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Increased expression of Nodal correlates with reduced patient survival in pancreatic cancer
Pancreatology 15 (2015) 156e161
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Pancreatology journal homepage: www.elsevier.com/locate/pan
Original article
Increased expression of Nodal correlates with reduced patient survival in pancreatic cancer Bo Kong a, 1, Weibin Wang a, b, 1, Irene Esposito c, 2, Helmut Friess a, € rg Kleeff a, * Christoph W. Michalski a, 3, Jo €t München, Munich, Germany Department of Surgery, Technische Universita Department of General Surgery, Peking Union Medical College Hospital, Beijing, China c €t München, Munich, Germany Institute of Pathology, Technische Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 12 February 2015
Background: Nodal (nodal growth differentiation factor) and its inhibitor Lefty (left right determination factor), which are ligands of the TGF (transforming growth factor) b superfamily, are responsible for the determination of left-right asymmetry in vertebrates. Nodal/Lefty signaling has been suggested to play a role in the development of metastatic melanoma and breast cancer. However, it remains unclear whether this pathway is also involved in human pancreatic ductal adenocarcinoma (PDAC). Methods: Pancreatic cancer patient specimens with clinical data (n ¼ 54) were used to investigate the clinical significance of Nodal-Lefty signaling. A set of in vitro assays were carried out in a human pancreatic cancer cell line (Colo-357) to assess the functional relevance of Nodal-Lefty signaling. Results: Nodal was absent in the human normal pancreas, while Lefty was present in islet cells. Though Nodal and Lefty expression were found in cancer cells at various expression levels, the cancer-associated tubular complexes were particularly positive for Lefty. Survival analysis revealed that high expression of Nodal correlated with reduced patient survival (median survival 17.8 vs 33.0 months, p ¼ 0.013). Cultured pancreatic cancer cell lines expressed Nodal and Lefty at different levels. In vitro functional assays revealed that treatment with human recombinant Nodal inhibited cell growth and increased invasion of Colo-357 pancreatic cancer cells whereas no effect was found upon treatment with recombinant Lefty. Conclusion: Nodal-Lefty signaling might be involved in the pathogenesis of PDAC as Nodal expression marks a subtype of PDAC with unfavorable prognosis. Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Nodal Lefty Pancreatic cancer Survival Transforming growth factor b Prognosis
Introduction Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with its incidence equaling its mortality rate [1]. Recent evidence suggests that aggressive tumor cells share many characteristics with embryonic stem cells (ESCs) in terms of cell plasticity [2]. In
* Corresponding author. Department of Surgery, Technische Universit€ at München, Ismaningerstrasse 22, 81675 Munich, Germany. Tel.: þ49 89 4140 5098; fax: þ49 89 4140 4870. E-mail address: [email protected] (J. Kleeff). 1 These authors contributed equally to the manuscript. 2 Current affiliation: Institute of Pathology, Medical University of Innsbruck, Innsbruck, Austria. 3 Current affiliation: Department of Surgery, University of Heidelberg, Heidelberg, Germany.
particular, the sequencing results of the PDAC genome uncovered that TGFb signaling, which has a prominent role in the earliest cell fate determination in embryogenesis, is altered in virtually all of human PDAC samples [3,4]. Apart from non-canonical signaling, TGFb superfamily ligands mainly signal through two branches: the SMAD2/3 branch, which transduces the signal of TGFb/Activin/Nodal via the type I receptors ALK4, ALK5 and ALK7 (activin receptor-like kinase 4/5/7) and the SMAD1/5 branch responsible for propagating signals from BMP/GDF/MIS (bone morphogenetic protein/growth differentiation factor/mülllerian inhibiting substance) via ALK1, ALK2, ALK3 and ALK6 receptors [5]. Upon direct phosphorylation through activated type I receptors, SMAD2/3 or SMAD1/5 form a complex with SMAD4, then further translocate to the nucleus to regulate gene expression [5]. Of note, the fine-tuned TGFb signaling essentially coordinates plasticity and differentiation events of human ESCs. As human
http://dx.doi.org/10.1016/j.pan.2015.02.001 1424-3903/Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
B. Kong et al. / Pancreatology 15 (2015) 156e161
ESCs undergo early differentiation, the activity of SMAD2/3 branch is decreasing while SMAD1/5 signaling is gradually activated [3]. Accordingly, deregulated TGFb signaling (e.g. TGFb1, BMP4) in cancer cells has been proposed to affect tumor cells plasticity by eliciting epithelial-to-mesenchymal transdifferentiation in PDAC [6e8]. Furthermore, components of TGFb signaling such as TGFb, ALK5 (Tb-R I), Tb-R II, BMP2, ALK3 (BMPR1A) and BMPR-II have been shown to be associated with patient prognosis, underscoring the clinical relevance of TGFb signaling in PDAC [9e12]. Nodal (nodal growth differentiation factor), belonging to the SMAD2/3 arm of TGFb signaling, induces the formation of mesoderm and endoderm and determines lefty-right asymmetry in vertebrates [13]. Nodal signals mainly through binding of ALK4/7 receptors, a process which is mediated and modulated by its coreceptor Cripto [14]. Lefty 1 and Lefty 2 (left right determination factor 1 and 2) are capable of binding to Cripto and preventing the assembly of Nodal/Cripto/receptor complex, therefore inhibiting Nodal signaling [15e17]. Moreover, a delicate feedback loop exists wherein Nodal signals induce the expression of its endogenous inhibitor Lefty to restrict Nodal signaling temporally and spatially during gastrulation [17e19]. Besides, Lefty expression can also be induced by TGFb in human pancreatic cancer cell lines, adding another layer of complexity to this feedback network between different members of the TGFb superfamily [20]. Recently, studies have demonstrated that both Nodal and Lefty are expressed in the mouse pancreas during embryogenesis and that Nodal-Lefty signaling is functionally relevant to expansion of epithelial cells in a mouse model of islet regeneration [21]. Given these findings in the mouse pancreas and the clinical significance of TGFb signaling in PDAC, we hypothesize that NodalLefty signaling might also be involved in human pancreatic carcinogenesis.
