Discrepancy Between Low Levels of mTOR Activity and High Levels of P-S6 in Primary Central Nervous System Lymphoma May Be Explained by PAS Domain-Containing Serine/Threonine-Protein Kinase-Mediated Phosphorylation

Discrepancy Between Low Levels of mTOR Activity and High Levels of P-S6 in Primary Central... Abstract The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6 phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4E-BP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1, and PASK antibodies. Finally, we examined the impact of PASK inhibition on S6 phosphorylation on BHD1 cell line. mTOR-pathway activity was significantly less frequent in PCNSL compared with DLBCL. p-S6 positivity was related to mTOR-pathway in DLBCL, but not in PCNSL. Among the other kinases potentially responsible for S6 phosphorylation, PASK proved to be positive in all cases of PCNSL and DLBCL. Inhibition of PASK resulted in reduced expression of p-S6 in BHD1-cells. This is the first study demonstrating an mTOR independent p-S6 activity in PCNSL and that PASK may contribute to the phosphorylation of S6. Our findings also suggest a potential role of PASK in the pathomechanism of PCNSL and in DLBCL. mTOR, PASK, Primary central nervous system lymphoma (PCNSL) INTRODUCTION Primary central nervous system lymphoma (PCNSL) is an uncommon and distinct subtype of nonHodgkin lymphoma, predominantly represented by diffuse large B-cell lymphoma (DLBCL) arising in the central nervous system (CNS). PCNSL differs from systemic DLBCL in many aspects, including molecular and clinical characteristics (1). Management of PCNSL includes high-dose methotrexate-based chemotherapy, with or without radiation, and more recently multi-agent chemo- or immunochemotherapy (2). Despite the efforts to find a more effective treatment, the overall survival of patients is still poor, around 40% at 3 years (2–4). Therefore, it is important to better understand the biology of PCNSL and to identify novel biomarkers that help stratifying patients to specific therapeutic regimens and improve survival. The distinction of DLBCL into cell of origin categories based on gene expression profiles reminiscent of germinal center B-cell (GC-) type or activated B-cell (ABC-) type has substantial biological, prognostic, and therapeutic implications, with patients in the ABC-type DLBCL group displaying inferior survival (5, 6). An ABC-like phenotype is typical in PCNSL, which might at least partly explain the poor prognosis of the disease (7). Dysregulation of the mTOR (mechanistic target of rapamycin) pathway, which is a key regulator of various cellular functions, such as protein synthesis, cell growth, division, and survival (8), has been recognized as a critical event in the development of different hematological malignancies (9–11) including systemic DLBCL (12–14). Generally, more than half of the DLBCL cases show aberrantly activated mTOR, which was reported to be more typical in GC-type cases in 1 study (14) and in ABC-type cases in others (12, 13). mTOR represents a therapeutic target, and mTOR inhibitors alone or in combination with other agents have demonstrated promising results in relapsed or refractory DLBCL (15). Despite the promising results with mTOR inhibitors in DLBCL and the need for better understanding the pathomechanisms of PCNSL, there is very limited data on mTOR pathway activity in PCNSL (16). Upon activation, p-mTOR phosphorylates key translational regulators, including initiation factor 4E-binding protein (4E-BP1) and 70 kDa S6 ribosomal protein kinase (p70S6K1) at T389, and the latter subsequently phosphorylates the ribosomal S6 protein (S6) (17, 18). Detection of p-S6 is commonly used as a marker of mTOR pathway activity. However, taking only p-S6 positivity into account regarding mTOR activity might be misleading, as S6 protein may also be phosphorylated independently of mTOR via other kinases, including phospho-ribosomal protein S6 kinase (p-RSK), p(T229)-p70S6K1 (through PDK1), and PAS domain-containing serine/threonine-protein kinase (PASK) (19–23). The primary aim of this study was to determine the mTOR pathway activity in PCNSL with an immunohistochemical approach. After demonstrating that the number of p-S6-positive cases largely exceeded the number of cases with mTOR activity in PCNSL, we also aimed to identify the mTOR independent alternative pathways leading to S6 phosphorylation in PCNSL. MATERIALS AND METHODS Patients Formalin fixed paraffin-embedded (FFPE) biopsy specimens from 31 cases of PCNSL and 51 cases of nodal DLBCL were included in this study. All cases were diagnosed at the 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary between 1992 and 2014. The cell of origin of the lymphomas was determined according to the Hans algorithm in 29 and 47 cases of PCNSL and DLBCL, respectively (24). The study was approved by the local ethical committee of the Semmelweis University (TUKEB-1552012) and it was conducted in accordance with the Declaration of Helsinki. Immunohistochemistry The 4-µm FFPE tissue sections were deparaffinized. After blocking endogenous peroxidase activity, antigen retrieval was performed in sodium citrate (pH = 6) in an electric pressure cooker for 20 minutes. Slides were incubated overnight at 4°C with the primary antibody for p-mTOR (#2976, 1:100, Cell Signaling Technology, Danvers, MA), p-S6 (#2211, 1:100, Cell Signaling Technology), p-4E-BP1 (#2855, 1:1600, Cell