Lung Cancer 86 (2014) 304–310
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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Notch1 controls cell invasion and metastasis in small cell lung carcinoma cell lines Wael Abdo Hassan a,b , Ryoji Yoshida c , Shinji Kudoh a , Koki Hasegawa a , Kanako Niimori-Kita a , Takaaki Ito a,∗ a
Department of Pathology and Experimental Medicine, Kumamoto University, Graduate School of Medical Sciences, Japan Department of Pathology, Faculty of Medicine, Suez Canal University, Egypt c Department of Oral and Maxillofacial Surgery, Kumamoto University, Graduate School of Medical Sciences, Japan b
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 7 October 2014 Accepted 11 October 2014 Keywords: Small cell lung carcinoma (SCLC) Notch1 signaling Epithelial mesenchymal transition (EMT) Cell motility Cell metastatic potential Tail vein xenograft mouse model
a b s t r a c t Introduction: Notch signaling plays a key role in a wide variety of human neoplasms, and it can be either oncogenic or anti-proliferative. Moreover, Notch function in regulating cancer is unpredictable, and its outcome is strictly context-dependent. Aim: To study the role of Notch1 signaling in human small cell lung carcinoma (SCLC) and its effect on cell invasion and metastasis. Materials and methods: We used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 SCLC cells. On the other hand, we up-regulated Notch1 in H69 and H1688 SCLC cells through transfection with venus Notch1 intracellular domain (v.NICD) plasmid. In addition, H69 cells with v.NICD were xenotransplanted into immune-compromised Rag2(−/−) Jak3(−/−) mice, for analysis of ex vivo tumor epithelial mesenchymal transition (EMT) phenotype and for detection of metastatic cancer cells in the lung tissues. Moreover, we examined the metastatic ability for H69AR and SBC3 cells transfected with siRNA against Notch1, compared to their subsequent controls, by use of tail vein xenograft mouse models. Results: Notch1 controls cell adhesion and EMT. Overexpression of Notch1 in SCLC switched off EMT, cell motility and cell metastatic potential. Conclusion: Our results demonstrate that activation of Notch1 signaling pathway may represent a new strategy for treating human SCLC. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer, with ∼1.3 million annual deaths worldwide, is the leading cause of cancer related death [1]. According to the histological classification, lung cancer is divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), which is divided into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma [2]. SCLC accounts for approximately 20% of all lung cancer cases, with high rate of relapse and failure of therapy [1,3]. One of the most important cell signaling systems is Notch pathway. Notch (Notch1–4) are trans-membrane proteins, which
∗ Corresponding author at: Department of Pathology and Experimental Medicine, Kumamoto University, Graduate School of Medical Sciences, Honjo 1-1-1, Chuo-ku, Kumamoto 860-8556, Japan. Tel.: +81 96 373 5086; fax: +81 96 373 5087. E-mail address: [email protected] (T. Ito). http://dx.doi.org/10.1016/j.lungcan.2014.10.007 0169-5002/© 2014 Elsevier Ireland Ltd. All rights reserved.
interact with ligands of the Delta (DLL1, DLL3 and DLL4) and/or Jagged/Serrate (Jagged1 and Jagged2) family. Ligand binding induces proteolytic cleavage of Notch receptor, releasing the Notch intracellular domain (NICD) into the cytoplasm, which enters the nucleus, and interacts with the transcription factor CBF1 (RBPjk), inducing the transcription of several genes: Hes1, cyclin D1, cMyc, Akt and others [4]. Notch signaling in tumorigenesis can be either oncogenic or anti-proliferative. In addition, opposite effect of Notch signaling has been observed in the same cancer cell line [5–8]. In lung carcinoma, Notch exhibits both tumor promoting and inhibiting functions, depending on cell type. Regarding SCLC, Notch1 signaling is suppressed [9,10] and overexpression of Notch1 resulted in inhibition of SCLC growth and suppression of neuroendocrine (NE) tumor phenotype, as we recently showed [11]. The present study investigates the role of Notch1 signaling in SCLC, focusing on its role in epithelial mesenchymal transition (EMT) process, in which tumor cells lose expression of cell adhesion proteins, such as E cadherin (E cad), by activation of a number of
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transcriptional factors, e.g. snail, slug and twist, and gain the expression of mesenchymal markers such as vimentin, leading to cancer cell metastasis [12,13]. Our findings explore a novel role of Notch1 in SCLC invasion and metastasis, which may provide new therapeutic strategies to combat SCLC.
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2 chain alpha (GL2) as a marker for cell motility, as previously described [14]. Cells were incubated with the appropriate secondary antibodies (Alexa Flour, Molecular Probes, Eugene, OR) and examined by fluorescent microscope (Olympus, Tokyo, Japan). 2.6. Invasion assay
2. Materials and methods 2.1. Cell lines H69, H1688 and H69AR cell lines were purchased from American Type Cell Collection (Rockville, MD). SBC3 cell lines were a generous gift from Dr. Makato Suzuki (Department of Respiratory Surgery, Graduate School of Medical Sciences, Kumamoto University). The growth media were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and supplied with 1% penicillin/streptomycin (Sigma Aldrich, Ontario, Canada). SBC3 cells were grown in EMEM with 10% FBS. H69AR cells were grown in RPMI 1640 medium, supplemented with 2 mM l-glutamine, 10 nM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and 20% FBS. H69 and H1688 cells were grown in similar RPMI 1640 medium, but supplemented with 10% FBS. All cells were incubated at 37 ◦ C in 5% CO2 and saturated humidity. 2.2. Transfection with siRNA 24 h before siRNA transfection was performed, H69AR and SBC3 cells were cultured in fresh medium, without antibiotics, in 60 mm dishes (Nunc, Waltham, MA, USA). The cells were then transfected with Notch1 specific siRNA and StealthTM RNAi Negative control (Invitrogen, Carlsbad, CA) using Lipofectamine RNAi MAX (Invitrogen) as described in manufacturer’s instruction. The sequences for siRNA were as follows: for Notch1, sense strand 5 -UCG CAU UGA CCA UUC AAA CUG GUGG-3 , antisense strand 5 -CCA CCA GUU UGA AUG GUC AAU GCGA-3 . The cells were harvested at 48 h post-transfection. 2.3. Construction of recombinant plasmid and transfection For construction of a recombinant plasmid bearing v.NICD gene (CMV-activated Notch1-venus-pA, generous gift from Dr. Mitsuru Morimoto; Laboratory of Lung Development and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan) and the control plasmid eukaryotic expression vector (PcDNA3.1-EGFP, Invitrogen), the purified products were transformed into Escherichia coli JM 2163, cultured in LB medium containing ampicillin (100 g/mL) overnight and then extracted using QIAGEN Plasmid Midi Kit as described in manufacturer’s instruction. For transfections, H69 and H1688 cells were seeded in 60 mm cell plates at a density of 1–2 × 105 cells/well in 5 mL medium and processed as previously described [11]. 2.4. Western blotting (WB) analysis Cells were prepared for WB as previously described [11]. List of primary antibodies used are given in Table S1. The membrane was then incubated with the appropriate secondary antibodies (Amersham Pharmacia Biotech, Buckinghamshire, UK), for 1 h and visualized with the ECL system (Santa Cruz, CA). 2.5. Immunofluorescence (IFA) analysis Cells were plated in 24-well plates and were treated as previously described [11]. List of primary antibodies used are given in Table S1. We used antibody against gamma-laminin