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mouse anti-human Nodal monoclonal antibody (1:2000 ab55676 Abcam Cambridge, UK) at 4 C overnight followed by incubation with horseradish peroxidase-labeled polymer conjugated with goat anti-rabbit and anti-mouse antibodies (Dako Envision System, Dako Cytomation GmbH, Hamburg, Germany). Diaminobenzidine was used as chromogen and nuclei were counterstained with Mayer's hematoxylin. To confirm antibody specificity, tissue sections were incubated in the absence of the primary antibodies with negative control rabbit or mouse IgG. Under these conditions, no immunostaining was detected. Inclusion criteria for the analysis were a histopathological confirmed diagnosis of PDAC and follow-up data for postoperative survival. The only exclusion criterion was a postoperative survival of less than 3 months, to exclude perioperative mortality. The stained specimens were analyzed independently by two researchers. Staining scores were calculated for both Nodal and Lefty by multiplying values for the intensity of staining (0 ¼ no staining, 1 ¼ weak or medium staining, 2 ¼ strong staining) with values for the stained area (1 ¼ less than one-third stained area, 2 ¼ between one and two-thirds stained area, 3 ¼ more than two-thirds stained area). The total histologic score (0e6) was expressed as a product of the intensity and area scores as previously described [11]. Immunoblot analysis Cultured pancreatic cancer cells were lysed in ice-cold RIPA buffer (#9806 Cell signaling, Danvers, MA) containing 1 tablet EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) for 10 min. The immunoblot was performed as previously described [23]. The following antibodies were used: rabbit anti-human Lefty polyclonal antibodies (1:100 ab71268 Abcam Cambridge, UK) or mouse anti-human Nodal monoclonal antibodies (1:800 ab55676 Abcam Cambridge, UK) rabbit antiGAPDH (1:5000 Santa Cruz Biotechnology) overnight at 4 C.
Materials and methods siRNA transfections Patients and tissue sampling Tissue samples were obtained from patients with pancreatic ductal adenocarcinoma who underwent resection at the Department of Surgery, Technical University of Munich, Germany. Normal pancreatic tissue samples were obtained during resections due to technical reasons like tumor infiltration of the periampullary area by another malignancy (i.e. colon cancer) or metastasis to the pancreas by kidney tumors. Tissue collection was approved by the Ethics Committee of the Technical University of Munich and informed consent was obtained from all patients. Tissue sampling and processing was performed as previously described [22]. Cell culture Eight pancreatic cancer cell lines (Aspc-1, Bxpc-3, Capan-1, Colo357, MiaPaca-2, Su86.86, Panc-1 and T3M4) were cultured as previously described [23]. Immortalized human pancreatic ductal epithelial (HPDE) cells were kindly provided by Dr. Ming-Sound Tsao (Ontario Cancer Institute, Canada) and cultured in keratinocyte medium (Life Technologies, Carlsbad, CA) with supplements as published before [24]. Immunohistochemistry Immunohistochemistry (IHC) and evaluation of histological score were performed as previously described [22]. Briefly, consecutive sections were incubated with rabbit anti-human Lefty polyclonal antibody (1:100 ab71268 Abcam Cambridge, UK) or
Synthetic siRNA oligonucleotides for human lefty 1 (SI00128709, CCA GAT AAT AAA GAC TTT GTA, SI00128716, CTG ACA AGT TAC CTC ACC TAA) Nodal (SI00120435, GTG CTC CTA GAT CAC CAT AAA, SI00120449, CTG CAT GTT CTC TTT ACT GAA) were obtained from Qiagen (Qiagen, Hilden, Germany) and transfected with Hiperfect transfection reagent according to the manufacturer's instructions. The efficacy of Nodal and Lefty 1 downregulation was assessed after 48 h of transfection. Proliferation assays Cell growth was determined using the 3-(4,5-dimethylthiazole2-yl)2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml in PBS; Sigma Aldrich, St. Louis, MO) colorimetric growth assay as previously described [23]. Cells were seeded at a density of 5000 cells/ well into 96-well plate. Daily growth of cells was measured by adding MTT solution (50 mg/well) for 4 h. Then, the culture solution was removed and the crystals were dissolved in acidic isopropanol (100 ml per well). The optical density was measured at 570 nm. T0 was defined as the optical density of cells 12 h after seeding into 96well plate. Then, the daily growth of cells (T24, T48 and T72) was normalized to T0. rNodal or Lefty 2 (all from R&D Systems; Wiesbaden-Nordenstadt, Germany) was added 12 h after seeding of the cells at increasing concentrations (for rNodal: 50, 200, 5000 ng/ml; for Lefty 2: 20, 50 ng/ml). PBS was used as a control. As for the siRNA transfection experiments, cells were seeded 24 h after transfection. All assays were performed in triplicates and were repeated three times.
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Invasion assays To assess cell invasion in vitro, 24-well Matrigel invasion chambers with 8-mm pore sizes (BD Biosciences, San Jose, CA) were used and were reconstituted with 600 ml serum-free DMEM medium in both the top and the bottom chambers for 2e4 h. Cells were trypsinized and were seeded into the top chamber at a density of 8 104 cells per well in 500 ml DMEM containing 0.5% fetal calf serum. The outer chambers contained 0.7 ml of medium (10% FCS). According to each experimental set up, rNodal (200 ng/ml) and rLefty 2 (50 ng/ml) were added to the top chambers, respectively. After incubation at 37 C for 24 h, cells remaining attached to the upper surface of the membrane were carefully removed with cotton swabs, while cells that reached the underside of the chamber were stained with H&E and were counted. Data are presented as fold of control by normalizing to the number of invading cell in control treated samples. All experiments were repeated three times. Statistical analysis For statistical analyses, the GraphPad Prism 5 Software (GraphPad, San Diego, CA) was used. The chi-square test (or Fisher's exact test) and unpaired t-test (age) were used to compare clinical
parameters between Nodal high and low patients. Unless otherwise stated, an unpaired t-test was used for group-wise comparisons. For the correlation analysis of Nodal high/low or Lefty positive/ negative expression and survival, as detected by analysis of immunohistochemical staining, the KaplaneMeier method was used, followed by a log-rank (ManteleCox) test. The level of statistical significance was set at p < 0.05. Results Localization of Lefty and Nodal in human pancreatic tissue In order to investigate the expression pattern of Lefty and Nodal in pancreatic tissues, staining was performed in consecutive sections of the normal pancreas (n ¼ 5) and PDAC tissues (n ¼ 54). Lefty immunoreactivity was clearly found in endocrine cells of the human normal pancreas (Fig. 1A) in accordance with reported results from mice [21], whereas neither Lefty nor Nodal were expressed in the normal ductal and acinar cells (Fig. 1A and B). In PDAC sections, islet cells preserved Lefty staining (Fig. 1C and D). Interestingly, tubular complexes (TC) adjacent to these islets as well as TC in atrophic areas at the border of the PDACassociated desmoplastic region were generally positive for Lefty staining as well (Fig. 1C and E). In contrast, in consecutive sections
Fig. 1. Localization of Lefty and Nodal in human pancreatic tissue. A, B, islet cells in the normal pancreas are immunopositive for Lefty but negative for Nodal (magnifications, 100). CeH, endocrine cells and tubular complexes in PDAC sections are Lefty positive whereas only cancer cells in the consecutive section show Nodal staining (magnifications, C and F: 50; D and E: 630; G and H: 400). I and M, PDAC with high levels of Nodal expression with positive Lefty staining (30% of the cases; magnifications, 100). J and N, PDAC with high levels of Nodal and no expression of Lefty (30% of the cases; magnifications, 100), K and O, Lefty-positive PDAC with low expression level of Nodal (9% of the cases; magnifications, 100). L and P, PDAC showing negative staining for Lefty and low expression of Nodal (31% of the cases; magnifications, 100).