Signaling Technology), p-p70S6K1 (T229, ab59208, 1:50, Abcam, Cambridge, UK), p-p70S6K1 (T389, ab126818, 1:1000, Abcam), p-RSK (sc-12445, 1:50, Santa Cruz Biotechnology, Dallas, TX), and PASK (ab154053, 1:100, Abcam). Novolink Polymer Detection System (Leica-Novocastra, Nussloch, Germany) was applied to detect antigens. The slides were visualized by diaminobenzidine (K3467, DAKO, Santa Clara, CA) and counterstained with hematoxylin. Immunostaining was evaluated by 2 independent pathologists. Because of performing multiple immunohistochemical reactions, only 26 out of 31 PCNSL cases contained sufficient amount of tissue for PASK immunohistochemistry. The intensity of the staining was scored from 0 to 2 (0 for absent staining, 1 for weak expression, and 2 for moderate to strong expression). The percentage of positive tumor cells was scaled from 1 to 3 (1 for <10%, 2 for 10%–50%, and 3 for >50% positive tumor cells) as described (14). The individual protein expression values were obtained by multiplying the scores of the intensity and percentage of positive tumor cells. The individual protein expression levels were defined as negative with a score 0, weak with 1–2, medium with 3–4, and high with 6. Generally, protein expression was considered positive above score 3. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive. Cell Culture and Treatment DLBCL cell line (MedB-1/BHD1) was cultured and treated in Iscove’s MDM (13390, Sigma, St. Louis, MO) and HyClone RPMI 1640 (GE Lifescience, Chicago, IL) (4:1) supplemented with 20% FCS (Biosera, Nuaille, France), 0.4% gentamicin (Sandoz, Holzkirchen, Germany) and l-glutamine (Gibco, Waltham, MA) at 37°C in a 5% CO2 atmosphere. Experiments were performed at 4 × 105/mL cell density. Cells were treated at 0 h with mTOR inhibitor rapamycin (50 ng/µL, Sigma), PASK inhibitor (1 µM, BioE-1115, Calbiochem, San Diego, CA) and both in combination, and incubated for 12 hours and 24 hours. Before the experiment several doses of PASK inhibitor (1, 10, and 50 µM) were tested. Because a 1-µM dose was proved to be the most effective, cells were treated with the lowest dose. Cells without treatment were used as control. Measuring p-S6 Expression (Western Blot and Flow Cytometry) After treatment, BHD1 cells were lysed in lysis buffer (50 mM Tris, 10% glycerol, 150 mM NaCl, 1% Nonidet-P40, 10 mM NaF, 1 mM PMSF, 0.5 mM NaVO3, pH 7.5). Protein concentrations were estimated by Quant-IT protein assay (Invitrogen, Carlsbad, CA). Equal amounts of proteins were separated in 12.5% SDS-PAGE gels and transferred onto PVDF membranes using the semidry blot system (BioRad, Hercules, CA). Membranes were incubated with anti-p-S6 (#2211, 1:1000, Cell Signaling Technology) primary antibody overnight at 4°C, followed by biotinylated secondary antibody and avidin-HRP complex (Vectastain Elite Universal ABC Kit, Vector Laboratories, Burlingame, CA), and detected by ECL (Advansta Inc., Menlo Park, CA). Anti-β-actin (A2228, 1:5000, Sigma) was used to confirm equal protein loading. The expression of p-S6 was defined by ImageJ 1.46r software (https://imagej.nih.gov, Accessed January 5, 2018). PerFix-nc Kit (B31167, Beckman Coulter, Brea, CA) was applied to promote intracellular staining of the cells, followed by incubation with p-S6 antibody conjugated to phycoerythrin (PE) (#5316, Cell Signaling Technology). The endogenous levels of p-S6 were detected with Navios flow cytometer (Beckman Coulter). The FACS results were analyzed by the Kaluza software (Beckman Coulter). Statistical Analysis Chi-square test or Fischer exact test were performed to analyze the connection between binary variables. Data were analyzed using SPSS version 20.0 software (IBM Corp., Armonk, NY). RESULTS Expression of mTOR Pathway-Related Proteins The majority of both PCNSL (83.9%; 26/31) and DLBCL cases (62.75%; 32/51) showed p-S6 expression. p-mTOR was positive in 12.9% (4/31) of PCNSL and in 54.9% (28/51) of DLBCL cases. p-p70S6K1 was positive in 6.5% (2/31) of PCNSL and in 31.4% (16/51) of DLBCL cases. p-4E-BP1 was positive in 12.9% (4/31) of PCNSL and in 29.4% (15/51) of DLBCL cases (Supplementary Data Table S1). Frequent mTOR-Independent p-S6 Expression in PCNSL In our cohort, only 25.8% (8/31) of the PCNSL cases, while 66.7% (34/51) of the DLBCL cases showed mTOR pathway activity by immunohistochemistry as defined by the criteria outlined in the materials and methods. The mTOR pathway activity was significantly less frequent in PCNSL compared with DLBCL (p < 0.001). As expected, the majority of the cases with active mTOR pathway also showed positivity for p-S6 in both groups (100% and 76.5%, respectively; Fig. 1). FIGURE 1. View largeDownload slide Distribution of mTOR signaling pathway activity and p-S6 expression. p-S6 positivity may represent mTOR pathway activity in diffuse large B-cell lymphoma (DLBCL); however, in primary central nervous system lymphoma (PCNSL) the number of p-S6-positive cases largely exceed the number of mTOR active cases. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive in the tumor cells. FIGURE 1. View largeDownload slide Distribution of mTOR signaling pathway activity and p-S6 expression. p-S6 positivity may represent mTOR pathway activity in diffuse large B-cell lymphoma (DLBCL); however, in primary central nervous system lymphoma (PCNSL) the number of p-S6-positive cases largely exceed the number of mTOR active cases. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive in the tumor cells. Interestingly, among the mTOR pathway inactive cases p-S6 was positive in 78.3% (18/23) of PCNSL and 35.3% (6/17) of DLBCL cases, respectively (Figs. 1 and2). In line with these results, positivity of p-S6 was significantly associated with the activated mTOR pathway in DLBCL (p = 0.005), but not in PCNSL (p = 0.198). FIGURE 2. View largeDownload slide Representative immunohistochemistry images of the mTOR pathway activity analysis in primary central nervous system lymphoma. The majority (58.1%; 18/31) of primary central nervous system lymphoma cases displays p-S6 positivity without mTOR pathway activity. Notably, in the cases considered mTOR pathway inactive only some reactive astrocytes, mitotic figures, and/or reactive lymphocytes were positive for p-mTOR, p-p70S6K1, and p-4E-BP1. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. All cases of PCNSL were PASK positive. (400× magnification). FIGURE 2. View largeDownload slide Representative immunohistochemistry images of the mTOR pathway activity analysis in primary central nervous system lymphoma. The majority (58.1%; 18/31) of primary central nervous system lymphoma cases displays p-S6 positivity without mTOR pathway activity. Notably, in the cases considered mTOR pathway inactive only some reactive astrocytes, mitotic figures, and/or reactive lymphocytes were positive for p-mTOR, p-p70S6K1, and p-4E-BP1. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. All cases of PCNSL were PASK positive. (400× magnification). These results indicate that p-S6 positivity does not necessarily represent mTOR pathway activity, especially in PCNSL, and suggest that other proteins independent of mTOR pathway may phosphorylate S6. Correlation of mTOR Pathway Activity and Cell of Origin The majority (78.7%; 37/47) of DLBCL cases showed ABC-like phenotype, 70.3% (26/37) of which were mTOR active as determined by our immunohistochemical markers. The remaining 21.3% (10/47) showed GC-like phenotype with 60% (6/10) representing mTOR active cases. All PCNSL cases analyzed for cell of origin fell into the ABC-subtype (29/29), consequently all cases with mTOR activity were also of ABC subtype (24.1%; 7/29; Fig. 3). mTOR pathway activity in PCNSL was also significantly less frequent when compared only to ABC-DLBCL (p < 0.001). FIGURE 3. View largeDownload slide Correlation of the mTOR pathway activity and cell of origin. The majority of diffuse large B-cell lymphoma (DLBCL) cases showed activated B-cell (ABC) phenotype and mTOR activity. All cases of primary central nervous system lymphoma (PCNSL) showed ABC phenotype with 24.1% mTOR activity. mTOR pathway activity in PCNSL was significantly less frequent when compared only to ABC-DLBCL (p < 0.001). The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. FIGURE 3. View largeDownload slide Correlation of the mTOR pathway activity and cell of origin. The majority of diffuse large B-cell lymphoma (DLBCL) cases showed activated B-cell (ABC) phenotype and mTOR activity. All cases of primary central nervous system lymphoma (PCNSL) showed ABC phenotype with 24.1% mTOR activity. mTOR pathway activity in PCNSL was significantly less frequent when compared only to ABC-DLBCL (p < 0.001). The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. The mTOR-Independent Kinases p-RSK and p(T229)-p70S6K1 Are Not Responsible for S6 Phosphorylation in PCNSL Three percent (1/31) and 0% (0/31) of the PCNSL cases showed positivity for p-RSK and p(T229)-p70S6K1, respectively. These results indicate that these mTOR-independent kinases do not play a major role in S6 phosphorylation in PCNSL. PASK May Contribute to S6 Phosphorylation in PCNSL and DLBCL Because it was proposed in the literature that PASK may also be involved in the phosphorylation of S6 (22), we tested its expression in our cohorts. All cases of PCNSL available for further immunohistochemistry (100%, 26/26) showed strong positivity for this kinase. For comparison, we performed PASK immunohistochemistry on the cases of DLBCL as well and found that all cases (100%, 51/51) of DLBCL were also PASK-positive. In order to confirm that PASK could contribute to S6 phosphorylation in PCNSL and DLBCL, we used the DLBCL cell line BHD1, in which S6 is known to be phosphorylated by mTOR (25). We treated this cell line with a PASK inhibitor and measured the subsequent changes in p-S6 levels. Treatment with the mTOR inhibitor rapamycin was used as a positive control. All values indicated below represent a mean value calculated from the results of 2 independent experiments. Inhibition of PASK in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blotting and flow cytometry analysis (Fig. 4). Densitometry performed on the blots showed that the level of p-S6 decreased to 61% compared with the untreated controls. Similarly, the X-mean value of p-S6 was reduced to 57% as measured by flow cytometry (Fig. 4). As expected, rapamycin also significantly reduced the level of p-S6 to 13% as confirmed by densitometry and to 28% by flow cytometry analysis (Fig. 4). The effect of the combined PASK inhibitor and rapamycin treatment was the most robust by reducing the protein level of p-S6 to 4% as revealed by densitometry and to 23% by flow cytometry analysis (Fig. 4). FIGURE 4. View largeDownload slide The effect of different treatments on p-S6 level. Inhibition of mTOR, PASK and both in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blot (A, C) and flow cytometry (B, C) analysis. The combined PASK inhibitor (PI) and rapamycin (R) treatment demonstrated the most robust effect. (C) The averages of 2 independent measurements with standard deviations are illustrated. FIGURE 4. View largeDownload slide The effect of different treatments on p-S6 level. Inhibition of mTOR, PASK and both in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blot (A, C) and flow cytometry (B, C) analysis. The combined PASK inhibitor (PI) and rapamycin (R) treatment demonstrated the most robust effect. (C) The averages of 2 independent measurements with standard deviations are illustrated. DISCUSSION In this study, we presented a comprehensive immunohistochemical analysis of mTOR pathway activity in PCNSL and DLBCL, and found PASK as a potential contributor of S6 phosphorylation in mTOR inactive cases. The importance of analyzing mTOR and other potentially targetable pathway activities in PCNSL is emphasized by the need to better understand the pathomechanism of the disease and to find more effective therapeutic targets to improve survival (2–4). This is the first study demonstrating that the mTOR pathway is probably inactive in the majority of PCNSL; therefore, mTOR inhibitor therapy would not add clinical benefit in this disease. A recent publication reported mTORC1 signaling pathway activity in a higher proportion of cases of PCNSL (16). There is no current consensus in the literature on how to assess mTOR activity in FFPE tissue and this study determined mTOR activity by a different methodology, analyzing the expression of Rheb, which is its upstream regulator, as well as the expression of the downstream molecules p-S6 and p-4E-BP1. This upstream regulator and these downstream molecules are known to reflect other pathway activities as well in addition to mTOR (20, 22, 26–28). In our study, in order to better focus on the mTOR pathway, we also analyzed p-mTOR and its other direct downstream substrate p-p70S6K1. Since only 4 of 31 cases of PCNSL were p-mTOR-positive, we further relaxed our criteria for putative mTOR pathway activity and also considered those cases mTOR active that were positive for at least 2 of the 3 downstream phospho-proteins. Even with these more permissive criteria, only a quarter of the PCNSL cases proved to be mTOR active in our cohort. We also found, however, that in contrast to DLBCL, cases of PCNSL with p-S6 expression largely outnumbered the cases with mTOR pathway activity. This suggests that the function of S6 protein is important for the development of the tumor and an mTOR independent alternative pathway may be involved in S6 activation. We decided to evaluate the possible mechanisms that may lead to S6 activation in PCNSL, and out of the 3 proteins known to represent mTOR independent ways of S6 phosphorylation (19–23), PASK protein was found to be expressed by the tumor cells in all examined cases of PCNSL. For comparison, we also evaluated PASK expression in cases of systemic DLBCL in which tumor cells also showed strong positivity, similarly to PCNSL. The capacity of PASK to phosphorylate S6 has been shown in vitro on recombinant proteins and in mouse embryonic fibroblasts (22). We investigated whether the same enzymatic reaction occurs in lymphoma cells as well. For this purpose, we chose to inhibit the function of PASK in a DLBCL cell line (BHD1) with high p-S6 levels due to mTOR activity (25). Inhibition of PASK led to a significant downregulation of p-S6 even in the presence of an active mTOR, suggesting that PASK indeed contributes to the phosphorylation of S6 in DLBCL cells as well. PASK is a nutrition sensing serine/threonine kinase (29, 30), with a substantial role in mammalian glucose homeostasis (31, 32) and lipid metabolism (33, 34). It is also involved in protein translation (22, 35) and is suggested to act as a coordinator between energy flux and translation (22). With the ability to control multiple essential metabolic pathways, PASK may be an attractive target for potential therapeutic approaches in DLBCL/PCNSL. In summary, this is the first study demonstrating that the increased p-S6 expression cannot entirely be explained by mTOR pathway activity in PCNSL and PASK may contribute to an mTOR independent S6 phosphorylation. Nevertheless, it is also possible that other known or novel unidentified kinases are also involved in this process. We also report, for the first time, frequent PASK expression in PCNSL as well as in nodal DLBCL, suggesting a potential role of PASK in the pathomechanism of PCNSL/DLBCL. However, further studies are needed to reveal if the signaling pathway related to PASK could be a potential target for therapy for the benefit of patients. ACKNOWLEDGMENT MedB-1/BHD1 cell line was kindly provided by Dr. P. Möller (Ulm, Germany). 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Mol Cell Biol  2003; 23: 6780– 9 http://dx.doi.org/10.1128/MCB.23.19.6780-6789.2003 Google Scholar CrossRef Search ADS PubMed  34 Zhang DD, Zhang JG, Wang YZet al.  , Per-Arnt-Sim Kinase (PASK): An emerging regulator of mammalian glucose and lipid metabolism. Nutrients  2015; 7: 7437– 50 http://dx.doi.org/10.3390/nu7095347 Google Scholar CrossRef Search ADS PubMed  35 Eckhardt K, Troger J, Reissmann Jet al.  , Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1. Cell Physiol Biochem  2007; 20: 227– 40 http://dx.doi.org/10.1159/000104169 Google Scholar CrossRef Search ADS PubMed  © 2018 American Association of Neuropathologists, Inc. All rights reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuropathology & Experimental Neurology Oxford University Press

