L1CAM–Integrin Interaction Induces Constitutive NF-Κb Activation In
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Oncogene (2010) 29, 4766–4778 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE L1CAM–integrin interaction induces constitutive NF-jB activation in pancreatic adenocarcinoma cells by enhancing IL-1b expression H Kiefel1, S Bondong1, N Erbe-Hoffmann1, J Hazin1, S Riedle1, J Wolf1, M Pfeifer1, A Arlt2, H Scha¨fer2, S Sebens Mu¨erko¨ster2 and P Altevogt1 1Translational Immunology, German Cancer Research Center, Heidelberg, Germany and 2Clinic for Internal Medicine, Laboratory of Molecular Gastroenterology and Hepatology, UKSH-Campus, University of Kiel, Kiel, Germany L1 cell adhesion molecule (L1CAM) overexpression is 2004; Jemal et al., 2007). Resection of the tumour is still often associated with bad prognosis in various human the only option that offers a chance for cure (Schneider carcinomas. Recent studies also suggest a role of L1CAM et al., 2005) hence, the prognosis for patients is poor and in pancreatic ductal adenocarcinomas (PDAC). To more than 80% die within 5 years after surgery. further address its contribution, we expressed functional Pancreatic cancer is often refractory to conventional domains of L1CAM in PT45-P1 PDAC cells. We found chemotherapeutic treatment because of the frequent that L1CAM that is full length (L1-FL), but neither the development of profound chemoresistance. This che- soluble ectodomain (L1ecto) nor the cytoplasmic part moresistant phenotype is often mediated by the con- (L1cyt), could enhance cell proliferation or tumour growth stitutive activation of the transcription factor NF-kB, in mice. Expression of L1-FL resulted in constitutive resulting from the autocrine production of interleukin- activation of NF-jB, which was abolished by L1CAM 1b (IL-1b) (Arlt et al., 2002; Muerkoster et al., 2005). knockdown. We showed that the expression of IL-1b was An important goal is to understand the molecular selectively upregulated by L1-FL, and increased IL-1b parameters involved not only in chemoresistance but levels were instrumental for sustained NF-jB activation. also in pancreatic carcinoma progression. IL-1b production and NF-jB activation were abolished by The L1 cell adhesion molecule (L1CAM) is a knockdown of a5-integrin and integrin-linked kinase, but 200–220 kDa transmembrane glycoprotein of the insensitive to depletion of L1CAM cleavage proteinases. immunoglobulin superfamily composed of six immuno- Supporting these data, PT45-P1 cells transduced with globulin-like domains and five fibronectin type III an L1CAM mutant deficient in integrin binding (L1-RGE) repeats, followed by a transmembrane region and a did not support the described L1-FL functions. Our highly conserved cytoplasmic tail (Moos et al., 1988). results suggest that membranous L1CAM interacts with L1CAM can interact with itself (homophilic) and with RGD-binding integrins, leading to sustained NF-jB activa- heterophilic ligands such as integrins, CD24, neurocan, tion by IL-1b production and autocrine/paracrine signalling. neuropilin-1 and other members of the neural cell The unravelling of this novel mechanism sheds new light on adhesion family (Brummendorf et al., 1998; Hortsch, the important role of L1CAM expression in PDAC cells. 2000). Many binding sites in the L1CAM molecule have Oncogene (2010) 29, 4766–4778; doi:10.1038/onc.2010.230; already been mapped. The RGD site(s) in the sixth published online 14 June 2010 immunoglobulin domain (one in human, two in mouse) support a5- and av-integrin-mediated cell binding Keywords: pancreatic tumour; L1CAM signalling; (Ruppert et al., 1995; Montgomery et al., 1996; integrins Oleszewski et al., 1999). Homophilic L1CAM interac- tion has been recently mapped to immunoglobulin domains 1–4 (Gouveia et al., 2008). L1CAM was initially described in the nervous system, Introduction in which it is important for cell migration and axon outgrowth. Subsequent work has shown overexpression Pancreatic cancer is a highly aggressive disease that is of L1CAM in a variety of human tumours, in which it is mostly detected in an advanced stage. Owing to its associated with bad prognosis and metastases formation exocrine functions, it frequently develops multiple (Thies et al., 2002; Fogel et al., 2003a, b; Boo et al., metastases and is the fourth leading cause of cancer- 2007; Kaifi et al., 2007). Furthermore, L1CAM was related death in the United States. (Neoptolemos et al., detected in tumour-associated endothelium (Issa et al., 2009; Maddaluno et al., 2009). Expression of L1CAM was also described in pancreatic ductal adenocarcino- Correspondence: Professor P Altevogt, Translational Immunology, mas (PDACs) (Sebens Muerkoster et al., 2007, 2009; D015, German Cancer Research Center, Im Neuenheimer Feld 280, Geismann et al., 2009). Accumulating evidence suggests Heidelberg D-69120, Baden Wu¨rtemberg, Germany. E-mail: [email protected] an important role of L1CAM in the malignancy of Received 28 October 2009; revised 19 April 2010; accepted 7 May 2010; this tumour (Sebens Muerkoster et al., 2007, 2009; published online 14 June 2010 Geismann et