TRKA signalling is an important survival factor for Hodgkin lymphoma derived cell lines

Available online at www.sciencedirect.com

Leukemia Research 32 (2008) 163–167

Brief communication

Autocrine NGF␤/TRKA signalling is an important survival factor for Hodgkin lymphoma derived cell lines Christoph Renn´e a,1 , Sarah Minner a,1 , Ralf K¨uppers b , Martin-Leo Hansmann a , Andreas Br¨auninger a,∗ a

Senckenberg Institute of Pathology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany b Institute for Cell Biology, University of Duisburg-Essen, Essen, Germany Received 28 March 2007; received in revised form 9 May 2007; accepted 19 May 2007 Available online 27 July 2007

Abstract Hodgkin-Reed/Sternberg cells, the tumor cells of Hodgkin lymphoma, aberrantly express several receptor tyrosine kinases, among them TRKA whose stimulation supports B cell survival. We show here high expression of TRKA in Hodgkin-Reed/Sternberg cell lines as compared to normal B cells and other B cell lymphomas, without major increases in TRKA gene dosage. A fraction of TRKA is constitutively activated, likely due to the coexpression of NGF␤, the TRKA high affinity ligand. The TRK inhibitor K-252a decreased survival of HodgkinReed/Sternberg cell lines accompanied by decreased AKT activation. Inclusion of TRK inhibitors in therapeutic regimens may thus be an interesting possibility. © 2007 Elsevier Ltd. All rights reserved. Keywords: Hodgkin lymphoma; Receptor tyrosine kinase; TRKA; NGFb; K-252a; AKT

1. Introduction Receptor tyrosine kinases (RTKs) are frequently involved in cellular transformation, and recently the high expression of seven RTKs in primary Hodgkin-Reed/Sternberg (HRS) cells in the majority of Hodgkin lymphoma (HL) cases has been observed [1]. Immunohistochemistry (IHC) with antibodies specific for the activated RTKs showed exemplarily the activation of two RTKs in large fractions of cases, and IHC for four of the RTK ligands indicated that the RTKs are likely to be activated in autocrine or paracrine fashions [1]. HRS cells are derived from B cells, and of the seven RTKs expressed by HRS cells only TRKA expression has been previously observed in non-neoplastic B cells [2]. B cells can also produce the TRKA high affinity ligand NGF␤, which is an autocrine survival factor for memory B cells [2]. Given the ∗ 1

Corresponding author. Tel.: +49 69 63015440; fax: +49 69 63015241. E-mail address: [email protected] (A. Br¨auninger). These authors contributed equally to this work.

0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2007.05.019

potential of NGF␤/TRKA signalling to support survival of B cells and the aberrantly high expression of TRKA on HRS cells in about 30% of cases as compared to normal B cells [1], TRKA signalling could be important for the survival of HRS cells. We thus analysed the role of TRKA for survival of four HRS-cell lines as models for primary HRS cells.

2. Materials and methods 2.1. Cell lines, tissue samples and peripheral blood B cells All cell lines were cultured as recommended (DSMZ, Braunschweig, Germany, and ATCC, Manassas, VA). Tissue samples were retrieved from the files of the Department of Pathology of the University of Frankfurt and were originally submitted for diagnostic purposes. For isolation of peripheral blood (PB) B cells mononuclear cells from two donors were isolated by Ficoll-Isopaque density centrifugation and B cells

