AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia … · 1 AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia and Lung Tumor Development. Running title: Ahnak Gene

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AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia and Lung Tumor

Development

Running title: Ahnak Gene Deficiency Promotes Mouse Lung Tumors

Jun Won Park1,2*

, Il Yong Kim1,2*

, Ji Won Choi 1, Hee Jung Lim

2, Jae Hoon Shin

1, Yo Na Kim

1, Seo

Hyun Lee1, Yeri Son

1, Mira Sohn

3, Jong Kyu Woo

1,2, Joseph H. Jeong

2, Cheolju Lee

4, Yun Soo Bae

3,

Je Kyung Seong1,2,5

*Jun Won Park and Il Yong Kim are equally contributed to this work.

1 Laboratory of Developmental Biology and Genomics, BK21 Program Plus for Advanced Veterinary

Science, and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul

National University, Seoul, Korea,

2 Korea Mouse Phenotyping Center (KMPC), Seoul, Korea

3Department of Life Sciences, Ewha Womans University, Seoul, Korea

4Center for Theragnosis, Korea Institute of Science and Technology, Seoul, Korea.

5Interdisciplinary Program for Bioinformatics, Program for Cancer Biology and BIO-MAX/N-Bio

Institute, Seoul National University, Seoul, Korea

* Corresponding author

Je Kyung Seong, DVM, PhD, KCLAM

Professor, Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine and

Interdisciplinary Program for Bioinformatics and Program for Cancer Biology, Seoul National

University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea

Director, Korea Mouse Phenotyping Center (KMPC)

Tel: +82-02-885-8395, Fax: +82-02-885-8397, E-mail: [email protected]

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed by authors.

Grant Supports

on April 14, 2020. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 3, 2018; DOI: 10.1158/1541-7786.MCR-17-0726

http://mcr.aacrjournals.org/

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This research was supported by the 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 Je Kyung Seong and Il Yong

Kim. Also it was partially supported by the Brain Korea 21 Plus Program and the Research Institute

for Veterinary Science of Seoul National University.

E-mail

Jun Won Park, DVM, PhD; [email protected]

Il Yong Kim, PhD; [email protected]

Ji Won Choi, MS; [email protected]

Hee Jung Lim, PhD; [email protected]

Jae Hoon Shin, PhD; [email protected]

Yo Na Kim, PhD; [email protected]

Seo Hyun Lee, MS; [email protected]

Yeri Son, PhD candidate; [email protected]

Mira Sohn, PhD; [email protected]

Jong Kyu Woo, PhD; [email protected]

Joseph H. Jeong, PhD; [email protected]

Cheolju Lee, PhD; [email protected].

Yun Soo Bae, PhD; [email protected]

Je Kyung Seong, DVM, PhD; [email protected]




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Abstract

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 non-neoplastic 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 (AMs) 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.




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Introduction

Lung cancer is the global leading cause of cancer-related mortality, and adenocarcinoma is the most

common histological 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, NFkappaB, 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 (700kDa) 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 4388 amino acids composed of 36 repeat units, and the

carboxyl-terminal region of 1003 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 TGF-β 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 down-regulation of Ahnak protein expression in human lung adenocarcinomas. Mechanistically,

we showed that Ahnak deficiency down-regulates TGF-β 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.




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Materials and Methods

Mice models

Ahnak-/- mice were generated by disruption of exon 5 in the Ahnak gene, as previously reported (10).

Genotyping was performed using genomic DNA isolated from tails according to methods previously

described (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). Six

week-old mice were injected intraperitoneally once weekly for 8 weeks with 1 mg/g of urethane

(Sigma, St Louis, MO) 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, Sunnyvale, CA, USA) were

used as previously described (19). 20 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 (IACUC) of

Seoul National University.

