CADM 1 associates with Hippo pathway core kinases; membranous co–expression of CADM 1 and LATS 2 in lung tumors predicts good prognosis
2019; Wiley; Volume: 110; Issue: 7 Linguagem: Inglês
10.1111/cas.14040
ISSN1349-7006
AutoresTakeshi Ito, Atsuko Nakamura, Ichidai Tanaka, Yumi Tsuboi, Teppei Morikawa, Jun Nakajima, Daiya Takai, Masashi Fukayama, Yoshitaka Sekido, Toshiro Niki, Daisuke Matsubara, Yoshinori Murakami,
Tópico(s)RNA Research and Splicing
ResumoCancer ScienceVolume 110, Issue 7 p. 2284-2295 ORIGINAL ARTICLEOpen Access CADM1 associates with Hippo pathway core kinases; membranous co–expression of CADM1 and LATS2 in lung tumors predicts good prognosis Takeshi Ito, Takeshi Ito Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorAtsuko Nakamura, Atsuko Nakamura Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorIchidai Tanaka, Ichidai Tanaka Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Aichi, JapanSearch for more papers by this authorYumi Tsuboi, Yumi Tsuboi Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorTeppei Morikawa, Teppei Morikawa Human Pathology Department, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorJun Nakajima, Jun Nakajima Department of Thoracic Surgery, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorDaiya Takai, Daiya Takai Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorMasashi Fukayama, Masashi Fukayama Human Pathology Department, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorYoshitaka Sekido, Yoshitaka Sekido Division of Molecular Oncology, Aichi Cancer Center Research Institute, Aichi, JapanSearch for more papers by this authorToshiro Niki, Toshiro Niki Division of Integrative Pathology, Jichi Medical University, Tochigi, JapanSearch for more papers by this authorDaisuke Matsubara, Corresponding Author Daisuke Matsubara VZV07574@nifty.com orcid.org/0000-0002-6233-6840 Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Division of Integrative Pathology, Jichi Medical University, Tochigi, Japan Correspondence Daisuke Matsubara, Division of Integrative Pathology, Jichi Medical University, Yakushiji, Shimotsukeshi, Tochigi, Japan. Email: VZV07574@nifty.comSearch for more papers by this authorYoshinori Murakami, Yoshinori Murakami orcid.org/0000-0002-2826-4396 Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this author Takeshi Ito, Takeshi Ito Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorAtsuko Nakamura, Atsuko Nakamura Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorIchidai Tanaka, Ichidai Tanaka Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Aichi, JapanSearch for more papers by this authorYumi Tsuboi, Yumi Tsuboi Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorTeppei Morikawa, Teppei Morikawa Human Pathology Department, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorJun Nakajima, Jun Nakajima Department of Thoracic Surgery, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorDaiya Takai, Daiya Takai Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorMasashi Fukayama, Masashi Fukayama Human Pathology Department, Graduate School of Medicine, The University of Tokyo, Tokyo, JapanSearch for more papers by this authorYoshitaka Sekido, Yoshitaka Sekido Division of Molecular Oncology, Aichi Cancer Center Research Institute, Aichi, JapanSearch for more papers by this authorToshiro Niki, Toshiro Niki Division of Integrative Pathology, Jichi Medical University, Tochigi, JapanSearch for more papers by this authorDaisuke Matsubara, Corresponding Author Daisuke Matsubara VZV07574@nifty.com orcid.org/0000-0002-6233-6840 Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Division of Integrative Pathology, Jichi Medical University, Tochigi, Japan Correspondence Daisuke Matsubara, Division of Integrative Pathology, Jichi Medical University, Yakushiji, Shimotsukeshi, Tochigi, Japan. Email: VZV07574@nifty.comSearch for more papers by this authorYoshinori Murakami, Yoshinori Murakami orcid.org/0000-0002-2826-4396 Molecular Pathology Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, JapanSearch for more papers by this author First published: 08 May 2019 https://doi.org/10.1111/cas.14040Citations: 10 Ito and Nakamura contributed equally to this work. Matsubara and Murakami are co–last authors. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Cell adhesion molecule-1 (CADM1) is a member of the immunoglobulin superfamily that functions as a tumor suppressor of lung tumors. We herein demonstrated that CADM1 interacts with Hippo pathway core kinases and enhances the phosphorylation of YAP1, and also that the membranous co–expression of CADM1 and LATS2 predicts a favorable prognosis in lung adenocarcinoma. CADM1 significantly repressed the saturation density elevated by YAP1 overexpression in NIH3T3 cells. CADM1 significantly promoted YAP1 phosphorylation on Ser 127 and downregulated YAP1 target gene expression at confluency in lung adenocarcinoma cell lines. Moreover, CADM1 was co–precipitated with multiple Hippo pathway components, including the core kinases MST1/2 and LATS1/2, suggesting the involvement of CADM1 in the regulation of the Hippo pathway through cell-cell contact. An immunohistochemical analysis of primary lung adenocarcinomas (n = 145) revealed that the histologically low-grade subtype frequently showed the membranous co–expression of CADM1 (20/22, 91% of low-grade; 61/91, 67% of intermediate grade; and 13/32, 41% of high-grade subtypes; P < 0.0001) and LATS2 (22/22, 100% of low-grade; 44/91, 48% of intermediate-grade; and 1/32, 3% of high-grade subtypes; P < 0.0001). A subset analysis of disease-free survival revealed that the membranous co–expression of CADM1 and LATS2 was a favorable prognosis factor (5-year disease-free survival rate: 83.8%), even with nuclear YAP1-positive expression (5-year disease-free survival rate: 83.7%), whereas nuclear YAP1-positive cases with the negative expression of CADM1 and LATS2 had a poorer prognosis (5-year disease-free survival rate: 33.3%). These results indicate that the relationship between CADM1 and Hippo pathway core kinases at the cell membrane is important for suppressing the oncogenic role of YAP1. 1 INTRODUCTION Lung cancer is the leading cause of cancer death in many developed countries, including the United States and Japan,1, 2 and adenocarcinoma is the most common histological subtype of primary lung cancer. The loss or inactivation of various tumor suppressor genes has been implicated in the development of lung adenocarcinoma, and the chromosome 11q23-encoded gene, CADM1 (cell adhesion molecule-1), was identified as a critical tumor suppressor by its inhibitory effects on tumor formation in human lung adenocarcinoma cell lines.3, 4 Cell adhesion molecule-1 is a member of the immunoglobulin superfamily of cell adhesion molecules (IgCAM). CADM1 is expressed at the lateral membrane in normal epithelial cells, and mediates cell-cell attachment by binding with CADM1 expressed in adjacent cells.5 CADM1 expression is frequently lost or reduced in concordance with tumor progression; lepidic growth components were positive for CADM1 expression, while invasive components of the same tumors were frequently negative for CADM1 in lung adenocarcinoma.6 Mao et al7 reported that high expression levels of CADM1 inhibited cell proliferation and induced apoptosis in lung adenocarcinoma cell lines. The cytoplasmic domain of CADM1 is an important region for conserving the tumor suppressive function of CADM1.8 However, the mechanisms underlying the anti–proliferative and pro–apoptotic activities of CADM1 have not yet been elucidated in detail. It has recently become increasingly apparent that abnormalities in upstream and downstream members of the Hippo pathway, which have been implicated in the cell contact inhibition of proliferation as well as organ size control, play important roles in the tumorigenesis of various human cancers.9 YAP1 is the main downstream effector of the Hippo pathway that promotes cell growth as a transcription cofactor and may be inactivated through its phosphorylation by the upstream kinases LATS1/2.10 In Drosophila, Echinoid, an