Cell invasion activity was measured with the BioCoat Matrigel Invasion Chamber (Becton Dickinson, Tokyo, Japan), according to the manufacturer’s protocol, as previously described [15]. In brief, cells transfected with Notch1 siRNA or with RNAi Negative control were inoculated on the coated matrigel of the culture inserts (upper chamber) at a density of 2 × 105 cells per 500 l of serum-free medium. The lower chamber contained the usual serum containing medium as chemo-attractant. At the end of the 48 h incubation at 37 ◦ C and a 5% CO2 atmosphere, the cells on the upper surface of the filter were completely removed with cotton swabs. The invaded cells that remained on the lower surface of the filter were fixed with methanol and stained with Diff-Quick (Sysmex, Hyogo, Japan). The numbers of stained cells in five randomly selected microscopic fields (100×) per filter were counted and the average number was calculated. The experiments were repeated at least three separate times. 2.7. Tumor xenograft growth in vivo and histopathological evaluation All animal work was done using Rag(−/−):Jak3(−/−) mice (generous gift from Prof. S. Okada; Kumamoto University) and in accordance to Institutional Animal Care and Use Committee guidelines. Two groups of mice (6 mice per group) were injected subcutaneously in the back; one group was injected with 1 × 106 stably transfected H69 cells with v.NICD, and the other group was injected with equal number of control cell population. Two months after, tumors were removed and fixed in 4% paraformaldehyde. In addition, mice lung tissues were removed and kept in liquid nitrogen for RNA extraction and analysis. Representative data were obtained from nine mice per experimental group. All samples were fixed with 10% formalin and embedded in paraffin. Tissue sets were stained with hematoxylin and eosin (H&E) staining and additional sections were used for immunohistochemical (IHC) staining, as previously described [15]. List of primary antibodies used are given in Table S1. The appropriate secondary antibody (Envision + System-HRP Labelled Polymer, Dako, Glostrup, Denmark) was then applied, followed by visualization with the Liquid DAB + Substrate Chromogen System (Dako). All slides were examined by the researcher and another pathologist. Notch1 and E cad were considered positive when cells displayed membranous or cytoplasmic staining. Snail was considered positive when cells showed nuclear or/and cytoplasmic staining. Slug was considered positive when cells showed nuclear staining. A semi-quantitative method to assess the staining intensity was used: a strong positive result was defined as strong immunoreactivity in 50% or more of tumor cells, a weak positive result was defined as weak immunoreactivity or staining of fewer than 50% of tumor cells, and tumors with no or minimal staining were scored as negative. 2.8. Mice tail vein injection H69AR and SBC3 cells were used in this experiment. 24 h after siRNA and negative control transfection, 1 × 106 cells from each group were intravenously injected via the lateral tail veins of Rag(−/−):Jak3(−/−) mice (6 mice per group) in 100 l of PBS. The process was repeated every 2 days to insure continuous infusion of siRNA transfected cells. After 4 weeks, all mice were sacrificed
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Fig. 1. Conforming successful knocking down (KD) and overexpression of Notch1. Western blotting (WB) analysis was performed in triplicate, using  actin as an internal control. Immunofluorescence (IFA) staining was performed in triplicate, using Alexa Fluors 488 as a green emitting signaling molecule for Notch1 specific antibody. All nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI, blue). Original magnification 200×. (A) WB analysis and IFA staining for Notch1 in cells transfected with Notch1 siRNA, 48 h post-transfection. WB analysis for Notch1, trans-membrane and NICD, showed significant decrease in all Notch1 components in cells transfected with Notch1 siRNA. In addition, IFA staining of Notch1 receptor showed decrease in the signal of the molecule in the membranes and cytosol of cells transfected with Notch1 siRNA. (B) WB analysis, IFA staining and IHC detection of Notch1 receptor in SCLC H69 cells stably transfected with venus Notch1 intracellular domain (v.NICD) plasmid. WB analysis showed induction of Notch1 in transfected cells. In addition, IFA staining of Notch1 showed the induction of signal of the molecule in the nuclei and cytoplasm of cells. IHC staining of Notch1 receptor in tissue sections from xenotransplanted H69 cells transfected with v.NICD plasmid showed the membranous immunoreactions (brown) for Notch1 in transfected cells; original magnification, 400×. Scale bar 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and their lungs were removed and stored in liquid nitrogen for RNA extraction and analysis. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from cells and mice lung tissues using Trizol reagent (Invitrogen) in accordance to the manufacturer’s instructions and then processed as previously described [11]. List of primers used and conditions of RT-PCR are given in Table S2. Amplified DNA fragments were separated in a 1.5% agarose gel and identified after ethidium bromide staining. The expression of human beta globin gene was used as a specific marker for the presence of human metastatic cancer cells in mice lung tissues, as previously described [16]. 2.10. Statistical analysis The results presented in tables were expressed as the means ± standard error of triplicate determinations. The differences in the mean values between the groups were analyzed by two-tailed statistical analysis using Student’s t-test. P value less than 0.05 was considered to be statistically significant. Statistical analysis was done using the JMP9 software program (SAS Institute Inc., Cary, NC).
3. Results 3.1. Knocking down (KD) Notch1 gene and Notch1 venus NICD (v.NICD) plasmid transfection The efficacy of siRNA against Notch1 in H69AR and SBC3 cells and the successful v.NICD plasmid transfection into H69 and H1688 cells were analyzed using WB and IFA (Fig. 1A). In addition, tissue samples from xenografted tumor cells revealed the expression of Notch1 in the membrane of H69 cells with v.NICD (Fig. 1B). 3.2. Effect of Notch1 signaling on cell morphology and adhesion H69AR, H1688 and SBC3 cells grow as flat cells in adherent aggregates to culture dish, while H69 cells grow as floating cells. In H69AR and SBC3 cells with KD Notch1, cells assumed a rounded shape and get detached from culture dish. On the other hand, H69 cells with v.NICD get attached to the culture dish (Fig. 2A). To further confirm this, WB, IFA and RT-PCR showed decreased E cad expression in cells with KD Notch1, and significant increase in its expression in H69 cells with v.NICD, both in vitro (Fig. 2B–D) and ex vivo (Fig. 2E). In case of H1688 cells, we could not detect any differences in either the cell morphology or E cad expression between v.NICD transfected cells and control cells (data not shown).
