Ablation of Liver X receptors α and β leads tospontaneous peripheral squamous cell lungcancer in miceYu-bing Daia, Yi-fei Miaoa,1, Wan-fu Wua,1, Yu Lib,1, Francesca D’Erricoa, Wen Sua, Alan R. Burnsc, Bo Huanga,Laure Maneixa, Margaret Warnera, and Jan-Åke Gustafssona,d,2

aCenter for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204; bDepartment ofPathology, Chongqing Medical University, Chongqing, China; cCollege of Optometry, University of Houston, Houston, TX 77204; and dCenter for InnovativeMedicine, Department of Biosciences and Nutrition, Novum, 14186 Stockholm, Sweden

Contributed by Jan-Åke Gustafsson, May 16, 2016 (sent for review March 12, 2016; reviewed by Brigid L. M. Hogan and Antonio Moschetta)

The etiology of peripheral squamous cell lung cancer (PSCCa) remainsunknown. Here, we show that this condition spontaneously de-velops in mice in which the genes for two oxysterol receptors, LiverX Receptor (LXR) α (Nr1h3) and β (Nr1h2), are inactivated. By 1 y ofage, most of these mice have to be euthanized because of severedyspnea. Starting at 3 mo, the lungs of LXRα,βDko mice, but not ofLXRα or LXRβ single knockout mice, progressively accumulate foamcells, so that by 1 y, the lungs are covered by a “golden coat.” Thereis infiltration of inflammatory cells and progressive accumulation oflipid in the alveolar wall, type 2 pneumocytes, and macrophages. By14 mo, there are three histological lesions: one resembling adeno-matous hyperplasia, one squamous metaplasia, and one squamouscell carcinoma characterized by expression of transformation-relatedprotein (p63), sex determining region Y-box 2 (Sox2), cytokeratin 14(CK14), and cytokeratin 13 (CK13) and absence of thyroid transcrip-tion factor 1 (TTF1), and prosurfactant protein C (pro-SPC). RNA se-quencing analysis at 12 mo confirmed a massive increase in markersofM1macrophages and lymphocytes. The data suggest a previouslyunidentified etiology of PSCCa: cholesterol dysregulation and M1macrophage-predominant lung inflammation combined with dam-age to, and aberrant repair of, lung tissue, particularly the peripheralparenchyma. The results raise the possibility that components of theLXR signaling may be useful targets in the treatment of PSCCa.

peripheral squamous cell lung carcinoma | Liver X receptor | lipidmetabolism | macrophage | inflammation

Lung cancer is the most common cancer and the leading causeof cancer-related death world-wide (1). Nonsmall cell lung

carcinomas (NSCLC), the major class of lung cancer, includessquamous cell lung carcinoma (SCCa) and adenocarcinoma(ADCa), which account for approximately 23% and 43% of lungcancer, respectively (2). On the basis of the site of the primarycancer, squamous cell lung cancer is described as central (CSCCa)or peripheral (PSCCa). The incidence of PSCCa is increasing andis reported to be approximately 50% of squamous cell lung cancer(3–6). Although the etiology of squamous cell lung cancer is notknown, smoking and chronic lung inflammation have been rec-ognized as predisposing factors (7). Smoking is a major risk factorfor chronic obstructive pulmonary disease, interstitial lung dis-eases, and all types of lung cancer, especially squamous cell lungcancer. Smoking exposure rapidly leads to lipid accumulation inpulmonary macrophages, a condition associated with abnormallung physiology, including dysregulated pulmonary innate andadaptive immune responses to the environment (8). Abnormallipid metabolism such as increase in total and esterified choles-terol, increased fatty acid synthesis, and accumulation of lipid-loaded macrophages (9–13) are characteristic of lung cancer.In mice, inactivation of the kinase activity of IKKα in the NF-κB

signaling pathway leads to SCCa (14), which can be prevented bydepleting macrophages or reintroducing WT (IKKα active) bonemarrow. The data point to dysfunctional macrophages as drivers

of SCCa. LXRα and LXRβ are oxysterol-activated transcriptionfactors regulating cholesterol homeostasis by (i) suppressing denovo biosynthesis of cholesterol; (ii) removing cholesterol fromcells via their stimulation of four members of the ATP-bindingcassette transporter (ABC) family, ABCA1, ABCG5, ABCG8,and ABCG1; and (iii) reducing the level of Niemann-Pick C1-Like1, a transporter that facilitates uptake of cholesterol into cells.LXRs are widely expressed in the immune system where they havea powerful antiinflammatory function. In the lung, LXRs protectagainst acute lung injury, chronic obstructive pulmonary disease,asthma, and bacterial infection (15–17).Through their roles in metabolism of cholesterol and lipids,