Discrepancy Between Low Levels of mTOR Activity and High Levels of P-S6 in Primary Central Nervous System Lymphoma May Be Explained by PAS Domain-Containing Serine/Threonine-Protein Kinase-Mediated Phosphorylation

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0022-3069
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Abstract

Abstract The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6 phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4E-BP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1, and PASK antibodies. Finally, we examined the impact of PASK inhibition on S6 phosphorylation on BHD1 cell line. mTOR-pathway activity was significantly less frequent in PCNSL compared with DLBCL. p-S6 positivity was related to mTOR-pathway in DLBCL, but not in PCNSL. Among the other kinases potentially responsible for S6 phosphorylation, PASK proved to be positive in all cases of PCNSL and DLBCL. Inhibition of PASK resulted in reduced expression of p-S6 in BHD1-cells. This is the first study demonstrating an mTOR independent p-S6 activity in PCNSL and that PASK may contribute to the phosphorylation of S6. Our findings also suggest a potential role of PASK in the pathomechanism of PCNSL and in DLBCL. mTOR, PASK, Primary central nervous system lymphoma (PCNSL) INTRODUCTION Primary central nervous system lymphoma (PCNSL) is an uncommon and distinct subtype of nonHodgkin lymphoma, predominantly represented by diffuse large B-cell lymphoma (DLBCL) arising in the central nervous system (CNS). PCNSL differs from systemic DLBCL in many aspects, including molecular and clinical characteristics (1). Management of PCNSL includes high-dose methotrexate-based chemotherapy, with or without radiation, and more recently multi-agent chemo- or immunochemotherapy (2). Despite the efforts to find a more effective treatment, the overall survival of patients is still poor, around 40% at 3 years (2–4). Therefore, it is important to better understand the biology of PCNSL and to identify novel biomarkers that help stratifying patients to specific therapeutic regimens and improve survival. The distinction of DLBCL into cell of origin categories based on gene expression profiles reminiscent of germinal center B-cell (GC-) type or activated B-cell (ABC-) type has substantial biological, prognostic, and therapeutic implications, with patients in the ABC-type DLBCL group displaying inferior survival (5, 6). An ABC-like phenotype is typical in PCNSL, which might at least partly explain the poor prognosis of the disease (7). Dysregulation of the mTOR (mechanistic target of rapamycin) pathway, which is a key regulator of various cellular functions, such as protein synthesis, cell growth, division, and survival (8), has been recognized as a critical event in the development of different hematological malignancies (9–11) including systemic DLBCL (12–14). Generally, more than half of the DLBCL cases show aberrantly activated mTOR, which was reported to be more typical in GC-type cases in 1 study (14) and in ABC-type cases in others (12, 13). mTOR represents a therapeutic target, and mTOR inhibitors alone or in combination with other agents have demonstrated promising results in relapsed or refractory DLBCL (15). Despite the promising results with mTOR inhibitors in DLBCL and the need for better understanding the pathomechanisms of PCNSL, there is very limited data on mTOR pathway activity in PCNSL (16). Upon activation, p-mTOR phosphorylates key translational regulators, including initiation factor 4E-binding protein (4E-BP1) and 70 kDa S6 ribosomal protein kinase (p70S6K1) at T389, and the latter subsequently phosphorylates the ribosomal S6 protein (S6) (17, 18). Detection of p-S6 is commonly used as a marker of mTOR pathway activity. However, taking only p-S6 positivity into account regarding mTOR activity might be misleading, as S6 protein may also be phosphorylated independently of mTOR via other kinases, including phospho-ribosomal protein S6 kinase (p-RSK), p(T229)-p70S6K1 (through PDK1), and PAS domain-containing serine/threonine-protein kinase (PASK) (19–23). The primary aim of this study was to determine the mTOR pathway activity in PCNSL with an immunohistochemical approach. After demonstrating that the number of p-S6-positive cases largely exceeded the number of cases with mTOR activity in PCNSL, we also aimed to identify the mTOR independent alternative pathways leading to S6 phosphorylation in PCNSL. MATERIALS AND METHODS Patients Formalin fixed paraffin-embedded (FFPE) biopsy specimens from 31 cases of PCNSL and 51 cases of nodal DLBCL were included in this study. All cases were diagnosed at the 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary between 1992 and 2014. The cell of origin of the lymphomas was determined according to the Hans algorithm in 29 and 47 cases of PCNSL and DLBCL, respectively (24). The study was approved by the local ethical committee of the Semmelweis University (TUKEB-1552012) and it was conducted in accordance with the Declaration of Helsinki. Immunohistochemistry The 4-µm FFPE tissue sections were deparaffinized. After blocking endogenous peroxidase activity, antigen retrieval was performed in sodium citrate (pH = 6) in an electric