Cell cultures, transfection, and co-culture

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 American Type Culture Collection (ATCC,

Manassas, VA, USA). 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) (11) using TransIT-

X2® Dynamic Delivery System (Mirus Bio LLC, Madison, WI, USA) according to manufacturer’s

instruction. pSBE-luc reporter plasmid (11) and Renilla luciferase-constitutively expressing vector

(Addgene; for internal control) were transfected into cells to measure TGF-β activities. Reporter

activities were evaluated via dual luciferase reporter assays (Promega, Madison, WI) 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 were isolated from mice according to previously described

methods (20,21). Briefly, after killing by CO2, mice were injected intraperitoneally with 10ml cold PBS

and the peritoneal fluid was withdrawn by syringe suction. Alveolar macrophages 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 alveolar macrophages from more than 5 mice were collected and

pooled together. Cell sorting by flow cytometry (SH800 cell sorter (Sony, Tokyo, Japan) using rat anti-




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mouse F4/80 (25-48-1; eBioscience, San Diego, CA, USA; PE-Cyanine7) was performed to enrich

macrophages from the peritoneal and bronchoalveolar suspension cells. After cell numbers were

determined by trypan-blue exclusion, 2x105 macrophages were seeded on 6-well cell culture plate in

DMEM (Welgene, Daegu, Korea) containing 2% FBS (Welgene, Daegu, Korea). After 3 hours of

seeding, cells were harvested for mRNA isolation. For co-culture experiments, MLE-12 cells, seeded

on six-well Trans-well inserts (Corning, Cambridge, MA) at 2×105

cells, were added to the 6-well

plates on which alveolar macrophages were seeded. The cell growth of MLE-12 cells was determined

using MTT assays after 48 hours of co-culture.

Histopathology, immunohistochemistry, immunofluorescence, and immunobotting

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, MountVernon, WA), immunohistochemistry (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, MD, USA) 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 M citrate buffer (pH 6.0) or EDTA unmasking solution (#14747, Cell

Signaling Technology, Beverly, MA). To perform IHC staining, the ImmPRESS Peroxidase Polymer kit

(Vector Laboratories, Burlingame, CA, USA) 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, CA, USA), rabbit anti-PDPN (sc-134483; Santa Cruz

Biotechnology), rat anti-F4/80 (sc-59171; Santa Cruz Biotechnology), mouse anti-Ki-67 (ab8191;

Abcam, Cambridge, MA, USA), rabbit anti-cyclinD1 (#2978; Cell Signaling Technology), mouse anti-

PCNA (sc-56; Santa Cruz Biotechnology), rabbit anti-phosphorylated IGF1R (#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-IGF1 (AF791; R&D Systems, Minneapolis, Minnesota, USA) 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 s.

Negative controls were performed by omitting the primary antibody. To perform IF staining, after

antigen unmasking and blocking with bovine serum albumin (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;




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Abcam), and rabbit anti-phosphorylated Smad3 (ab52903; Abcam). Then, slides were incubated for 2

hour at room temperature with 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), 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, Budapest, Hungary) and evaluated on Case Viewer Software (3D

HISTECH) using a 40x objective for at least 5 spots per mouse with a minimum of three mice in each

group.

To perform immunoblotting, harvested cells were lysed in lysed in RIPA buffer (GenDEPOT, Barker,

TX, USA) 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-cyclinD1 (#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, Guro, Seoul). Densitometry calculation was performed by ImageJ 1.49v software

developed by the National Institutes of Health.

RNA extraction and quantitative real-time RT-PCR

Total RNA from both lung tissues and cell lines was extracted by TRIzol (Ambion, Austin, TX, USA)

according to the manufacturer’s instructions. First-strand cDNA was synthesized using the Acculower

RT Premix (Bioneer, Daejeon, Korea) 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, Taunton, MA, USA). ActB was used as the endogeneous

reference control for all transcripts. All quantitative Real-Time RT-PCR (qRT–PCR) experiments were

repeated at least three independent times. Primers used are

F: 5`-CTTCTGGGCCTGCTGTTCA-3`

R: 5`-CCAGCCTACTCATTGGGATCA-3` for Mcp1,

F: 5`-AGCACAGAAAGCATGATCCG-3`

R: 5`-CTGATGAGAGGGAGGCCATT-3` for Tnf,




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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, Vancouver, BC, Canada) as previously reported (23). Cells were stained

with rat anti-mouse F4/80 (25-48-1; eBioscience, San Diego, CA, USA; 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, Tokyo, Japan).

