AHNAK is known to be a tumor suppressor in breast cancer due to its ability to activate the TGFβ signaling pathway. However, the role of AHNAK in lung tumor development and progression remains unknown. Here, the Ahnak gene was disrupted to determine its effect on lung tumorigenesis and the mechanism by which it triggers lung tumor development was investigated. First, AHNAK protein expression was determined to be decreased in human lung adenocarcinomas compared with matched nonneoplastic lung tissues. Then, Ahnak−/− mice were used to investigate the role of AHNAK in pulmonary tumorigenesis. Ahnak−/− mice showed increased lung volume and thicker alveolar walls with type II pneumocyte hyperplasia. Most importantly, approximately 20% of aged Ahnak−/− mice developed lung tumors, and Ahnak−/− mice were more susceptible to urethane-induced pulmonary carcinogenesis than wild-type mice. Mechanistically, Ahnak deficiency promotes the cell growth of lung epithelial cells by suppressing the TGFβ signaling pathway. In addition, increased numbers of M2-like alveolar macrophages (AM) were observed in 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 lesions in Ahnak-null mice. Collectively, AHNAK suppresses type II pneumocyte proliferation and inhibits tumor-promoting M2 alternative activation of macrophages in mouse lung tissue. These results suggest that AHNAK functions as a novel tumor suppressor in lung cancer.

Implications: The tumor suppressor function of AHNAK, in murine lungs, occurs by suppressing alveolar epithelial cell proliferation and modulating lung microenvironment. Mol Cancer Res; 16(8); 1287–98. ©2018 AACR.

Lung cancer is the global leading cause of cancer-related mortality, and adenocarcinoma is the most common histologic type (1). A number of genes commonly altered in human lung adenocarcinomas have been identified (2), and their roles in lung tumorigenesis have been evaluated using genetically engineered mouse (GEM) models (3). In particular, GEM models expressing activating oncogenic mutants of the KRAS, EGFR, BRAF, and PIK3CA pathways developed lung tumors, and the combination of these mutants had synergistic effects on tumorigenesis and tumor progression (3). In addition, knocking out several other genes, including Myc, Rac1, NF-κB, or Gata2, in the GEM models above abrogated tumor development and progression, implying oncogenic roles as additional hits in lung tumorigenesis (3, 4). In parallel, ablation of several tumor suppressor genes, including Trp53, Rb, and Lkb1, in the GEM models accelerated tumor development and progression, thereby validating their roles as tumor suppressors in lung tumorigenesis (3).

Ahnak is an exceptionally large protein (700 kDa) that was initially identified from human neuroblastomas and skin epithelial cells (5, 6). Ahnak is known to be a scaffolding protein that regulates cytoskeletal structure formation, muscle regeneration, calcium homeostasis, and signaling (7, 8). Structurally, Ahnak is divided into three distinct regions: the amino-terminal region of 500 amino acids, the large central region of about 4,388 amino acids composed of 36 repeat units, and the carboxyl-terminal region of 1,003 amino acids (9). It has been suggested that the central repeat unit (CRU) supports the structural integrity and scaffolding activity of Ahnak (9–12). CRU plays the role of a molecular linker for calcium homeostasis by interacting with phospholipase C and protein kinase C (9, 10). In addition, CRU interacts with R-Smad proteins through MH2 domain and stimulates Smad3 nuclear translocation and markedly inhibits c-Myc promoter activity (11). In regards to cancer biology, the role of Ahnak in tumorigenesis is controversial. For example, Ahnak functions as a tumor suppressor in breast cancer by inhibiting cell growth via potentiation of the TGFβ signaling pathway (11). However, Ahnak is also associated with tumor development and progression (13–15).

In our previous studies, we reported that Ahnak−/− mice exhibit enhanced insulin sensitivity, higher energy expenditure, upregulated lipolysis of white adipose tissues, and impaired adipocyte differentiation (8, 16, 17). Intriguingly, we found pneumocyte hyperplasia 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 expression in human lung adenocarcinomas. Mechanistically, we showed that Ahnak deficiency downregulates TGFβ signaling in pneumocytes. In addition, we revealed that increased numbers of M2-like AMs in lungs of Ahnak−/− mice contribute to the pneumocyte hyperplasia and the formation of tumor microenvironment. Taken together, these findings suggest that Ahnak functions as a tumor suppressor in murine lungs by suppressing alveolar epithelial cell proliferation and modulating the lung microenvironment.

Mice models

Ahnak−/− mice were generated by disruption of exon 5 in the Ahnak gene, as reported previously (10). Genotyping was performed using genomic DNA isolated from tails according to methods described previously (10). Ahnak−/− mice were housed in a specific pathogen-free condition. To induce lung tumors in mice using urethane, we used a modified protocol derived from a previous report (18). 6-week-old mice were injected intraperitoneally once weekly for 8 weeks with 1 mg/g of urethane (Sigma) dissolved in 0.9% NaCl. The mice were sacrificed at 23 weeks after the initial urethane injection. Tumors were visually counted on the Tellyesniczky's fixative-cleared lungs by three blinded readers under a dissecting microscope. Tumor diameter was measured by micro-CT images using PET/CT scanner (eXplore Vista PET/CT Pre-Clinical, GE Healthcare). To deplete macrophages in lungs of mice, clodronate liposomes (F70101C-A, FormuMax Scientific) were used as described previously (19). Twenty-week-old mice were treated intranasally with clodronate liposomes or negative control liposomes every 3 days for 3 weeks at doses of 24 μL. All experiments were performed according to the “Guide for Animal Experiments” (Edited by Korean Academy of Medical Sciences) and approved by the Institutional Animal Care and Use Committee of Seoul National University (Seoul, Korea).

