The LIM-domain only protein 4 contributes to lung epithelial cell proliferation but is not essential for tumor progression

Background The lung is constantly exposed to environmental challenges and must rapidly respond to external insults. Mechanisms involved in the repair of the damaged lung involve expansion of different epithelial cells to repopulate the injured cellular compartment. However, factors regulating cell proliferation following lung injury remain poorly understood. Here we studied the role of the transcriptional regulator Lmo4 during lung development, in the regulation of adult lung epithelial cell proliferation following lung damage and in the context of oncogenic transformation. Methods To study the role of Lmo4 in embryonic lung development, lung repair and tumorigenesis, we used conditional knock-out mice to delete Lmo4 in lung epithelial cells from the first stages of lung development. The role of Lmo4 in lung repair was evaluated using two experimental models of lung damage involving chemical and viral injury. The role of Lmo4 in lung tumorigenesis was measured using a mouse model of lung adenocarcinoma in which the oncogenic K-Ras protein has been knocked into the K-Ras locus. Overall survival difference between genotypes was tested by log rank test. Difference between means was tested using one-way ANOVA after assuring that assumptions of normality and equality of variance were satisfied. Results We found that Lmo4 was not required for normal embryonic lung morphogenesis. In the adult lung, loss of Lmo4 reduced epithelial cell proliferation and delayed repair of the lung following naphthalene or flu-mediated injury, suggesting that Lmo4 participates in the regulation of epithelial cell expansion in response to cellular damage. In the context of K-RasG12D-driven lung tumor formation, Lmo4 loss did not alter overall survival but delayed initiation of lung hyperplasia in K-RasG12D mice sensitized by naphthalene injury. Finally, we evaluated the expression of LMO4 in tissue microarrays of early stage non-small cell lung cancer and observed that LMO4 is more highly expressed in lung squamous cell carcinoma compared to adenocarcinoma. Conclusions Together these results show that the transcriptional regulator Lmo4 participates in the regulation of lung epithelial cell proliferation in the context of injury and oncogenic transformation but that Lmo4 depletion is not sufficient to prevent lung repair or tumour formation. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0228-0) contains supplementary material, which is available to authorized users.


Background
The regulator of transcription LMO4 is a member of the LIM-domain only (LMO) protein family that act as molecular adaptors, providing a scaffold for multiprotein complexes of DNA-binding factors and transcriptional regulatory proteins [1][2][3][4]. LMO4 modulates neuronal progenitor cell proliferation and inhibits differentiation of mammary epithelial cells, suggesting that it may participate in the regulation of progenitor cell function [2,5]. LMO4 is highly expressed in epithelial cells, often at location of active mesenchymal-epithelial interactions, such as hair follicles, teeth, epidermis, mammary gland, kidney, and lung [6][7][8][9] suggesting that LMO4 may modulate the function of DNA-binding proteins in signaling pathways involved in mesenchymal-epithelial cross-talk. LMO4 interacts with Smad proteins and increases the effect of TGFβ on epithelial cell growth [10]. Mice lacking Lmo4 die perinatally from complex phenotypic abnormalities, with approximately 50 % of mice exhibiting exencephaly. Mice that do not display exencephaly are pale at birth, gasping for air and die shortly after birth [11]. Deregulation of LMO4 has been described in several tumor types including breast, prostate, pancreas, lung and squamous cell carcinoma of the oral cavity [2,[12][13][14][15][16]. LMO4 is overexpressed in more than 50 % of breast cancers and high nuclear expression of LMO4 has been found to be a predictor of worse outcome in breast cancer patients [3]. Down-regulation of LMO4 reduced proliferation of human breast cancer cells and increased differentiation of mouse mammary epithelial cells while overexpression of LMO4 in non-invasive, immortalised human cells promoted cell motility and invasion [3], suggesting that Lmo4 may play a role in epithelial cell proliferation and differentiation.
The lung is constantly exposed to toxic agents and pathogens present in the environment and must activate a number of molecular and cellular processes to induce rapid cell proliferation and repair. It is becoming evident that distinct types of lung stem/progenitor cells are activated depending on the type of injury inflicted to the lung [17]. Experimental protocols using chemical agents, viruses, or diffusible gases, have been used in mouse models to study the activation of lung stem cells in response to environmental challenges. Each model is unique in the type of injury caused as reflected by the degree of immune cell infiltration, fibrosis and the cell types affected [18,19]. Specifically, administration of H1N1 flu virus to animals leads to broad damage of epithelial cell types in the lung including club cells, ciliated cells, as well as alveolar cells. Damage and repair of the lung after H1N1 infection occurs in the absence of fibrosis as opposed to bleomycin treatment that induces extensive fibrosis [20]. A rare population of distal lung basal progenitor cells with regenerative capacity has been identified and shown to be responsible for the repair of the lung after H1N1 injury. Lineage tracing experiments and transplantation assays demonstrated that these rare distal basal cells expanded after injury and gave rise to club cells, ciliated cells and alveolar cells [21,22]. Vaughan et al. indicated that these progenitor cells did not express lung lineage specific markers but were present in a subset of cells expressing integrin β4 (CD104) but negative for the club cell marker CC10 [22]. In the non-injured lung, cells expressing the laminin receptor integrin α6β4 were shown to be enriched for cells with colony forming capacity in vitro [23,24], suggesting they behaved as progenitor cells in the distal lung. Chemical injury induced by administration of naphthalene, a component found in tobacco smoke, leads to the ablation of the large majority of club cells (Cyp2f2 + ). Only a small number of these cells, named variant club cells, that do not express Cyp2f2 resist injury and are thought to be the progenitor cells responsible for repair of the airways [25,26]. Naphthalene injury has also been shown to accelerate tumour growth when combined with oncogenic alterations such as expression of K-Ras G12D [27]. Factors involved in the regulation of the different classes of progenitor cells in the lung remain poorly characterised.
The observation that Lmo4 knockout mice display breathing difficulty at birth and that LMO4 is overexpressed in advanced lung cancer prompted us to explore its role in lung morphogenesis, adult lung repair and cancer. We used conditional knock-out mice to ablate Lmo4 expression in the lung epithelium from E9.5 and found that Shh-cre;Lmo4 fl/fl mice were viable and healthy. However, we observed that Lmo4 loss reduced proliferation of adult lung epithelial cells and delayed repair following virus-induced and chemical-induced lung injury. We then examined the role of Lmo4 in lung tumorigenesis by deleting Lmo4 in mice expressing the oncogenic K-Ras G12D . Our results showed that in the context of naphthalene-induced sensitization of K-Ras G12D -driven carcinogenesis, loss of Lmo4 reduced cell proliferation and delayed the onset of transformation but did not affect overall survival or tumor latency.