164

C. Renn´e et al. / Leukemia Research 32 (2008) 163–167

were separated by magnetic cell separation using the MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). 2.2. Immunohistochemical staining and Western blot analysis IHC for TRKA was performed as previously described [1]. For WB analysis cells or 10 ␮m sections of frozen tissues were lysed in L¨ammli buffer (8 min, 100 ◦ C), and lysates corresponding to 1 × 105 cells separated by SDS-PAGE, blotted onto PDVF membranes (BioRad, Munich, Germany) and incubated overnight at 4 ◦ C with primary antibodies (1:1000 of TRKA C-14, NGF␤ M-20 and Actin C-11, Santa Cruz Biotechnology; phospho (p)-TRKA T 9691, Sigma–Aldrich, Munich, Germany; AKT #9272 and p-AKT (Ser473) #4051, Cell Signaling, Beverly, MA). Proteins were visualized using the ECL plus system and the ECL Advance system for NGF␤ (Amersham Biosciences, Freiburg, Germany). For p-TRKA analysis cell culture media were supplemented with 2 mM NaVO4 prior to lysis to enrich p-tyrosine containing proteins. Specificity of the p-TRKA antibody was tested by pretreatment of the WB membrane with alkaline phosphatase (15 U/ml, 12 h, 37 ◦ C, Roche, Mannheim, Germany). 2.3. Fluorescence in situ hybridisation (FISH) BAC clones for TRKA (RP11-107D16, RP11-66D17 and RP11-356J7; Ensembl databank) were obtained from BACPAC Resources (Oakland, CA). Probes were prepared as described [3], tested on normal metaphases and hybridized together with a chromosome 1 centromere probe to cytospins of the cell lines as decribed [3]. Slides were analysed using a Zeiss Axioskop-2 fluorescence microscope (Zeiss, G¨ottingen, Germany). 2.4. NGFβ stimulation and K-252a treatment For NGF␤ stimulation cells grown in complete media (10% FCS for L428, KMH2 and HDLM2, 20% FCS for L1236) or media with only 1% FCS for 48 h were washed once in PBS and then incubated with 250 ng/ml rNGF␤ in PBS at 37 ◦ C (Peprotech, Hamburg, Germany) for 10 min and then lysed. For inhibition of TRKA phosphorylation cells were incubated with 50 ␮g/ml K-252a (Merck, Darmstadt, Germany) for 2 h prior to NGF␤ stimulation. For K-252a sensitivity tests cells were grown in 96 well tissue culture plates (starting with 1 × 105 cells/ml) with various concentrations of K-252a. After incubation for 24 h, MTT (Calbiochem, Darmstadt, Germany) was added to 0.5 mg/ml. After 4 h cells were lysed (5% SDS, 12 h, 37 ◦ C) and absorbance measured with an EL311SX microplate reader (Biotek Instruments, Winooski, VT). To determine the effects of K-252a on AKT phosphorylation status cells were grown at 2 × 105 cells/ml for 6 h with or without 200 ␮M K252a in complete medium without exogeneous NGF␤ and then harvested and lysed.

2.5. NGFβ ELISA 4 × 106 cells/ml were cultured for 24 h and supernatants were cleared by centrifugation (5 min, 13,000×g) and filtration (Millex GS 0.22 ␮m, Millipore, Bedford, MA). NGF EMax ELISAs (Promega Corporation, Madison, WI) with supernatants were performed following the manufacturers instructions.

3. Results TRKA expression in B cells has been previously demonstrated by WB analysis and FACS [2]. Using IHC, TRKA expression in B cells of lymph nodes, tonsils and spleens was, however, below the detection limit, and among B cell lymphomas high TRKA expression was largely restricted to HRS cells of HL cases (Fig. 1A–C and [1]). WB analysis with HRS-cell lines, CD19+ PB B cells, whole tissue sections from tonsils and lymph nodes and cell lines derived from other B cell lymphomas revealed strong TRKA expression in three of the four HRS-cell lines while TRKA expression in CD19+ PB B cells, tonsils and lymph nodes was only detectable after prolonged exposure of the WB (Fig. 1D and not shown). Among the B cell non-Hodgkin lymphoma lines analysed only one showed elevated TRKA expression. Gene dosage effects can contribute to the overexpression of genes, and we thus performed FISH analyses for the TRKA locus (chromosome 1p21–22) with the HRS-cell lines (Fig. 2). In KMH2 and L1236 minor gains of the TRKA locus were observed. Both cell lines are characterized as hypotriploid (DSMZ) and in both cell lines four signals were observed with the TRKA specific probes. In order to analyse the functionality and activation status of TRKA expressed on HRS-cell lines, we performed WB analysis with a p-TRKA specific antibody. In all four HRScell lines and PC12, a rat neuronal cell line expressing high levels of TRKA, a low level of TRKA phosphorylation was observed (Fig. 3A). Control experiments showed specificity of this antibody for phosphorylated TRKA, as pretreatment of the WB membrane with a phosphatase completely abolished detection (Fig. 3B). NGF␤ stimulation caused a strong increase of TRKA-phosphorylation in all cell lines, which was prevented by preincubation of the cells with the TRK inhibitor K-252a [4]. Given the constitutive activation of a fraction of TRKA expressed on HRS-cell lines, we investigated whether the HRS-cell lines expressed NGFß and could thus stimulate TRKA in an autocrine fashion. WB analysis with cell lines lysates and ELISAs with culture supernatants showed that indeed all four cell lines expressed and secreted NGF␤ (Fig. 3C and D). In order to investigate the role of TRKA signalling for HRS-cell survival all four HRS and two control cell lines, neither expressing TRKA nor NGF␤ at amounts comparable to HRS cells, were incubated with various concentrations