Cell cultures, transfection, and coculture

Human lung cancer cell lines (H460 and H23), mouse lung epithelial cell line MLE-12, and mouse macrophage-like cell line RAW264.7 were purchased from ATCC. All cell lines were cultured at 37°C in a 5% CO2 humidified incubator in the media according to ATCC recommendations. H460, H23 cells, and RAW264.7 cells were transfected with pcDNA-HA or pcDNA3-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 constitutively expressing vector (Addgene; for internal control) were transfected into cells to measure TGFβ activities. Reporter activities were evaluated via dual luciferase reporter assays (Promega) according to the manufacturer's instructions. To establish Ahnak KO RAW264.7 cells, we transfected RAW264.7 cells with Cas9 Ahnak CRISPR/Cas9 KO plasmid (sc-425992; Santa Cruz Biotechnology) and selected KO clones according to the manufacturer's protocol.

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

Histopathology, IHC, immunofluorescence, and immunoblotting

Mouse lung tissues were perfused and fixed in 4% paraformaldehyde overnight, processed in a routine manner, and embedded in paraffin. Hematoxylin and eosin stain (H&E), Masson's trichrome stain (SSK5005; BBC Biochemical), IHC, and immunofluorescence (IF) were performed on 4-μm thick serial sections from mouse tissue paraffin blocks. Light microscopic examinations were performed on H&E slides by pathologists to evaluate mouse lung lesions. Mouse lung tumors were classified according to classification of mouse lung tumors (22).

A tissue microarray (TMA) slide (LC1504; US Biomax Inc.) was applied to IHC for Ahnak. Dewaxed and rehydrated paraffin sections were subjected to antigen retrieval by heating the sections to 100°C for 20 minutes in 0.01 mol/L citrate buffer (pH 6.0) or EDTA unmasking solution (#14747, Cell Signaling Technology). To perform IHC staining, the ImmPRESS Peroxidase Polymer Kit (Vector Laboratories) was used for immunostaining according to the manufacturer's protocol. The slides were incubated with the following primary antibodies: goat anti-SP-C (sc-7726; Santa Cruz Biotechnology), rabbit anti-PDPN (sc-134483; Santa Cruz Biotechnology), rat anti-F4/80 (sc-59171; Santa Cruz Biotechnology), mouse anti-Ki-67 (ab8191; Abcam), rabbit anti-cyclin D1 (#2978; Cell Signaling Technology), mouse anti-PCNA (sc-56; Santa Cruz Biotechnology), rabbit anti-phosphorylated IGF-1R (#3918, Cell Signaling Technology), rabbit anti-phosphorylated EGFR (#4407, Cell Signaling Technology), rabbit anti-phosphorylated STAT3 (#9145, Cell Signaling Technology), rabbit anti-phosphorylated ERK (#4370, Cell Signaling Technology), rabbit anti-phosphorylated AKT (#9272, Cell Signaling Technology), goat anti–IGF-1 (AF791; R&D Systems), and mouse anti-Ahnak (ab68556; Abcam). The slides were subjected to colorimetric detection with ImmPact DAB substrate (SK-4105, Vector Laboratories). The slides were counterstained with Meyer's hematoxylin for 10 seconds. Negative controls were performed by omitting the primary antibody. To perform IF staining, after antigen unmasking and blocking with BSA, the slides were incubated overnight at 4°C with following primary antibodies: goat anti-SP-C (sc-7726; Santa Cruz Biotechnology), rabbit anti-PDPN (sc-134483; Santa Cruz Biotechnology), mouse anti-Ki-67 (ab8191; Abcam), and rabbit anti-phosphorylated Smad3 (ab52903; Abcam). Then, slides were incubated for 2 hours at room temperature with the following secondary antibodies; donkey anti-goat IgG (H+L), Alexa Fluor 568 (A11057; Thermo Fisher Scientific), donkey anti-mouse IgG (H+L), Alexa Fluor 488 (A21202; Thermo Fisher Scientific), and donkey anti-rabbit IgG (H+L), and Alexa Fluor 647 (A31573; Thermo Fisher Scientific). Slides were mounted with Vectashield mounting media (H-1200; Vector Laboratories). To perform scoring for H&E, IHC, and IF results, slides were scanned by Pannoramic SCAN slide scanner (3D HISTECH) and evaluated on Case Viewer Software (3D HISTECH) using a 40× objective for at least 5 spots per mouse with a minimum of 3 mice in each group.

To perform immunoblotting, harvested cells were lysed in lysed in RIPA buffer (GenDEPOT) with protease inhibitors (Xpert protease inhibitor cocktail solution, GenDEPOT). Total cell extracts were fractionated by electrophoresis on a gradient SDS polyacrylamide gel and transferred onto a PVDF membrane. The following 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, Santa Cruz Biotechnology), rabbit anti-AKT (#9272, Cell Signaling Technology), rabbit anti-ERK (#4695, Cell Signaling Technology), phosphorylated ERK (#4370, Cell Signaling Technology), rabbit anti-phosphorylated AKT (#9272, Cell Signaling Technology), mouse anti–α-tubulin (sc-8037, Santa Cruz Biotechnology), and rabbit anti–HA-Tag (#3724, Cell Signaling Technology). Immunodetection was performed by using an Enhanced Chemiluminescence Detection Kit (AbClon). Densitometry calculation was performed by ImageJ 1.49v software developed by the NIH (Bethesda, MD).