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Nodal staining was absent both in endocrine cells and in TC (Fig. 1F and G) but positive in a subset of cancer cells (Fig. 1H). Cancer cells were weakly positive for Lefty in 10 cases (19%, Fig. 1I and K) and moderate to strong in 11 cases (20%, Fig. 1I and K); Lefty staining was absent in the cancer cells of 33 cases (61%, Fig. 1J and L). In contrast, Nodal staining was moderate to strong in the cancer cells of 32 cases (60%, Fig. 1M and N) but weak in 22 cases (40%, Fig. 1O and P). Due to relatively small number of moderate to strong Lefty expressing PDAC cases (histological score 3e6), we stratified these cases into Lefty-negative (histological score 0, Leftyneg) and Lefty-positive group (histological score 1e6, Leftypos) but Nodal low (histological score 0e2, Nodallow) and nodal high group (histological score 3e6, Nodalhigh ). In total, 50% (16/32, 30% of all) of Nodalhigh cases showed positive Lefty staining (Fig. 1I, J, M, and N), while only 23% (5/22, 9% of all) of Nodallow cases exhibiting detectable expression of Lefty in the cancer cells (Fig. 1K, L, O and P).
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Table 1 Demographic data and clinical characteristics comparison between Nodal high and low patients.
Gender Age (mean, year) Resection (R) Tumor size (T) Lymph node (N) Metastasis (M) Grade (G)
Nodal low (n ¼ 22)
Nodal high (n ¼ 32)
P value
Male (10) Female (12) 62.4 R0 (19) R1 (3) T1-T2 (1) T3 (21) N0 (4) N1 (18) M0 (21) M1 (1) G1-G2 (15) G3 (7)
Male (22) Female (10) 67.1 R0 (28) R1 (4) T1-T2 (2) T3 (30) N0 (4) N1 (28) M0 (29) M1 (3) G1-G2 (23) G3 (9)
0.10 0.04 1.0 1.0 0.70 0.64 0.77
Pancreatic cancer cell lines express Lefty and Nodal Nodal expression rather than Lefty correlates with survival Since previous studies in melanoma and breast cancer revealed that Nodal expression was correlated with clinical progression of malignancy [25e27], we set out to determine whether Nodal expression also affects the prognosis of PDAC patients. Therefore, KaplaneMeier analysis was used to compared survival difference between Nodallow and Nodalhigh patients. This analysis revealed that patients with low Nodal expression lived significantly longer than patients with high Nodal expression (median survival 33.0 vs 17.8 months, p ¼ 0.013, Fig. 2A). Furthermore, other than age (4.7 years difference in the mean age) the clinical parameters were comparable between these two groups (Table 1). However, there was no survival difference between Lefty negative and positive patients (median survival 26.8 vs 20.7 months, p ¼ 0.870, Fig. 2B).
Immunoblot assays were employed to analyze Lefty and Nodal expression in eight cultured pancreatic cancer cell lines (Aspc-1, Bxpc-3, Capan-1, Colo-357, MiaPaCa-2, Panc-1, Su86.86 and T3M4) as well as in HPDE cells. This analysis revealed that cultured cancer cell lines (Fig. 3A and B) and HPDE cells (Fig. 3C and D) expressed various levels of Lefty and Nodal. Antibody specificity for Lefty and Nodal was confirmed by performing siRNA transfection assays in Colo-357 cells. Specifically, Nodal siRNA transfection led to down-regulation of protein appearing at around 40 kDa in accordance with the predicted size of human Nodal (Fig. 3E, left panel). Correspondingly, Lefty 1 siRNA transfection reduced expression of a protein appearing at the predicted size (41 kDa) of human lefty 1. Interestingly, we observed that the intensity of a band greater than 55 kDa also decreased (Fig. 3E, right panel). Nodal affects proliferation and invasion Colo-357 cells In an attempt to address functional the relevance of Nodal and Lefty in carcinogenesis of PDAC, Colo-357 cells maintaining an intact TGFb pathway were chosen for further investigation [28]. Though lower dose of human recombinant Nodal (rNodal, 50 and 200 ng/ml) had no obvious effect, higher dose (5000 ng/ml) inhibited growth of Colo-357 cells (Fig. 4A, left panel) consistent with results obtained from rat pancreatic tumor cells AR42J [21]. In addition, rNodal (200 ng/ml) also increased invasiveness of Colo357 cells where no effect on cell growth was observed at this dose (Fig. 4A, left and right panel). In contrast, treatment of human recombinant Lefty 2 (rLefty) had no effect either on cell growth or invasion (Fig. 4B). In addition, silencing endogenous Nodal or Lefty in Colo-357 cells by siRNA transfection had no additional influence on either cell growth (Fig. 4C and D). Discussion
Fig. 2. Nodal expression but not Lefty correlates with survival. A, survival proportions in patients with low and high Nodal expression; p ¼ 0.013. B, survival proportions in patients with Lefty negative and positive staining. No significant (n.s.) differences were found between the groups.
Though tubular complexes are regularly found in human specimen of chronic pancreatitis and pancreatic cancer, the cell of origin for such lesions remains controversial: ductal, centroacinar/acinar and the islet compartment have been considered to be candidates [29e32]. Indeed, these tubular complexes have been described as key histological changes in many genetically engineered mice (involving all lineages) including loss of tumor suppressors (e.g. Pten (phosphatase and tensin homolog) and Lkb1 (serine/threonine kinase 11), overexpression of growth factors (e.g. TGF-a (transforming growth factor, alpha)), transformation of oncogenic Kras as
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Fig. 3. Pancreatic cancer cell lines unanimously express Lefty and Nodal. A, B. Expression (immunoblot) of Nodal (A) and Lefty (B) in pancreatic cancer cell lines. All 8 tested cell lines (Aspc-1 (As), Bxpc-3 (Bx), Capan-1 (Ca), Colo357 (Co), Mia-PaCa2 (Mi), Panc-1 (Pa), Su86.86 (Su), T3M4 (T3)) express Nodal and Lefty at various level. C, D. Similarly, Nodal (C) and Lefty (D) expression can also be detected in HPDE and Colo357 (internal control) cells. E. Nodal (left panel) and Lefty (right panel) silencing with specific siRNA or negative control siRNA (control) at 72 h.