regulation of the extracellular milieu, and the cell cycle, LXRs havebeen reported to protect against cancer (18). Although LXRα andLXRβ are expressed in pneumocytes and bronchial epithelium (19),little has been reported on the role of LXRs in lung carcinogenesis.In the present study, we examined the lungs of LXRα; LXRβ doublenull mutant (LXRα,βDko) mice on the C57BL/6 background thatwere fed a regular diet. We found chronic lung inflammation anddisturbed lung lipid homeostasis characterized by progressive andmassive accumulation of lipid in type 1 and type 2 pneumocytes,fibroblasts, and macrophages. By 14 mo of age, mice spontaneouslydeveloped lesions resembling peripheral squamous cell carcinoma.We conclude that LXRs normally play a key role in maintaininglung epithelial homeostasis and preventing epithelial hyperplasia,metaplasia, and squamous cell cancer.

Significance

Lung cancer is the leading cause of cancer death worldwide.Lung squamous cell carcinoma (SCCa) is classified as central orperipheral, according to the location of the primary tumor.Peripheral SCCa (PSCCa) is becoming as common as central SCCa,but the reasons for this increase are unclear. Smoking andchronic lung inflammation are recognized as risk factors for lungcancer. LXRα,βDko mice accumulate lipid in their lungs and de-velop M1-macrophage-predominant chronic lung inflammationand eventually lesions resembling PSCCa. This mouse model mayprovide a tool to study the etiology and progression of lungPSCCa and suggests a need for examination of LXR signaling inhuman PSCCa.

Author contributions: Y.-b.D., M.W., and J.-Å.G. designed research; Y.-b.D., Y.-f.M., W.-f.W.,Y.L., F.D., W.S., A.R.B., B.H., L.M., and M.W. performed research; Y.-b.D., Y.-f.M., W.-f.W.,Y.L., F.D., W.S., M.W., and J.-Å.G. analyzed data; and Y.-b.D., M.W., and J.-Å.G. wrotethe paper.

Reviewers: B.L.M.H., Duke University Medical Center; and A.M., University of Bari, Italy.

The authors declare no conflict of interest.1Y.-f.M, W.-f.W., and Y.L. contributed equally to this work.2To whom correspondence should be addressed. Email: jgustafsson@uh.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1607590113/-/DCSupplemental.

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ResultsSquamous Cell Lung Carcinoma-Like Lesions Develop in LXRα,βDko

Mice Fed Regular Diet. The overall survival of (LXRα,βDko) (n =5) and wild-type (WT) mice (n = 5) was followed up to 20 mo.After 12 mo of age, LXRα,βDko mice began to die, whereas all WTmice were still alive at 20 mo of age. The overall survival of theLXRα,βDko mice was 16.5 ± 2.9 mo. The difference in survivalrates was significant (Fig. S1, P = 0.0133). LXRα,βDko mice werefound dead in their cages or had to be euthanized because ofsevere dyspnea. Both LXRα (Fig. S2A) and LXRβ (Fig. S2C) areexpressed in the nuclei of alveolar macrophages, type 2 pneu-mocytes, and type 1 pneumocytes. In the lungs of LXRα,βDko

mice, no LXRα or LXRβ protein was detectable (Fig. S2 B andD).Multiple lesions with a high density of disorganized epithelial

cells were found in the lungs of all LXRα,βDko mice at 14 mo ofage. The lesions were present in the alveolar space (Fig. 1 B andE) and in the lung parenchyma (Fig. 1 C and F). Significantly,many of the cells within the lesions were Ki67+ and had hyper-chromatic, atypical nuclei of median to large size with irregularnuclear contours, prominent nucleoli, and abnormal mitoticfigures (Fig. 1 E, F, H, and I), all characteristics of cancer tissue.By contrast, the lung parenchyma of WT littermates had a normal

structure with clear alveolar spaces (Fig. 1 A andD) and few Ki67+

nuclei (Fig. 1G). To further analyze the lesions, we used sequen-tial sections for immunohistochemical staining for transformation-related protein 63 (p63), cytokeratin 14 (CK14), cytokeratin 13(CK13), sex determining region Y-box 2 (Sox2), thyroid tran-scription factor 1 (TTF1), and prosurfactant protein C (pro-SPC).The lesions were p63, CK14, CK13, and Sox2 positive, but nega-tive for TTF1 and pro-SPC (Fig. 1 J–O), indicating SCCa andnot adenocarcinoma.