RNA extraction and qRT-PCR

Total RNA from both lung tissues and cell lines was extracted by TRIzol (Ambion) according to the manufacturer's instructions. First-strand cDNA was synthesized using the Acculower RT Premix (Bioneer) according to the manufacturer's instructions. PCR reactions were performed on 7500 Real Time PCR System (Applied Biosystems) using SensiFAST SYBR Green PCR Master Mix (BIO-94020; Bioline). ActB was used as the endogenous reference control for all transcripts. All qRT-PCR experiments were repeated at least three independent times. Primers used are as follows:

  • F: 5′-CTTCTGGGCCTGCTGTTCA-3′

  • R: 5′-CCAGCCTACTCATTGGGATCA-3′ for Mcp1,

  • F: 5′-AGCACAGAAAGCATGATCCG-3′

  • R: 5′-CTGATGAGAGGGAGGCCATT-3′ for Tnf,

  • F: 5′-GAGGATACCACTCCCAACAGACC-3′

  • R: 5′-AAGTGCATCATCGTTGTTCATACA-3′ for Il6,

  • F: 5′-AGACAGGCATTGTGGATGAG-3′

  • R: 5′-TGAGTCTTGGGCATGTCAGT-3′ for Igf1,

  • F: 5′-TGGCTGTGTCCTGACATCAG-3′

  • R: 5′-GAAGACAGATCTGGCTGCATC-3′ for Egf,

  • F: 5′-TGACGGCACAGAGCTATTGA-3′

  • R: 5′-TTCGTTGCTGTGAGGACGTT-3′ for Il4,

  • F: 5′-GCTCTTACTGACTGGCATGAG-3′

  • R: 5′-CGCAGCTCTAGGAGCATGTG-3′ for Il10,

  • F: 5′-GAGGTCTTTACGGATGTCAACG-3′

  • R: 5′-GGTCATCACTATTGGCAACGAG-3′ for Actb.

FACS analysis and cell sorting

To 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 IgG isotype controls for 1 hour at 4°C in the dark room. To detect Ahnak expression in cells, the cells were fixed with 4% paraformaldehyde and then permeabilized with ice-cold methanol. The cells were incubated with mouse anti-Ahnak (ab68556; Abcam) for 1 hour at 4°C in a dark room. The secondary antibody was donkey anti-mouse IgG (H+L), Alexa Fluor 488 (A21202; Thermo Fisher Scientific). The cells were analyzed and sorted by a SH800 cell sorter (Sony).

Statistical analysis

Statistical analysis was performed by GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Analyses were performed using a Student t test. P values of less than 0.05 were considered statistically significant. Results are presented as mean ± SEMs.

Ahnak protein expression is downregulated in human lung cancers

We performed IHC analysis using an antibody against Ahnak of a TMA slide containing 50 cases of human lung adenocarcinomas and matched normal lung tissues. In normal lung tissues, Ahnak protein expression was mainly observed in the membrane and/or cytoplasm of pneumocytes and AMs (Fig. 1A). However, Ahnak protein expression was significantly downregulated in lung cancer cells compared with normal lung tissues (Fig. 1A and B; Supplementary Fig. S1). According to previously published data (24), AHNAK mRNA levels were also significantly lower in all types of lung cancer tissues than those in normal lung tissues (Fig. 1C). In addition, analysis of the Cancer Cell Line Encyclopedia database revealed that lung cancer cell lines have relatively lower AHNAK mRNA expression compared with other cancer cell lines (Fig. 1D; ref. 25). AHNAK mRNA expression is highest in normal lung tissue among human tissues (15), and Ahnak mRNA was abundantly expressed in mouse lung tissue (26), suggesting physiologically important roles of Ahnak in lung. Collectively, these findings suggest that downregulation of Ahnak gene expression is associated with lung tumorigenesis.

Figure 1.

Downregulation of Ahnak in human lung adenocarcinomas. A, Representative images of downregulated Ahnak expression in human lung cancer tissues and matched normal lung tissues from a TMA slide containing 50 lung adenocarcinoma cases. Original magnification, ×40; inset, ×1,000. Scale bar, 50 μm. B, IHC scoring of Ahnak expression in human lung adenocarcinomas and matched normal lung tissues from the TMA slide. Ahnak expression was scored according to the intensity of staining: 0, negative staining; 1, weakly positive staining; 2, moderately positive staining; 3, strongly positive staining. C,AHNAK mRNA expression in lung cancer 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 Cancer Cell Line Encyclopedia (CCLE) database (GSE36139). *, P < 0.05 by unpaired, two-tailed Student t test in B.

Figure 1.

Downregulation of Ahnak in human lung adenocarcinomas. A, Representative images of downregulated Ahnak expression in human lung cancer tissues and matched normal lung tissues from a TMA slide containing 50 lung adenocarcinoma cases. Original magnification, ×40; inset, ×1,000. Scale bar, 50 μm. B, IHC scoring of Ahnak expression in human lung adenocarcinomas and matched normal lung tissues from the TMA slide. Ahnak expression was scored according to the intensity of staining: 0, negative staining; 1, weakly positive staining; 2, moderately positive staining; 3, strongly positive staining. C,AHNAK mRNA expression in lung cancer 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 Cancer Cell Line Encyclopedia (CCLE) database (GSE36139). *, P < 0.05 by unpaired, two-tailed Student t test in B.