well as tissue injury [33e38]. Furthermore, since tubular complexes are frequently observed prior to formation of PanIN lesions or directly before PDAC development, they have been proposed as precursor lesions of PDAC as well [29,39]. It is likely that tubular complexes formation is a default process for pancreas remodeling in response to intrinsic (e.g. oncogenic, energy and growth) and extrinsic (e.g. inflammation and tissue damage) stress. In the current study, we observed that Lefty (rather than Nodal) is expressed distinctively in tubular complexes of human pancreatic cancer specimen. This is consistent with findings in a mouse model of islet regeneration where it is described that a strong expression of Lefty
is observed in a subset of duct-like structures [21]. However, it remains obscure how and why tubular complexes express Lefty. It is also not clear whether Lefty expression is functionally relevant to the formation of tubular complexes or rather acts as feedback response to strong Nodal signaling in adjacent cancer cells. In addition, Lefty expression in tubular complexes (especially in the absence of active RAS/MEK/ERK signaling) can potentially be induced by TGFb present within the tumor microenvironment [20,40]. Previous studies have demonstrated that Nodal is present in human metastatic melanoma and breast cancer cells, and that it
Fig. 4. Nodal affects proliferation and invasion Colo-357 cells. A. Proliferation of Colo-357 cells as assessed by MTT assays following rNodal treatment: Lower dose (50 and 200 ng/ml) had no effect whereas high dose (5000 ng/ml) inhibited cell growth. Data from three independent experiments are expressed as mean ± SEM (left panel). In a matrigel invasion assay, rNodal (200 ng/ml) increases cell invasion of Colo-357 cells (right panel). Values shown are the mean ± SEM fold change of the negative control obtained from three independent experiments. B. rLefty has no effect on either growth (20 and 50 ng/ml) or invasiveness (50 ng/ml) of Colo-357 cells. No significant (n.s.) differences were found between the groups. C, D Proliferation of Colo-357 cells as assessed by MTT assays following down-regulation of Nodal and Lefty expression via siRNA transfection shows no effect. Results from three independent experiments are expressed as mean ± SEM.
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correlates with clinical progression. In line, inhibition of Nodal signaling by synthetic TGFb inhibitors or human ESCs-derived Lefty reduces cancer cell invasiveness and tumorigenicity [25,27]. Similarly, Lefty may account for the tumor suppressive activity of human liver stem cells by inhibiting nodal signaling [41]. Accordingly, we also observed the presence of Nodal expression in a majority of PDAC samples, and high expression of Nodal was associated with shorter patient survival. In addition, rNodal promotes cancer cells invasion in vitro. However, the reason why a subgroup of cancer cells acquires high expression levels of Nodal remains elusive. In contrast to findings in other tumors [25,27], there was no corresponding loss of Lefty (at least in 50% of cases) in the cancer cells with high Nodal expression as well as in cultured cell lines. Additionally, pancreatic cancer cells do not respond to treatment of rLefty in terms of cell growth and invasion. Hence, we speculate that due to constant exposure to Lefty (endocrine cell-derived), pancreatic cancer cells may have evolved an undefined mechanism (e.g. by overexpressing co-receptor Cripto for Nodal activation) to circumvent this feedback loop existing in human ESCs [42]. To conclude, we provide evidence that both Nodal and its natural inhibitor Lefty are expressed by pancreatic cancer cells, and high expression of Nodal is linked to reduced patient survival suggesting a clinical significance of this signaling pathway. Acknowledgments We thank Felicitas Altmayr, Tanja Rossmann-Bloeck, Manja Thorwirth and Carmen Marthen for excellent technical support. References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64: 9e29. [2] Hendrix MJ, Seftor EA, Seftor RE, Kasemeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer 2007;7:246e55. [3] James D, Levine AJ, Besser D, Hemmati-Brivanlou A. Tgfbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 2005;132:1273e82. [4] Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008;321:1801e6. [5] Shi Y, Massague J. Mechanisms of tgf-beta signaling from cell membrane to the nucleus. Cell 2003;113:685e700. [6] Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C, et al. Transforming growth factor beta1 treatment leads to an epithelialmesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation. Cancer Res 2001;61:4222e8. [7] Hamada S, Satoh K, Hirota M, Kimura K, Kanno A, Masamune A, et al. Bone morphogenetic protein 4 induces epithelial-mesenchymal transition through msx2 induction on pancreatic cancer cell line. J Cell Physiol 2007;213:768e74. [8] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelialmesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704e15. [9] Friess H, Yamanaka Y, Buchler M, Berger HG, Kobrin MS, Baldwin RL, et al. Enhanced expression of the type ii transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type iii receptor expression. Cancer Res 1993;53:2704e7. [10] Kleeff J, Maruyama H, Ishiwata T, Sawhney H, Friess H, Buchler MW, et al. Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo. Gastroenterology 1999;116:1202e16. [11] Kong B, Michalski CW, Hong X, Valkovskaya N, Rieder S, Abiatari I, et al. Azgp1 is a tumor suppressor in pancreatic cancer inducing mesenchymal-toepithelial transdifferentiation by inhibiting tgf-beta-mediated erk signaling. Oncogene 2010;29:5146e58. [12] Wagner M, Kleeff J, Friess H, Buchler MW, Korc M. Enhanced expression of the type ii transforming growth factor-beta receptor is associated with decreased survival in human pancreatic cancer. Pancreas 1999;19:370e6. [13] Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol 2003;19:589e621. [14] Yeo C, Whitman M. Nodal signals to smads through cripto-dependent and cripto-independent mechanisms. Mol Cell 2001;7:949e57.
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Overexpression of heparin-binding egf-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia. Gastroenterology 2003;124:1020e36. [35] Morris JP, Cano DA, Sekine S, Wang SC, Hebrok M. Beta-catenin blocks krasdependent reprogramming of acini into pancreatic cancer precursor lesions in mice. J Clin Invest;120:508e520. [36] Schmid RM, Kloppel G, Adler G, Wagner M. Acinar-ductal-carcinoma sequence in transforming growth factor-alpha transgenic mice. Ann N Y Acad Sci 1999;880:219e30. [37] Stanger BZ, Stiles B, Lauwers GY, Bardeesy N, Mendoza M, Wang Y, et al. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer Cell 2005;8:185e95. [38] Wagner M, Luhrs H, Kloppel G, Adler G, Schmid RM. Malignant transformation of duct-like cells originating from acini in transforming growth factor transgenic mice. Gastroenterology 1998;115:1254e62. [39] De La OJ, Murtaugh LC. Notch and kras in pancreatic cancer: at the crossroads of mutation, differentiation and signaling. Cell Cycle 2009;8:1860e4. [40] Saito A, Ochiai H, Okada S, Miyata N, Azuma T. Suppression of lefty expression in induced pluripotent cancer cells. Faseb J 2013;27:2165e74. [41] Cavallari C, Fonsato V, Herrera MB, Bruno S, Tetta C, Camussi G. Role of lefty in the anti tumor activity of human adult liver stem cells. Oncogene 2013;32: 819e26. [42] Friess H, Yamanaka Y, Buchler M, Kobrin MS, Tahara E, Korc M. Cripto, a member of the epidermal growth factor family, is over-expressed in human pancreatic cancer and chronic pancreatitis. Int J Cancer 1994;56:668e74.