IgCAM member, was shown to function as an upstream regulator of the Hippo pathway. The loss of Echinoid compromises the phosphorylation of Yorkie (YAP1 in mammals), resulting in elevated Yorkie activity that drives tissue overgrowth.11 In the present study, we showed that CADM1 associates with the Hippo pathway core kinases, MST1/2 and LATS1/2, and may increase the phosphorylation of YAP1 through cell-cell contact. We also demonstrated, through an immunohistochemical analysis, that LATS2 was significantly co–expressed with CADM1 in the membranes of primary lung adenocarcinomas, and that the membranous co–expression of CADM1 and LATS2 correlated with a better prognosis. This is the first study to show that CADM1 is involved in the regulation of the Hippo signaling pathway through cell-cell contact. 2 MATERIALS AND METHODS 2.1 Cell lines The human lung adenocarcinoma cell line HCC827 was obtained from the RIKEN BioResource Center (Tsukuba, Japan) and the NCI-H292 and NCI-H1838 cell lines were from the ATCC (Manassas, VA, USA). The mouse fibroblast cell line NIH3T3 was obtained from the ATCC. The human embryonic kidney cell line 293FT was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Plat-A retroviral packaging cell line was a kind gift from Dr Toshio Kitamura (Institute of Medical Science, University of Tokyo). HCC827, NCI-H292 and NCI-H1838 cells were cultured in RPMI 1640 (Nacalai Tesque, kyoto, Japan) supplemented with 10% FBS (Biowest, Nuaillé, France), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). NIH3T3 and 293FT cells were cultured in DMEM (Nacalai Tesque) supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Plat-A cells were cultured in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 1 μg/mL puromycin (Wako Pure Chemical Industries, Tokyo, Japan) and 10 μg/mL blasticidin (Kaken Pharmaceutical, Tokyo, Japan). Cells were maintained at 37°C in a humidified 5% CO2 incubator (Panasonic, Osaka, Japan). 2.2 Antibodies for Western blotting Rabbit monoclonal anti–LATS1 (C66B5), anti–phospho-LATS1 (Thr1079) (D57D3), anti–phospho-YAP1 (Ser397) (D1E7Y) and anti–YAP (D8H1X) antibodies and a rabbit polyclonal anti–phospho-YAP (Ser127) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti–LATS2 antibodies were obtained from Novus Biologicals (Centennial, CO, USA) and Atlas Antibodies (Bromma, Sweden). The rabbit polyclonal anti–CADM1 (C-18) antibody was previously described.12 Goat polyclonal anti–GAPDH (V-18), rat monoclonal anti–HA (3F10) and mouse monoclonal anti–V5 (E10/V4RR) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), Roche (Basel, Switzerland) and Thermo Fisher Scientific, respectively. 2.3 Western blotting Cell lysates were extracted using RIPA buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L EDTA, .1% SDS and .5% sodium deoxycholate) containing a protease inhibitor cocktail (200 μmol/L AEBSF, 10 μmol/L leupeptin and 1 μmol/L pepstatin A) and phosphatase inhibitor cocktail (10 mmol/L NaF and 1 mmol/L Na3VO4). Protein samples were prepared by mixing lysates with 4 × sample buffer (.25 mol/L Tris-HCl [pH 6.8], 40% glycerol, 8% SDS, 20% 2-mercaptoethanol and .02% bromophenol blue) and then boiling at 95°C for 5 minutes. Equal amounts of total protein were fractionated in 5%-10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, VT, USA), and incubated with primary antibodies in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan). Primary antibody binding was detected using the Pierce Western Blotting Substrate (Thermo Fisher Scientific) with HRP-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA). Signals were visualized using ImageQuant LAS 4000 Mini (GE Healthcare) and quantified with ImageJ software. 