Statistics

Statistical analysis was performed by GraphPadPrism 4 (GraphPad Software, San Diego, CA,

http://www.graphpad.com). Analyses were performed using a Student’s t test. P values of less than

0.05 were considered statistically significant. Results are presented as mean ± SEMs.

Results

Ahnak protein expression is down-regulated in human lung cancers

We performed immunohistochemical (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




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pneumocytes and AMs (Fig. 1A). However, Ahnak protein expression was significantly down-

regulated in lung cancer cells compared with normal lung tissues (Fig. 1A, 1B, and Supplementary Fig.

1). 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 (CCLE) database revealed that lung cancer cell lines have relatively

lower AHNAK mRNA expression compared to other cancer cell lines (Fig. 1D) (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 down-regulation of Ahnak gene expression is

associated with lung tumorigenesis.

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 and Supplementary Fig. 2). Histologically, the alveolar septa of

Ahnak-/- lungs were thickened, which was accompanied by dilated alveolar space (Fig. 2B and 2C).

Ahank-/- lungs at embryonic day 18.5 also showed reduced airspace with denser cellularity than WT

lungs (Supplementary Fig. 3), 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 and Supplementary Fig. 4). Importantly, IHC analysis revealed that SP-C-

positive alveolar type II pneumocytes (AT2) were significantly increased in Ahnak-/- lungs in

comparison to 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 to WT lungs (Supplementary Fig. 4). 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 down-regulation of cyclin D (11), cyclin D1 was up-

regulated in Ahnak-/- lungs (Fig. 3A). These findings were confirmed by western blot using whole lung

cell lysates from WT and Ahnak-/- mice (Fig. 3B). Co-immunofluorescence (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 3D). Taken together, lung lesions in Ahnak-/-

mice are characterized by hyperplastic AT2 cells due to their increased cell proliferation.




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Ahnak-/- pneumocytes show down-regulation of the TGF-β signaling pathway

It is known that Ahnak inhibits cell growth by potentiating TGF-β-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 4B). We confirmed that phosphorylation of Smad3 is reduced in Ahnak-/- whole

lung tissues using western blot analysis (Fig. 4C). Since 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 TGF-β

reporter activity and decreased cell growth (Fig. 4E and 4F). Taken together, these results suggest

that Ahnak inhibits the cell growth of lung epithelial cells by activating the TGF-β signaling pathway.

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 tumors 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-positive 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 cyclinD1 (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.

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 histopathological 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, 5F, and 5G). Moreover, higher

numbers of macrophages were prominent both inside and surrounding the tumor tissues in Ahnak-/-




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mice compared to 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 160kD 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,

TNF-α, IL-12, IFNs, etc. and for M2, IL-10, IL-4, IL-13, etc.) and their immune functions (for M1, pro-

inflammatory and for M2, anti-inflammatory) (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 pro-inflammatory cytokine TNF was significantly lower in

Ahnak-/- AMs than in WT AMs, while anti-inflammatory cytokines such as IL-4 and IL-10 were

significantly higher (Fig. 6D). Notably, Ahnak-/- AMs also produced more growth factors such as

Insulin-like growth factor 1 (IGF-1) (Fig. 6D and Supplementary Fig. 5C) and epithelial growth factor

(EGF) (Fig. 6D) than WT AMs. Activation of the IGF and EGF signaling pathways in Ahnak-/- lungs

was also confirmed by IHC (Supplementary Fig. 5A and 5B). 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.

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 IL-4 treatment. In

response to LPS treatment, Ahnak-/- peritoneal macrophages exhibited reduced levels of TNF and

increased levels of MCP1 and IL-6 compared with those of WT peritoneal macrophages (Fig. 6F).