Histology and staining
For histological examination, lungs were fixed in 4 % paraformaldehyde in phosphate-buffered saline (PBS), embedded in paraffin, sectioned (2 μm) and stained with haematoxylin and eosin. For immunohistochemistry, sections were blocked in 10 % serum prior to incubation with specific antibodies or an isotype-matched control anti-rat (Pharmingen) or anti-rabbit (Cell Signaling) antibody diluted in PBS, 0.1 % tween containing 5 % serum, followed by biotin-conjugated secondary anti-rat or antirabbit antibody (Vector). Signal was amplified using Vectastain Elite ABC Reagent (Vector) for 30 min followed by 3, 3′-diaminobenzidine (DAKO).
Images of BrdU stained lung sections from Naphthalene injury and K-Ras G12D models were acquired on Aperio ScanScope AT Slide Scanner (Leica Biosystems) and analysed with FIJI image processing software (http:// fiji.sc). For Naphthalene injury model, BrdU positive cells were scored manually on the scanned images and number of positive cells in each airway was normalised to the length of airway circumference. Number of BrdU positive cells in hyperplastic airways at the week 12 time point was quantified using a custom FIJI script based on colour deconvolution and thresholding. The number of BrdU positive cells was normalised to the area of the corresponding hyperplastic airway and statistical analysis was carried out at the level of individual airways. For quantification of proliferating cells in hyperplastic airways at day six time point, number of BrdU positive cells normalised to area was calculated as above and averaged for each mouse based on ten airways per mouse. Statistical analysis was then performed at the mouse level. Tumour areas in K-Ras G12D model was measured manually on the scanned images. Measurements were log transformed and statistical analysis was carried out at the level of individual tumours.

RNA isolation and Real Time PCR
Total RNA was extracted from lung tissue using the miR-Neasy Mini Kit (Qiagen) according to manufacturer's instructions. DNase treatment was performed using TURBO DNA-free Kit based on the company's instructions. cDNA was generated using SuperScript III (Invitrogen) according to the manufacturer's instructions. qPCR was performed using the Sensimix SYBR Hi-Rox kit (Bioline) and the following primers for Lmo4: forward, 5′-CATGTTCT ACCTGCCGGAAT-3′ reverse: 5′-TCTGGTCTGGCAG TAGTGGA-3′. PCR was carried out in the Rotorgene RG-6000 and expression levels were normalized against the expression of 18S rRNA using the following primers: forward, 5′-GTAACCCGTTGAACCCCATT-3′; and reverse 5′-CCATCCAATCGGTAGTAGCG-3′.