Progressive Lipid Accumulation in Lungs of LXRα,βDko Mice. At thelevel of gross morphology, focal golden spots were observedalong the margin of LXRα,βDko lungs at 3 mo of age (Fig. 2F), andby 7 mo of age, these lesions had spread from the periphery towardthe center (Fig. 2G, black arrows). At 14 mo, the lungs of WT andLXRαko mice showed normal gross morphology (Fig. 2 A and B),whereas in LXRβko mice, there were scattered golden patches onthe surface (Fig. 2C, black arrow). Strikingly, in LXRα,βDko mice,most of the lung was covered by a “golden coat” of lipid (Fig. 2 Dand H, black arrows).We examined the time course of appearance of lipid laden

cells within the lung by two methods; Oil Red O and HCSLipidTOX Deep Red staining and transmission electron micros-copy (TEM). In the lungs of WT mice, there was no obviousstaining with Oil Red O between 3 and 14 mo of age (Fig. 3 A–Cat 20× magnification), although fibroblasts with lipid inclusionscould be seen in the alveolar wall at 100× magnification. Bycontrast, in the lungs of LXRα,βDko mice (Fig. 3D), lipid depositswere clearly evident along the pleura and in the alveolar wallsclose to the pleura at 3 mo of age and by 7 and 14 mo, foci ofdensely packed cells including enlarged foamy cells completelyobscured normal alveolar structures (Fig. 3 E and F). In the lungsof 3-mo-old LXRα,βDko mice before the appearance of foam cellsin the alveolar space, there was lipid accumulation in type 2pneumocytes and in the alveolar wall (Fig. 3 G and H). As lipiddeposition increased, lipid-loaded foamy cells began to accumu-late in the alveolar space (Fig. 3 I and J). Thus, as evidenced bymicroscopic observation, type 2 pneumocytes are the first re-sponders in these mice, with lipid accumulation occurring beforethe invasion of macrophages. Massive lipid accumulation was seenin cells surrounding the hyperplasic epithelial lesions in 14-mo-oldmice (Fig. 3 K and L) and in epithelial cells in the bronchioles(Fig. 3 M and N). To identify the cell types with lipid accumula-tion, we combined staining with HCS LipidTOX Deep Red andimmunofluorescence for the macrophage marker CD206, and thetype 2 pneumocyte marker, pro-SPC. In the lungs of 3-mo-oldLXRα,βDko mice, CD206 and pro-SPC were coexpressed with HCSLipidTOX Deep Red (Fig. 3 O–Q). TEM was used to observe the

Fig. 1. Tumors in the lungs of 14-mo-old LXRα,βDko mice. In 14-mo-oldLXRα,βDko mouse lungs (B and C), the parenchyma close to pleura and thealveolar spaces were filled with disorganized cells with hyperchromaticatypical nuclei, prominent nucleoli, and irregular mitotic figures. (E and F)High-magnification insets show abnormal mitoses. A few Ki67+ cells arepresent in the normal WT lung (A and G), but many more Ki67+ cells arepresent in the epithelial lesions of LXRα,βDko mice (H and I). Yellow linescircle the lesion field in B and C. E and F are magnified areas of labeledboxes. Immunohistochemical study to differentiate between squamous celllung cancer and adenocarcinoma in sequential sections with different lungcancer markers. Cells in the lesions of the LXRα,βDko mouse lung were CK14+

(J), p63+ (K), Sox2+ (L), CK13+ (M). They did not express pro-SPC (N) or TTF1(O), but there were pro-SPC and TTF1-positive cells scattered around thelesion. (Scale bars: A–C, 100 μm; D–O, 50 μm.)

Fig. 2. Gross morphological changes in the lungs of WT, LXRαko, LXRβko,and LXRα,βDko mice fed with regular diet. The lungs of 14-mo-old WT mice(A) and those of LXRαko (B) showed a normal gross appearance. There weregolden patches in the periphery of the lungs of LXRβko mice (C), and thesepatches covered most of the lung of 14-mo-old LXRα,βDko mice (D). From 3to 14 mo of age, there was a progressive expansion of the golden lesionfrom the margin of lung toward the center (E–H). Arrows show the marginsof the lesions.

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ultrastructures of subpleural lungs of 3-mo-old WT and LXRα,βDko

mice. In WT lung, the alveolar macrophage and type 1 and type2 pneumocytes did not show obvious lipid inclusions and somefibroblasts showed small size lipid droplets in cytoplasm (Fig. 3R–U); in LXRα,βDko mice, the alveolar macrophages and type 1and type 2 pneumocytes showed obvious lipid accumulation,the type 2 pneumocytes also showed abnormal lamellar bodies,

and the lipofibroblasts showed increased size of lipid dropletsaround the nucleus (Fig. 3 V–Y).