Although IL-6 is known for a pro-inflammatory cytokine, roles of IL-6 in macrophage polarization were

context-dependent and IL-6-promoting M2 programming has been reported (28,29). In parallel, we

overexpressed 4CRUs in CRISPR/Cas9-mediated Ahnak knockout (KO) RAW264.7 cells. IL-4, IL-10,

IL-6 and IGF-1 expressions by LPS or IL-4 treatment were suppressed in restored cells (Fig. 6G).

Phosphorylated Akt was reduced in LPS or IL-4-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 while loss of Akt1 augments M1

activation (30). Taken together, these results suggest that Ahnak gene deficiency induces the




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transition of AM cytokine profiles in favor of M2-like macrophage programming and monocyte

recruitment.

Ahnak-/- alveolar macrophages 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-H and Fig. 7I). clodronate liposome treatment for 3

weeks attenuated the thickness of alveolar septa in Ahnak-/- lungs (Fig. 7A, 7B, 7C, and 7J)

compared with that of control liposome-treated Ahnak-/- lungs (Fig. 7E, 7F, 7G, 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 co-cultured with isolated AMs from

Ahnak-/- mice or AMs from WT mice. MLE-12 cells grew more rapidly when co-cultured 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 co-cultured with Ahnak-/- AMs than those co-cultured with

WT AMs (Supplementary Fig. 5D). The treatment with recombinant IGF-1 and EGF, which were highly

produced in Ahnak-/- AMs, enhanced the growth of lung cancer cells (Supplementary Fig. 5E) and

upregulated core cell cycle regulators (Supplementary Fig. 5F). Taken together, all these results

suggest that Ahnak-/- AMs enhance the proliferative activity of pneumocytes in the mice.

Discussion

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. Firstly, down-regulation of the TGF-β signaling in the pneumocytes of Ahnak-/- lungs

might promote lung epithelial proliferation. It was previously shown that activation of the TGF-β

pathway promotes cell cycle arrest and apoptosis in early-stage tumors (31). In addition, cyclin D1

and p21, downstream target molecules of the TGF-β 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 up-regulation of cyclin D1. Secondly,

increased M2-like AMs in Ahnak-/- lungs might contribute to tumorigenesis, since 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




13

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 activations 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

present data. First, Ahnak deficiency induced the polarization of macrophages to anti-inflammatory

M2 phenotypes via the activation of Akt signaling (30). Second, the up-regulation of MCP1 and IL-4 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 (7,9). 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 (10). 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




14

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 TGF-β

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.

Acknowledgement

This research was supported by the 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 Je Kyung Seong and Il Yong

Kim. Also it was partially supported by the Brain Korea 21 Plus Program and the Research Institute

for Veterinary Science of Seoul National University.

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Figure Legends

Figure 1.

Down-regulation of Ahnak in human lung adenocarcinomas. A, Representative images of down-

regulated Ahnak expression in human lung cancer tissues and matched normal lung tissues from a

TMA slide containing 50 lung adenocarcinoma cases. Original magnification, x40; Insert, x1000.

Bar=50µm. B, Immunohistochemical 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

histological subtypes based on published data sets (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, 2-tailed Student’s t test in B.

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 airspaces (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 upper panels are magnified in

lower panels. 14 week-old Ahnak-/- mice showed hypercellularity and excessive connective tissues.

Bar=200µm. D, There was an increased number of SP-C-positive AT2 cells per HPF (high power field,

200x) 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.

Bar=50µm. 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, 2-

tailed Student’s t test in D.

Figure 3.

Proliferation of AT2 cells in Ahnak-/- lungs. A, IHC analysis for cell cycle regulators such as Ki-67,

PCNA, and cyclinD1 in WT and Ahnak-/- lungs. Right graphs show the IHC scoring for each marker in




18

lung tissues. The scorings were calculated as the percentage of cells exhibiting strong nuclear

staining per HPF (high power field, 400x). n=3 Ahnak-/- mice, n=3 WT mice at 10 weeks of age.