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Ahnak−/− mice show high proliferative activity in alveolar type II pneumocytes

At 6, 10, 14, and 18 weeks of age, Ahnak−/− lungs showed increased size and weight than age-matched wild-type (WT) lungs (Fig. 2A; Supplementary Fig. S2). Histologically, the alveolar septa of Ahnak−/− lungs were thickened, which was accompanied by dilated alveolar space (Fig. 2B and C). Ahank−/− lungs at embryonic day 18.5 also showed reduced airspace with denser cellularity than WT lungs (Supplementary Fig. S3), suggesting that Ahnak deficiency might affect lung in developmental stages. The thickened walls showed high cellularity and excessive connective tissues including collagen deposition (Fig. 2C; Supplementary Fig. S4). Importantly, IHC analysis revealed that SP-C–positive alveolar type II pneumocytes (AT2) were significantly increased in Ahnak−/− lungs in comparison with WT lungs (Fig. 2D), whereas PDPN-positive alveolar type I pneumocytes (AT1) lining alveolar spaces were significantly decreased and formed a discontinuous pattern (Fig. 2D). Western blot analysis confirmed an increased expression of SP-C proteins and reduced expression of PDPN proteins in Ahnak−/− whole lung tissues (Fig. 2E). Notably, we did not detect an increase in infiltrated CD45 (a leukocyte common antigen marker)–positive cells and CD3 (a T-cell marker)–positive cells in Ahnak−/− lungs compared with WT lungs (Supplementary Fig. S4). Furthermore, IHC analysis of both Ki-67 and PCNA, markers of cell proliferation, showed increased positive cells in Ahnak−/− lungs compared with WT lungs (Fig. 3A). Consistent with previous results showing that introduction of CRU of Ahnak results in cell-cycle arrest through the downregulation of cyclin D (11), cyclin D1 was upregulated in Ahnak−/− lungs (Fig. 3A). These findings were confirmed by Western blot analysis 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 lung hyperplasia (Fig. 3C and D). Taken together, lung lesions in Ahnak−/− mice are characterized by hyperplastic AT2 cells due to their increased cell proliferation.

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 compared with WT mice. The scoring for air spaces (mm2) and thickness were performed using Case Viewer Software. n = 3 Ahnak−/− mice, n = 3 WT mice at each time point. 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 μm. D, There was an increased number of SP-C–positive AT2 cells per HPF (high-power field, 200×) in 10-week-old Ahnak−/− lungs. PDPN-positive AT1 cells incompletely lined alveolar walls in Ahnak−/− lungs according to H-score system [(1 × (% weakly positive cells) + 2 × (% moderately positive cells) + 3 × (% strongly positive cells)]. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. Scale bar, 50 μm. E, Western blot analysis using whole lung tissues confirmed the increased SP-C and decreased PDPN expression in Ahnak−/− lungs compared with WT lungs. *, P < 0.05 by unpaired, two-tailed Student t test in D.

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 compared with WT mice. The scoring for air spaces (mm2) and thickness were performed using Case Viewer Software. n = 3 Ahnak−/− mice, n = 3 WT mice at each time point. 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 μm. D, There was an increased number of SP-C–positive AT2 cells per HPF (high-power field, 200×) in 10-week-old Ahnak−/− lungs. PDPN-positive AT1 cells incompletely lined alveolar walls in Ahnak−/− lungs according to H-score system [(1 × (% weakly positive cells) + 2 × (% moderately positive cells) + 3 × (% strongly positive cells)]. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. Scale bar, 50 μm. E, Western blot analysis using whole lung tissues confirmed the increased SP-C and decreased PDPN expression in Ahnak−/− lungs compared with WT lungs. *, P < 0.05 by unpaired, two-tailed Student t test in D.

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Figure 3.

Proliferation of AT2 cells in Ahnak−/− lungs. A, IHC analysis for cell-cycle regulators such as Ki-67, PCNA, and cyclin D1 in WT and Ahnak−/− lungs. Right graphs show the 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×). n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. Scale bar, 100 μm. B, Western blot analysis for PCNA and cyclin D1 in Ahnak−/− and WT whole lung tissues at 6, 10, 14, and 18 weeks of age. C and D, IF analysis for SP-C and PDPN pneumocyte markers and a proliferation marker Ki-67 in Ahnak−/− lungs at 10 weeks of age. C, Representative images of the IF staining. SP-C+/Ki-67+ cells, arrow. PDPN+/Ki-67+ cells, arrowhead. SP-C+/PDPN+/PCNA+ cells, asterisk. Scale bar, 40 μm. D, Proportion of SP-C–positive and/or PDPN-positive cells in Ki-67–positive cells. SP-C–positive AT2 cells accounted for more than half of Ki-67–positive cells. Similar results were obtained from 3 mice. *, P < 0.05 by unpaired, two-tailed Student t test in A.

Figure 3.

Proliferation of AT2 cells in Ahnak−/− lungs. A, IHC analysis for cell-cycle regulators such as Ki-67, PCNA, and cyclin D1 in WT and Ahnak−/− lungs. Right graphs show the 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×). n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. Scale bar, 100 μm. B, Western blot analysis for PCNA and cyclin D1 in Ahnak−/− and WT whole lung tissues at 6, 10, 14, and 18 weeks of age. C and D, IF analysis for SP-C and PDPN pneumocyte markers and a proliferation marker Ki-67 in Ahnak−/− lungs at 10 weeks of age. C, Representative images of the IF staining. SP-C+/Ki-67+ cells, arrow. PDPN+/Ki-67+ cells, arrowhead. SP-C+/PDPN+/PCNA+ cells, asterisk. Scale bar, 40 μm. D, Proportion of SP-C–positive and/or PDPN-positive cells in Ki-67–positive cells. SP-C–positive AT2 cells accounted for more than half of Ki-67–positive cells. Similar results were obtained from 3 mice. *, P < 0.05 by unpaired, two-tailed Student t test in A.