C. Renn´e et al. / Leukemia Research 32 (2008) 163–167

165

Fig. 1. Expression of TRKA in normal and malignant B cells. (A–C) IHC for TRKA shows strong expression in HRS cells (A) while TRKA expression is beyond the detection limit in B cells of primary and secondary follicles of tonsils (B), lymph nodes (C) and also marginal zones of spleens (data not shown). (D) WB analysis of TRKA expression in four HRS-cell lines (L428, L1236, HDLM2 and KMH2), two diffuse large B cell lymphoma cell lines (OCI-LY3 and 7), one mediastinal large B cell lymphoma cell line (Karpas 1106P), three Burkitt lymphoma cell lines (RAJI, BJAB and RAMOS), the rat neuronal cell line PC12, which expresses high amounts of TRKA, two tonsils, two lymph nodes and MACS enriched CD19+ B cells from PB of two healthy donors. Actin was used to control loading of equal amounts of proteins.

of the TRK inhibitor K-252a for 24 h and survival of cell lines was measured (Fig. 4A). While all cell lines showed sensitivity towards K-252a, which is at higher concentrations also an inhibitor of CaM kinase II, myosin light chain kinase, PKA and PKC, the TRKA expressing HRS-cell lines showed the greatest sensitivity. To address the role of NGF␤/TRKA signalling for HRScell line survival at the molecular level, we analysed the activation (i.e. phosphorylation) status of AKT, which is a

major activator of pro-survival pathways, after serum starvation and subsequent NGF␤ application in two HRS-cell lines. In both cell lines NGF␤ application caused significant increases in p-AKT (Fig. 4B). In addition, incubation of both HRS-cell lines with K-252a for 6 h in media without exogeneous NGF␤ caused a significant decrease in AKT phosphorylation (Fig. 4C), indicating that autocrine NGF␤/TRKA signalling is an important survival factor for HRS-cell lines.

Fig. 2. FISH analysis of metaphase spreads of HRS-cell lines. TRKA specific probes are shown in green, the chromosome 1 centromere probe is shown in red. (A) In KMH2 1 chromosome 1 copy contained TRKA while two chromosomes 1 lacked a TRKA signal and three TRKA signals were observed on other chromosomes. (B) For L1236 a duplication of TRKA on one of the three chromosomes one was observed.

166

C. Renn´e et al. / Leukemia Research 32 (2008) 163–167

Fig. 3. Constitutive activation of TRKA and expression of NGF␤ in HRS-cell lines. (A) WB analysis with a p-TRKA specific antibody shows that in all four HRS-cell lines and the PC12 control a fraction of TRKA is phosphorylated without application of exogeneous NGF␤. Stimulation with NGF␤ caused strong increases of p-TRKA and preincubation of cells with the TRKA inhibitor K-252a prevented increases in p-TRKA upon NGF␤ application. (B) Pretreatment of WB membranes on which lysates of NGF␤ stimulated PC12 cells were blotted with alkaline phosphatase eliminates detection of p-TRKA and thus shows the specificity of the p-TRKA antibody for phosphorylated TRKA. WB analysis for actin shows that amounts of proteins in lysates remained unchanged during phosphatase treatment and that equal amounts were loaded. (C) Western blot analysis for NGF␤ with cell culture lysates revealed that the HRS-cell lines expressed NGF␤. Recombinant human NGF␤ (rhNGF␤) was used as control. (D) Also a NGF␤ specific ELISA with cell culture supernatants showed that HRS-cell lines expressed and secreted NGF␤. RhNGF␤ was used as positive control, medium without cells as negative control and two B cell non-Hodgkin lymphoma cell lines were included for comparisons. All measurements were performed in triplicate.

4. Discussion Although TRKA expression in normal B cells is below the detection limit of IHC, it has been shown that NGF␤ induced TRKA signalling is an important survival factor for memory B cells [2]. Given the strong TRKA expression detected by IHC in HRS cells [1], we speculated that TRKA signalling could be an important survival factor for the B cell derived HRS cells and thus analysed the role of TRKA in HRS-cell lines. We show here that the strong HRS-cell specific TRKA expression as compared to normal and malignant B cells derived from other lymphomas is maintained in HRS-cell lines. This high expression is, in contrast to the high expression of HER2 in mammary carcinomas or JAK2 in HL [5,6], not accompanied by major gene dosage gains of TRKA. Interestingly enough, a fraction of TRKA was constitutively activated in all four HRS-cell lines, and as we also observed the expression and secretion of NGF␤ by all four HRS-cell lines, this constitutive activation is most likely due to autocrine stimulation by NGF␤. Finally, we could show that the survival of HRS-cell lines is significantly inhibited by K-252a [4], the only commercially available TRKA