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Fig. 2. Effect of Notch1 on cell morphology, adhesion and epithelial–mesenchymal transition (EMT) markers (E cad, snail, slug, vimentin and twist). (A) Representative photos of H69AR and SBC3 cells transfected with Notch1 siRNA, and H69 cells with v.NICD gene, through light microscope. Note the loss of cell to cell adhesion (black arrows) in cells transfected with Notch1 siRNA and the adhesion of H69 cells with v.NICD gene (white arrow) to the culture dish in. Original magnification, 200×. (B) WB analysis of EMT markers. Note the decreased E cadherin (E cad) and the increased snail, slug and vimentin (vim) expressions in H69AR and SBC3 cells transfected with Notch1 siRNA. On the other hand, note the increased E cad and the decreased snail, slug and vimentin expressions in H69 cells with v.NICD. The expression of  actin was used as an internal control. The experiment was performed in triplicate. (C) Representative images of IFA staining for E cad and vim in cells transfected with Notch1 siRNA. The changes in the signal of E cad (Alexa Fluors 488, green) and that of vim (Alexa Fluors 568, red) were clearly detected in the membrane and cytoplasm of cells. Original magnification, 200×. (D) RT-PCR showing the level of mRNA expressions of EMT related molecules. Note the decrease in expression of E cad in H69AR and SBC3 cells with Notch1 siRNA, coupled with increase in snail, slug and twist. In H69 cells with v.NICD, the level of E cad was increased, while that of snail, slug and twist were decreased. The expression of GAPDH was used as an internal control. The experiment was performed in triplicate. (E) Representative photos for IHC staining of E cad, snail and slug, in xenotransplanted H69 cells transfected with v.NICD gene. Note the membranous expression of E cad, the decreased cytoplasmic expression of snail and the absence of nuclear expression of slug in H69 cells with v.NICD gene. Original magnification, 400×. Scale bar 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. Effect of Notch1 on cell motility and invasion ability. (A) IFA and IHC staining for gamma laminin 2 chain alpha protein (GL2), as a cell motility marker. The IFA images show the induction of the signal of GL2 (Alexa Fluors 488, green) in the cytosol of cells transfected with Notch1 siRNA, original magnification, 400×. The IHC images show the decreased cytoplasmic reaction to GL2 (brown) in H69 cells with v.NICD gene; original magnification 400×, scale bar 20 m. (B) In vitro matrigel trans-membrane invasion assay. Cells were incubated with either negative control or Notch1-specific siRNA for 24 h. Cells that penetrated the matrigel-coated membrane were fixed and stained. The number of invaded cells obtained from three independent experiments were counted and statistically analyzed. The bar graphs represent the average number of cells on the underside of the membranes. The Y axis shows the number of cells invading the underside of the membranes. The photos are representative fields of invasive cells on the membranes. Original magnification, 100×. Inhibiting Notch1 in H69AR and SBC3 cells increased the number of invading cells. Mean ± s.d. of three independent experiments. P value analyzed by Student’s t-test. (C) RT-PCR showing the difference in the level of mRNA expression of human beta globin gene in mice that were xenotransplanted with H69 cells with v.NICD gene compared to their control group (upper figure) and in mice that were injected via their tail veins with SBC3 cells transfected with Notch1-specific siRNA compared to their control group (lower figure). Six mice per group were represented. The expression of mice GAPDH was used as an internal control. Note the decrease in the expression of human beta globin gene in the six cases of H69 cells with v.NICD gene. In case of SBC3 cells, the first case (C-1, si-1) did not show significant difference, while the rest of cases showed significant increase in the expression of human beta globin gene in cells transfected with Notch1-specific siRNA. C, control; N, mice xenotransplanted with cells transfected with v.NICD gene; si, mice injected with cells transfected with siRNA against Notch1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Table 1 Invasion assay of cells. Cell
Negative control
H69AR SBC3
60.3 ± 7.41 71 ± 9.4
siNotch1 147.7 ± 10.40 147 ± 23.7
3.3. Effect of Notch1 signaling on epithelial mesenchymal transition (EMT) We detected the expression of snail, slug, vimentin and twist as EMT related molecules. Regarding H69AR and SBC3 cells with KD Notch1, we noticed increased expression of snail, slug and vimentin by WB, increased vimentin expression by IFA and increased expression of snail, slug and twist by RT-PCR (Fig. 2B–D). To further prove these findings, we observed decreased EMT markers expression in H69 cells with v.NICD, both in vitro (Fig. 2B and D) and ex vivo (Fig. 2E). In H1688 cells, no significant changes were noticed in EMT related molecules between v.NICD transfected cells and control cells (data not shown). 3.4. Effect of Notch1 signaling on cell motility and invasion Depending on our results, we proposed that Notch1 could affect cell motility and thus cell invasion ability. The cytoplasmic expression of GL2 was increased in H69AR and SBC3 cells with KD Notch1, as seen by IFA, while it decreased in tissue sections from H69 cells with v.NICD gene (Fig. 3A). Moreover, in vitro matrigel invasion assay showed statistically significant increase in number of invasive H69AR and SBC3 cells transfected with KD Notch1 compared to control cells (P value = 0.01 respectively) (Fig. 3B and Table 1). However, we could not observe any change in expression level of GL2 protein in H1688 cells with v.NICD gene and control cells (data not shown). To confirm our in vitro results, we extracted the total RNA of lung tissues from mice that were xenotransplanted with H69 cells with v.NICD and from the control group, and also from mice that were injected with H69AR and SBC3 with KD Notch1 through their lateral tail veins and their subsequent controls. We examined the expression of human beta globin gene in the mice lungs, as a specific marker for the presence of human metastatic cancer cells [14]. We found that the expression of human beta globin gene was significantly reduced in all lungs of the mice that were xenotransplanted with H69 cells transfected with v.NICD gene (n = 6/6), while its expression was significantly increased in 5 mice lungs that were injected with SBC3 with KD Notch1, compared to their subsequent control groups (n = 5/6) (Fig. 3C). However, we could not detect any change in its expression in mice lungs that were injected with H69AR cells with KD Notch1 (data not shown). Moreover, analysis of tissue samples from mice lungs using H&E and IHC staining did not detect any visible metastatic cancer cells, probably due to the small number of metastatic cells (data not shown). 4. Discussion The role of Notch signaling in oncogenesis is far from fully understood, due to the complex nature of Notch signaling and that difference in strength, timing, cell type, and context of the signal could affect its outcome [17]. Our present report provides an evaluation of Notch1 expression in human SCLC, focusing on its role in cell EMT and invasion. We previously showed that induction of Notch1 in SCLC cells induce an epithelial-like cell arrangement with decreased neuro-endocrine (NE) markers [11]. In the present study, we confirmed the role of Notch1 signaling in mediating cell adhesion in SCLC cells, as previously described in H69 cells [18]. However,
Fig. 4. Molecular pathway for Notch1 signaling in SCLC. Effect of Notch1 expression in SCLC. H69, H69AR and SBC3 cells are representatives of SCLC. In H69, H69AR and SBC3 cells, Notch1 activation leads to expression of E cadherin, which in turn promotes cell adhesion and inhibits the expression of EMT molecules (snail, slug, twist and vimentin), leading to decreased expression of gamma laminin 2 chain alpha and thus cell motility, invasion ability and metastatic spread are inhibited. Vim, vimentin; EMT, epithelial mesenchymal transition.