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Original article
Increased expression of Nodal correlates with reduced patient survival in pancreatic cancer Bo Kong a, 1, Weibin Wang a, b, 1, Irene Esposito c, 2, Helmut Friess a, € rg Kleeff a, * Christoph W. Michalski a, 3, Jo €t München, Munich, Germany Department of Surgery, Technische Universita Department of General Surgery, Peking Union Medical College Hospital, Beijing, China c €t München, Munich, Germany Institute of Pathology, Technische Universita a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Available online 12 February 2015
Background: Nodal (nodal growth differentiation factor) and its inhibitor Lefty (left right determination factor), which are ligands of the TGF (transforming growth factor) b superfamily, are responsible for the determination of left-right asymmetry in vertebrates. Nodal/Lefty signaling has been suggested to play a role in the development of metastatic melanoma and breast cancer. However, it remains unclear whether this pathway is also involved in human pancreatic ductal adenocarcinoma (PDAC). Methods: Pancreatic cancer patient specimens with clinical data (n ¼ 54) were used to investigate the clinical significance of Nodal-Lefty signaling. A set of in vitro assays were carried out in a human pancreatic cancer cell line (Colo-357) to assess the functional relevance of Nodal-Lefty signaling. Results: Nodal was absent in the human normal pancreas, while Lefty was present in islet cells. Though Nodal and Lefty expression were found in cancer cells at various expression levels, the cancer-associated tubular complexes were particularly positive for Lefty. Survival analysis revealed that high expression of Nodal correlated with reduced patient survival (median survival 17.8 vs 33.0 months, p ¼ 0.013). Cultured pancreatic cancer cell lines expressed Nodal and Lefty at different levels. In vitro functional assays revealed that treatment with human recombinant Nodal inhibited cell growth and increased invasion of Colo-357 pancreatic cancer cells whereas no effect was found upon treatment with recombinant Lefty. Conclusion: Nodal-Lefty signaling might be involved in the pathogenesis of PDAC as Nodal expression marks a subtype of PDAC with unfavorable prognosis. Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Nodal Lefty Pancreatic cancer Survival Transforming growth factor b Prognosis
Introduction Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with its incidence equaling its mortality rate [1]. Recent evidence suggests that aggressive tumor cells share many characteristics with embryonic stem cells (ESCs) in terms of cell plasticity [2]. In
* Corresponding author. Department of Surgery, Technische Universit€ at München, Ismaningerstrasse 22, 81675 Munich, Germany. Tel.: þ49 89 4140 5098; fax: þ49 89 4140 4870. E-mail address: [email protected] (J. Kleeff). 1 These authors contributed equally to the manuscript. 2 Current affiliation: Institute of Pathology, Medical University of Innsbruck, Innsbruck, Austria. 3 Current affiliation: Department of Surgery, University of Heidelberg, Heidelberg, Germany.
particular, the sequencing results of the PDAC genome uncovered that TGFb signaling, which has a prominent role in the earliest cell fate determination in embryogenesis, is altered in virtually all of human PDAC samples [3,4]. Apart from non-canonical signaling, TGFb superfamily ligands mainly signal through two branches: the SMAD2/3 branch, which transduces the signal of TGFb/Activin/Nodal via the type I receptors ALK4, ALK5 and ALK7 (activin receptor-like kinase 4/5/7) and the SMAD1/5 branch responsible for propagating signals from BMP/GDF/MIS (bone morphogenetic protein/growth differentiation factor/mülllerian inhibiting substance) via ALK1, ALK2, ALK3 and ALK6 receptors [5]. Upon direct phosphorylation through activated type I receptors, SMAD2/3 or SMAD1/5 form a complex with SMAD4, then further translocate to the nucleus to regulate gene expression [5]. Of note, the fine-tuned TGFb signaling essentially coordinates plasticity and differentiation events of human ESCs. As human
http://dx.doi.org/10.1016/j.pan.2015.02.001 1424-3903/Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
B. Kong et al. / Pancreatology 15 (2015) 156e161
ESCs undergo early differentiation, the activity of SMAD2/3 branch is decreasing while SMAD1/5 signaling is gradually activated [3]. Accordingly, deregulated TGFb signaling (e.g. TGFb1, BMP4) in cancer cells has been proposed to affect tumor cells plasticity by eliciting epithelial-to-mesenchymal transdifferentiation in PDAC [6e8]. Furthermore, components of TGFb signaling such as TGFb, ALK5 (Tb-R I), Tb-R II, BMP2, ALK3 (BMPR1A) and BMPR-II have been shown to be associated with patient prognosis, underscoring the clinical relevance of TGFb signaling in PDAC [9e12]. Nodal (nodal growth differentiation factor), belonging to the SMAD2/3 arm of TGFb signaling, induces the formation of mesoderm and endoderm and determines lefty-right asymmetry in vertebrates [13]. Nodal signals mainly through binding of ALK4/7 receptors, a process which is mediated and modulated by its coreceptor Cripto [14]. Lefty 1 and Lefty 2 (left right determination factor 1 and 2) are capable of binding to Cripto and preventing the assembly of Nodal/Cripto/receptor complex, therefore inhibiting Nodal signaling [15e17]. Moreover, a delicate feedback loop exists wherein Nodal signals induce the expression of its endogenous inhibitor Lefty to restrict Nodal signaling temporally and spatially during gastrulation [17e19]. Besides, Lefty expression can also be induced by TGFb in human pancreatic cancer cell lines, adding another layer of complexity to this feedback network between different members of the TGFb superfamily [20]. Recently, studies have demonstrated that both Nodal and Lefty are expressed in the mouse pancreas during embryogenesis and that Nodal-Lefty signaling is functionally relevant to expansion of epithelial cells in a mouse model of islet regeneration [21]. Given these findings in the mouse pancreas and the clinical significance of TGFb signaling in PDAC, we hypothesize that NodalLefty signaling might also be involved in human pancreatic carcinogenesis.