inhibitor, and that the inhibition of survival is accompanied by decreases in AKT activation status. Although K-252a affects several other kinases at higher concentrations and the effects observed may thus not only be due to inhibition of TRKA signalling [7], the NGF␤/TRKA coexpressing HRScell lines showed greater sensitivity than cell lines without TRKA expression, indicating that TRKA signalling is an important survival factor for HRS-cell lines. We previously demonstrated that inhibition of signalling of another RTK aberrantly expressed on HRS cells, PDGFRA, reduced proliferation of a HRS-cell line coexpressing the ligand PDGFA and the receptor PDGFRA [1]. While this effect was only seen in media with reduced serum concentrations (1%) and proliferation of the cell line thus only partly depended on PDGFRA signalling, the effects of K-252a were observed in media with the serum concentrations recommended for the cell lines, indicating that HRS-cell line survival may be dependent on TRKA signalling. Due to the fact that TRKA is activated in a considerable fraction of HL cases, likely by paracrine stimulation with NGF␤ produced by the large numbers of granulocytes in HL infiltrates [1], it may be an interesting option to test the effects of the recently

C. Renn´e et al. / Leukemia Research 32 (2008) 163–167

167

supported by the Deutsche Forschungs Gemeinschaft through grants KU 1315/2-2 and BR 1238/6-2. There are no financial conflicts of interest to disclose. Contributions. Christoph Renn´e and Sarah Minner designed and performed research. Ralf K¨uppers and MartinLeo Hansmann designed research and analysed data. Andreas Br¨auninger designed research, analysed data and wrote the manuscript.

References

Fig. 4. Sensitivity of HRS-cell lines towards the TRK inhibitor K-252a is accompanied by decreases in AKT activation status. (A) Incubation with the TRKA inhibitor K-252a inhibited survival of HRS-cell lines. The read out for survival was the activity of an enzyme of the mitochondrial respiratory chain in a MTT assay. Mean values of six replicates are shown. (B) Application of exogeneous NGF␤ to KMH2 and L1236 HRS-cell lines after 48 h serum starvation caused increases in p-AKT, demonstrating that NGF␤/TRKA signalling in HRS-cell lines activates a major pro-survival pathway. (C) HRS-cell lines grown with 200 ␮M K-252a or DMSO (in which K-252a is dissolved) alone for 6 h without any exogeneous NGF␤ show significant decreases in AKT activation, indicating that autocrine NGF␤/TRKA signalling causes significant AKT activation in the cell lines analysed.

developed highly specific and potent TRK inhibitors on HL [8].

Acknowledgements We would like to thank Ekaterini Hadzoglou and Sabine Albrecht for excellent technical assistance. This work was

[1] Renn´e C, Willenbrock K, K¨uppers R, Hansmann ML, Br¨auninger A. Autocrine- and paracrine-activated receptor tyrosine kinases in classic Hodgkin lymphoma. Blood 2005;105:4051–9. [2] Torcia M, Bracci-Laudiero L, Lucibello M, et al. Nerve growth factor is an autocrine survival factor for memory B lymphocytes. Cell 1996;85:345–56. [3] Martin-Subero JI, Harder L, Gesk S, et al. Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci. Int J Cancer 2002;98:470–4. [4] Ruggeri BA, Miknyoczki SJ, Singh J, Hudkins RL. Role of neurotrophintrk interactions in oncology: the anti-tumor efficacy of potent and selective trk tyrosine kinase inhibitors in pre-clinical tumor models. Curr Med Chem 1999;6:845–57. [5] Zhou BP, Hung MC. Dysregulation of cellular signaling by HER2/neu in breast cancer. Semin Oncol 2003;30:38–48. [6] Renn´e C, Willenbrock K, Martin-Subero JI, et al. High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma. Leukemia 2007;21:780–7. [7] Kase H, Iwahashi K, Nakanishi S, et al. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun 1987;142:436– 40. [8] Undevia SD, Vogelzang NJ, Mauer AM, Janisch L, Mani S, Ratain MJ. Phase I clinical trial of CEP-2563 dihydrochloride, a receptor tyrosine kinase inhibitor, in patients with refractory solid tumors. Invest New Drugs 2004;22:449–58.