overexpression of Notch1 in H1688 cells had no effect on cell adhesive growth pattern, which indicates that these cells could depend on other signaling pathway to maintain cell adhesion. Moreover, we showed that Notch1 controls EMT, cell motility and invasion, and that induction of Notch1 in H69 cells resulted in suppression of EMT markers and inhibits the expression of gamma-laminin 2 chain alpha (GL2) protein, which contributes to cell motility and invasion [14]. In addition, we showed that in mice lungs that were xenotransplanted with H69 cells with Notch1 induction, there was significant decrease in the metastatic cells, when compared to the control group, through analysis of human beta globin gene in mice lung, as a marker of metastatic human cancer cells [16]. Also in mice lungs that were injected with SBC3 with KD Notch1 via their lateral tail veins, the expression of human beta globin gene was increased, indicating increase in the metastatic cells. On the other hand, overexpressing Notch1 in H1688 cells had no effect on cell adhesion or EMT phenotype. Also, we could not significantly compare the invasive ability of both H69 and H1688 cells with overexpressed Notch1 with control cells, using in vitro matrigel invasion assay, as the results were not informative, probably due to the floating nature of H69 cells, the weak adhesive ability of H69 cells with overexpressed Notch1 and the limited invasive ability of H1688 cells. Moreover, we could not detect significant changes in the expression of human beta globin gene in mice lungs that were injected with H69AR cells with KD Notch1, compared to their subsequent control group. In addition, our results contradict a previous study by Kuramoto et al. [19], which showed that SBC3 cells with blocked DLL4 had reduced Notch1 expression and decreased metastasis in mice liver, but not in the kidney, lymph node or bone. Moreover, they noticed no effect on cell growth or proliferation. Their results contradict the present study, which showed that inhibiting Notch1 in SBC3 by siRNA technology, switched on EMT phenotype, enhanced cell motility and invasion, both in vitro and in vivo. Moreover, we previously showed that SBC3 cells with KD Notch1 have enhanced cell proliferation, despite apoptotic activation [11]. These discrepancies could be due to the fact that DLL4 – as other Notch ligands – has an intrinsic ligand signaling activity, independent of Notch, and may contribute to the complexity of Notch signaling [20]. 5. Summary and conclusion The high rate of relapse and failure of chemotherapy in SCLC necessitate new pharmacologic modalities. This study
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demonstrates that induction of Notch1 signaling has an inhibitory function in SCLC cells, in the context of cell invasion and metastasis (Fig. 4). However, due to the complex nature of the Notch signaling in tumorigenesis, more research is needed for better identification of other Notch signaling components which could be involved in inhibiting SCLC cell metastasis. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We thank Dr. Mitsuru Morimoto (Laboratory of Lung Development and Regeneration, RIKEN Center for Development Biology, Kobe, Japan) for his contribution. We also would like to thank Mrs. Motoko Kagayama and Mrs. Takako Maeda for their skillful technical assistance. This study was partially supported by a Grantin-Aid for Scientific Research (C; No. 23220010) from Ministry of Education, Culture, Sports, Science and Technology of Japan. Wael Abdo Hassan is a MEXT fellowship recipient. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2014.10.007. References [1] Bunn Jr PA. Worldwide overview of the current status of lung cancer diagnosis and treatment. Arch Pathol Lab Med 2012;136:1478. [2] Travis W, Brambilla E, Müller-Hermelink H, Harris C. Pathology and genetics of tumours of the lung, pleura, thymus and heart. World Health Organization classification of tumours. Lyon, France: IARC Press; 2004. [3] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. [4] Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene 2008;27:5124–31.
[5] Yu H, Zhao X, Huang S, Jiang L, Qian G, Ge S. Blocking Notch1 signaling by RNA interference can induce growth inhibition in HeLa cells. Int J Gynecol Cancer 2007;17:511–6. [6] Wang L, Qin H, Chen B, Xin X, Li J, Han H. Overexpressed active Notch1 induces cell growth arrest of HeLa cervical carcinoma cells. Int J Gynecol Cancer 2007;17:1283–92. [7] Zheng Q, Qin H, Zhang H, Li J, Hou L, Wang H, et al. Notch signaling inhibits growth of the human lung adenocarcinoma cell line A549. Oncol Rep 2007;17:847–52. [8] Ji X, Wang Z, Geamanu A, Sarkar FH, Gupta SV. Inhibition of cell growth and induction of apoptosis in non-small cell lung cancer cells by delta-tocotrienol is associated with notch-1 down-regulation. J Cell Biochem 2011;112: 2773–83. [9] Ito T, Udaka N, Okudela K, Yazawa T, Kitamura H. Mechanisms of neuroendocrine differentiation in pulmonary neuroendocrine cells and small cell carcinoma. Endocr Pathol 2003;14:133–9. [10] Kunnimalaiyaan M, Chen H. Tumor suppressor role of Notch1 signaling in neuroendocrine tumors. Oncologist 2007;12:535–42. [11] Wael H, Yoshida R, Kudoh S, Hasegawa K, Niimori-Kita K, Ito T. Notch1 signaling controls cell proliferation, apoptosis and differentiation in lung carcinoma. Lung Cancer 2014;85:131–40. [12] Xie M, Zhang L, He CS, Xu F, Liu JL, Hu ZH, et al. Activation of Notch-1 enhances epithelial–mesenchymal transition in gefitinib-acquired resistant lung cancer cells. J Cell Biochem 2012;113:1501–13. [13] Thiery J, Acloque H, Huang R, Nieto M. Epithelial–mesenchymal transitions in development and disease. Cell 2009;139:871–90. [14] Hintermann E, Quaranta V. Epithelial cell motility on laminin-5: regulation by matrix assembly, proteolysis, integrins and erbB receptors. Matrix Biol 2004;23:75–85. [15] Yoshida R, Nagata M, Nakayama H, Niimori-Kita K, Hassan W, Tanaka T, et al. The pathological significance of Notch1 in oral squamous cell carcinoma. Lab Invest 2013;93:1068–81. [16] Watanabe M, Takahashi Y, Ohta T, Mai M, Sasaki T, Seiki M. Inhibition of metastasis in human gastric cancer cells transfected with tissue inhibitor of metalloproteinase 1 gene in nude mice. Cancer 1996;77: 1676–80. [17] Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999;284: 770–6. [18] Sriuranpong V, Borges M, Ravi R, Arnold DR, Nelkin BD, Baylin SB, et al. Notch signaling induces cell cycle arrest in small cell lung cancer cells. Cancer Res 2001;61:3200–5. [19] Kuramoto T, Goto H, Mitsuhashi A, Tabata S, Ogawa H, Uehara H, et al. Dll4-Fc, an inhibitor of Dll4-Notch signaling, suppresses liver metastasis of small cell lung cancer cells through the downregulation of the NF-kappa-B activity. Mol Cancer Ther 2012;11:2578–87. [20] D’Souza B, Miyamoto A, Weinmaster G. The many facets of Notch ligands. Oncogene 2008;27:5148–67.