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Ahnak−/− pneumocytes show downregulation of the TGFβ signaling pathway

It is known that Ahnak inhibits cell growth by potentiating TGFβ-induced transcriptional activity via its direct interaction with Smad3 (11). We found that the nuclear expression of phosphorylated Smad3 (S423/S425) in SP-C–positive AT2 cells was significantly reduced in Ahnak−/− lungs compared with WT lungs (Fig. 4A and B). We confirmed that phosphorylation of Smad3 is reduced in Ahnak−/− whole lung tissues using Western blot analysis (Fig. 4C). Because the CRU of Ahnak is the scaffolding motif responsible for proper modulation of its signaling pathways (11), we overexpressed four CRUs (4CRUs) in human lung cancer H460 and H23 cell lines (Fig. 4D). These showed increased TGFβ reporter activity and decreased cell growth (Fig. 4E and F). Taken together, these results suggest that Ahnak inhibits the cell growth of lung epithelial cells by activating the TGFβ signaling pathway.

Figure 4.

Downregulation of the TGFβ signaling in Ahnak−/− lungs. A, IF analysis for SP-C, phosphorylated Smad3 (phosphorylation sites, S423+S425), and Ki-67 in lung tissues from 10-week-old Ahnak−/− and WT mice. Arrows indicate SP-C–positive AT2 cells showing nuclear expression of phosphorylated Smad3. Scale bar, 50 μm. B, Scoring of presence of nuclear phosphorylated Smad3 in SP-C–positive AT2 cells based on IF staining. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. 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- and 18-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 TGFβ signaling activation. After 3 days of transfection with HA-4CRU-Ahnak, luciferase reporter activities were measured. Cells were treated with 5 ng/mL TGFβ for 6 hours. Relative light units (RLU) is a ratio of firefly luciferase units normalized to Renilla luciferase units. F, A hemocytometer-based trypan blue dye exclusion assay was conducted to quantify cells and measure viability. Growth inhibition was observed after transfection with H4-4CRU. *, P < 0.05 by unpaired, two-tailed Student t test in B, E, and F. The data are means ± SEM of three independent experiments.

Figure 4.

Downregulation of the TGFβ signaling in Ahnak−/− lungs. A, IF analysis for SP-C, phosphorylated Smad3 (phosphorylation sites, S423+S425), and Ki-67 in lung tissues from 10-week-old Ahnak−/− and WT mice. Arrows indicate SP-C–positive AT2 cells showing nuclear expression of phosphorylated Smad3. Scale bar, 50 μm. B, Scoring of presence of nuclear phosphorylated Smad3 in SP-C–positive AT2 cells based on IF staining. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. 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- and 18-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 TGFβ signaling activation. After 3 days of transfection with HA-4CRU-Ahnak, luciferase reporter activities were measured. Cells were treated with 5 ng/mL TGFβ for 6 hours. Relative light units (RLU) is a ratio of firefly luciferase units normalized to Renilla luciferase units. F, A hemocytometer-based trypan blue dye exclusion assay was conducted to quantify cells and measure viability. Growth inhibition was observed after transfection with H4-4CRU. *, P < 0.05 by unpaired, two-tailed Student t test in B, E, and F. The data are means ± SEM of three independent experiments.

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Ahnak−/− mice spontaneously develop lung tumors and show high susceptibility to urethane-induced lung carcinogenesis

Notably, approximately 20% of aged Ahnak−/− mice (2 out of 9 one-year-old Ahnak−/− mice) developed lung tumors. Grossly, these tumors appear as a solitary gray or white nodule that slightly protrudes from the lung surface (Fig. 5A). Histologically, one lung tumor was adenoma and the other was adenocarcinoma. The adenoma was unencapsulated but well-demarcated with its dense cellular neoplasm, which was composed of well-to-moderately differentiated polygonal cells in a tubulopapillary pattern (Fig. 5B). The adenocarcinoma had no clear margin and showed solid growth pattern, which was composed of moderately-to-poorly differentiated neoplastic cells (Fig. 5B). In addition, there were a large number of infiltrated foamy macrophages, rare T cells (CD3+ cells) and neutrophils (polymorphonuclear cells). Co-IF staining revealed that most of tumor cells were SP-C positive (Fig. 5C), raising the possibility that the origin of tumor cells might be AT2 cells. Tumor cells from all the cases were E-cadherin (an epithelial cell marker)-positive and showed higher proliferative activity compared with normal lung tissue evidenced by IHC for Ki-67 and cyclin D1 (Fig. 5D). Ki-67 and cyclin D1 immunoreactivities were stronger in the adenocarcinoma than in the adenoma (Fig. 5D), indicating higher proliferative activity in adenocarcinoma.

Figure 5.

Development of spontaneous lung tumors and higher susceptibility to carcinogen-induced pulmonary carcinogenesis in Ahnak−/− mice. A, A representative gross picture 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−/− mice bearing a pulmonary adenoma (#1) and an adenocarcinoma (#2) based on H&E staining. Original magnification, ×40. Boxed areas are magnified in inset. Scale bar, 50 μm. C, Co-IF analysis for SP-C and PDPN. The dotted line indicates tumor margin. Scale bar, 200 μm. Right, boxed area is magnified. Scale bar, 50 μm. 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 μm. E, Representative photographs of urethane-induced lung tumors in WT and Ahnak−/− mice. Scale bar, 5 mm. F, Increased numbers and diameters 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 by unpaired, two-tailed Student t test in F.