Bar=100µm. B, Western blot analysis for PCNA and cyclinD1 in Ahnak-/- and WT whole lung tissues

at 6, 10, 14, and 18 weeks of age. C-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, arrow head. SP-C+/PDPN+/PCNA+ cells,

asterisk. Bar=40µm. 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 three mice. * P<0.05 by unpaired, 2-tailed Student’s t test in (A).

Figure 4.

Down-regulation of the TGF-β 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. Bar=50µm. 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 2000 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 Hemaagglutinin (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 TGF-β signaling activation. After 3 days of transfection with HA-4CRU-Ahnak, luciferase

reporter activities were measured. Cells were treated with 5 ng/ml TGF-β 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, 2-

tailed Student’s t test in B, E, and F. The data are means ± s.e.m. of three independent experiments.

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

one-year-old Ahnak-/- mouse (#1). Arrow heads indicate tumor margin. Bar=1cm. B, Histopathological

findings of Ahnak-/- mice bearing a pulmonary adenoma (#1) and an adenocarcinoma (#2) based on

H&E staining. Original magnification, x40. Boxed areas are magnified in inserts. Bar=50µm. C, Co-IF

analysis for SP-C and PDPN. The dotted line indicates tumor margin. Bar=200µm. Boxed area is

magnified in the right panel. Bar=50 µm. Ki-67 staining was done to denote proliferation. D,




19

Immunohistochemistry for E-cadherin, Ki-67, and cyclinD1 was performed to characterize

spontaneous tumors in Ahnak-/- mice. Bar=50µm. E, Representative photographs of urethane-

induced lung tumors in WT and Ahnak-/- mice. Bar=5mm. 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, x100. *, P<0.05; **, P<0.01; ***, P<0.001 by unpaired,

2-tailed Student’s t test in F.

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. Bar=100µm. The numbers of F4/80-positive

alveolar macrophages (AMs) in Ahnak-/- and WT lungs at the indicated time points. F4/80-positive

cells were counted and averaged in 400x 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, Quantitative real-time RT-PCR analysis for mRNA expressions of cytokines,

chemokines, and growth factors in Ahnak-/- AMs from 14 week-old mice. E, Quantitative real-time RT-

PCR analysis for mRNA expressions of these factors in Ahnak-/- and WT whole lung tissues from 14

week-old mice. F, TNF, IL-6, and MCP1 induction after 48 hours of 10 ng/ml LPS treatment was

measured by quantitative real-time RT-PCR analysis. The cells from peritoneal spaces were enriched

for peritoneal macrophages by selecting F4/80-positive cell populations via FACS sorting. G,

Quantitative real-time RT-PCR analysis to evaluate mRNA expression changes of IL-4, IL-10, IL-6,

and IGF-1 in CRISPR/Cas9-mediated Ahnak KO RAW264.7 cells after transfection of the 4CRU-

Ahnak vector. Cells were treated with 10ng/ml LPS or 10ng/ml IL-4 for 48 hours. Similar results were

obtained in three independent experiments. * P<0.05 by unpaired, 2-tailed Student’s t test in A, B, D,

E, F, and G. H, Western blot analysis for phosphorylated Akt expression in LPS or IL-4-treated Ahnak

KO RAW264.7 cells after the restoration of 4CRU-Ahnak. Bottom, quantification of western blots was

performed using ImageJ software.

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, B, and C; three




20

independent mice) and control liposome (E, F, and G). Bar=200µm. 16 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 400x 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 (200x). Bar=100µm. L, MTT assay

for MLE-12 at 48 hours after co-culture with WT or Ahnak-/- alveolar macrophages (AMs). Left,

schematic drawing of the co-culture system. * P<0.05 by unpaired, 2-tailed Student’s t test in (I, J, K,

and L).

























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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AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia … · 1 AHNAK Loss in Mice Promotes Type II Pneumocyte Hyperplasia and Lung Tumor Development. Running title: Ahnak Gene