ahnaklossinmicepromotestypeiipneumocyte hyperplasia and lung tumor … · 2018. 6. 6. ·...

Oncogenes and Tumor Suppressors

AHNAKLoss inMicePromotesType IIPneumocyteHyperplasia and Lung Tumor DevelopmentJun Won Park1,2, Il Yong Kim1,2, Ji Won Choi1, Hee Jung Lim2, Jae Hoon Shin1,Yo Na Kim1, Seo Hyun Lee1, Yeri Son1, Mira Sohn3, Jong Kyu Woo1,2,Joseph H. Jeong2, Cheolju Lee4, Yun Soo Bae3, and Je Kyung Seong1,2,5

Abstract

AHNAK is known to be a tumor suppressor in breast cancerdue to its ability to activate the TGFb signaling pathway.However, the role of AHNAK in lung tumor development andprogression remains unknown. Here, the Ahnak gene wasdisrupted to determine its effect on lung tumorigenesis andthe mechanism by which it triggers lung tumor developmentwas investigated. First, AHNAK protein expression was deter-mined to be decreased in human lung adenocarcinomas com-pared with matched nonneoplastic lung tissues. Then, Ahnak�/� mice were used to investigate the role of AHNAK in pul-monary tumorigenesis. Ahnak�/� mice showed increased lungvolume and thicker alveolar walls with type II pneumocytehyperplasia. Most importantly, approximately 20% of agedAhnak�/� mice developed lung tumors, and Ahnak�/� micewere more susceptible to urethane-induced pulmonary carci-nogenesis than wild-type mice. Mechanistically, Ahnak defi-

ciency promotes the cell growth of lung epithelial cells bysuppressing the TGFb signaling pathway. In addition, increasednumbers of M2-like alveolar macrophages (AM) were observedin Ahnak�/� lungs, and the depletion of AMs in Ahnak�/�

lungs alleviated lung hyperplastic lesions, suggesting that M2-like AMs promoted the progression of lung hyperplastic lesionsin Ahnak-null mice. Collectively, AHNAK suppresses type IIpneumocyte proliferation and inhibits tumor-promoting M2alternative activation of macrophages in mouse lung tissue.These results suggest that AHNAK functions as a novel tumorsuppressor in lung cancer.

Implications: The tumor suppressor function of AHNAK, inmurine lungs, occurs by suppressing alveolar epithelial cell pro-liferation andmodulating lung microenvironment.Mol Cancer Res;1–12. �2018 AACR.

IntroductionLung cancer is the global leading cause of cancer-related mor-

tality, and adenocarcinoma is the most common histologic type(1). A number of genes commonly altered in human lung ade-nocarcinomas have been identified (2), and their roles in lungtumorigenesis have been evaluated using genetically engineeredmouse (GEM) models (3). In particular, GEMmodels expressingactivating oncogenic mutants of the KRAS, EGFR, BRAF, andPIK3CA pathways developed lung tumors, and the combinationof these mutants had synergistic effects on tumorigenesis and

tumor progression (3). In addition, knocking out several othergenes, including Myc, Rac1, NF-kB, or Gata2, in the GEM modelsabove abrogated tumor development and progression, implyingoncogenic roles as additional hits in lung tumorigenesis (3, 4). Inparallel, ablation of several tumor suppressor genes, includingTrp53, Rb, and Lkb1, in the GEM models accelerated tumordevelopment and progression, thereby validating their roles astumor suppressors in lung tumorigenesis (3).

Ahnak is an exceptionally large protein (700 kDa) that wasinitially identified from human neuroblastomas and skin epithe-lial cells (5, 6). Ahnak is known to be a scaffolding protein thatregulates cytoskeletal structure formation, muscle regeneration,calcium homeostasis, and signaling (7, 8). Structurally, Ahnak isdivided into three distinct regions: the amino-terminal region of500 amino acids, the large central region of about 4,388 aminoacids composed of 36 repeat units, and the carboxyl-terminalregion of 1,003 amino acids (9). It has been suggested that thecentral repeat unit (CRU) supports the structural integrity andscaffolding activity of Ahnak (9–12). CRU plays the role of amolecular linker for calcium homeostasis by interacting withphospholipase C and protein kinase C (9, 10). In addition, CRUinteracts with R-Smad proteins throughMH2 domain and stimu-lates Smad3 nuclear translocation and markedly inhibits c-Mycpromoter activity (11). In regards to cancer biology, the role ofAhnak in tumorigenesis is controversial. For example, Ahnakfunctions as a tumor suppressor in breast cancer by inhibitingcell growth via potentiation of the TGFb signaling pathway (11).However, Ahnak is also associated with tumor development andprogression (13–15).

1Laboratory of Developmental Biology and Genomics, BK21 Program Plus forAdvanced Veterinary Science, and Research Institute for Veterinary Science,College of Veterinary Medicine, Seoul National University, Seoul, Korea. 2KoreaMouse Phenotyping Center (KMPC), Seoul, Korea. 3Department of Life Sciences,EwhaWomans University, Seoul, Korea. 4Center for Theragnosis, Korea Instituteof Science and Technology, Seoul, Korea. 5Interdisciplinary Program forBioinformatics, Program for Cancer Biology and BIO-MAX/N-Bio Institute, SeoulNational University, Seoul, Korea.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

J.W. Park and I.Y. Kim contributed equally to this article.

Corresponding Author: Je Kyung Seong, College of Veterinary Medicine, SeoulNational University, Sinrym-dong, Seoul 151-742, Republic of Korea (South).Phone: 82-2-885-8395; Fax: 82-2-885-8397; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0726

�2018 American Association for Cancer Research.