Lipid Dysregulation in the Lung Induces Chronic Lung Inflammation.At 12 mo of age, there was no evident inflammation in the lungsof WT mice (Fig. 4A). However, in the lungs of LXRα,βDko mice,there was lymphoid hyperplasia around the vessels and CD3+

inflammatory T cells infiltrating the parenchyma (Fig. 4 B, C, G,and J). Scattered CD206+ macrophages were found in the alveolarspace and interstitium of WT mice (Fig. 4E), whereas large clus-ters of macrophages occupied the alveolar spaces in LXRα,βDko

mice (Fig. 4 H and K). Myeloperoxidase (MPO), a neutrophilmarker, was highly expressed in lung macrophages of LXRα,βDko

mice (Fig. 4 I and L), but not in WT lungs (Fig. 4F). There weremany cells expressing the inflammatory cytokines IL-1β and TNF-α in lungs of LXRα,βDko (Fig. 5 D–I) but not in WT mice (Fig. 5A, C, G, and I). IL-6 was expressed in the cytoplasm of macro-phages of both LXRα,βDko and WT mice (Fig. 5 B and H).

Evidence for Lung Injury and Alveolar Remodeling in Response toChronic Lung Inflammation. With Masson’s trichrome stain, therewas no indication of fibrosis in either WT (Fig. S3 A and D) orLXRα,βDko mice (Fig. S3 B, C, E, and F). However, histologicalexamination showed evidence for extensive remodeling of thealveolar regions of the lung. For example, type I pneumocytes,which express high levels of caveolin-1 (Cav-1), normally cover

Fig. 3. Progressive accumulation of lipid in the lung of LXRα,βDko mice withage. There was no positive staining for lipid with Oil Red O in the lungs ofWT mice from 3 to 14 mo of age (A–C). In lungs of 3-mo-old LXRα,βDko mice,scattered lipid deposits were found along the pleura and the alveolar walls(D). In 7- and 14-mo-old LXRα,βDko mice, there were dense lesions full ofenlarged foamy cells (E and F). At 3 mo of age, there was lipid accumulationin type 2 pneumocytes (black asterisk) and in the alveolar wall (white arrow).In some areas, the alveolar space was clear (G and H), but in others, therewas an accumulation of lipid-loaded foamy cells (black arrow) (I and J). In14-mo-old mice, there was lipid accumulation around the tumor masses (Kand L), and foamy cells were present in the bronchioles (M and N). L and Nare magnified views of the boxes in K and M. In 3-mo-old LXRα,βDko mice,pro-SPC (type 2 pneumocyte marker) positive cells (white arrowhead)stained positively for HCS LipidTOX Deep Red (neutral lipid) (O and P). CD206(macrophage marker) cells also stained positively for HCS LipidTOX DeepRed (pink arrowheads) (Q). EM is to show different cells close to pleura inlung of WT mice and mutants at 3 mo of age. (R–U) WT lung: alveolarmacrophage (R); type 1 cell, black arrowhead and lipofibroblast with smalllipid inclusion, yellow star (S); type 2 cell, yellow arrowhead (T ); fibroblastwithout lipid inclusion (U). (V–Y ) LXRα,βDko lung: alveolar macrophage,white arrow (V ); type 1 cell with lipid inclusion, white arrow (W ); type 2cell with abnormal lamellar bodies, yellow arrowhead and lipid inclusionshown by white arrow (X ); lipofibroblast, white arrow (Y ). (Scale bars:A–F, K, M, and O–Q, 50 μm; G–J, L, and N, 20 μm; R, 0.5 μm; V, 2 μm; S–Uand W–Y, 1 μm.)

Fig. 4. Increased inflammatory cell infiltration in the lung of LXRα,βDko miceat 12 mo of age. In the lungs of 12-mo-old LXRα,βDko (B and C) but not WTmice (A), there was lymphoid hyperplasia around the vessels and in-flammatory cells infiltrating into the parenchyma with foamy cells filling thealveolar space. A few CD3+ T cells (D) were found along the WT vessels, butthere was a clear increase in the number of CD3+ T-cells in LXRα,βDko lungs(G). In WT mice, scattered CD206+ macrophages were found in the lunginterstitium (E) and on the alveolar wall and few MPO+ cells were detectable(F). In LXRα,βDko mice, clustered CD206+ macrophages with enlarged cellbodies filled the alveolar space (H) and MPO was found in the cytoplasm ofmacrophages (I). The densities of CD3+ T cells, CD206+ macrophages, and MPO+

cells in lungs of WT and LXRα,βDko mice are depicted in J–L. (Scale bars: 50 μm.)*P < 0.05, **P < 0.01, ***P < 0.001.