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Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Notch1 controls cell invasion and metastasis in small cell lung carcinoma cell lines Wael Abdo Hassan a,b , Ryoji Yoshida c , Shinji Kudoh a , Koki Hasegawa a , Kanako Niimori-Kita a , Takaaki Ito a,∗ a
Department of Pathology and Experimental Medicine, Kumamoto University, Graduate School of Medical Sciences, Japan Department of Pathology, Faculty of Medicine, Suez Canal University, Egypt c Department of Oral and Maxillofacial Surgery, Kumamoto University, Graduate School of Medical Sciences, Japan b
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 7 October 2014 Accepted 11 October 2014 Keywords: Small cell lung carcinoma (SCLC) Notch1 signaling Epithelial mesenchymal transition (EMT) Cell motility Cell metastatic potential Tail vein xenograft mouse model
a b s t r a c t Introduction: Notch signaling plays a key role in a wide variety of human neoplasms, and it can be either oncogenic or anti-proliferative. Moreover, Notch function in regulating cancer is unpredictable, and its outcome is strictly context-dependent. Aim: To study the role of Notch1 signaling in human small cell lung carcinoma (SCLC) and its effect on cell invasion and metastasis. Materials and methods: We used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 SCLC cells. On the other hand, we up-regulated Notch1 in H69 and H1688 SCLC cells through transfection with venus Notch1 intracellular domain (v.NICD) plasmid. In addition, H69 cells with v.NICD were xenotransplanted into immune-compromised Rag2(−/−) Jak3(−/−) mice, for analysis of ex vivo tumor epithelial mesenchymal transition (EMT) phenotype and for detection of metastatic cancer cells in the lung tissues. Moreover, we examined the metastatic ability for H69AR and SBC3 cells transfected with siRNA against Notch1, compared to their subsequent controls, by use of tail vein xenograft mouse models. Results: Notch1 controls cell adhesion and EMT. Overexpression of Notch1 in SCLC switched off EMT, cell motility and cell metastatic potential. Conclusion: Our results demonstrate that activation of Notch1 signaling pathway may represent a new strategy for treating human SCLC. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer, with ∼1.3 million annual deaths worldwide, is the leading cause of cancer related death [1]. According to the histological classification, lung cancer is divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), which is divided into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma [2]. SCLC accounts for approximately 20% of all lung cancer cases, with high rate of relapse and failure of therapy [1,3]. One of the most important cell signaling systems is Notch pathway. Notch (Notch1–4) are trans-membrane proteins, which
∗ Corresponding author at: Department of Pathology and Experimental Medicine, Kumamoto University, Graduate School of Medical Sciences, Honjo 1-1-1, Chuo-ku, Kumamoto 860-8556, Japan. Tel.: +81 96 373 5086; fax: +81 96 373 5087. E-mail address: [email protected] (T. Ito). http://dx.doi.org/10.1016/j.lungcan.2014.10.007 0169-5002/© 2014 Elsevier Ireland Ltd. All rights reserved.
interact with ligands of the Delta (DLL1, DLL3 and DLL4) and/or Jagged/Serrate (Jagged1 and Jagged2) family. Ligand binding induces proteolytic cleavage of Notch receptor, releasing the Notch intracellular domain (NICD) into the cytoplasm, which enters the nucleus, and interacts with the transcription factor CBF1 (RBPjk), inducing the transcription of several genes: Hes1, cyclin D1, cMyc, Akt and others [4]. Notch signaling in tumorigenesis can be either oncogenic or anti-proliferative. In addition, opposite effect of Notch signaling has been observed in the same cancer cell line [5–8]. In lung carcinoma, Notch exhibits both tumor promoting and inhibiting functions, depending on cell type. Regarding SCLC, Notch1 signaling is suppressed [9,10] and overexpression of Notch1 resulted in inhibition of SCLC growth and suppression of neuroendocrine (NE) tumor phenotype, as we recently showed [11]. The present study investigates the role of Notch1 signaling in SCLC, focusing on its role in epithelial mesenchymal transition (EMT) process, in which tumor cells lose expression of cell adhesion proteins, such as E cadherin (E cad), by activation of a number of
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transcriptional factors, e.g. snail, slug and twist, and gain the expression of mesenchymal markers such as vimentin, leading to cancer cell metastasis [12,13]. Our findings explore a novel role of Notch1 in SCLC invasion and metastasis, which may provide new therapeutic strategies to combat SCLC.