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In our previous studies, we reported that Ahnak�/�mice exhibitenhanced insulin sensitivity, higher energy expenditure, upregu-lated lipolysis of white adipose tissues, and impaired adipocytedifferentiation (8, 16, 17). Intriguingly, we found pneumocytehyperplasia and lung tumor development in Ahnak�/� mice.In this study, we assessed roles of Ahnak in lung tumorigenesis.We first confirmed the downregulation of Ahnak protein expres-sion in human lung adenocarcinomas. Mechanistically, weshowed that Ahnak deficiency downregulates TGFb signaling inpneumocytes. In addition, we revealed that increased numbersof M2-like AMs in lungs of Ahnak�/� mice contribute to thepneumocyte hyperplasia and the formation of tumor microen-vironment. Taken together, these findings suggest that Ahnakfunctions as a tumor suppressor in murine lungs by suppressingalveolar epithelial cell proliferation and modulating the lungmicroenvironment.

Materials and MethodsMice models

Ahnak�/� mice were generated by disruption of exon 5 in theAhnak gene, as reported previously (10). Genotyping was per-formed using genomic DNA isolated from tails according tomethods described previously (10). Ahnak�/� mice were housedin a specific pathogen-free condition. To induce lung tumors inmice using urethane, we used a modified protocol derived from aprevious report (18). 6-week-old mice were injected intraperito-neally once weekly for 8 weeks with 1 mg/g of urethane (Sigma)dissolved in 0.9% NaCl. The mice were sacrificed at 23 weeksafter the initial urethane injection. Tumors were visually countedon the Tellyesniczky's fixative-cleared lungs by three blindedreaders under a dissecting microscope. Tumor diameter wasmeasured by micro-CT images using PET/CT scanner (eXploreVista PET/CT Pre-Clinical, GE Healthcare). To deplete macro-phages in lungs of mice, clodronate liposomes (F70101C-A,FormuMax Scientific) were used as described previously (19).Twenty-week-old mice were treated intranasally with clodronateliposomes or negative control liposomes every 3 days for 3 weeksat doses of 24 mL. All experiments were performed accordingto the "Guide for Animal Experiments" (Edited by Korean Acad-emy of Medical Sciences) and approved by the InstitutionalAnimal Care and Use Committee of Seoul National University(Seoul, Korea).

Cell cultures, transfection, and cocultureHuman lung cancer cell lines (H460 and H23), mouse lung

epithelial cell line MLE-12, and mouse macrophage-like cellline RAW264.7 were purchased from ATCC. All cell lines werecultured at 37�C in a 5% CO2 humidified incubator in themedia according to ATCC recommendations. H460, H23 cells,and RAW264.7 cells were transfected with pcDNA-HA orpcDNA3-HA-4CRU of Ahnak (amino acid residues 4105–4633; ref. 11) using TransIT-X2 Dynamic Delivery System(Mirus Bio LLC) according to the manufacturer's instructions.pSBE-luc reporter plasmid (11) and Renilla luciferase constitu-tively expressing vector (Addgene; for internal control) weretransfected into cells to measure TGFb activities. Reporteractivities were evaluated via dual luciferase reporter assays(Promega) according to the manufacturer's instructions. Toestablish Ahnak KO RAW264.7 cells, we transfected RAW264.7cells with Cas9 Ahnak CRISPR/Cas9 KO plasmid (sc-425992;

Santa Cruz Biotechnology) and selected KO clones according tothe manufacturer's protocol.

Peritoneal and alveolar macrophages (AM) were isolatedfrom mice according to previously described methods(20, 21). Briefly, after killing by CO2, mice were injectedintraperitoneally with 10 mL cold PBS, and the peritoneal fluidwas withdrawn by syringe suction. AMs were isolated frombronchoalveolar lavages with 35 mL/g of PBS using a 20 gneedle inserted into the trachea. Peritoneal macrophages from3 mice and AMs from more than 5 mice were collected andpooled together. Cell sorting by flow cytometry (SH800 cellsorter) using rat anti-mouse F4/80 (25-48-1; eBioscience; PE-Cyanine7) was performed to enrich macrophages from theperitoneal and bronchoalveolar suspension cells. After cellnumbers were determined by trypan blue exclusion, 2 � 105macrophages were seeded on 6-well cell culture plate in DMEM(Welgene) containing 2% FBS (Welgene). After 3 hours ofseeding, cells were harvested for mRNA isolation. For cocultureexperiments, MLE-12 cells, seeded on 6-well Trans-well inserts(Corning) at 2 � 105 cells, were added to the 6-well plates onwhich AMs were seeded. The cell growth of MLE-12 cells wasdetermined using MTT assays after 48 hours of coculture.

Histopathology, IHC, immunofluorescence, andimmunoblotting

Mouse lung tissues were perfused and fixed in 4% parafor-maldehyde overnight, processed in a routine manner, andembedded in paraffin. Hematoxylin and eosin stain (H&E),Masson's trichrome stain (SSK5005; BBC Biochemical), IHC,and immunofluorescence (IF) were performed on 4-mm thickserial sections from mouse tissue paraffin blocks. Light micro-scopic examinations were performed on H&E slides by pathol-ogists to evaluate mouse lung lesions. Mouse lung tumorswere classified according to classification of mouse lungtumors (22).

A tissue microarray (TMA) slide (LC1504; US Biomax Inc.)was applied to IHC for Ahnak. Dewaxed and rehydrated par-affin sections were subjected to antigen retrieval by heatingthe sections to 100�C for 20 minutes in 0.01 mol/L citratebuffer (pH 6.0) or EDTA unmasking solution (#14747,Cell Signaling Technology). To perform IHC staining, theImmPRESS Peroxidase Polymer Kit (Vector Laboratories) wasused for immunostaining according to the manufacturer'sprotocol. The slides were incubated with the following primaryantibodies: goat anti-SP-C (sc-7726; Santa Cruz Biotechnolo-gy), rabbit anti-PDPN (sc-134483; Santa Cruz Biotechnology),rat anti-F4/80 (sc-59171; Santa Cruz Biotechnology), mouseanti-Ki-67 (ab8191; Abcam), rabbit anti-cyclin D1 (#2978;Cell Signaling Technology), mouse anti-PCNA (sc-56; SantaCruz Biotechnology), rabbit anti-phosphorylated IGF-1R(#3918, Cell Signaling Technology), rabbit anti-phosphorylat-ed EGFR (#4407, Cell Signaling Technology), rabbit anti-phos-phorylated STAT3 (#9145, Cell Signaling Technology), rabbitanti-phosphorylated ERK (#4370, Cell Signaling Technology),rabbit anti-phosphorylated AKT (#9272, Cell Signaling Tech-nology), goat anti–IGF-1 (AF791; R&D Systems), and mouseanti-Ahnak (ab68556; Abcam). The slides were subjected tocolorimetric detection with ImmPact DAB substrate (SK-4105,Vector Laboratories). The slides were counterstained withMeyer's hematoxylin for 10 seconds. Negative controls wereperformed by omitting the primary antibody. To perform IF