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mouse anti-human Nodal monoclonal antibody (1:2000 ab55676 Abcam Cambridge, UK) at 4 C overnight followed by incubation with horseradish peroxidase-labeled polymer conjugated with goat anti-rabbit and anti-mouse antibodies (Dako Envision System, Dako Cytomation GmbH, Hamburg, Germany). Diaminobenzidine was used as chromogen and nuclei were counterstained with Mayer's hematoxylin. To confirm antibody specificity, tissue sections were incubated in the absence of the primary antibodies with negative control rabbit or mouse IgG. Under these conditions, no immunostaining was detected. Inclusion criteria for the analysis were a histopathological confirmed diagnosis of PDAC and follow-up data for postoperative survival. The only exclusion criterion was a postoperative survival of less than 3 months, to exclude perioperative mortality. The stained specimens were analyzed independently by two researchers. Staining scores were calculated for both Nodal and Lefty by multiplying values for the intensity of staining (0 ¼ no staining, 1 ¼ weak or medium staining, 2 ¼ strong staining) with values for the stained area (1 ¼ less than one-third stained area, 2 ¼ between one and two-thirds stained area, 3 ¼ more than two-thirds stained area). The total histologic score (0e6) was expressed as a product of the intensity and area scores as previously described [11]. Immunoblot analysis Cultured pancreatic cancer cells were lysed in ice-cold RIPA buffer (#9806 Cell signaling, Danvers, MA) containing 1 tablet EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) for 10 min. The immunoblot was performed as previously described [23]. The following antibodies were used: rabbit anti-human Lefty polyclonal antibodies (1:100 ab71268 Abcam Cambridge, UK) or mouse anti-human Nodal monoclonal antibodies (1:800 ab55676 Abcam Cambridge, UK) rabbit antiGAPDH (1:5000 Santa Cruz Biotechnology) overnight at 4 C.
Materials and methods siRNA transfections Patients and tissue sampling Tissue samples were obtained from patients with pancreatic ductal adenocarcinoma who underwent resection at the Department of Surgery, Technical University of Munich, Germany. Normal pancreatic tissue samples were obtained during resections due to technical reasons like tumor infiltration of the periampullary area by another malignancy (i.e. colon cancer) or metastasis to the pancreas by kidney tumors. Tissue collection was approved by the Ethics Committee of the Technical University of Munich and informed consent was obtained from all patients. Tissue sampling and processing was performed as previously described [22]. Cell culture Eight pancreatic cancer cell lines (Aspc-1, Bxpc-3, Capan-1, Colo357, MiaPaca-2, Su86.86, Panc-1 and T3M4) were cultured as previously described [23]. Immortalized human pancreatic ductal epithelial (HPDE) cells were kindly provided by Dr. Ming-Sound Tsao (Ontario Cancer Institute, Canada) and cultured in keratinocyte medium (Life Technologies, Carlsbad, CA) with supplements as published before [24]. Immunohistochemistry Immunohistochemistry (IHC) and evaluation of histological score were performed as previously described [22]. Briefly, consecutive sections were incubated with rabbit anti-human Lefty polyclonal antibody (1:100 ab71268 Abcam Cambridge, UK) or
Synthetic siRNA oligonucleotides for human lefty 1 (SI00128709, CCA GAT AAT AAA GAC TTT GTA, SI00128716, CTG ACA AGT TAC CTC ACC TAA) Nodal (SI00120435, GTG CTC CTA GAT CAC CAT AAA, SI00120449, CTG CAT GTT CTC TTT ACT GAA) were obtained from Qiagen (Qiagen, Hilden, Germany) and transfected with Hiperfect transfection reagent according to the manufacturer's instructions. The efficacy of Nodal and Lefty 1 downregulation was assessed after 48 h of transfection. Proliferation assays Cell growth was determined using the 3-(4,5-dimethylthiazole2-yl)2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml in PBS; Sigma Aldrich, St. Louis, MO) colorimetric growth assay as previously described [23]. Cells were seeded at a density of 5000 cells/ well into 96-well plate. Daily growth of cells was measured by adding MTT solution (50 mg/well) for 4 h. Then, the culture solution was removed and the crystals were dissolved in acidic isopropanol (100 ml per well). The optical density was measured at 570 nm. T0 was defined as the optical density of cells 12 h after seeding into 96well plate. Then, the daily growth of cells (T24, T48 and T72) was normalized to T0. rNodal or Lefty 2 (all from R&D Systems; Wiesbaden-Nordenstadt, Germany) was added 12 h after seeding of the cells at increasing concentrations (for rNodal: 50, 200, 5000 ng/ml; for Lefty 2: 20, 50 ng/ml). PBS was used as a control. As for the siRNA transfection experiments, cells were seeded 24 h after transfection. All assays were performed in triplicates and were repeated three times.
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Invasion assays To assess cell invasion in vitro, 24-well Matrigel invasion chambers with 8-mm pore sizes (BD Biosciences, San Jose, CA) were used and were reconstituted with 600 ml serum-free DMEM medium in both the top and the bottom chambers for 2e4 h. Cells were trypsinized and were seeded into the top chamber at a density of 8 104 cells per well in 500 ml DMEM containing 0.5% fetal calf serum. The outer chambers contained 0.7 ml of medium (10% FCS). According to each experimental set up, rNodal (200 ng/ml) and rLefty 2 (50 ng/ml) were added to the top chambers, respectively. After incubation at 37 C for 24 h, cells remaining attached to the upper surface of the membrane were carefully removed with cotton swabs, while cells that reached the underside of the chamber were stained with H&E and were counted. Data are presented as fold of control by normalizing to the number of invading cell in control treated samples. All experiments were repeated three times. Statistical analysis For statistical analyses, the GraphPad Prism 5 Software (GraphPad, San Diego, CA) was used. The chi-square test (or Fisher's exact test) and unpaired t-test (age) were used to compare clinical
parameters between Nodal high and low patients. Unless otherwise stated, an unpaired t-test was used for group-wise comparisons. For the correlation analysis of Nodal high/low or Lefty positive/ negative expression and survival, as detected by analysis of immunohistochemical staining, the KaplaneMeier method was used, followed by a log-rank (ManteleCox) test. The level of statistical significance was set at p < 0.05. Results Localization of Lefty and Nodal in human pancreatic tissue In order to investigate the expression pattern of Lefty and Nodal in pancreatic tissues, staining was performed in consecutive sections of the normal pancreas (n ¼ 5) and PDAC tissues (n ¼ 54). Lefty immunoreactivity was clearly found in endocrine cells of the human normal pancreas (Fig. 1A) in accordance with reported results from mice [21], whereas neither Lefty nor Nodal were expressed in the normal ductal and acinar cells (Fig. 1A and B). In PDAC sections, islet cells preserved Lefty staining (Fig. 1C and D). Interestingly, tubular complexes (TC) adjacent to these islets as well as TC in atrophic areas at the border of the PDACassociated desmoplastic region were generally positive for Lefty staining as well (Fig. 1C and E). In contrast, in consecutive sections
Fig. 1. Localization of Lefty and Nodal in human pancreatic tissue. A, B, islet cells in the normal pancreas are immunopositive for Lefty but negative for Nodal (magnifications, 100). CeH, endocrine cells and tubular complexes in PDAC sections are Lefty positive whereas only cancer cells in the consecutive section show Nodal staining (magnifications, C and F: 50; D and E: 630; G and H: 400). I and M, PDAC with high levels of Nodal expression with positive Lefty staining (30% of the cases; magnifications, 100). J and N, PDAC with high levels of Nodal and no expression of Lefty (30% of the cases; magnifications, 100), K and O, Lefty-positive PDAC with low expression level of Nodal (9% of the cases; magnifications, 100). L and P, PDAC showing negative staining for Lefty and low expression of Nodal (31% of the cases; magnifications, 100).