2.4 Retroviral gene transfer The Igκ secretion leader sequence followed by CADM1 lacking its signal peptide sequence (45-442 a.a.) with an N-terminal HA tag (HA-CADM1) was cloned into the EcoRI–SalI site of a pBABE-puro vector, which was a gift from Drs Hartmut Land, Jay Morgenstern and Bob Weinberg (Addgene plasmid #1764).13 YAP1 was cloned into the EcoRI–NotI site of a pMX-IRES-GFP vector.14 Retroviral vectors were then transfected into Plat-A cells using Lipofectamine LTX (Thermo Fisher Scientific). After 48 hours, NIH3T3 cells were infected with the retrovirus by incubating with the culture supernatant of Plat-A cells. CADM1-expressing cells were obtained by puromycin selection at a concentration of 10 μg/mL. YAP1-expressing cells were obtained by sorting GFP-positive cells with FACS Aria II (BD Biosciences, Franklin Lakes, NJ, USA). 2.5 Lentiviral gene transfer HA-CADM1 was cloned in a pENTR/D-TOPO vector (Thermo Fisher Scientific). The lentiviral expression vector was then obtained by Gateway recombination with pLenti6-V5/DEST (Thermo Fisher Scientific). The vector obtained and ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) were co–transfected into 293FT cells using Polyethylenimine Max (Polysciences, Warrington, UK). After 48 hours, HCC827 cells were infected with the lentivirus by incubating with the culture supernatant of 293FT cells containing 5 μg/mL Polybrene (Nacalai Tesque). HCC827 cells expressing HA-CADM1 were selected by 20 μg/mL blasticidin. 2.6 Immunoprecipitation The coding sequences of WWC1/KIBRA, LATS1, LATS2, MST1, MST2, SAV1, YAP1 and YWHAH/14-3-3η without stop codons were cloned into the pENTR/D-TOPO vector. The templates used for cloning are listed in Table S1. Gateway Entry clones of the AMOT (100073116), AMOTL1 (100002205) and NF2 (100009338) genes without stop codons were purchased from DNAFORM (Yokohama, Japan). Expression vectors were obtained by Gateway recombination with a pHEK-V5 destination vector, which was generated by replacing the human IgG2-Fc fragment of the pHEK-Fc vector15 with the V5 peptide sequence. Expression vectors and a pHEK293 Enhancer Vector (Takara Bio, Kusatsu, Japan) were transfected into 293FT cells using Polyethylenimine Max in 10-cm dishes. After 24 hours, cells were collected and lysed with lysis buffer (25 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1% NP-40, 1 mmol/L EDTA and 5% glycerol) containing protease and phosphatase inhibitor cocktails. Lysates were centrifuged at 20,600 g at 4°C for 10 minutes, supernatants were pre–cleared with normal rabbit IgG (R&D Systems) and protein A-sepharose (GE Healthcare) at 4°C for 1 hour, and then incubated with the antibody against CADM1 or normal rabbit IgG at 4°C overnight. After incubating with protein A-sepharose at 4°C for 2 hours, sepharose was washed 4 times with lysis buffer, suspended in a sample buffer, boiled at 95°C for 5 minutes, and then incubated on ice. Samples were then subjected to SDS-PAGE followed by western blotting. 2.7 Real-time quantitative PCR Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) from cells collected 3 days after seeding at 1 × 106 cells on a 6-cm dish. First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Kit (Toyobo). Real-time PCR was performed using the ABI 7300 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with the SYBR Green PCR Master Mix (Applied Biosystems). Relative gene expression was calculated using the ddCt method. The sequences of primers used to detect gene expression were as follows: for ANKRD1, sense 5′-AAGCAGGAGGATCTGAAGACACTT-3′ and antisense 5′-GTTGTTTCTCGCTTTTCCACTGT-3′; for CYR61, sense 5′-GCGTTTCCCTTCTACAGGCT-3′ and antisense 5′-TTCTCCAATCGTGGCTGCAT-3′; for GAPDH, sense 5′-CAACGGATTTGGTCGTATTGG-3′ and antisense 5′-GCAACAATATCCACTTTACCAGAGTTAA-3′. 2.8 Tissue microarrays We used 7 different tissue microarrays (TMAs) that were produced to accommodate primary lung adenocarcinoma tissue core sections collected from patients (n = 166) who had undergone surgical resection at the University of Tokyo Hospital between June 2005 and September 2008. Core sections were carefully selected from histologically predominant invasive components in the case of adenocarcinoma with mixed subtypes by pathologists in the Department of Pathology, the University of Tokyo. Informed consent was obtained from all patients, and the study was approved by the Institutional Ethics Review Committee. Of 