al., 2009). The mechanism of L1CAM- L1CAM and NF-kappaB H Kiefel et al 4767 mediated tumour progression is not clearly established. processed further in both PT45-P1-L1-FL and Panc-1 Previous studies showed that L1CAM augments tumour cells (Supplementary Figure 1b). There was no effect of growth in NOD/Scid mice, enhances cell motility on treatment observed in L1cyt-expressing cells (data not extracellular matrix proteins and increases matrigel shown). invasion (Mechtersheimer et al., 2001; Thelen et al., L1ecto was secreted in large amounts into the medium 2002; Silletti et al., 2004; Gast et al., 2005; Gavert et al., (Figure 1e) and was detected by western blot (Figure 1d) 2005; Meier et al., 2006). Interference with L1CAM in cell lysates and L1CAM-specific capture enzyme- expression by genetic manipulation or by selective linked immunosorbent assay (ELISA) (Figure 1e). As knockdown was found to be growth inhibitory (Primiano expected, L1-FL cells released significantly less soluble et al., 2003; Bao et al., 2008). Studies from different L1CAM into the medium (Figure 1e). The amount laboratories showed L1CAM-dependent gene expres- of soluble L1CAM from L1-FL in the supernatant sion in mouse and human carcinoma cells (Silletti et al., was increased by PMA treatment, whereas there was 2004; Gavert et al., 2007; Gast et al., 2008a) that is no effect on the release of L1ecto (Supplementary relevant for understanding the mechanisms of L1CAM Figure 1a). These results implicate that L1CAM tumour-promoting effects. processing in pancreatic cells is similar to that of other To further analyse the mechanisms of L1CAM carcinoma cells. signalling, we expressed functional domains of L1CAM in PT45-P1 PDAC cells. We show that only L1-FL shows nuclear translocation that is increased L1CAM that is full-length (L1-FL), but neither the by PMA treatment soluble L1CAM (L1ecto) nor the cytoplasmic part In a recent study, we have shown that the C-terminal (L1cyt), can promote cell proliferation and tumour L1CAM fragment translocates into the nucleus depend- growth in vivo. Only L1-FL-expressing cells support ing on proteolysis by ADAM10 and presenilin (Riedle NF-kB activation and upregulate various genes includ- et al., 2009). We examined nuclear translocation in ing the prototype NF-kB inducer IL-1b. The L1CAM- PT45-P1 cells expressing L1-FL and L1cyt by fluores- mediated upregulation of IL-1b expression seems to be cence microscopy, using pcytL1 to the cytoplasmic tail instrumental for NF-kB activation and requires the of L1CAM (Riedle et al., 2009). Increased nuclear RGD motive in L1CAM and integrin signalling. Our staining of L1CAM was observed following treatment work identifies important novel downstream effects of of L1-FL cells with PMA (Figure 1f). PMA-induced L1CAM that help to understand the molecular mechan- nuclear translocation of L1-FL was clearly blocked by isms leading to tumour progression promoted by pretreatment with DAPT (Figure 1f and Supplementary L1CAM. Figure 2). In contrast, no effect of the inhibitor or PMA was observed in PT45-P1 cells expressing L1cyt, suggesting that L1cyt could reach the nucleus without Results proteolytic processing (Figure 1f and Supplementary Figure 2). Characterization of PT45-P1 cells overexpressing L1CAM domains We transduced PT45-P1 pancreatic adenocarcinoma PT45-P1 expressing L1CAM full-length augments cell cells with functional domains of L1-FL, cytoplasmic proliferation and tumour growth in vivo L1CAM (L1cyt) or L1CAM ectodomain (L1ecto), as Next, we analysed the impact of L1CAM domains on outlined (Figure 1a). The selected cells stably expressed cellular functions in PDAC cells. Therefore, we tested L1CAM fragments as detected by quantitative real-time cell proliferation of PT45-P1 cells expressing various (qRT)–PCR (Figure 1b) and by fluorescence-activated L1CAM constructs. A clear growth-promoting effect of cell sorting analysis using antibodies to the cytoplasmic L1-FL compared with mock-transfected cells was noted (mAb 74 5H7) or the extracellular portion of L1CAM after 96 h (Figure 2a). Despite the significantly higher (mAb L1-11A and L1-9.3) (Figure 1c). L1-FL and L1cyt amounts of released L1ecto, only a marginal effect was were visualized by western blot analysis of cell lysates observed in L1ecto cells. (Figure 1d). L1-FL also showed detectable amounts of To test whether enhanced proliferation in vitro L1-32 (Figure 1d), representing the membrane-retained resulted in augmented tumour growth in vivo,we cleavage fragment resulting from ADAM10-mediated injected the different cell lines into NOD/Scid mice. ectodomain shedding of L1CAM (Gutwein et al., 2000; Moreover, in vivo, only PT45-P1 cells expressing L1-FL Mechtersheimer et al., 2001). Further analysis showed showed significantly enhanced tumour growth com- that cleavage of L1-FL was augmented by Phorbol 12- pared with mock-transduced cells (Figures 2b and c). myristate 13-acetate (PMA) treatment and was blocked In contrast, tumour growth of L1ecto and L1cyt cells by the metalloproteinase inhibitor BB-3644 (Supple- was comparable to that of PT45-P1 mock-transduced mentary Figures 1a and b).