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Nodal staining was absent both in endocrine cells and in TC (Fig. 1F and G) but positive in a subset of cancer cells (Fig. 1H). Cancer cells were weakly positive for Lefty in 10 cases (19%, Fig. 1I and K) and moderate to strong in 11 cases (20%, Fig. 1I and K); Lefty staining was absent in the cancer cells of 33 cases (61%, Fig. 1J and L). In contrast, Nodal staining was moderate to strong in the cancer cells of 32 cases (60%, Fig. 1M and N) but weak in 22 cases (40%, Fig. 1O and P). Due to relatively small number of moderate to strong Lefty expressing PDAC cases (histological score 3e6), we stratified these cases into Lefty-negative (histological score 0, Leftyneg) and Lefty-positive group (histological score 1e6, Leftypos) but Nodal low (histological score 0e2, Nodallow) and nodal high group (histological score 3e6, Nodalhigh ). In total, 50% (16/32, 30% of all) of Nodalhigh cases showed positive Lefty staining (Fig. 1I, J, M, and N), while only 23% (5/22, 9% of all) of Nodallow cases exhibiting detectable expression of Lefty in the cancer cells (Fig. 1K, L, O and P).
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Table 1 Demographic data and clinical characteristics comparison between Nodal high and low patients.
Gender Age (mean, year) Resection (R) Tumor size (T) Lymph node (N) Metastasis (M) Grade (G)
Nodal low (n ¼ 22)
Nodal high (n ¼ 32)
P value
Male (10) Female (12) 62.4 R0 (19) R1 (3) T1-T2 (1) T3 (21) N0 (4) N1 (18) M0 (21) M1 (1) G1-G2 (15) G3 (7)
Male (22) Female (10) 67.1 R0 (28) R1 (4) T1-T2 (2) T3 (30) N0 (4) N1 (28) M0 (29) M1 (3) G1-G2 (23) G3 (9)
0.10 0.04 1.0 1.0 0.70 0.64 0.77
Pancreatic cancer cell lines express Lefty and Nodal Nodal expression rather than Lefty correlates with survival Since previous studies in melanoma and breast cancer revealed that Nodal expression was correlated with clinical progression of malignancy [25e27], we set out to determine whether Nodal expression also affects the prognosis of PDAC patients. Therefore, KaplaneMeier analysis was used to compared survival difference between Nodallow and Nodalhigh patients. This analysis revealed that patients with low Nodal expression lived significantly longer than patients with high Nodal expression (median survival 33.0 vs 17.8 months, p ¼ 0.013, Fig. 2A). Furthermore, other than age (4.7 years difference in the mean age) the clinical parameters were comparable between these two groups (Table 1). However, there was no survival difference between Lefty negative and positive patients (median survival 26.8 vs 20.7 months, p ¼ 0.870, Fig. 2B).
Immunoblot assays were employed to analyze Lefty and Nodal expression in eight cultured pancreatic cancer cell lines (Aspc-1, Bxpc-3, Capan-1, Colo-357, MiaPaCa-2, Panc-1, Su86.86 and T3M4) as well as in HPDE cells. This analysis revealed that cultured cancer cell lines (Fig. 3A and B) and HPDE cells (Fig. 3C and D) expressed various levels of Lefty and Nodal. Antibody specificity for Lefty and Nodal was confirmed by performing siRNA transfection assays in Colo-357 cells. Specifically, Nodal siRNA transfection led to down-regulation of protein appearing at around 40 kDa in accordance with the predicted size of human Nodal (Fig. 3E, left panel). Correspondingly, Lefty 1 siRNA transfection reduced expression of a protein appearing at the predicted size (41 kDa) of human lefty 1. Interestingly, we observed that the intensity of a band greater than 55 kDa also decreased (Fig. 3E, right panel). Nodal affects proliferation and invasion Colo-357 cells In an attempt to address functional the relevance of Nodal and Lefty in carcinogenesis of PDAC, Colo-357 cells maintaining an intact TGFb pathway were chosen for further investigation [28]. Though lower dose of human recombinant Nodal (rNodal, 50 and 200 ng/ml) had no obvious effect, higher dose (5000 ng/ml) inhibited growth of Colo-357 cells (Fig. 4A, left panel) consistent with results obtained from rat pancreatic tumor cells AR42J [21]. In addition, rNodal (200 ng/ml) also increased invasiveness of Colo357 cells where no effect on cell growth was observed at this dose (Fig. 4A, left and right panel). In contrast, treatment of human recombinant Lefty 2 (rLefty) had no effect either on cell growth or invasion (Fig. 4B). In addition, silencing endogenous Nodal or Lefty in Colo-357 cells by siRNA transfection had no additional influence on either cell growth (Fig. 4C and D). Discussion
Fig. 2. Nodal expression but not Lefty correlates with survival. A, survival proportions in patients with low and high Nodal expression; p ¼ 0.013. B, survival proportions in patients with Lefty negative and positive staining. No significant (n.s.) differences were found between the groups.