166 core sections, 12 were missing from TMA sections, and only 9 invasive adenocarcinoma cases (except minimally invasive adenocarcinoma cases) showed lepidic growth components without invasive lesions on TMA sections because of repeated slicing; therefore, the immunohistochemical analysis was performed using the data of 145 cases. Patients (n = 145) included 78 men and 67 women, ranging in age between 34 and 86 years (average 66.2 years). Each case was reassigned for its TNM classification and pathological stage based on the 7th Edition of the TNM Classification for Lung Cancer16: 18 were Stage 0, 89 Stage I (43 Stage IA, 46 Stage IB), 10 Stage II (5 Stage IIA, 5 Stage IIB), 22 Stage III (17 Stage IIIA, 5 Stage IIIB), and 4 Stage IV. The stages of 3 cases were unknown (2 were more than Stage I). A total of 145 cases included 16 non–mucinous adenocarcinomas in situ, 9 minimally invasive adenocarcinomas and 120 invasive adenocarcinomas. Each case was classified by 2 pathologists (D. M. and T. M.) based on the predominant histopathological subtype in the invasive lesion on TMA sections, except for cases of adenocarcinoma in situ and minimally invasive adenocarcinoma that showed lepidic growth components without invasive lesions on TMA sections, as follows: lepidic growth components (without an invasive lesion) (n = 22), papillary adenocarcinoma (n = 60), acinar adenocarcinoma (n = 31), solid adenocarcinoma (n = 30) and invasive mucinous adenocarcinoma (n = 2). These cases were also classified into 3 grades: low-grade containing lepidic growth (n = 22), intermediate-grade containing acinar and papillary adenocarcinomas (n = 91), and high-grade containing solid and invasive mucinous adenocarcinomas (n = 32), by referring to the histopathological grading described previously by Yoshizawa et al17 with a slight modification. 2.9 Immunohistochemistry Tissue sections for CADM1, LATS2 and YAP1 were initially treated with .3% hydrogen peroxide in methanol for 30 minutes to block endogenous peroxidase activity, and then autoclaved in 10 mmol/L citrate buffer (pH 6.0) at 120°C for 10 minutes. Sections were then preincubated with 10% normal horse serum in PBS incubated with a rabbit polyclonal anti–CADM1 (C-18) antibody at a dilution of 1:1000, a mouse monoclonal anti–human LATS2 (HPA039191) antibody from Atlas Antibodies at a dilution of 1∶25, and a rabbit monoclonal anti–human YAP1 (ab52771) antibody from Abcam at a dilution of 1:200 at 4°C overnight. The CADM1 antibody was detected with the DAKO Envision System-HRP Anti–rabbit system, the LATS2 antibody was detected with the DAKO LSAB2 streptavidin-peroxidase system, and the YAP1 antibody was detected with the DAKO Envision™ + Dual Link System, according to the manufacturer's instructions. 3,3′-Diaminobenzidine tetrahydrochloride (DAB) was used as a chromogen, whereas hematoxylin was used as a light counterstain. 2.10 Evaluation of immunohistochemistry Immunohistochemical staining was evaluated independently by 2 pathologists (D. M. and T. M.) through light microscopic observations and without knowledge of the clinical data of each patient. Cases of disagreement were reviewed jointly to reach a consensus score. Membranous staining was assessed for CADM1. Immunohistochemical results were subdivided into 2 categories, positive and negative, with cut-off values of 30% of tumor cells for CADM1, by referring to a previous study.6 Membranous and cytoplasmic staining was assessed for LATS2. Immunohistochemical results were subdivided into 2 categories, positive and negative, with cut-off values of 30% of tumor cells for LATS2. LATS2-positive cases were also divided into membrane-positive cases (with or without cytoplasmic expression) and cytoplasm-positive cases (without membranous expression). Cytoplasmic and nuclear staining was assessed for YAP1. Immunoreactivity was evaluated semi-quantitatively based on the intensity and estimated percentage of tumor cells that were stained. Intensity was quantified as follows: 1+, weak staining (detection required a high magnification); 2+, moderate staining (readily detected at a medium magnification); 3+, strong staining (readily detected at a low magnification). The percentages of positive cells were scored into 5 categories: 0, 0%; 1, 1%-25%; 2, 26%-50%; 3, 51%-75%; 4, 76%-100%. The product of the intensity and percentage scores was used as the final staining score. Final scores for nuclear/cytoplasmic YAP1 staining were defined as negative (final staining score <5) and positive (final staining score ≥5). 