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approximately 95% of the alveolar surface and are tightly apposedto capillary vessels that are also Cav-1 positive (Fig. 6A). In 12-mo-old LXRα,βDko mice, Cav-1 staining was seen in the endothelialcells but the type 1 pneumocyte staining was discontinuous inareas where there was infiltration of macrophages (Fig. 6B). Inneighboring areas, the luminal sides of the alveoli were lined bysimple cuboidal epithelial cells that did not express Cav-1 (Fig.6C). Immunohistochemistry for pro-SPC indicated some of thecuboidal cells populating the remodeled alveoli are type 2 pneu-mocytes. At 7 mo of age, there was evidence for hyperplasia ofthese cells (Fig. 7B), and by 12 mo, pro-SPC+ cells were observedlining alveoli-like structures (Fig. 7C). Remarkably, by 14 mo, theepithelial cells covering the alveolar surface did not express pro-SPC (Fig. 7D) but expressed CK14 (Fig. 7H). Earlier, in 12-mo-old LXRα,βDko, only scattered CK14+ cells were present on theluminal surface of those alveoli that were full of foamy cells (Fig.7 E and G). Immunofluorescence staining showed that the CK14+

cells in lung parenchyma were also positive for p63, a marker forbasal cells in the lung (Fig. 7 I–L). The time course of appearanceof these cells was followed in more detail below.

Appearance of Ectopic CK14+p63+ Cells in the Lungs of LXRα,βDko

Mice. In WT adult mice, no CK14+ or p63+ cells were detect-able in the epithelium of the bronchioles or lung parenchyma (Fig.S4 A, B, E, and F). However, in 12-mo-old LXRα,βDko mice, p63+

cells were located basally in some bronchioles (Fig. S4C). Signif-icantly, these cells were not CK14+ (Fig. S4D). p63+ cells werealso seen in terminal bronchioles in the neighborhood of the areasof focal hyperplasia and PSCCa-like lesions. Analysis of sequentialsections showed that only some of the p63+ cells expressed CK14(Fig. S4 G and H), whereas all CK14+ cells were p63+ (Fig. S4I–L). In some areas near terminal bronchioles, clusters of p63+

cells appeared to be infiltrating into the lung parenchyma (Fig. S4O and P). In some areas at 14 mo of age, there were “glandular-like structures” composed of epithelial cells positive for p63,CK14, Sox2, and TTF1 but negative for pro-SPC (Fig. S5 F–J).Within the lesions designated histologically as squamous

metaplasia with dysplasia, all layers of squamous epitheliaexpressed CK14 and Sox2. At 14 mo, approximately one-third ofthese epithelial cells expressed p63, but not TTF1 or pro-SPC(Fig. S5 K–O).

RNA Sequencing of WT and LXRα,βDko Mice at 3 and 12 Mo ofAge (Figs. S6–S8). Comparison of RNA transcripts in lungs ofLXRα,βDko and WT mice at 3 mo of age revealed few differ-ences. However, in lungs of 12-mo-old LXRα,βDko mice, therewas clear evidence for M1 macrophage-predominant immuneinvasion and a down-regulation of markers of Club cells. Themost up-regulated genes were (i): the cell surface markersCD180, 209, 274, 300, 37, 3, 4, 48, 5, 74, 79, and 8; (ii) thecytokines CXCl1, 2, 3, 5, 10, 12, 13, 16, and 19; (iii) cytokinereceptors CXCR1 and 5; (iv) interleukins 1a, 1b, 6, 16, 18, 33,34; (v) IL2RB; (vi) metalloproteases MMP 2, 8, 12, 14, and19; (vii) NF-κB2; (viii) the tyrosine receptor binding proteinTyrobp and its partner Trem2; and (ix) the acute phase se-creted serum amyloid A protein Saa3 (Figs. S6–S8). The mostdown-regulated genes in the RNAseq were markers of clubcells (Cyp2f2, Scgb1a1, Scgb3a2) (Fig. S8). Some genes werechosen to validate the RNA sequencing, which is summarizedin Fig. S9. Despite the massive accumulation of lipids in thedouble mutant mice, there was no up-regulation of the ABCtransporters (Figs. S6 and S9B); scavenger receptors CD36,LDLR, SREBP1, and SCAP; or the cholesterol metabolizinggenes CYP27A and CYP46A1 (Figs. S6 and S9 B and F).