Though tubular complexes are regularly found in human specimen of chronic pancreatitis and pancreatic cancer, the cell of origin for such lesions remains controversial: ductal, centroacinar/acinar and the islet compartment have been considered to be candidates [29e32]. Indeed, these tubular complexes have been described as key histological changes in many genetically engineered mice (involving all lineages) including loss of tumor suppressors (e.g. Pten (phosphatase and tensin homolog) and Lkb1 (serine/threonine kinase 11), overexpression of growth factors (e.g. TGF-a (transforming growth factor, alpha)), transformation of oncogenic Kras as
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Fig. 3. Pancreatic cancer cell lines unanimously express Lefty and Nodal. A, B. Expression (immunoblot) of Nodal (A) and Lefty (B) in pancreatic cancer cell lines. All 8 tested cell lines (Aspc-1 (As), Bxpc-3 (Bx), Capan-1 (Ca), Colo357 (Co), Mia-PaCa2 (Mi), Panc-1 (Pa), Su86.86 (Su), T3M4 (T3)) express Nodal and Lefty at various level. C, D. Similarly, Nodal (C) and Lefty (D) expression can also be detected in HPDE and Colo357 (internal control) cells. E. Nodal (left panel) and Lefty (right panel) silencing with specific siRNA or negative control siRNA (control) at 72 h.
well as tissue injury [33e38]. Furthermore, since tubular complexes are frequently observed prior to formation of PanIN lesions or directly before PDAC development, they have been proposed as precursor lesions of PDAC as well [29,39]. It is likely that tubular complexes formation is a default process for pancreas remodeling in response to intrinsic (e.g. oncogenic, energy and growth) and extrinsic (e.g. inflammation and tissue damage) stress. In the current study, we observed that Lefty (rather than Nodal) is expressed distinctively in tubular complexes of human pancreatic cancer specimen. This is consistent with findings in a mouse model of islet regeneration where it is described that a strong expression of Lefty
is observed in a subset of duct-like structures [21]. However, it remains obscure how and why tubular complexes express Lefty. It is also not clear whether Lefty expression is functionally relevant to the formation of tubular complexes or rather acts as feedback response to strong Nodal signaling in adjacent cancer cells. In addition, Lefty expression in tubular complexes (especially in the absence of active RAS/MEK/ERK signaling) can potentially be induced by TGFb present within the tumor microenvironment [20,40]. Previous studies have demonstrated that Nodal is present in human metastatic melanoma and breast cancer cells, and that it
Fig. 4. Nodal affects proliferation and invasion Colo-357 cells. A. Proliferation of Colo-357 cells as assessed by MTT assays following rNodal treatment: Lower dose (50 and 200 ng/ml) had no effect whereas high dose (5000 ng/ml) inhibited cell growth. Data from three independent experiments are expressed as mean ± SEM (left panel). In a matrigel invasion assay, rNodal (200 ng/ml) increases cell invasion of Colo-357 cells (right panel). Values shown are the mean ± SEM fold change of the negative control obtained from three independent experiments. B. rLefty has no effect on either growth (20 and 50 ng/ml) or invasiveness (50 ng/ml) of Colo-357 cells. No significant (n.s.) differences were found between the groups. C, D Proliferation of Colo-357 cells as assessed by MTT assays following down-regulation of Nodal and Lefty expression via siRNA transfection shows no effect. Results from three independent experiments are expressed as mean ± SEM.
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correlates with clinical progression. In line, inhibition of Nodal signaling by synthetic TGFb inhibitors or human ESCs-derived Lefty reduces cancer cell invasiveness and tumorigenicity [25,27]. Similarly, Lefty may account for the tumor suppressive activity of human liver stem cells by inhibiting nodal signaling [41]. Accordingly, we also observed the presence of Nodal expression in a majority of PDAC samples, and high expression of Nodal was associated with shorter patient survival. In addition, rNodal promotes cancer cells invasion in vitro. However, the reason why a subgroup of cancer cells acquires high expression levels of Nodal remains elusive. In contrast to findings in other tumors [25,27], there was no corresponding loss of Lefty (at least in 50% of cases) in the cancer cells with high Nodal expression as well as in cultured cell lines. Additionally, pancreatic cancer cells do not respond to treatment of rLefty in terms of cell growth and invasion. Hence, we speculate that due to constant exposure to Lefty (endocrine cell-derived), pancreatic cancer cells may have evolved an undefined mechanism (e.g. by overexpressing co-receptor Cripto for Nodal activation) to circumvent this feedback loop existing in human ESCs [42]. To conclude, we provide evidence that both Nodal and its natural inhibitor Lefty are expressed by pancreatic cancer cells, and high expression of Nodal is linked to reduced patient survival suggesting a clinical significance of this signaling pathway. Acknowledgments We thank Felicitas Altmayr, Tanja Rossmann-Bloeck, Manja Thorwirth and Carmen Marthen for excellent technical support. References [1] Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin 2014;64: 9e29. [2] Hendrix MJ, Seftor EA, Seftor RE, Kasemeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer 2007;7:246e55. [3] James D, Levine AJ, Besser D, Hemmati-Brivanlou A. Tgfbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 2005;132:1273e82. [4] Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Angenendt P, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 2008;321:1801e6. [5] Shi Y, Massague J. Mechanisms of tgf-beta signaling from cell membrane to the nucleus. Cell 2003;113:685e700. [6] Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C, et al. Transforming growth factor beta1 treatment leads to an epithelialmesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation. Cancer Res 2001;61:4222e8. [7] Hamada S, Satoh K, Hirota M, Kimura K, Kanno A, Masamune A, et al. Bone morphogenetic protein 4 induces epithelial-mesenchymal transition through msx2 induction on pancreatic cancer cell line. J Cell Physiol 2007;213:768e74. [8] Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelialmesenchymal transition generates cells with properties of stem cells. Cell 2008;133:704e15. [9] Friess H, Yamanaka Y, Buchler M, Berger HG, Kobrin MS, Baldwin RL, et al. Enhanced expression of the type ii transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type iii receptor expression. Cancer Res 1993;53:2704e7. [10] Kleeff J, Maruyama H, Ishiwata T, Sawhney H, Friess H, Buchler MW, et al. Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo. Gastroenterology 1999;116:1202e16. [11] Kong B, Michalski CW, Hong X, Valkovskaya N, Rieder S, Abiatari I, et al. Azgp1 is a tumor suppressor in pancreatic cancer inducing mesenchymal-toepithelial transdifferentiation by inhibiting tgf-beta-mediated erk signaling. Oncogene 2010;29:5146e58. [12] Wagner M, Kleeff J, Friess H, Buchler MW, Korc M. Enhanced expression of the type ii transforming growth factor-beta receptor is associated with decreased survival in human pancreatic cancer. Pancreas 1999;19:370e6. [13] Schier AF. Nodal signaling in vertebrate development. Annu Rev Cell Dev Biol 2003;19:589e621. [14] Yeo C, Whitman M. Nodal signals to smads through cripto-dependent and cripto-independent mechanisms. Mol Cell 2001;7:949e57.
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