2.11 Statistical analysis The χ2-test was used to evaluate clinicopathological correlations, except for histopathological grades. The Mann-Whitney U-test was used to evaluate histopathological grades, with each grade being scored as follows: low-grade 1, intermediate-grade 2, and high-grade 3. Survival curves were generated using the Kaplan–Meyer method and differences in survival were analyzed using the Wilcoxon method. Results were considered to be significant if the P-value was <0.05. All statistical calculations were performed using the StatView computer program (Abacus Concepts). 3 RESULTS 3.1 CADM1 repressed the saturation density elevated by YAP1 overexpression in NIH3T3 cell lines We compared cell growth among NIH3T3 cell lines transfected with CADM1 and YAP1 together (+CADM1/YAP1), CADM1 and the control vector pMX (+CADM1/vec), the control vector pBABE and YAP1 (+vec/YAP1), and both control vectors (+vec/vec) (Figure 1). As previously reported,18 the overexpression of YAP1 promoted cell growth and elevated the saturation density of NIH3T3 cell lines, and the saturation density elevated by YAP1 was significantly repressed by CADM1 (Figure 1B). These results suggested that CADM1 is involved in cell density sensing through cell-cell adhesion, and that the inactivation of CADM1 induces the loss of contact inhibition, leading to cancer development. Figure 1Open in figure viewerPowerPoint CADM1 is involved in regulating the contact inhibition of growth and phosphorylation of YAP1 in confluent cells. A, Generation of NIH3T3 cells overexpressing CADM1 and/or YAP1. The protein expression levels of CADM1, YAP1 and GAPDH in NIH3T3 cells transfected with either CADM1 or YAP1 (+CADM1/vec and +vec/YAP1), both (+CADM1/YAP1), or neither (+vec/vec) were confirmed by western blotting. B, Saturation density of NIH3T3 cells transfected with either CADM1 or YAP1, both, or neither. Cells were seeded at 2 × 105 cells on 6-cm dishes and the cell number was counted every day until day 7. Mean ± SE of the cell number were shown (n = 4, *P < 0.05, **P < 0.01, paired t test). C, The phosphorylation status of YAP1 (ser 127) in HCC827 cells transfected with an empty vector (+vector) or CADM1 expression vector (+CADM1). Cells were seeded at 2 × 105 or 1 × 106 cells on 6-cm dishes and harvested after a 3-d culture. The signals of YAP1 and p-YAP1 obtained by western blotting (left) were quantified using ImageJ software. The ratio of p-YAP1/YAP1 in each cell was shown as a bar graph (mean ± SD, n = 7, **P < 0.01, paired t test) (right). D, The mRNA expression levels of the YAP1 target genes, ANKRD1 and CYR61, in HCC827 + vector and HCC827 + CADM1 cells were quantified by real-time PCR. Mean ± SD of the relative expression were shown (n = 4, *P < 0.05, paired t test) 3.2 Involvement of CADM1 in the control of contact inhibition through the Hippo pathway We used the lung adenocarcinoma cell line HCC827 with low levels of endogenous CADM1 expression. We established HCC827 cells overexpressing CADM1 and examined the effects of CADM1 on: (i) the phosphorylation of YAP1 on Ser 127 and Ser 397; and (ii) the expression levels of YAP1 target genes. YAP1 phosphorylation on Ser 127 correlated with its nuclear-cytoplasmic translocation, while that on Ser 397 correlated with its degradation.19 Under a high cell density, CADM1 significantly promoted the phosphorylation of YAP1 on Ser 127 (Figure 1C) and downregulated YAP1 target gene expression (Figure 1D) from those in the control. However, no significant difference was observed in the level of YAP1 phosphorylation on Ser 127 under a low cell density (Figure 1C). The induction of YAP1 phosphorylation on Ser 127 by CADM1 at confluence was also confirmed in a different lung adenocarcinoma cell line, NCI-H292 (Figure S1). Figure S2 also shows no significant effect of CADM1 on the phosphorylation of YAP1 on Ser 397 regardless of cell density, suggesting that CADM1 is not involved in the degradation of YAP1. 