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staining, after antigen unmasking and blocking with BSA, theslides were incubated overnight at 4�C with following primaryantibodies: goat anti-SP-C (sc-7726; Santa Cruz Biotechnolo-gy), rabbit anti-PDPN (sc-134483; Santa Cruz Biotechnology),mouse anti-Ki-67 (ab8191; Abcam), and rabbit anti-phosphor-ylated Smad3 (ab52903; Abcam). Then, slides were incubatedfor 2 hours at room temperature with the following secondaryantibodies; donkey anti-goat IgG (HþL), Alexa Fluor 568(A11057; Thermo Fisher Scientific), donkey anti-mouse IgG(HþL), Alexa Fluor 488 (A21202; Thermo Fisher Scientific),and donkey anti-rabbit IgG (HþL), and Alexa Fluor 647(A31573; Thermo Fisher Scientific). Slides were mounted withVectashield mounting media (H-1200; Vector Laboratories). Toperform scoring for H&E, IHC, and IF results, slides werescanned by Pannoramic SCAN slide scanner (3D HISTECH)and evaluated on Case Viewer Software (3D HISTECH) using a40� objective for at least 5 spots per mouse with a minimum of3 mice in each group.

Toperform immunoblotting, harvested cellswere lysed in lysedin RIPA buffer (GenDEPOT) with protease inhibitors (Xpertprotease inhibitor cocktail solution, GenDEPOT). Total cellextracts were fractionated by electrophoresis on a gradient SDSpolyacrylamide gel and transferred onto a PVDF membrane. Thefollowing primary antibodies were used; rabbit anti-cyclin D1(#2978; Cell Signaling Technology), mouse anti-PCNA (sc-56;Santa Cruz Biotechnology),mouse anti-GAPDH (sc-32233, SantaCruz Biotechnology), rabbit anti-AKT (#9272, Cell SignalingTechnology), rabbit anti-ERK (#4695, Cell Signaling Technolo-gy), phosphorylated ERK (#4370, Cell Signaling Technology),rabbit anti-phosphorylated AKT (#9272, Cell Signaling Technol-ogy), mouse anti–a-tubulin (sc-8037, Santa Cruz Biotechnolo-gy), and rabbit anti–HA-Tag (#3724, Cell Signaling Technology).Immunodetection was performed by using an Enhanced Chemi-luminescence Detection Kit (AbClon). Densitometry calculationwas performed by ImageJ 1.49v software developed by the NIH(Bethesda, MD).

RNA extraction and qRT-PCRTotal RNA from both lung tissues and cell lines was extracted

by TRIzol (Ambion) according to the manufacturer's instruc-tions. First-strand cDNA was synthesized using the AcculowerRT Premix (Bioneer) according to the manufacturer's instruc-tions. PCR reactions were performed on 7500 Real Time PCRSystem (Applied Biosystems) using SensiFAST SYBR Green PCRMaster Mix (BIO-94020; Bioline). ActB was used as the endog-enous reference control for all transcripts. All qRT-PCR experi-ments were repeated at least three independent times. Primersused are as follows:

F: 50-CTTCTGGGCCTGCTGTTCA-30

R: 50-CCAGCCTACTCATTGGGATCA-30 for Mcp1,F: 50-AGCACAGAAAGCATGATCCG-30

R: 50-CTGATGAGAGGGAGGCCATT-30 for Tnf,F: 50-GAGGATACCACTCCCAACAGACC-30

R: 50-AAGTGCATCATCGTTGTTCATACA-30 for Il6,F: 50-AGACAGGCATTGTGGATGAG-30

R: 50-TGAGTCTTGGGCATGTCAGT-30 for Igf1,F: 50-TGGCTGTGTCCTGACATCAG-30

R: 50-GAAGACAGATCTGGCTGCATC-30 for Egf,F: 50-TGACGGCACAGAGCTATTGA-30

R: 50-TTCGTTGCTGTGAGGACGTT-30 for Il4,

F: 50-GCTCTTACTGACTGGCATGAG-30

R: 50-CGCAGCTCTAGGAGCATGTG-30 for Il10,F: 50-GAGGTCTTTACGGATGTCAACG-30

R: 50-GGTCATCACTATTGGCAACGAG-30 for Actb.

FACS analysis and cell sortingTo prepare the lung cell suspension, we performed enzymatic

digestion of lung tissues using dispase (STEMCELL Technologies)as reported previously (23). Cells were stained with rat anti-mouse F4/80 (25-48-1; eBioscience; PE-Cyanine7), E-cadherin(46-3259; eBioscience; PerCP-eFluor710), CD31 (12-0311;eBioscience; PE), CD45 (553079; BD Pharmingen; FITC),CD16/32 (553142; BD Pharmingen; for blocking), and rat IgGisotype controls for 1 hour at 4�C in the dark room. To detectAhnak expression in cells, the cells were fixed with 4% parafor-maldehyde and then permeabilized with ice-cold methanol. Thecells were incubated with mouse anti-Ahnak (ab68556; Abcam)for 1 hour at 4�C in a dark room. The secondary antibody wasdonkey anti-mouse IgG (HþL), Alexa Fluor 488 (A21202;Thermo Fisher Scientific). The cells were analyzed and sorted bya SH800 cell sorter (Sony).