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2 chain alpha (GL2) as a marker for cell motility, as previously described [14]. Cells were incubated with the appropriate secondary antibodies (Alexa Flour, Molecular Probes, Eugene, OR) and examined by fluorescent microscope (Olympus, Tokyo, Japan). 2.6. Invasion assay
2. Materials and methods 2.1. Cell lines H69, H1688 and H69AR cell lines were purchased from American Type Cell Collection (Rockville, MD). SBC3 cell lines were a generous gift from Dr. Makato Suzuki (Department of Respiratory Surgery, Graduate School of Medical Sciences, Kumamoto University). The growth media were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and supplied with 1% penicillin/streptomycin (Sigma Aldrich, Ontario, Canada). SBC3 cells were grown in EMEM with 10% FBS. H69AR cells were grown in RPMI 1640 medium, supplemented with 2 mM l-glutamine, 10 nM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate and 20% FBS. H69 and H1688 cells were grown in similar RPMI 1640 medium, but supplemented with 10% FBS. All cells were incubated at 37 ◦ C in 5% CO2 and saturated humidity. 2.2. Transfection with siRNA 24 h before siRNA transfection was performed, H69AR and SBC3 cells were cultured in fresh medium, without antibiotics, in 60 mm dishes (Nunc, Waltham, MA, USA). The cells were then transfected with Notch1 specific siRNA and StealthTM RNAi Negative control (Invitrogen, Carlsbad, CA) using Lipofectamine RNAi MAX (Invitrogen) as described in manufacturer’s instruction. The sequences for siRNA were as follows: for Notch1, sense strand 5 -UCG CAU UGA CCA UUC AAA CUG GUGG-3 , antisense strand 5 -CCA CCA GUU UGA AUG GUC AAU GCGA-3 . The cells were harvested at 48 h post-transfection. 2.3. Construction of recombinant plasmid and transfection For construction of a recombinant plasmid bearing v.NICD gene (CMV-activated Notch1-venus-pA, generous gift from Dr. Mitsuru Morimoto; Laboratory of Lung Development and Regeneration, RIKEN Center for Developmental Biology, Kobe, Japan) and the control plasmid eukaryotic expression vector (PcDNA3.1-EGFP, Invitrogen), the purified products were transformed into Escherichia coli JM 2163, cultured in LB medium containing ampicillin (100 g/mL) overnight and then extracted using QIAGEN Plasmid Midi Kit as described in manufacturer’s instruction. For transfections, H69 and H1688 cells were seeded in 60 mm cell plates at a density of 1–2 × 105 cells/well in 5 mL medium and processed as previously described [11]. 2.4. Western blotting (WB) analysis Cells were prepared for WB as previously described [11]. List of primary antibodies used are given in Table S1. The membrane was then incubated with the appropriate secondary antibodies (Amersham Pharmacia Biotech, Buckinghamshire, UK), for 1 h and visualized with the ECL system (Santa Cruz, CA). 2.5. Immunofluorescence (IFA) analysis Cells were plated in 24-well plates and were treated as previously described [11]. List of primary antibodies used are given in Table S1. We used antibody against gamma-laminin
Cell invasion activity was measured with the BioCoat Matrigel Invasion Chamber (Becton Dickinson, Tokyo, Japan), according to the manufacturer’s protocol, as previously described [15]. In brief, cells transfected with Notch1 siRNA or with RNAi Negative control were inoculated on the coated matrigel of the culture inserts (upper chamber) at a density of 2 × 105 cells per 500 l of serum-free medium. The lower chamber contained the usual serum containing medium as chemo-attractant. At the end of the 48 h incubation at 37 ◦ C and a 5% CO2 atmosphere, the cells on the upper surface of the filter were completely removed with cotton swabs. The invaded cells that remained on the lower surface of the filter were fixed with methanol and stained with Diff-Quick (Sysmex, Hyogo, Japan). The numbers of stained cells in five randomly selected microscopic fields (100×) per filter were counted and the average number was calculated. The experiments were repeated at least three separate times. 2.7. Tumor xenograft growth in vivo and histopathological evaluation All animal work was done using Rag(−/−):Jak3(−/−) mice (generous gift from Prof. S. Okada; Kumamoto University) and in accordance to Institutional Animal Care and Use Committee guidelines. Two groups of mice (6 mice per group) were injected subcutaneously in the back; one group was injected with 1 × 106 stably transfected H69 cells with v.NICD, and the other group was injected with equal number of control cell population. Two months after, tumors were removed and fixed in 4% paraformaldehyde. In addition, mice lung tissues were removed and kept in liquid nitrogen for RNA extraction and analysis. Representative data were obtained from nine mice per experimental group. All samples were fixed with 10% formalin and embedded in paraffin. Tissue sets were stained with hematoxylin and eosin (H&E) staining and additional sections were used for immunohistochemical (IHC) staining, as previously described [15]. List of primary antibodies used are given in Table S1. The appropriate secondary antibody (Envision + System-HRP Labelled Polymer, Dako, Glostrup, Denmark) was then applied, followed by visualization with the Liquid DAB + Substrate Chromogen System (Dako). All slides were examined by the researcher and another pathologist. Notch1 and E cad were considered positive when cells displayed membranous or cytoplasmic staining. Snail was considered positive when cells showed nuclear or/and cytoplasmic staining. Slug was considered positive when cells showed nuclear staining. A semi-quantitative method to assess the staining intensity was used: a strong positive result was defined as strong immunoreactivity in 50% or more of tumor cells, a weak positive result was defined as weak immunoreactivity or staining of fewer than 50% of tumor cells, and tumors with no or minimal staining were scored as negative. 2.8. Mice tail vein injection H69AR and SBC3 cells were used in this experiment. 24 h after siRNA and negative control transfection, 1 × 106 cells from each group were intravenously injected via the lateral tail veins of Rag(−/−):Jak3(−/−) mice (6 mice per group) in 100 l of PBS. The process was repeated every 2 days to insure continuous infusion of siRNA transfected cells. After 4 weeks, all mice were sacrificed
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Fig. 1. Conforming successful knocking down (KD) and overexpression of Notch1. Western blotting (WB) analysis was performed in triplicate, using  actin as an internal control. Immunofluorescence (IFA) staining was performed in triplicate, using Alexa Fluors 488 as a green emitting signaling molecule for Notch1 specific antibody. All nuclei were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI, blue). Original magnification 200×. (A) WB analysis and IFA staining for Notch1 in cells transfected with Notch1 siRNA, 48 h post-transfection. WB analysis for Notch1, trans-membrane and NICD, showed significant decrease in all Notch1 components in cells transfected with Notch1 siRNA. In addition, IFA staining of Notch1 receptor showed decrease in the signal of the molecule in the membranes and cytosol of cells transfected with Notch1 siRNA. (B) WB analysis, IFA staining and IHC detection of Notch1 receptor in SCLC H69 cells stably transfected with venus Notch1 intracellular domain (v.NICD) plasmid. WB analysis showed induction of Notch1 in transfected cells. In addition, IFA staining of Notch1 showed the induction of signal of the molecule in the nuclei and cytoplasm of cells. IHC staining of Notch1 receptor in tissue sections from xenotransplanted H69 cells transfected with v.NICD plasmid showed the membranous immunoreactions (brown) for Notch1 in transfected cells; original magnification, 400×. Scale bar 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
and their lungs were removed and stored in liquid nitrogen for RNA extraction and analysis. 2.9. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from cells and mice lung tissues using Trizol reagent (Invitrogen) in accordance to the manufacturer’s instructions and then processed as previously described [11]. List of primers used and conditions of RT-PCR are given in Table S2. Amplified DNA fragments were separated in a 1.5% agarose gel and identified after ethidium bromide staining. The expression of human beta globin gene was used as a specific marker for the presence of human metastatic cancer cells in mice lung tissues, as previously described [16]. 2.10. Statistical analysis The results presented in tables were expressed as the means ± standard error of triplicate determinations. The differences in the mean values between the groups were analyzed by two-tailed statistical analysis using Student’s t-test. P value less than 0.05 was considered to be statistically significant. Statistical analysis was done using the JMP9 software program (SAS Institute Inc., Cary, NC).