Figure 5.

Development of spontaneous lung tumors and higher susceptibility to carcinogen-induced pulmonary carcinogenesis in Ahnak−/− mice. A, A representative gross picture 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−/− mice bearing a pulmonary adenoma (#1) and an adenocarcinoma (#2) based on H&E staining. Original magnification, ×40. Boxed areas are magnified in inset. Scale bar, 50 μm. C, Co-IF analysis for SP-C and PDPN. The dotted line indicates tumor margin. Scale bar, 200 μm. Right, boxed area is magnified. Scale bar, 50 μm. 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 μm. E, Representative photographs of urethane-induced lung tumors in WT and Ahnak−/− mice. Scale bar, 5 mm. F, Increased numbers and diameters 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 by unpaired, two-tailed Student t test in F.

Close modal

As this low number of tumor-bearing mice was insufficient to achieve statistical significance, we utilized the urethane-induced lung carcinogenesis model to evaluate the effect of Ahnak gene deficiency on lung tumorigenesis (18). With this model, we determined that both WT and Ahnak−/− mice formed pulmonary adenomas. We did not find any histopathologic differences, including cell differentiation and invasiveness, between tumor cells arising from wild and from Ahnak−/− lungs. However, the number and size of the urethane-induced lung tumors in the Ahnak−/− mice were significantly greater than those in those in the WT mice (Fig. 5E–G). Moreover, higher numbers of macrophages were prominent both inside and surrounding the tumor tissues in Ahnak−/− mice compared with those of the WT mice. Overall, these data suggest that Ahnak gene deficiency promotes spontaneous lung tumorigenesis and increases susceptibility to carcinogen-induced lung carcinogenesis.

Ahnak gene deficiency leads to increased numbers of M2-like alveolar macrophages in Ahnak−/− lungs

IHC analysis of F4/80, a 160-kDa glycoprotein expressed by murine macrophages, revealed significantly increased numbers of AMs in Ahnak−/− lungs compared with WT lungs (Fig. 6A). We also confirmed increased numbers of AMs in bronchoalveolar lavage fluid (BALF) of Ahnak−/− lungs (Fig. 6B). In general, macrophages are classified into two subtypes, classically activated M1 macrophages and alternatively activated M2 macrophages, based on their cytokine expression patterns (for M1, TNFα, IL12, IFNs, etc. and for M2, IL10, IL4, IL13, etc.) and their immune functions (for M1, proinflammatory and for M2, anti-inflammatory; ref. 27). Therefore, to delineate the identity of the enhanced population of AMs in Ahnak−/− lungs, we performed flow cytometry (FACS) analysis. Interestingly, Ahnak−/− AMs exhibited higher expression of CD206, a well-known M2 marker, than WT AMs (Fig. 6C). In addition, the mRNA expression level of proinflammatory cytokine TNF was significantly lower in Ahnak−/− AMs than in WT AMs, whereas anti-inflammatory cytokines such as IL4 and IL10 were significantly higher (Fig. 6D). Notably, Ahnak−/− AMs also produced more growth factors such as insulin-like growth factor 1 (IGF-1; Fig. 6D; Supplementary Fig. S5C) and EGF (Fig. 6D) than WT AMs. Activation of the IGF and EGF signaling pathways in Ahnak−/− lungs was also confirmed by IHC (Supplementary Fig. S5A and S5B). The cytokine expression profile of Ahnak−/− whole lung tissues versus WT lung tissues reflected that of Ahnak−/− AMs (Fig. 6E). Collectively, our data revealed that M2-like AMs producing growth factors were accumulated in Ahnak−/− lung.

Figure 6.

Increased numbers of M2-like alveolar macrophages in Ahnak−/− lungs. A, Representative IHC images of F4/80 in WT, Ahnak−/−, and tumor-bearing lungs. Scale bar, 100 μm. The numbers of F4/80-positive AM in Ahnak−/− and WT lungs at the indicated time points. F4/80-positive cells were counted and averaged in 400× high-power fields (HPF) of lungs. n = 3 Ahnak−/− mice, n = 3 WT mice at each time point. B, Increased AMs in bronchi alveolar lavage fluid (BALF) in Ahnak−/− lungs versus WT lungs. F4/80-positive macrophages from BALF were counted by flow cytometry. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. C, FACS analysis for CD206, a M2 macrophage marker, in Ahnak−/− and WT AMs of 10-week-old mice. Single-cell suspensions were obtained from whole lung tissues after dispase digestion. Macrophages were defined as both CD45 and F4/80-positive cells. D, qRT-PCR analysis for mRNA expressions of cytokines, chemokines, and growth factors in Ahnak−/− AMs from 14-week-old mice. E, qRT-PCR analysis for mRNA expressions of these factors in Ahnak−/− and WT whole lung tissues 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 cells from peritoneal spaces were enriched for peritoneal macrophages by selecting F4/80-positive cell populations via FACS sorting. G, qRT-PCR analysis to evaluate mRNA 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. Cells were treated with 10 ng/mL LPS or 10 ng/mL IL4 for 48 hours. Similar results were obtained in three independent experiments. *, P < 0.05 by unpaired, two-tailed Student t test in A, B, D, E, F, and G. H, Western blot analysis for phosphorylated Akt expression in LPS or IL4-treated Ahnak KO RAW264.7 cells after the restoration of 4CRU-Ahnak. Bottom, quantification of Western blots was performed using ImageJ software.