pressure cooker for 20 minutes. Slides were incubated overnight at 4°C with the primary antibody for p-mTOR (#2976, 1:100, Cell Signaling Technology, Danvers, MA), p-S6 (#2211, 1:100, Cell Signaling Technology), p-4E-BP1 (#2855, 1:1600, Cell Signaling Technology), p-p70S6K1 (T229, ab59208, 1:50, Abcam, Cambridge, UK), p-p70S6K1 (T389, ab126818, 1:1000, Abcam), p-RSK (sc-12445, 1:50, Santa Cruz Biotechnology, Dallas, TX), and PASK (ab154053, 1:100, Abcam). Novolink Polymer Detection System (Leica-Novocastra, Nussloch, Germany) was applied to detect antigens. The slides were visualized by diaminobenzidine (K3467, DAKO, Santa Clara, CA) and counterstained with hematoxylin. Immunostaining was evaluated by 2 independent pathologists. Because of performing multiple immunohistochemical reactions, only 26 out of 31 PCNSL cases contained sufficient amount of tissue for PASK immunohistochemistry. The intensity of the staining was scored from 0 to 2 (0 for absent staining, 1 for weak expression, and 2 for moderate to strong expression). The percentage of positive tumor cells was scaled from 1 to 3 (1 for <10%, 2 for 10%–50%, and 3 for >50% positive tumor cells) as described (14). The individual protein expression values were obtained by multiplying the scores of the intensity and percentage of positive tumor cells. The individual protein expression levels were defined as negative with a score 0, weak with 1–2, medium with 3–4, and high with 6. Generally, protein expression was considered positive above score 3. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive. Cell Culture and Treatment DLBCL cell line (MedB-1/BHD1) was cultured and treated in Iscove’s MDM (13390, Sigma, St. Louis, MO) and HyClone RPMI 1640 (GE Lifescience, Chicago, IL) (4:1) supplemented with 20% FCS (Biosera, Nuaille, France), 0.4% gentamicin (Sandoz, Holzkirchen, Germany) and l-glutamine (Gibco, Waltham, MA) at 37°C in a 5% CO2 atmosphere. Experiments were performed at 4 × 105/mL cell density. Cells were treated at 0 h with mTOR inhibitor rapamycin (50 ng/µL, Sigma), PASK inhibitor (1 µM, BioE-1115, Calbiochem, San Diego, CA) and both in combination, and incubated for 12 hours and 24 hours. Before the experiment several doses of PASK inhibitor (1, 10, and 50 µM) were tested. Because a 1-µM dose was proved to be the most effective, cells were treated with the lowest dose. Cells without treatment were used as control. Measuring p-S6 Expression (Western Blot and Flow Cytometry) After treatment, BHD1 cells were lysed in lysis buffer (50 mM Tris, 10% glycerol, 150 mM NaCl, 1% Nonidet-P40, 10 mM NaF, 1 mM PMSF, 0.5 mM NaVO3, pH 7.5). Protein concentrations were estimated by Quant-IT protein assay (Invitrogen, Carlsbad, CA). Equal amounts of proteins were separated in 12.5% SDS-PAGE gels and transferred onto PVDF membranes using the semidry blot system (BioRad, Hercules, CA). Membranes were incubated with anti-p-S6 (#2211, 1:1000, Cell Signaling Technology) primary antibody overnight at 4°C, followed by biotinylated secondary antibody and avidin-HRP complex (Vectastain Elite Universal ABC Kit, Vector Laboratories, Burlingame, CA), and detected by ECL (Advansta Inc., Menlo Park, CA). Anti-β-actin (A2228, 1:5000, Sigma) was used to confirm equal protein loading. The expression of p-S6 was defined by ImageJ 1.46r software (https://imagej.nih.gov, Accessed January 5, 2018). PerFix-nc Kit (B31167, Beckman Coulter, Brea, CA) was applied to promote intracellular staining of the cells, followed by incubation with p-S6 antibody conjugated to phycoerythrin (PE) (#5316, Cell Signaling Technology). The endogenous levels of p-S6 were detected with Navios flow cytometer (Beckman Coulter). The FACS results were analyzed by the Kaluza software (Beckman Coulter). Statistical Analysis Chi-square test or Fischer exact test were performed to analyze the connection between binary variables. Data were analyzed using SPSS version 20.0 software (IBM Corp., Armonk, NY). RESULTS Expression of mTOR Pathway-Related Proteins The majority of both PCNSL (83.9%; 26/31) and DLBCL cases (62.75%; 32/51) showed p-S6 expression. p-mTOR was positive in 12.9% (4/31) of PCNSL and in 54.9% (28/51) of DLBCL cases. p-p70S6K1 was positive in 6.5% (2/31) of PCNSL and in 31.4% (16/51) of DLBCL cases. p-4E-BP1 was positive in 12.9% (4/31) of PCNSL and in 29.4% (15/51) of DLBCL cases (Supplementary Data Table S1). Frequent mTOR-Independent p-S6 Expression in PCNSL In our cohort, only 25.8% (8/31) of the PCNSL cases, while 66.7% (34/51) of the DLBCL cases showed mTOR pathway activity by immunohistochemistry as defined by the criteria outlined in the materials and methods. The mTOR pathway activity was significantly less frequent in PCNSL compared with DLBCL (p < 0.001). As expected, the majority of the cases with active mTOR pathway also showed positivity for p-S6 in both groups (100% and 76.5%, respectively; Fig. 1). FIGURE 1. View largeDownload slide Distribution of mTOR signaling pathway activity and p-S6 expression. p-S6 positivity may represent mTOR pathway activity in diffuse large B-cell lymphoma (DLBCL); however, in primary central nervous system lymphoma (PCNSL) the number of p-S6-positive cases largely exceed the number of mTOR active cases. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive in the tumor cells. FIGURE 1. View largeDownload slide Distribution of mTOR signaling pathway activity and p-S6 expression. p-S6 positivity may represent mTOR pathway activity in diffuse large B-cell lymphoma (DLBCL); however, in primary central nervous system lymphoma (PCNSL) the number of p-S6-positive cases largely exceed the number of mTOR active cases. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-S6, and p-4E-BP1) were positive in the tumor cells. Interestingly, among the mTOR pathway inactive cases p-S6 was positive in 78.3% (18/23) of PCNSL and 35.3% (6/17) of DLBCL cases, respectively (Figs. 1 and2). In line with these results, positivity of p-S6 was significantly associated with the activated mTOR pathway in DLBCL (p = 0.005), but not in PCNSL (p = 0.198). FIGURE 2. View largeDownload slide Representative immunohistochemistry images of the mTOR pathway activity analysis in primary central nervous system lymphoma. The majority (58.1%; 18/31) of primary central nervous system lymphoma cases displays p-S6 positivity without mTOR pathway activity. Notably, in the cases considered mTOR pathway inactive only some reactive astrocytes, mitotic figures, and/or reactive lymphocytes were positive for p-mTOR, p-p70S6K1, and p-4E-BP1. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. All cases of PCNSL were PASK positive. (400× magnification). FIGURE 2. View largeDownload slide Representative immunohistochemistry images of the mTOR pathway activity analysis in primary central nervous system lymphoma. The majority (58.1%; 18/31) of primary central nervous system lymphoma cases displays p-S6 positivity without mTOR pathway activity. Notably, in the cases considered mTOR pathway inactive only some reactive astrocytes, mitotic figures, and/or reactive lymphocytes were positive for p-mTOR, p-p70S6K1, and p-4E-BP1. The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. All cases of PCNSL were PASK positive. (400× magnification). These results indicate that p-S6 positivity does not necessarily represent mTOR pathway activity, especially in PCNSL, and suggest that other proteins independent of mTOR pathway may phosphorylate S6. Correlation of mTOR Pathway Activity and Cell of Origin The majority (78.7%; 37/47) of DLBCL cases showed ABC-like phenotype, 70.3% (26/37) of which were mTOR active as determined by our immunohistochemical markers. The remaining 21.3% (10/47) showed GC-like phenotype with 60% (6/10) representing mTOR active cases. All PCNSL cases analyzed for cell of origin fell into the ABC-subtype (29/29), consequently all cases with mTOR activity were also of ABC subtype (24.1%; 7/29; Fig. 3). mTOR pathway activity in PCNSL was also significantly less frequent when compared only to ABC-DLBCL (p < 0.001). FIGURE 3. View largeDownload slide Correlation of the mTOR pathway activity and cell of origin. The majority of diffuse large B-cell lymphoma (DLBCL) cases showed activated B-cell (ABC) phenotype and mTOR activity. All cases of primary central nervous system lymphoma (PCNSL) showed ABC phenotype with 24.1% mTOR activity. mTOR pathway activity in PCNSL was significantly less frequent when compared only to ABC-DLBCL (p < 0.001). The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. FIGURE 3. View largeDownload slide Correlation of the mTOR pathway activity and cell of origin. The majority of diffuse large B-cell lymphoma (DLBCL) cases showed activated B-cell (ABC) phenotype and mTOR activity. All cases of primary central nervous system lymphoma (PCNSL) showed ABC phenotype with 24.1% mTOR activity. mTOR pathway activity in PCNSL was significantly less frequent when compared only to ABC-DLBCL (p < 0.001). The mTOR pathway was considered active if p-mTOR or at least 2 out of the 3 downstream phospho-proteins (p(T389)-p70S6K1, p-4E-BP1, and p-S6) were positive in the tumor cells. The mTOR-Independent Kinases p-RSK and p(T229)-p70S6K1 Are Not Responsible for S6 Phosphorylation in PCNSL Three percent (1/31) and 0% (0/31) of the PCNSL cases showed positivity for p-RSK and p(T229)-p70S6K1, respectively. These results indicate that these mTOR-independent kinases do not play a major role in S6 phosphorylation in PCNSL. PASK May Contribute to S6 Phosphorylation in PCNSL and DLBCL Because it was proposed in the literature that PASK may also be involved in the phosphorylation of S6 (22), we tested its expression in our cohorts. All cases of PCNSL available for further immunohistochemistry (100%, 26/26) showed strong positivity for this kinase. For comparison, we performed PASK immunohistochemistry on the cases of DLBCL as well and found that all cases (100%, 51/51) of DLBCL were also PASK-positive. In order to confirm that PASK could contribute to S6 phosphorylation in PCNSL and DLBCL, we used the DLBCL cell line BHD1, in which S6 is known to be phosphorylated by mTOR (25). We treated this cell line with a PASK inhibitor and measured the subsequent changes in p-S6 levels. Treatment with the mTOR inhibitor rapamycin was used as a positive control. All values indicated below represent a mean value calculated from the results of 2 independent experiments. Inhibition of PASK in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blotting and flow cytometry analysis (Fig. 4). Densitometry performed on the blots showed that the level of p-S6 decreased to 61% compared with the untreated controls. Similarly, the X-mean value of p-S6 was reduced to 57% as measured by flow cytometry (Fig. 4). As expected, rapamycin also significantly reduced the level of p-S6 to 13% as confirmed by densitometry and to 28% by flow