3. Results 3.1. Knocking down (KD) Notch1 gene and Notch1 venus NICD (v.NICD) plasmid transfection The efficacy of siRNA against Notch1 in H69AR and SBC3 cells and the successful v.NICD plasmid transfection into H69 and H1688 cells were analyzed using WB and IFA (Fig. 1A). In addition, tissue samples from xenografted tumor cells revealed the expression of Notch1 in the membrane of H69 cells with v.NICD (Fig. 1B). 3.2. Effect of Notch1 signaling on cell morphology and adhesion H69AR, H1688 and SBC3 cells grow as flat cells in adherent aggregates to culture dish, while H69 cells grow as floating cells. In H69AR and SBC3 cells with KD Notch1, cells assumed a rounded shape and get detached from culture dish. On the other hand, H69 cells with v.NICD get attached to the culture dish (Fig. 2A). To further confirm this, WB, IFA and RT-PCR showed decreased E cad expression in cells with KD Notch1, and significant increase in its expression in H69 cells with v.NICD, both in vitro (Fig. 2B–D) and ex vivo (Fig. 2E). In case of H1688 cells, we could not detect any differences in either the cell morphology or E cad expression between v.NICD transfected cells and control cells (data not shown).
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Fig. 2. Effect of Notch1 on cell morphology, adhesion and epithelial–mesenchymal transition (EMT) markers (E cad, snail, slug, vimentin and twist). (A) Representative photos of H69AR and SBC3 cells transfected with Notch1 siRNA, and H69 cells with v.NICD gene, through light microscope. Note the loss of cell to cell adhesion (black arrows) in cells transfected with Notch1 siRNA and the adhesion of H69 cells with v.NICD gene (white arrow) to the culture dish in. Original magnification, 200×. (B) WB analysis of EMT markers. Note the decreased E cadherin (E cad) and the increased snail, slug and vimentin (vim) expressions in H69AR and SBC3 cells transfected with Notch1 siRNA. On the other hand, note the increased E cad and the decreased snail, slug and vimentin expressions in H69 cells with v.NICD. The expression of  actin was used as an internal control. The experiment was performed in triplicate. (C) Representative images of IFA staining for E cad and vim in cells transfected with Notch1 siRNA. The changes in the signal of E cad (Alexa Fluors 488, green) and that of vim (Alexa Fluors 568, red) were clearly detected in the membrane and cytoplasm of cells. Original magnification, 200×. (D) RT-PCR showing the level of mRNA expressions of EMT related molecules. Note the decrease in expression of E cad in H69AR and SBC3 cells with Notch1 siRNA, coupled with increase in snail, slug and twist. In H69 cells with v.NICD, the level of E cad was increased, while that of snail, slug and twist were decreased. The expression of GAPDH was used as an internal control. The experiment was performed in triplicate. (E) Representative photos for IHC staining of E cad, snail and slug, in xenotransplanted H69 cells transfected with v.NICD gene. Note the membranous expression of E cad, the decreased cytoplasmic expression of snail and the absence of nuclear expression of slug in H69 cells with v.NICD gene. Original magnification, 400×. Scale bar 20 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. Effect of Notch1 on cell motility and invasion ability. (A) IFA and IHC staining for gamma laminin 2 chain alpha protein (GL2), as a cell motility marker. The IFA images show the induction of the signal of GL2 (Alexa Fluors 488, green) in the cytosol of cells transfected with Notch1 siRNA, original magnification, 400×. The IHC images show the decreased cytoplasmic reaction to GL2 (brown) in H69 cells with v.NICD gene; original magnification 400×, scale bar 20 m. (B) In vitro matrigel trans-membrane invasion assay. Cells were incubated with either negative control or Notch1-specific siRNA for 24 h. Cells that penetrated the matrigel-coated membrane were fixed and stained. The number of invaded cells obtained from three independent experiments were counted and statistically analyzed. The bar graphs represent the average number of cells on the underside of the membranes. The Y axis shows the number of cells invading the underside of the membranes. The photos are representative fields of invasive cells on the membranes. Original magnification, 100×. Inhibiting Notch1 in H69AR and SBC3 cells increased the number of invading cells. Mean ± s.d. of three independent experiments. P value analyzed by Student’s t-test. (C) RT-PCR showing the difference in the level of mRNA expression of human beta globin gene in mice that were xenotransplanted with H69 cells with v.NICD gene compared to their control group (upper figure) and in mice that were injected via their tail veins with SBC3 cells transfected with Notch1-specific siRNA compared to their control group (lower figure). Six mice per group were represented. The expression of mice GAPDH was used as an internal control. Note the decrease in the expression of human beta globin gene in the six cases of H69 cells with v.NICD gene. In case of SBC3 cells, the first case (C-1, si-1) did not show significant difference, while the rest of cases showed significant increase in the expression of human beta globin gene in cells transfected with Notch1-specific siRNA. C, control; N, mice xenotransplanted with cells transfected with v.NICD gene; si, mice injected with cells transfected with siRNA against Notch1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Table 1 Invasion assay of cells. Cell
Negative control
H69AR SBC3
60.3 ± 7.41 71 ± 9.4
siNotch1 147.7 ± 10.40 147 ± 23.7
3.3. Effect of Notch1 signaling on epithelial mesenchymal transition (EMT) We detected the expression of snail, slug, vimentin and twist as EMT related molecules. Regarding H69AR and SBC3 cells with KD Notch1, we noticed increased expression of snail, slug and vimentin by WB, increased vimentin expression by IFA and increased expression of snail, slug and twist by RT-PCR (Fig. 2B–D). To further prove these findings, we observed decreased EMT markers expression in H69 cells with v.NICD, both in vitro (Fig. 2B and D) and ex vivo (Fig. 2E). In H1688 cells, no significant changes were noticed in EMT related molecules between v.NICD transfected cells and control cells (data not shown). 