DiscussionWe have shown here a previously unidentified mechanism fordevelopment of squamous cell lung cancer-like lesions in mice,namely the inactivation of both LXRα and LXRβ. We havepresented evidence that LXRα and LXRβ are essential for lipidhomeostasis in the lung. Without these two receptors, there is amassive accumulation of foam cells in the lung accompanied bychronic lung inflammation and eventual development through-out the alveolar region of extensive epithelial dysplasia, squa-mous metaplasia, and squamous cell cancer-like lesions thatresult in death when mice are approximately 14 mo old. Thefindings that, on a regular diet, there is a massive accumulationof lipids in pulmonary type 1 and type 2 pneumocytes, fibroblasts,and macrophages of LXRα,βDko mice identifies a critical defect inthe ability of these mice to excrete cholesterol from the lung.Lipid deposits along the alveolar wall indicate that there may beproblems in lipid disposition in type 1 pneumocytes. Abca1 andAbcg1, which are LXR-target genes, facilitate cholesterol effluxfrom cells and are important for the maintenance of normal lunglipid composition, structure, and function. It has been reportedthat Abca1 or Abcg1 knockout mice develop enlarged foamymacrophages and lipid-filled type 2 pneumocytes with abnormal

Fig. 5. Increased cytokines in the lung of 12-mo-old LXRα,βDko mice. Mac-rophages in the alveoli of 12-mo-old LXRα,βDko but not WT mice (A) stronglyexpressed IL-1β (D). Few scattered IL-6–expressing cells (B) but no TNF-α+ cells(C) were detected in WT mice. However, in LXRα,βDko mice, clusters of IL-6+

cells were found in alveoli (E), and macrophages strongly expressed TNF-α(F). The densities of cytokine (IL-1β, IL-6, and TNF-α) positive cells are depicted inlung of WT and LXRα,βDko mice (G–I). LXRα,βDko (Scale bars: 50 μm.) *P < 0.05,**P < 0.01.

Fig. 6. Injury and abnormal repair in the lungs of LXRα,βDko mice. In lungsof WT mice, there was a continuous band of caveolin-1 staining along thealveolar wall (A), but this staining of caveolin-1 was discontinuous inLXRα,βDko mice (B). Regular lung structure was lost (black arrow), andalveoli were filled with foamy cells (white arrow). The luminal side of thealveoli around the injured lung parenchyma was lined by simple cuboidalepithelial cells, which did not express caveolin-1 (C, green arrow). Caveolin-1 expression was maintained in the capillaries (C, yellow arrow). (Scale bars:20 μm.)

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lung lipid profiles. However, no lung cancer was found in thesemice (20–22). In our studies, lipid deposition was much moresevere than that reported in the Abc knockout mice and lesionsoccurred at an earlier age. Despite the massive lipid accumu-lation, there was no induction of RNA for either Abca1 orAbcg1 in the lungs of LXRα,βDko mice, clearly showing that theessential role for LXR in cholesterol homeostasis of lung isnot Abca1 or Abcg1 dependent.Lipid metabolism is an important modulator of innate and

adaptive immune response (23). Modified LDL and LPS activateToll-like receptors of macrophages, which suppress LXR activityon its target genes, Abca1 and Abcg1, causing decreased macro-phage cholesterol efflux (24) and directly triggering proinflammatorysignaling to produce cytokines and chemokines. Deficiency ofABCA1 and ABCG1 leads to accumulation of free and esterifiedcholesterol in macrophages. Cholesterol-laden macrophages releaseinflammatory cytokines and matrix metalloproteinases and inducelung inflammation (25).Several mouse models show that macrophages play a vital role

in the development of SCCa (14, 26). The susceptibility to andincidence of spontaneous lung tumors, and the chemical in-duction of lung tumors, vary largely between mouse-inbredstrains. The C57BL/6 background is known to confer resistanceto spontaneous and chemically induced lung tumors (27), and ithas been argued that C57BL/6 mice may not be the appropriatebackground for studying the contribution of inflammation tolung cancer development (28). Despite this resistance of C57BL/6 mice, the LXRα,βDko mice on the C57BL/6 background de-veloped lung cancer-like lesions after 12 mo of age. It is possiblethat the defect in lipid metabolism was the factor that over-came the resistance of this mouse strain to the development oflung cancer.In this report, we have followed the morphological, histological,