Statistical analysisStatistical analysis was performed by GraphPad Prism 4

(GraphPad Software, http://www.graphpad.com). Analyses wereperformed using a Student t test. P values of less than 0.05were considered statistically significant. Results are presented asmean � SEMs.

ResultsAhnak protein expression is downregulated in human lungcancers

We performed IHC analysis using an antibody against Ahnakof a TMA slide containing 50 cases of human lung adenocarci-nomas and matched normal lung tissues. In normal lungtissues, Ahnak protein expression was mainly observed inthe membrane and/or cytoplasm of pneumocytes and AMs(Fig. 1A). However, Ahnak protein expression was significantlydownregulated in lung cancer cells compared with normallung tissues (Fig. 1A and B; Supplementary Fig. S1). Accordingto previously published data (24), AHNAK mRNA levels werealso significantly lower in all types of lung cancer tissues thanthose in normal lung tissues (Fig. 1C). In addition, analysis ofthe Cancer Cell Line Encyclopedia database revealed that lungcancer cell lines have relatively lower AHNAKmRNA expressioncompared with other cancer cell lines (Fig. 1D; ref. 25). AHNAKmRNA expression is highest in normal lung tissue amonghuman tissues (15), and Ahnak mRNA was abundantlyexpressed in mouse lung tissue (26), suggesting physiologicallyimportant roles of Ahnak in lung. Collectively, these findingssuggest that downregulation of Ahnak gene expression is asso-ciated with lung tumorigenesis.

Ahnak�/� mice show high proliferative activity in alveolar typeII pneumocytes

At 6, 10, 14, and 18 weeks of age, Ahnak�/� lungs showedincreased size andweight than age-matched wild-type (WT) lungs(Fig. 2A; Supplementary Fig. S2).Histologically, the alveolar septa

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of Ahnak�/� lungs were thickened, which was accompanied bydilated alveolar space (Fig. 2B and C). Ahank�/� lungs atembryonic day 18.5 also showed reduced airspace with densercellularity than WT lungs (Supplementary Fig. S3), suggestingthat Ahnak deficiency might affect lung in developmentalstages. The thickened walls showed high cellularity and exces-sive connective tissues including collagen deposition (Fig. 2C;Supplementary Fig. S4). Importantly, IHC analysis revealedthat SP-C–positive alveolar type II pneumocytes (AT2) weresignificantly increased in Ahnak�/� lungs in comparison withWT lungs (Fig. 2D), whereas PDPN-positive alveolar type Ipneumocytes (AT1) lining alveolar spaces were significantlydecreased and formed a discontinuous pattern (Fig. 2D). West-ern blot analysis confirmed an increased expression of SP-Cproteins and reduced expression of PDPN proteins in Ahnak�/�

whole lung tissues (Fig. 2E). Notably, we did not detect anincrease in infiltrated CD45 (a leukocyte common antigenmarker)–positive cells and CD3 (a T-cell marker)–positive cellsin Ahnak�/� lungs compared with WT lungs (SupplementaryFig. S4). Furthermore, IHC analysis of both Ki-67 and PCNA,markers of cell proliferation, showed increased positive cells inAhnak�/� lungs compared with WT lungs (Fig. 3A). Consistentwith previous results showing that introduction of CRU ofAhnak results in cell-cycle arrest through the downregulationof cyclin D (11), cyclin D1 was upregulated in Ahnak�/� lungs(Fig. 3A). These findings were confirmed by Western blotanalysis using whole lung cell lysates from WT and Ahnak�/�

mice (Fig. 3B). Coimmunofluorescence (co-IF) staining of SP-C–positive or PDPN-positive cells for Ki-67 revealed that SP-C–positive AT2 are highly proliferative and therefore contribute to

Figure 1.

Downregulation ofAhnak in human lung adenocarcinomas.A,Representative images of downregulatedAhnak expression in human lung cancer tissues andmatchednormal lung tissues from a TMA slide containing 50 lung adenocarcinoma cases. Original magnification, �40; inset, �1,000. Scale bar, 50 mm. B, IHC scoring ofAhnak expression in human lung adenocarcinomas and matched normal lung tissues from the TMA slide. Ahnak expression was scored according to theintensity of staining: 0, negative staining; 1, weakly positive staining; 2, moderately positive staining; 3, strongly positive staining. C,AHNAKmRNA expression in lungcancer tissues according to histologic subtypes based on published datasets (GSE83227). AdenoC, adenocarcinoma; SCLC, small-cell lung carcinoma;NSCLC, non–small cell lung carcinoma; SCC, squamous cell carcinoma. D, AHNAK mRNA expression in various types of cancer cell lines obtained from the CancerCell Line Encyclopedia (CCLE) database (GSE36139). � , P < 0.05 by unpaired, two-tailed Student t test in B.

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lung hyperplasia (Fig. 3C and D). Taken together, lung lesionsin Ahnak�/� mice are characterized by hyperplastic AT2 cellsdue to their increased cell proliferation.

Ahnak�/� pneumocytes show downregulation of the TGFbsignaling pathway

It is known that Ahnak inhibits cell growth by potentiatingTGFb-induced transcriptional activity via its direct interactionwith Smad3 (11). We found that the nuclear expression ofphosphorylated Smad3 (S423/S425) in SP-C–positive AT2 cellswas significantly reduced in Ahnak�/� lungs compared with WTlungs (Fig. 4A and B). We confirmed that phosphorylation ofSmad3 is reduced in Ahnak�/� whole lung tissues using Westernblot analysis (Fig. 4C). Because the CRU of Ahnak is the scaffold-ing motif responsible for proper modulation of its signalingpathways (11), we overexpressed four CRUs (4CRUs) in humanlung cancer H460 and H23 cell lines (Fig. 4D). These showedincreased TGFb reporter activity anddecreased cell growth (Fig. 4Eand F). Taken together, these results suggest that Ahnak inhibitsthe cell growth of lung epithelial cells by activating the TGFbsignaling pathway.