3.4. Effect of Notch1 signaling on cell motility and invasion Depending on our results, we proposed that Notch1 could affect cell motility and thus cell invasion ability. The cytoplasmic expression of GL2 was increased in H69AR and SBC3 cells with KD Notch1, as seen by IFA, while it decreased in tissue sections from H69 cells with v.NICD gene (Fig. 3A). Moreover, in vitro matrigel invasion assay showed statistically significant increase in number of invasive H69AR and SBC3 cells transfected with KD Notch1 compared to control cells (P value = 0.01 respectively) (Fig. 3B and Table 1). However, we could not observe any change in expression level of GL2 protein in H1688 cells with v.NICD gene and control cells (data not shown). To confirm our in vitro results, we extracted the total RNA of lung tissues from mice that were xenotransplanted with H69 cells with v.NICD and from the control group, and also from mice that were injected with H69AR and SBC3 with KD Notch1 through their lateral tail veins and their subsequent controls. We examined the expression of human beta globin gene in the mice lungs, as a specific marker for the presence of human metastatic cancer cells [14]. We found that the expression of human beta globin gene was significantly reduced in all lungs of the mice that were xenotransplanted with H69 cells transfected with v.NICD gene (n = 6/6), while its expression was significantly increased in 5 mice lungs that were injected with SBC3 with KD Notch1, compared to their subsequent control groups (n = 5/6) (Fig. 3C). However, we could not detect any change in its expression in mice lungs that were injected with H69AR cells with KD Notch1 (data not shown). Moreover, analysis of tissue samples from mice lungs using H&E and IHC staining did not detect any visible metastatic cancer cells, probably due to the small number of metastatic cells (data not shown). 4. Discussion The role of Notch signaling in oncogenesis is far from fully understood, due to the complex nature of Notch signaling and that difference in strength, timing, cell type, and context of the signal could affect its outcome [17]. Our present report provides an evaluation of Notch1 expression in human SCLC, focusing on its role in cell EMT and invasion. We previously showed that induction of Notch1 in SCLC cells induce an epithelial-like cell arrangement with decreased neuro-endocrine (NE) markers [11]. In the present study, we confirmed the role of Notch1 signaling in mediating cell adhesion in SCLC cells, as previously described in H69 cells [18]. However,
Fig. 4. Molecular pathway for Notch1 signaling in SCLC. Effect of Notch1 expression in SCLC. H69, H69AR and SBC3 cells are representatives of SCLC. In H69, H69AR and SBC3 cells, Notch1 activation leads to expression of E cadherin, which in turn promotes cell adhesion and inhibits the expression of EMT molecules (snail, slug, twist and vimentin), leading to decreased expression of gamma laminin 2 chain alpha and thus cell motility, invasion ability and metastatic spread are inhibited. Vim, vimentin; EMT, epithelial mesenchymal transition.
overexpression of Notch1 in H1688 cells had no effect on cell adhesive growth pattern, which indicates that these cells could depend on other signaling pathway to maintain cell adhesion. Moreover, we showed that Notch1 controls EMT, cell motility and invasion, and that induction of Notch1 in H69 cells resulted in suppression of EMT markers and inhibits the expression of gamma-laminin 2 chain alpha (GL2) protein, which contributes to cell motility and invasion [14]. In addition, we showed that in mice lungs that were xenotransplanted with H69 cells with Notch1 induction, there was significant decrease in the metastatic cells, when compared to the control group, through analysis of human beta globin gene in mice lung, as a marker of metastatic human cancer cells [16]. Also in mice lungs that were injected with SBC3 with KD Notch1 via their lateral tail veins, the expression of human beta globin gene was increased, indicating increase in the metastatic cells. On the other hand, overexpressing Notch1 in H1688 cells had no effect on cell adhesion or EMT phenotype. Also, we could not significantly compare the invasive ability of both H69 and H1688 cells with overexpressed Notch1 with control cells, using in vitro matrigel invasion assay, as the results were not informative, probably due to the floating nature of H69 cells, the weak adhesive ability of H69 cells with overexpressed Notch1 and the limited invasive ability of H1688 cells. Moreover, we could not detect significant changes in the expression of human beta globin gene in mice lungs that were injected with H69AR cells with KD Notch1, compared to their subsequent control group. In addition, our results contradict a previous study by Kuramoto et al. [19], which showed that SBC3 cells with blocked DLL4 had reduced Notch1 expression and decreased metastasis in mice liver, but not in the kidney, lymph node or bone. Moreover, they noticed no effect on cell growth or proliferation. Their results contradict the present study, which showed that inhibiting Notch1 in SBC3 by siRNA technology, switched on EMT phenotype, enhanced cell motility and invasion, both in vitro and in vivo. Moreover, we previously showed that SBC3 cells with KD Notch1 have enhanced cell proliferation, despite apoptotic activation [11]. These discrepancies could be due to the fact that DLL4 – as other Notch ligands – has an intrinsic ligand signaling activity, independent of Notch, and may contribute to the complexity of Notch signaling [20]. 5. Summary and conclusion The high rate of relapse and failure of chemotherapy in SCLC necessitate new pharmacologic modalities. This study
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demonstrates that induction of Notch1 signaling has an inhibitory function in SCLC cells, in the context of cell invasion and metastasis (Fig. 4). However, due to the complex nature of the Notch signaling in tumorigenesis, more research is needed for better identification of other Notch signaling components which could be involved in inhibiting SCLC cell metastasis. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements We thank Dr. Mitsuru Morimoto (Laboratory of Lung Development and Regeneration, RIKEN Center for Development Biology, Kobe, Japan) for his contribution. We also would like to thank Mrs. Motoko Kagayama and Mrs. Takako Maeda for their skillful technical assistance. This study was partially supported by a Grantin-Aid for Scientific Research (C; No. 23220010) from Ministry of Education, Culture, Sports, Science and Technology of Japan. Wael Abdo Hassan is a MEXT fellowship recipient. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2014.10.007. References [1] Bunn Jr PA. Worldwide overview of the current status of lung cancer diagnosis and treatment. Arch Pathol Lab Med 2012;136:1478. [2] Travis W, Brambilla E, Müller-Hermelink H, Harris C. Pathology and genetics of tumours of the lung, pleura, thymus and heart. World Health Organization classification of tumours. Lyon, France: IARC Press; 2004. [3] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90. [4] Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene 2008;27:5124–31.
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