, Cambier JC, Rickert R, Hertz M. B-cell antigen receptor competence regulate selection and survival. Immunol Rev 2000;176:154–70. [2] Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 2014;15:155–62. [3] Laplante M, Sabatini DM. MTOR signaling in growth control and disease. Cell 2012;149:274–93. [4] Ghayad SE, Cohen PA. Inhibitors of the PI3K/Akt/mTOR pathway: new hope for breast cancer patients. Recent Pat Anticancer Drug Discov 2010;5:29–57. [5] Wysocki PJ. MTOR in renal cell cancer: modulator of tumor biology and therapeutic target. Expert Rev Mol Diagn 2009;9:231–41. [6] Younes A, Samad N. Utility of mTOR inhibition in hematologic malignancies. Oncologist 2011;16:730–1. [7] Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N. Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta 2010;1804: 433–9. [8] Rivera-Rodriguez N, Cabanillas F. Recent advances in the management of mantle cell lymphoma. Curr Opin Oncol 2013;25:716–21. [9] Arita A, McFarland DC, Myklebust JH, Parekh S, Petersen B, Gabrilove J, et al. Signaling pathways in lymphoma: pathogenesis and therapeutic targets. Future Oncol 2013;9:1549–71.
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