and genetic changes in LXRα,βDko mice and their WT littermatesfrom 3 to 14 mo of age. Mice were always fed a normal diet andwere kept under specific pathogen-free conditions. By 3 mo of age,the LXRα,βDko mice began to accumulate lipids in distal parts ofthe lung close to the pleural surface. Over the next 12 mo, therefollowed a progressive and massive lipid deposition in type 2pneumocytes, in the alveolar wall, and in macrophages. This lipidaccumulation of lung was accompanied by loss of Cav-1+ type 1pneumocytes, the apparent proliferation and dysplasia of type 2pneumocytes, and the eventual appearance of pro-SPC negativecuboidal epithelial cells lining the alveoli. As the mice aged,

the number of small lesions increased, and by 14 mo, 100% ofLXRα,βDko mice had developed extensive squamous cell carci-noma-like lesions involving p63, CK14, CK13, Sox2-positive, andTTF1 and pro-SPC-negative epithelial cells throughout the lungparenchyma. An important question, which will require futurestudies, including genetic lineage tracing, is the origin of thesecells, which have some characteristics of basal cells normally pre-sent in the pseudostratified epithelium of the trachea and largeproximal airways in the mouse lung. One possibility is that theyderive from type 2 pneumocytes, which have been shown to pro-liferate and regenerate type 1 cells in alveolar injury models (29–31). Another possibility is that they arise from the same cells thatgenerate “pods” of p63+ CK5+ cells after H1N1 influenza virusinfection (32–34). At present, the precise origin of these cells is amatter of debate, but includes club cells, basal-like cells, and/orlineage-negative progenitors in the distal airways (32–35). How-ever, following influenza infection, these cells do not undergoextensive dysplasia or malignant transformation.In conclusion, we have shown that inactivation of both Liver X

receptors induced progressive lung lipid accumulation, chronicmacrophage-predominant lung inflammation, and lesions re-sembling squamous cell lung cancer. Lung lipid dysregulation andlipid-induced chronic lung inflammation in LXRα,βDko mice couldboth have contributed to the generation and apparent transfor-mation of p63+CK14+ cells in response to extensive alveolardamage (36). There are several mouse models of lung squa-mous cell lung cancer, but none for PSCCa as described here inthe LXRα,βDko mouse. Defective Liver X receptor signaling hasnot been studied in human PSCCa, and the LXRα,βDko mousemay help us to understand the etiology, progression, and un-derlying mechanisms of human peripheral SCCa.

Materials and MethodsAnimals and Tissue Preparation. Male C57BL/6 WT, LXRαko, LXRβko, andLXRα,βDko mice were used for experiments. The generation of LXRαko, LXRβko,and LXRα,βDko mice on the C57BL/6 genetic background has been described(37). Mice were housed in a room of standard temperature (22 ± 1 °C) with aregular 12-h light, 12-h dark cycle and given free access to water and standardrodent chow. The animal care and use program complied with the standardsand recommendations set forth in the National Research Council’s 1996 Guidefor the Care and Use of Laboratory Animals and in accordance with theUniversity of Houston guidelines on the use of laboratory animals.

All mice were anesthetized deeply with CO2. Blood was withdrawn frominferior vena cava or right ventricle. The right hilum of the lung was ligated;the right lung was removed and frozen in liquid nitrogen. The trachea wasintubated, and left lung was inflated with 4% (wt/vol) paraformaldehyde in0.1 M PBS (pH 7.4). The mice were transcardially perfused with 0.1 M PBSfollowed by 4% (wt/vol) paraformaldehyde. The trachea was ligated, andthe heart and lung were postfixed in the same fixative at 4 °C overnight.Half of the left lung was used for paraffin sections and the other half forfrozen sections. Paraffin sections (5 μm) were for hematoxylin and eosinstains, Masson’s Trichrome Stain, immunohistochemistry, and immunofluo-rescence studies. Frozen sections (5 μm) were prepared for Oil Red O stainingand HCS LipidTOX Deep Red neutral lipid staining.