Ahnak�/� mice spontaneously develop lung tumors and showhigh susceptibility to urethane-induced lung carcinogenesis

Notably, approximately 20% of aged Ahnak�/� mice (2 out of9 one-year-old Ahnak�/� mice) developed lung tumors. Grossly,these tumors appear as a solitary gray orwhite nodule that slightlyprotrudes from the lung surface (Fig. 5A).Histologically, one lungtumor was adenoma and the other was adenocarcinoma. Theadenomawas unencapsulated but well-demarcatedwith its densecellular neoplasm, which was composed of well-to-moderatelydifferentiated polygonal cells in a tubulopapillary pattern(Fig. 5B). The adenocarcinoma had no clear margin and showedsolid growth pattern, which was composed of moderately-to-poorly differentiated neoplastic cells (Fig. 5B). In addition, therewere a large number of infiltrated foamymacrophages, rare T cells(CD3þ cells) and neutrophils (polymorphonuclear cells). Co-IFstaining revealed that most of tumor cells were SP-C positive(Fig. 5C), raising the possibility that the origin of tumorcells might be AT2 cells. Tumor cells from all the cases were E-cadherin (an epithelial cell marker)-positive and showed higherproliferative activity comparedwith normal lung tissue evidencedby IHC for Ki-67 and cyclin D1 (Fig. 5D). Ki-67 and cyclin D1

Figure 2.

Thicker alveolar septa due to increased AT2 cells. A, Enlarged lungs were observed in Ahnak�/� mice compared with WT mice at 6, 10, 14, and 18 weeks of age.Lung weights after normalization to body weights were calculated. B, Dilated airspaces and thicker alveolar septa were observed in Ahnak�/� mice comparedwith WT mice. The scoring for air spaces (mm2) and thickness were performed using Case Viewer Software. n ¼ 3 Ahnak�/� mice, n ¼ 3 WT mice at each timepoint. C, Representative H&E pictures of pulmonary lesions of Ahnak�/� lungs. Red boxed areas in the top panels are magnified in the bottom panels.Fourteen-week-old Ahnak�/� mice showed hypercellularity and excessive connective tissues. Scale bar, 200 mm. D, There was an increased number ofSP-C–positive AT2 cells per HPF (high-power field, 200�) in 10-week-old Ahnak�/� lungs. PDPN-positive AT1 cells incompletely lined alveolar walls inAhnak�/� lungs according to H-score system [(1 � (% weakly positive cells) þ 2 � (% moderately positive cells) þ 3 � (% strongly positive cells)].n ¼ 3 Ahnak�/� mice, n ¼ 3 WT mice at 10 weeks of age. Scale bar, 50 mm. E,Western blot analysis using whole lung tissues confirmed the increased SP-C anddecreased PDPN expression in Ahnak�/� lungs compared with WT lungs. � , P < 0.05 by unpaired, two-tailed Student t test in D.






immunoreactivities were stronger in the adenocarcinoma than inthe adenoma (Fig. 5D), indicating higher proliferative activity inadenocarcinoma.

As this low number of tumor-bearing mice was insufficient toachieve statistical significance, we utilized the urethane-inducedlung carcinogenesis model to evaluate the effect of Ahnak genedeficiency on lung tumorigenesis (18). With this model, wedetermined that bothWT and Ahnak�/�mice formed pulmonaryadenomas. We did not find any histopathologic differences,including cell differentiation and invasiveness, between tumorcells arising from wild and from Ahnak�/� lungs. However, thenumber and size of the urethane-induced lung tumors in theAhnak�/�micewere significantly greater than those in those in theWTmice (Fig. 5E–G). Moreover, higher numbers of macrophageswere prominent both inside and surrounding the tumor tissues inAhnak�/� mice compared with those of the WT mice. Overall,these data suggest that Ahnak gene deficiency promotes sponta-neous lung tumorigenesis and increases susceptibility to carcin-ogen-induced lung carcinogenesis.

Ahnak gene deficiency leads to increased numbers of M2-likealveolar macrophages in Ahnak�/� lungs

IHC analysis of F4/80, a 160-kDa glycoprotein expressed bymurinemacrophages, revealed significantly increased numbers ofAMs in Ahnak�/� lungs compared with WT lungs (Fig. 6A). Wealso confirmed increased numbers of AMs in bronchoalveolarlavage fluid (BALF) of Ahnak�/� lungs (Fig. 6B). In general,macrophages are classified into two subtypes, classically activatedM1 macrophages and alternatively activated M2 macrophages,based on their cytokine expression patterns (for M1, TNFa, IL12,IFNs, etc. and for M2, IL10, IL4, IL13, etc.) and their immunefunctions (for M1, proinflammatory and for M2, anti-inflamma-tory; ref. 27). Therefore, to delineate the identity of the enhancedpopulation of AMs in Ahnak�/� lungs, we performed flow cyto-metry (FACS) analysis. Interestingly, Ahnak�/� AMs exhibitedhigher expression of CD206, a well-known M2 marker, than WTAMs (Fig. 6C). In addition, the mRNA expression level of proin-flammatory cytokine TNF was significantly lower in Ahnak�/�

AMs than in WT AMs, whereas anti-inflammatory cytokines such

Figure 3.