cytometry analysis (Fig. 4). The effect of the combined PASK inhibitor and rapamycin treatment was the most robust by reducing the protein level of p-S6 to 4% as revealed by densitometry and to 23% by flow cytometry analysis (Fig. 4). FIGURE 4. View largeDownload slide The effect of different treatments on p-S6 level. Inhibition of mTOR, PASK and both in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blot (A, C) and flow cytometry (B, C) analysis. The combined PASK inhibitor (PI) and rapamycin (R) treatment demonstrated the most robust effect. (C) The averages of 2 independent measurements with standard deviations are illustrated. FIGURE 4. View largeDownload slide The effect of different treatments on p-S6 level. Inhibition of mTOR, PASK and both in BHD1 cell culture greatly reduced the level of p-S6 as confirmed by Western blot (A, C) and flow cytometry (B, C) analysis. The combined PASK inhibitor (PI) and rapamycin (R) treatment demonstrated the most robust effect. (C) The averages of 2 independent measurements with standard deviations are illustrated. DISCUSSION In this study, we presented a comprehensive immunohistochemical analysis of mTOR pathway activity in PCNSL and DLBCL, and found PASK as a potential contributor of S6 phosphorylation in mTOR inactive cases. The importance of analyzing mTOR and other potentially targetable pathway activities in PCNSL is emphasized by the need to better understand the pathomechanism of the disease and to find more effective therapeutic targets to improve survival (2–4). This is the first study demonstrating that the mTOR pathway is probably inactive in the majority of PCNSL; therefore, mTOR inhibitor therapy would not add clinical benefit in this disease. A recent publication reported mTORC1 signaling pathway activity in a higher proportion of cases of PCNSL (16). There is no current consensus in the literature on how to assess mTOR activity in FFPE tissue and this study determined mTOR activity by a different methodology, analyzing the expression of Rheb, which is its upstream regulator, as well as the expression of the downstream molecules p-S6 and p-4E-BP1. This upstream regulator and these downstream molecules are known to reflect other pathway activities as well in addition to mTOR (20, 22, 26–28). In our study, in order to better focus on the mTOR pathway, we also analyzed p-mTOR and its other direct downstream substrate p-p70S6K1. Since only 4 of 31 cases of PCNSL were p-mTOR-positive, we further relaxed our criteria for putative mTOR pathway activity and also considered those cases mTOR active that were positive for at least 2 of the 3 downstream phospho-proteins. Even with these more permissive criteria, only a quarter of the PCNSL cases proved to be mTOR active in our cohort. We also found, however, that in contrast to DLBCL, cases of PCNSL with p-S6 expression largely outnumbered the cases with mTOR pathway activity. This suggests that the function of S6 protein is important for the development of the tumor and an mTOR independent alternative pathway may be involved in S6 activation. We decided to evaluate the possible mechanisms that may lead to S6 activation in PCNSL, and out of the 3 proteins known to represent mTOR independent ways of S6 phosphorylation (19–23), PASK protein was found to be expressed by the tumor cells in all examined cases of PCNSL. For comparison, we also evaluated PASK expression in cases of systemic DLBCL in which tumor cells also showed strong positivity, similarly to PCNSL. The capacity of PASK to phosphorylate S6 has been shown in vitro on recombinant proteins and in mouse embryonic fibroblasts (22). We investigated whether the same enzymatic reaction occurs in lymphoma cells as well. For this purpose, we chose to inhibit the function of PASK in a DLBCL cell line (BHD1) with high p-S6 levels due to mTOR activity (25). Inhibition of PASK led to a significant downregulation of p-S6 even in the presence of an active mTOR, suggesting that PASK indeed contributes to the phosphorylation of S6 in DLBCL cells as well. PASK is a nutrition sensing serine/threonine kinase (29, 30), with a substantial role in mammalian glucose homeostasis (31, 32) and lipid metabolism (33, 34). It is also involved in protein translation (22, 35) and is suggested to act as a coordinator between energy flux and translation (22). With the ability to control multiple essential metabolic pathways, PASK may be an attractive target for potential therapeutic approaches in DLBCL/PCNSL. In summary, this is the first study demonstrating that the increased p-S6 expression cannot entirely be explained by mTOR pathway activity in PCNSL and PASK may contribute to an mTOR independent S6 phosphorylation. Nevertheless, it is also possible that other known or novel unidentified kinases are also involved in this process. We also report, for the first time, frequent PASK expression in PCNSL as well as in nodal DLBCL, suggesting a potential role of PASK in the pathomechanism of PCNSL/DLBCL. However, further studies are needed to reveal if the signaling pathway related to PASK could be a potential target for therapy for the benefit of patients. ACKNOWLEDGMENT MedB-1/BHD1 cell line was kindly provided by Dr. P. Möller (Ulm, Germany). 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Journal of Neuropathology & Experimental NeurologyOxford University Press

Published: Apr 1, 2018

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