Figure 6.

Increased numbers of M2-like alveolar macrophages in Ahnak−/− lungs. A, Representative IHC images of F4/80 in WT, Ahnak−/−, and tumor-bearing lungs. Scale bar, 100 μm. The numbers of F4/80-positive AM in Ahnak−/− and WT lungs at the indicated time points. F4/80-positive cells were counted and averaged in 400× high-power fields (HPF) of lungs. n = 3 Ahnak−/− mice, n = 3 WT mice at each time point. B, Increased AMs in bronchi alveolar lavage fluid (BALF) in Ahnak−/− lungs versus WT lungs. F4/80-positive macrophages from BALF were counted by flow cytometry. n = 3 Ahnak−/− mice, n = 3 WT mice at 10 weeks of age. C, FACS analysis for CD206, a M2 macrophage marker, in Ahnak−/− and WT AMs of 10-week-old mice. Single-cell suspensions were obtained from whole lung tissues after dispase digestion. Macrophages were defined as both CD45 and F4/80-positive cells. D, qRT-PCR analysis for mRNA expressions of cytokines, chemokines, and growth factors in Ahnak−/− AMs from 14-week-old mice. E, qRT-PCR analysis for mRNA expressions of these factors in Ahnak−/− and WT whole lung tissues 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 cells from peritoneal spaces were enriched for peritoneal macrophages by selecting F4/80-positive cell populations via FACS sorting. G, qRT-PCR analysis to evaluate mRNA 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. Cells were treated with 10 ng/mL LPS or 10 ng/mL IL4 for 48 hours. Similar results were obtained in three independent experiments. *, P < 0.05 by unpaired, two-tailed Student t test in A, B, D, E, F, and G. H, Western blot analysis for phosphorylated Akt expression in LPS or IL4-treated Ahnak KO RAW264.7 cells after the restoration of 4CRU-Ahnak. Bottom, quantification of Western blots was performed using ImageJ software.

Close modal

To investigate roles of Ahnak in cytokine production during macrophage polarization, we induced macrophages to differentiate either into M1 in response to LPS or M2 following IL4 treatment. In response to LPS treatment, Ahnak−/− peritoneal macrophages exhibited reduced levels of TNF and increased levels of MCP1 and IL6 compared with those of WT peritoneal macrophages (Fig. 6F). Although IL6 is known for a proinflammatory cytokine, roles of IL6 in macrophage polarization were context dependent and IL6-promoting M2 programming has been reported (28, 29). In parallel, we overexpressed 4CRUs in CRISPR/Cas9–mediated Ahnak knockout (KO) RAW264.7 cells. IL4, IL10, IL6, and IGF-1 expressions by LPS or IL4 treatment were suppressed in restored cells (Fig. 6G). Phosphorylated Akt was reduced in LPS or IL4-treated Ahnak KO RAW264.7 cells after the restoration of 4CRU-Ahnak (Fig. 6H). Akt signaling is implicated in macrophage activation, and it has been suggested that the activation of Akt promotes M2 polarization, whereas loss of Akt1 augments M1 activation (30). Taken together, these results suggest that Ahnak gene deficiency induces the transition of AM cytokine profiles in favor of M2-like macrophage programming and monocyte recruitment.

Ahnak−/− AMs confer enhanced lung pneumocyte proliferation

To assess whether cytokines and/or growth factors released from a larger AM population in Ahnak−/− lungs contribute to pneumocyte cell proliferation, we depleted AMs in Ahnak−/− lungs by intranasal administration of clodronate liposome (Fig. 7A–I). Clodronate liposome treatment for 3 weeks attenuated the thickness of alveolar septa in Ahnak−/− lungs (Fig. 7A–C and 7J) compared with that of control liposome-treated Ahnak−/− lungs (Fig. 7E–G and 7J). Consistent with this observation, Ki-67 staining was decreased in Ahnak−/− lungs after the depletion of AMs, indicative of decreased proliferative activity (Fig. 7K). To recapitulate this in an in vitro system, MLE-12 cells, an immortalized mouse lung epithelial cell line, were cocultured with isolated 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 factors secreted from Ahnak−/− AMs affected the cell growth. Ras–Raf–ERK and PI3K–Akt pathways play a central role in driving many of the phenotypic changes induced by growth factors. Western blot analysis showed higher expression of phosphorylated Akt and Erk in MLE-12 cells cocultured with Ahnak−/− AMs than those cocultured with WT AMs (Supplementary Fig. S5D). The treatment with recombinant IGF-1 and EGF, which were highly produced in Ahnak−/− AMs, enhanced the growth of lung cancer cells (Supplementary Fig. S5E) and upregulated core cell-cycle regulators (Supplementary Fig. S5F). Taken together, all these results suggest that Ahnak−/− AMs enhance the proliferative activity of pneumocytes in the mice.

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 μm. Sixteen-week-old Ahnak−/− (n = 3) and WT (n = 3) mice were treated with clodronate 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 reduced macrophages 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 μm. 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.

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 μm. Sixteen-week-old Ahnak−/− (n = 3) and WT (n = 3) mice were treated with clodronate 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 reduced macrophages 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 μm. 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.