Proliferation of AT2 cells in Ahnak�/� lungs.A, IHC analysis for cell-cycle regulators such as Ki-67, PCNA, and cyclin D1 inWT andAhnak�/� lungs. Right graphs showthe IHC scoring for each marker in lung tissues. The scorings were calculated as the percentage of cells exhibiting strong nuclear staining per HPF (high-power field,400�). n¼ 3 Ahnak�/�mice, n¼ 3WTmice at 10 weeks of age. Scale bar, 100 mm. B,Western blot analysis for PCNA and cyclin D1 in Ahnak�/� andWTwhole lungtissues at 6, 10, 14, and 18weeks of age.C andD, IF analysis for SP-C and PDPNpneumocytemarkers and a proliferationmarker Ki-67 in Ahnak�/� lungs at 10weeks ofage. C, Representative images of the IF staining. SP-Cþ/Ki-67þ cells, arrow. PDPNþ/Ki-67þ cells, arrowhead. SP-Cþ/PDPNþ/PCNAþ cells, asterisk. Scale bar, 40 mm.D,Proportion of SP-C–positive and/or PDPN-positive cells in Ki-67–positive cells. SP-C–positive AT2 cells accounted formore than half of Ki-67–positive cells. Similarresults were obtained from 3 mice. � , P < 0.05 by unpaired, two-tailed Student t test in A.

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as IL4 and IL10 were significantly higher (Fig. 6D). Notably,Ahnak�/� AMs also produced more growth factors such as insu-lin-like growth factor 1 (IGF-1; Fig. 6D; Supplementary Fig. S5C)and EGF (Fig. 6D) than WT AMs. Activation of the IGF and EGFsignaling pathways in Ahnak�/� lungs was also confirmed by IHC(Supplementary Fig. S5A and S5B). The cytokine expressionprofile of Ahnak�/� whole lung tissues versus WT lung tissues

reflected that of Ahnak�/� AMs (Fig. 6E). Collectively, our datarevealed that M2-like AMs producing growth factors were accu-mulated in Ahnak�/� lung.

To investigate roles of Ahnak in cytokine production duringmacrophage polarization, we induced macrophages to differ-entiate either into M1 in response to LPS or M2 following IL4treatment. In response to LPS treatment, Ahnak�/� peritoneal

Figure 4.

Downregulation of the TGFb signaling in Ahnak�/� lungs. A, IF analysis for SP-C, phosphorylated Smad3 (phosphorylation sites, S423þS425), and Ki-67 in lungtissues from 10-week-old Ahnak�/� andWTmice. Arrows indicate SP-C–positive AT2 cells showing nuclear expression of phosphorylated Smad3. Scale bar, 50 mm.B, Scoring of presence of nuclear phosphorylated Smad3 in SP-C–positive AT2 cells based on IF staining. n ¼ 3 Ahnak�/� mice, n ¼ 3 WT mice at 10 weeks ofage. More than 2,000 SP-C–positive cells were evaluated per mouse. C,Western blot analysis for phosphorylated and total Smad3 in whole lung tissues from 6- and18-week-old Ahnak�/� and WT mice. D, Human lung cancer cell lines were transfected with hemagglutinin (HA)-tagged 4CRU of Ahnak (pcDNA3-HA-4CRU).Successful transfection was confirmed by Western blot analysis for HA. E, Dual luciferase reporter assay for the TGFb signaling activation. After 3 days oftransfectionwith HA-4CRU-Ahnak, luciferase reporter activitiesweremeasured. Cellswere treatedwith 5 ng/mL TGFb for 6 hours. Relative light units (RLU) is a ratioof firefly luciferase units normalized to Renilla luciferase units. F, A hemocytometer-based trypan blue dye exclusion assay was conducted to quantifycells andmeasure viability. Growth inhibitionwas observed after transfectionwith H4-4CRU. � , P < 0.05 by unpaired, two-tailed Student t test inB, E, and F. The dataare means � SEM of three independent experiments.






macrophages exhibited reduced levels of TNF and increasedlevels of MCP1 and IL6 compared with those of WT peritonealmacrophages (Fig. 6F). Although IL6 is known for a proin-flammatory cytokine, roles of IL6 in macrophage polarizationwere context dependent and IL6-promoting M2 programminghas been reported (28, 29). In parallel, we overexpressed4CRUs in CRISPR/Cas9–mediated Ahnak knockout (KO)RAW264.7 cells. IL4, IL10, IL6, and IGF-1 expressions by LPSor IL4 treatment were suppressed in restored cells (Fig. 6G).Phosphorylated Akt was reduced in LPS or IL4-treated AhnakKO RAW264.7 cells after the restoration of 4CRU-Ahnak (Fig.6H). Akt signaling is implicated in macrophage activation, andit has been suggested that the activation of Akt promotes M2polarization, whereas loss of Akt1 augments M1 activation(30). Taken together, these results suggest that Ahnak gene

deficiency induces the transition of AM cytokine profiles infavor of M2-like macrophage programming and monocyterecruitment.

Ahnak�/� AMs confer enhanced lung pneumocyte proliferationTo assess whether cytokines and/or growth factors released

from a larger AM population in Ahnak�/� lungs contribute topneumocyte cell proliferation, we depleted AMs in Ahnak�/�

lungs by intranasal administration of clodronate liposome(Fig. 7A–I). Clodronate liposome treatment for 3 weeks attenu-ated the thickness of alveolar septa in Ahnak�/� lungs (Fig. 7A–Cand7J) comparedwith that of control liposome-treatedAhnak�/�

lungs (Fig. 7E–G and 7J). Consistent with this observation, Ki-67staining was decreased in Ahnak�/� lungs after the depletion ofAMs, indicative of decreased proliferative activity (Fig. 7K). To

Figure 5.

Development of spontaneous lung tumors and higher susceptibility to carcinogen-induced pulmonary carcinogenesis in Ahnak�/� mice. A, A representative grosspicture of a spontaneous lung tumor in a 1-year-old Ahnak�/�mouse (#1). Arrowheads, tumor margin. Scale bar, 1 cm. B, Histopathologic findings of Ahnak�/�micebearing a pulmonary adenoma (#1) and an adenocarcinoma (#2) based on H&E staining. Original magnification, �40. Boxed areas are magnified in inset.Scale bar, 50mm.C,Co-IF analysis for SP-C andPDPN. The dotted line indicates tumormargin. Scale bar, 200mm.Right, boxed area ismagnified. Scale bar, 50mm.Ki-67 staining was done to denote proliferation. D, IHC for E-cadherin, Ki-67, and cyclin D1 was performed to characterize spontaneous tumors in Ahnak�/� mice.Scale bar, 50 mm. E, Representative photographs of urethane-induced lung tumors in WT and Ahnak�/� mice. Scale bar, 5 mm. F, Increased numbers anddiameters of urethane-induced tumors in Ahnak�/� mice versus WT mice. The sizes and numbers of lung tumors were measured by micro-CT images.G, Representative H&E images of urethane-induced tumors in WT and Ahnak�/� lungs. Original magnification, �100. � , P < 0.05; �� , P < 0.01; �� , P < 0.001 byunpaired, two-tailed Student t test in F.