Immunohistochemistry and Immunofluorescence. Paraffin sections weredeparaffinized in xylene, rehydrated through graded alcohol, and processedfor antigen retrieval in 10 mM citrate buffer (pH 6.0) for 10 min at 97 °C withpretreatment module. Sections were incubated in 0.3% H2O2 in 50% (vol/vol)methanol for 20 min at room temperature to quench endogenous peroxidase.To block nonspecific binding, sections were incubated in 3% BSA for 30 min,and then biotin-blocking system (Dako) was used to block endogenous biotin.The sections were incubated with antibodies specific for p63 (1:1000;Abcam, ab124762), Sox2 (1:1000; Santa Cruz Biotechnology, Sc17320), CK13(1:500; Thermofisher Pierce, MA1-5764), CK14 (1:1500; Abcam, ab7800), CK8(1:500; Abcam, ab59400), TTF1 (1:1000; Abcam, ab133638), pro-SPC (1:3000;Chemicon, AB3768), CD3 (1:1000; Santa Cruz Biotechnology, Sc-1127), CD206(1:1000; Abcam, ab64693), TNFα (1:500; Abcam, ab1793), IL1β (1:1000; Abcam,ab9722), IL6 (1:500; Santa Cruz Biotechnology, E209), MPO (1:1000; Abcam,ab45977), Caveolin 1 (1:1000; BD Transduction Laboratories, 610060), Ki67(1:1000; Abcam, ab15580), and anti-LXR α and β (1:1000; made in laboratory of

Fig. 7. Loss of pro-SPC and gain of CK14 in cuboidal epithelia of alveoli inLXRα,βDko mice. The lung of WT mice showed well-distributed type 2pneumocytes expressing pro-SPC (A). In 7-mo-old LXRα,βDko mice, clusters ofpro-SPC cells were seen (B). In 12-mo-old LXRα,βDko mice, pro-SPC+ cellscovered alveolar walls (C), but by 14 mo, these cells did not express pro-SPC(D), but expressed CK14 and p63 (white arrows) (G–L). CK14+ cells were notdetected in the alveolar region of WT mice (E) nor in the lesions of 7-mo-oldLXRα,βDko mice (F). (Scale bars: 20 μm.)

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J.-Å.G.). Primary antibodies were replaced with BSA as negative controls.Sections were incubated with biotinylated secondary antibody (1:200) for 1 hat room temperature, and then the Vectastain Avidin-Biotin Complex kit(Vector Laboratories) was used according to the manufacturer’s instruction,followed by 3,3-diaminobenzidine staining (Thermo Scientific) and counter-staining with Mayer’s hematoxylin (Sigma-Aldrich).

For immunofluorescence, quenching of endogenous peroxidase andblocking of endogenous biotin were omitted. Sections were incubated withanti-p63, anti-CK14, anti-CD206, and anti-pro SPC. Primary antibodies weredetected with fluorescent secondary antibodies. Sections were counter-stained with Vectashield mounting medium (Vector Laboratories).

Staining for Neutral Lipid. Frozen sections were stained with Oil Red O stainKit (American MasterTech., KTORO) according to the manufacturer’s in-struction. For fluorescence staining of neutral lipid, the frozen sectionswere stained with HCS LipidTOX Deep Red neutral lipid stain (1:1000,Invitrogen).

RNA Sequencing and Gene Expression Analysis. Total RNA of inferior lobe ofright lung was isolated by using the Qiagen RNeasy lipid tissue mini kitfollowing manufacturer’s protocol. After paired-end sequencing of samplesusing the Illumina HiSEq 2000 platform, TopHat v2.0.9 was used to align thesequence reads to the mm10 reference genome. The FastQC package v0.11.2

was used to assess the read quality. Partek Genomics Suite v6.6 was used toassign reads to genes by using RefSeq transcript annotations, generatingreads per kilobase of exon model per million mapped reads (38). Forquantitative PCR (qPCR), the RNA was reverse-transcribed by using randomhexamer primers and a revertAid first-strand cDNA synthesis kit (Invitrogen).qPCR was performed by using iTaq Universal SYBR Green Supermix (Bio-Rad). The sequences of qPCR primers are listed in Table S1.

Data Analysis. Data are expressed as mean ± SD. Statistical comparisons weremade by using Student’s t test or two-way ANOVA followed by Bonferroni posthoc test. Survival percent was analyzed by using the Kaplan–Meiermethod andwas compared by using the log rank test. P < 0.05 was considered to indicatestatistical significance.

ACKNOWLEDGMENTS.We thank our colleagues Dr. Kaberi Das and ChristopherA. Brooks for assistance with mice handling, Dr. Bilqees Bhatti for Masson’sTrichrome staining, and Margaret Gondo (College of Optometry, University ofHouston) for processing samples of TEM. This study was supported by EmergingTechnology Fund of Texas grants under Agreement 18 No. 300-9-1958, CancerPrevention and Research Institute of Texas Grant RP110444-P1, the RobertA. Welch Fund Grant E-0044, the Swedish Science Council, the Center for In-novative Medicine, and National Eye Institute Grants P30EY007551-29 andEY018239 (to A.R.B.).

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