Close modal

In this study, we propose that Ahnak functions as a tumor suppressor in lungs. In particular, we showed that approximately 20% of aged Ahnak−/− mice spontaneously developed lung tumors. Lung tumor development in the absence of Ahnak could be attributable to several underlying molecular mechanisms. First, downregulation of the TGFβ signaling in the pneumocytes of Ahnak−/− lungs might promote lung epithelial proliferation. It was previously shown that activation of the TGFβ pathway promotes cell-cycle arrest and apoptosis in early-stage tumors (31). In addition, cyclin D1 and p21, downstream target molecules of the TGFβ signaling, are frequently altered in human lung adenocarcinomas, ultimately contributing to cell-cycle progression 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 contribute to tumorigenesis, as macrophage depletion by liposomal clodronate attenuated proliferative activities in Ahnak−/− lungs. Tumor-associated macrophages polarized to the M2 phenotype play key roles in tumor progression in lung cancer (32). It was also reported that macrophage depletion by liposomal clodronate attenuates urethane-induced lung tumorigenesis during both the tumor development and progression stages in mice (19). In addition to this increased number of M2-like AMs, Ahnak−/− AMs produced 2.5 times more IGF-1 than WT AMs. It is known that aberrant IGF-1 is associated with various types of cancers, including lung cancer (33), and AMs are one of the main producers of IGF-1 in pathogenic conditions, such as lung injury and cancer (34). Furthermore, AM-derived IGF-1 induced the proliferation of neoplastic murine lung epithelial cells (35). This is supported by our finding that activation of phospho-AKT and phospho-ERK, two downstream signaling molecules of IGF1 and EGF signaling pathways, is increased in Ahnak−/− lung cancers.

The increased number of M2-like AMs in Ahnak−/− lungs is possibly the result of upregulated recruitment signals and/or proliferation of resident macrophages (36). Although detailed mechanism 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 macrophages to anti-inflammatory M2 phenotypes via the activation of Akt signaling (30). Second, the upregulation of MCP1 and IL4 in Ahnak−/− AMs might trigger the recruitment of macrophages to alveolar spaces (36). Third, Ahnak deficiency in other stromal cells, such as fibroblasts and endothelial cells, might create tumor-supportive microenvironments, in which M2-like AMs are recruited and/or nourished. Further studies will be needed to determine the exact functions of Ahnak gene in AM polarization, cytokine production, and AM recruitment.

Studies have suggested possible roles for Ahnak in mediating various signaling events, such as the actin cytoskeleton network, PI3K/AKT and MAPK/ERK signaling pathways, DNA damage signaling, cell contacts, and calcium channel regulation, which are involved in carcinogenesis (37, 38). Thus, Ahnak deficiency may promote the neoplastic and malignant transformation of lung epithelial cells by targeting multiple pathways in addition to the Smad pathway. For example, there are possibilities that Ahnak deficiency may affect Kras mutations and/or may provoke the activation of oncogenic signaling pathways, such as KRAS signaling in cell-intrinsic processes, because urethane-induced lung tumors frequently harbor activating mutations in the KRAS oncogene (39). Thus, further studies are necessary to explore the signaling pathways that are disrupted in Ahnak-deficient lung epithelial cells. In addition to perturbations in cell-intrinsic processes, Ahnak might lead to the disruption of several pathways pertaining to the interaction between the tissue microenvironments in various cell types. Indeed, we also found excessive connective tissue, including collagen deposits, in the lungs of Ahnak−/− mice. Although this could result from the stimulation of fibroblasts by the various growth factors produced by increased M2-like AMs, it is also possible that Ahnak deficiency directly affected the fibroblasts' collagen production. To specify the role of Ahnak and clearly elucidate which types of cells play important roles in lung tumor development in Ahnak−/− mice, further studies using cell- or tissue-specific Ahnak knockout mice may be helpful. In addition, Ahnak−/− lungs possess features similar to lungs with human idiopathic pulmonary fibrosis (IPF), which is characterized by the progressive deposition within the interstitial space of an extracellular matrix that includes collagen, as well as the accumulation of M2 macrophages in the lung (40). Thus, we suggest that elucidating the mechanism of the involvement of Ahnak in the formation of lung lesions may be helpful for finding potential therapeutic targets for the treatment of IPF.

In this study, we demonstrate that Ahnak plays a critical role as a novel tumor suppressor in lung tumor development. Ahnak appears to suppress AT2 cell proliferation by activating the TGFβ signaling pathway. Ahnak also seems to inhibit the transition of M1 to M2 macrophage in lung environments, thereby suppressing the development of tumor-promoting microenvironments. Taken together, we have identified Ahnak as a novel lung tumor suppressor in this study.

No potential conflicts of interest were disclosed.

Conception and design: J.W. Park, I.Y. Kim, H.J. Lim, J.K. Seong

Development of methodology: J.W. Park, I.Y. Kim, J.W. Choi, H.J. Lim, J.H. Shin, Y. Son, J.K. Woo, C. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.W. Park, H.J. Lim, J.H. Shin, J.K. Woo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.W. Park, I.Y. Kim, J.W. Choi, H.J. Lim

Writing, review, and/or revision of the manuscript: J.W. Park, I.Y. Kim, J.W. Choi, J.H. Jeong, J.K. Seong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.N. Kim, S.H. Lee, M. Sohn

Study supervision: Y.S. Bae, J.K. Seong

This research was supported by research grants (2013M3A9D5072550; Korea Mouse Phenotyping Project, 2012M3A9B6055344, 2012M3A9D1054622 and 2013M3A9B6046417) from National Research Foundation (NRF) funded by the Ministry of Science and ICT, Korea and from Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (grant no. HI13C2148; to J.K. Seong and I.Y. Kim). Also, it was partially supported by the Brain Korea 21 Plus Program and the Research Institute for Veterinary Science of Seoul National University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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