Park et al.





recapitulate this in an in vitro system, MLE-12 cells, an immor-talized mouse lung epithelial cell line, were cocultured withisolated AMs from Ahnak�/� mice or AMs from WT mice.MLE-12 cells grew more rapidly when cocultured with AMs from

Ahnak�/� mice than WT mice (Fig. 7L), indicating that factorssecreted from Ahnak�/� AMs affected the cell growth. Ras–Raf–ERK and PI3K–Akt pathways play a central role in drivingmany ofthe phenotypic changes induced by growth factors. Western blot

Figure 6.

Increased numbers ofM2-like alveolarmacrophages inAhnak�/� lungs.A,Representative IHC images of F4/80 inWT, Ahnak�/�, and tumor-bearing lungs. Scale bar,100 mm. The numbers of F4/80-positive AM in Ahnak�/� andWT lungs at the indicated time points. F4/80-positive cells were counted and averaged in 400� high-power fields (HPF) of lungs. n ¼ 3 Ahnak�/� mice, n ¼ 3 WT mice at each time point. B, Increased AMs in bronchi alveolar lavage fluid (BALF) in Ahnak�/� lungsversus WT lungs. F4/80-positive macrophages from BALF were counted by flow cytometry. n ¼ 3 Ahnak�/� mice, n ¼ 3 WT mice at 10 weeks of age. C, FACSanalysis for CD206, a M2 macrophage marker, in Ahnak�/� and WT AMs of 10-week-old mice. Single-cell suspensions were obtained from whole lungtissues after dispase digestion. Macrophages were defined as both CD45 and F4/80-positive cells. D, qRT-PCR analysis for mRNA expressions of cytokines,chemokines, andgrowth factors inAhnak�/�AMs from 14-week-oldmice.E,qRT-PCRanalysis formRNAexpressionsof these factors inAhnak�/�andWTwhole lungtissues from 14-week-old mice. F, TNF, IL6, and MCP1 induction after 48 hours of 10 ng/mL LPS treatment was measured by qRT-PCR analysis. The cellsfrom peritoneal spaces were enriched for peritoneal macrophages by selecting F4/80-positive cell populations via FACS sorting. G, qRT-PCR analysis to evaluatemRNA expression changes of IL4, IL10, IL6, and IGF-1 in CRISPR/Cas9–mediated Ahnak KO RAW264.7 cells after transfection of the 4CRU-Ahnak vector.Cellswere treatedwith 10 ng/mL LPS or 10 ng/mL IL4 for 48 hours. Similar resultswere obtained in three independent experiments. � ,P

analysis showed higher expression of phosphorylated Akt and Erkin MLE-12 cells cocultured with Ahnak�/� AMs than those cocul-turedwithWTAMs (Supplementary Fig. S5D). The treatmentwithrecombinant IGF-1 and EGF, which were highly produced inAhnak�/� AMs, enhanced the growth of lung cancer cells (Sup-plementary Fig. S5E) and upregulated core cell-cycle regulators(Supplementary Fig. S5F). Taken together, all these results suggestthat Ahnak�/� AMs enhance the proliferative activity of pneumo-cytes in the mice.

DiscussionIn this study, we propose that Ahnak functions as a tumor

suppressor in lungs. In particular, we showed that approximately20% of aged Ahnak�/� mice spontaneously developed lungtumors. Lung tumor development in the absence of Ahnak couldbe attributable to several underlyingmolecularmechanisms. First,downregulation of the TGFb signaling in the pneumocytes ofAhnak�/� lungs might promote lung epithelial proliferation. Itwas previously shown that activation of the TGFb pathwaypromotes cell-cycle arrest and apoptosis in early-stage tumors(31). In addition, cyclin D1 and p21, downstream target mole-cules of the TGFb signaling, are frequently altered in human lungadenocarcinomas, ultimately contributing to cell-cycle progres-sion in lung cancer (2). Consistent with these previous findings,Ahnak�/� lungs also showed the upregulation of cyclin D1.

Second, increased M2-like AMs in Ahnak�/� lungs might con-tribute to tumorigenesis, as macrophage depletion by liposomalclodronate attenuated proliferative activities in Ahnak�/� lungs.Tumor-associated macrophages polarized to the M2 phenotypeplay key roles in tumor progression in lung cancer (32). It was alsoreported that macrophage depletion by liposomal clodronateattenuates urethane-induced lung tumorigenesis during both thetumor development and progression stages in mice (19). Inaddition to this increased number of M2-like AMs, Ahnak�/�

AMs produced 2.5 times more IGF-1 than WT AMs. It is knownthat aberrant IGF-1 is associated with various types of cancers,including lung cancer (33), and AMs are one of the main pro-ducers of IGF-1 in pathogenic conditions, such as lung injury andcancer (34). Furthermore, AM-derived IGF-1 induced the prolif-eration of neoplastic murine lung epithelial cells (35). This issupported by our finding that activation of phospho-AKT andphospho-ERK, two downstream signaling molecules of IGF1 andEGF signaling pathways, is increased in Ahnak�/� lung cancers.