3.3 Interaction between CADM1 and Hippo pathway core molecules The regulatory mechanisms of the Hippo pathway by CADM1 have not yet been elucidated in detail; therefore, we performed immunoprecipitation assays to examine the relationship between CADM1 and Hippo pathway core molecules. The results obtained showed that CADM1 was coprecipitated with NF2, KIBRA, SAV1, MST1/2, LATS1/2, AMOTL1 and 14-3-3η, but not with YAP1 or AMOT (Figure 2). These results suggest that CADM1 forms complexes with Hippo pathway core molecules at the cell membrane. We also confirmed that endogenous CADM1 and LATS2 interacted in the lung adenocarcinoma cell line H1838 using an immunoprecipitation assay (Figure S3). We speculated that CADM1 recruits the Hippo pathway core kinases MST1/2 and LATS1/2 to the cell membrane through scaffold protein complexes containing NF2 and KIBRA, which activates a kinase cascade reaction. Figure 2Open in figure viewerPowerPoint CADM1 interacts with multiple Hippo pathway components; 293FT cells were transiently transfected with V5-tagged Hippo pathway core molecules (NF2, KIBRA, AMOT, AMOTL1, MST1/2, LATS1/2, SAV1, YAP1 and 14-3-3η) and cell lysates were immunoprecipitated by an anti–CADM1 antibody. The co–precipitation of CADM1 with these molecules was detected by western blotting with an anti–V5 antibody. Red arrows indicate positive signals. Co–precipitation experiments were performed at least twice 3.4 Immunohistochemical expression of LATS2 and CADM1 in primary lung adenocarcinoma tissues and their relationships with histological types, clinicopathological factors, and disease-free survival The junctional localization of LATS1/2 plays an important role in the regulation of the Hippo pathway. LATS1/2 has been shown to directly phosphorylate YAP1,20, 21 and the recruitment of LATS1/2 to the cell membrane is needed to promote the phosphorylation of LATS1/2.22 However, the membranous expression of LATS1/2 in primary lung tumors has not yet been investigated. To confirm whether CADM1 forms a complex with Hippo pathway core molecules in primary lung adenocarcinomas, we selected LATS2 as a representative of Hippo pathway core kinases interacting with CADM1, and examined the immunohistochemical expression of LATS2, particularly in the cell membrane, as well as its relationships with: (i) CADM1 expression; (ii) histopathological subtypes; (iii) clinicopathological factors; and (iv) disease-free survival using the TMA sections of 145 primary lung adenocarcinoma cases. Reactive type II pneumocytes constantly stained positive in the membrane for LATS2 (Figure 3A) and were weakly positive in the membrane for CADM1, as previously reported,6 and, thus, served as excellent internal positive controls. Figure 3Open in figure viewerPowerPoint A, Reactive type II pneumocytes as an internal positive control for LATS2, showing membranous LATS2-positive staining. The red arrow indicates a reactive type II pneumocyte. B, Reactive type II pneumocytes as an internal positive control for YAP1, showing nuclear YAP1-positive staining. The red arrow indicates a reactive type II pneumocyte. C, Fibroblasts as an internal positive control for YAP1, showing nuclear and cytoplasmic YAP1-positive staining. A red circle surrounds fibroblasts Among 145 cases, 66 (46%) were judged to show the membranous expression of LATS2, 35 (24%) cytoplasmic expression, but no membranous expression of LATS2, and 44 (30%) the completely negative expression of LATS2, while 92 cases (63%) were judged as showing the membranous expression of CADM1 and 53 (37%) the negative expression of membranous
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