The increased number of M2-like AMs in Ahnak�/� lungs ispossibly the result of upregulated recruitment signals and/orproliferation of resident macrophages (36). Although detailedmechanism studies need to be performed in a follow-up study,we propose several possibilities based on our current data. First,Ahnak deficiency induced the polarization of macrophagesto anti-inflammatory M2 phenotypes via the activation of Aktsignaling (30). Second, the upregulation of MCP1 and IL4 in

Figure 7.

Reduced thickness of alveolar walls after macrophage depletion in Ahnak�/�mice. A–H, Representative H&E staining images of clodronate-treated Ahnak�/� lungs(A–C; 3 independent mice) and control liposome (E–G). Scale bar, 200 mm. Sixteen-week-old Ahnak�/� (n ¼ 3) and WT (n ¼ 3) mice were treated withclodronate or control liposome every 3 days for 3 weeks. D and H, There were no effects of clodronate treatment on WT lungs. I, IHC for F4/80 showing reducedmacrophages in Ahnak�/� lungs after clodronate treatment. F4/80-positive cells were counted and averaged in 400� high-power fields of lungs.J, Reduced thickness of alveolar walls in Ahnak�/� lungs after clodronate treatment. Alveolar wall thickness was measured by Case Viewer Software. K,Representative IHC images for Ki-67 in Ahnak�/� lungs after clodronate treatment. The right graph shows scoring results for Ki-67–positive cells per high-power field(200�). Scale bar, 100 mm. L, MTT assay for MLE-12 at 48 hours after coculture with WT or Ahnak�/� AMs. Left, schematic drawing of the coculture system.� , P < 0.05 by unpaired, two-tailed Student t test in I–L.

Park et al.





Ahnak�/� AMs might trigger the recruitment of macrophages toalveolar spaces (36). Third, Ahnak deficiency in other stromalcells, such asfibroblasts and endothelial cells,might create tumor-supportive microenvironments, in which M2-like AMs arerecruited and/or nourished. Further studies will be needed todetermine the exact functions of Ahnak gene in AM polarization,cytokine production, and AM recruitment.

Studies have suggested possible roles for Ahnak in mediatingvarious signaling events, such as the actin cytoskeleton net-work, PI3K/AKT and MAPK/ERK signaling pathways, DNAdamage signaling, cell contacts, and calcium channel regula-tion, which are involved in carcinogenesis (37, 38). Thus,Ahnak deficiency may promote the neoplastic and malignanttransformation of lung epithelial cells by targeting multiplepathways in addition to the Smad pathway. For example, thereare possibilities that Ahnak deficiency may affect Kras muta-tions and/or may provoke the activation of oncogenic signalingpathways, such as KRAS signaling in cell-intrinsic processes,because urethane-induced lung tumors frequently harbor acti-vating mutations in the KRAS oncogene (39). Thus, furtherstudies are necessary to explore the signaling pathways that aredisrupted in Ahnak-deficient lung epithelial cells. In addition toperturbations in cell-intrinsic processes, Ahnak might lead tothe disruption of several pathways pertaining to the interactionbetween the tissue microenvironments in various cell types.Indeed, we also found excessive connective tissue, includingcollagen deposits, in the lungs of Ahnak�/� mice. Although thiscould result from the stimulation of fibroblasts by the variousgrowth factors produced by increased M2-like AMs, it is alsopossible that Ahnak deficiency directly affected the fibroblasts'collagen production. To specify the role of Ahnak and clearlyelucidate which types of cells play important roles in lungtumor development in Ahnak�/� mice, further studies usingcell- or tissue-specific Ahnak knockout mice may be helpful. Inaddition, Ahnak�/� lungs possess features similar to lungs withhuman idiopathic pulmonary fibrosis (IPF), which is charac-terized by the progressive deposition within the interstitialspace of an extracellular matrix that includes collagen, as wellas the accumulation of M2 macrophages in the lung (40). Thus,we suggest that elucidating the mechanism of the involvementof Ahnak in the formation of lung lesions may be helpful forfinding potential therapeutic targets for the treatment of IPF.

In this study,wedemonstrate that Ahnakplays a critical role as anovel tumor suppressor in lung tumor development. Ahnakappears to suppress AT2 cell proliferation by activating the TGFbsignaling pathway. Ahnak also seems to inhibit the transition ofM1 toM2macrophage in lung environments, thereby suppressingthedevelopment of tumor-promotingmicroenvironments. Takentogether, we have identified Ahnak as a novel lung tumor sup-pressor in this study.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.W. Park, I.Y. Kim, H.J. Lim, J.K. SeongDevelopment of methodology: J.W. Park, I.Y. Kim, J.W. Choi, H.J. Lim,J.H. Shin, Y. Son, J.K. Woo, C. LeeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.W. Park, H.J. Lim, J.H. Shin, J.K. WooAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.W. Park, I.Y. Kim, J.W. Choi, H.J. LimWriting, review, and/or revision of the manuscript: J.W. Park, I.Y. Kim,J.W. Choi, J.H. Jeong, J.K. SeongAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.N. Kim, S.H. Lee, M. SohnStudy supervision: Y.S. Bae, J.K. Seong

AcknowledgmentsThis research was supported by research grants (2013M3A9D5072550;

Korea Mouse Phenotyping Project, 2012M3A9B6055344, 2012M3A9-D1054622 and 2013M3A9B6046417) from National Research Foundation(NRF) funded by the Ministry of Science and ICT, Korea and from HealthTechnology R&D Project through the Korea Health Industry DevelopmentInstitute (KHIDI) funded by the Ministry of Health & Welfare (grant no.HI13C2148; to J.K. Seong and I.Y. Kim). Also, it was partially supported bythe Brain Korea 21 Plus Program and the Research Institute for VeterinaryScience of Seoul National University.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 4, 2017; revised January 24, 2018; accepted April 19,2018; published first May 3, 2018.

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Park et al.




Published OnlineFirst May 3, 2018.Mol Cancer Res Jun Won Park, Il Yong Kim, Ji Won Choi, et al. Hyperplasia and Lung Tumor DevelopmentAHNAK Loss in Mice Promotes Type II Pneumocyte

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