Fluorescent Activated Cells Sorting (FACS)
For FACS analysis, lungs were digested in collagenase (1mg/ml collagenase in 3ml DPBS + 0.2g/L glucose per lung) at 37°C for 60 min while shaking at 165 rpm, followed by red blood cell lysis with 0.64 % ammonium chloride at 37°C for 3 min [31]. Cells were resuspended in blocking solution (anti-FcR and Rat IgG) and incubated on ice for 10 min. Cells were stained with conjugated antibodies CD31-PECy7, CD45-PECy7, EpCAM-APC, CD104-FITC and CD24-PE (Biolegend) as described previously [32]. Cells were then washed and resuspended in PI solution. Cells were sorted on an ARIA II (Beckton Dickinson).

Tissue microarray analysis
Tissue microarrays (2 mm cores, 3 per patient) were created from 34 squamous cell carcinomas, 40 adenocarcinomas and 10 otherwise classified Non Small Cell Lung Cancer (NSCLC) including large cell carcinomas cases collected from patients who underwent lung cancer surgery. Samples were obtained via the Victorian Cancer Biobank (Melbourne Australia). This study has Ethical Committee approval (Walter and Eliza Hall Institute Human Research Ethic Committee number 10/04). The scoring system for intensity was as follows: 0 -no staining; one -weak staining; two -moderate staining; and three -strong staining; and the scoring system for percentage was as follows: 0, no cells staining positive for LMO4; 1 -≤ 10 % cells staining positive; 2 -11 % to 50 % positive cells; 3 -51 % to 80 % positive cells; and 4 -≥ 80 % positive cells. The final histoscore (range from 0 to 12) was obtained from the product of the nuclear staining intensity and percentage of stained cells and averaged between three separate tissue cores from each of the patients. Difference in LMO4 histoscores between the cancer types was tested using Wilcoxon rank sum test.

Statistical analysis
All statistical analysis was carried out using R software [33]. Overall survival difference between genotypes was tested by log rank test. To determine survival difference between each pair of the genotypes, log rank test was carried out on all pair-wise comparisons and resulting p values were adjusted for multiple comparisons using Holm method.
Unless otherwise specified, difference between means was tested using one-way ANOVA after assuring that assumptions of normality and equality of variance were satisfied. In cases, where residual errors did not follow normal distribution, variables were log transformed. Where necessary, p values were adjusted for multiple testing using Tukey HSD method.
Differences in the number of BrdU positive cells in the airways following Naphthalene injury (in normal and K-Ras G12D lungs) were analysed using Wilcoxon rank sum test to accommodate multiple zero values.
Gene expression data for lung adenocarcinomas and squamous cell carcinomas was obtained from the respective public RNA-seq datasets (IlluminaHiSeq_RNASeq) of TCGA Research Network (http://cancergenome.nih.gov, accessed on 18/07/2014) [34,35]. Raw count data was filtered to exclude lowly expressed genes (average log counts per million (cpm) < 0) and normalised using TMM normalisation in edgeR [36]. Log cpm values for LMO4 were calculated across the samples using normalised library sizes and difference in LMO4 expression between the cancer types was tested using one-way ANOVA.

Results
Lung-specific Lmo4 conditional knock-out mice display normal lung development We first examined the expression of Lmo4 during embryonic lung morphogenesis, after birth and in adult. Quantitative RT-PCR results showed low levels of Lmo4 expression in the early phase of embryonic development from E11.5 to E15.5. Lmo4 expression peaks at E16.5 and then decreases in adult tissue (Additional file 1: Figure S1A). To further analyse the expression pattern of Lmo4 during embryonic lung development we performed immunofluorescence co-staining for Lmo4 and Nkx2.1, a marker of lung epithelial cells. Dynamic changes in the expression pattern of Lmo4 were observed (Fig. 1a). At E13.5, Lmo4 expression is detected in both the mesenchymal and endodermal compartments of the lung (Fig. 1a). At E16.5, strong Lmo4 expression is observed in the endoderm of the conducting airways while its expression is reduced in the distal branches from which the alveoli will form (Fig. 1a). At E18.5 the expression of Lmo4 becomes restricted to the cells lining the airways (Fig. 1a). Further evaluation of Lmo4 expression in adult lung shows that Lmo4 is expressed in club cells and ciliated cells, as shown by co-staining with CC10 and FoxJ1, markers of club cells and ciliated cells respectively (Fig. 1b). These results were confirmed by FACS analysis where lung epithelial cells were sorted based on the expression of EpCAM, CD104 (integrin β4) and CD24 (Heat Stable Antigen) as previously described by McQualter et al. [24] (Fig. 1c). Using these markers, EpCAM + epithelial cells can be subdivided into CD104 + cells enriched in alveolar cells, CD104 + CD24 lo cells enriched for club and progenitor cells and CD104 + CD24 hi cells enriched in ciliated cells [24]. Using Lmo4 LacZKi/+ mice in which the LacZ transgene has been knocked into the Lmo4 locus [9] we observed β-galactosidase staining in CD104 + CD24 lo and CD104 + CD24 hi cells indicating that Lmo4 is expressed in CD104-positive cells (Fig. 1d).
To explore the role of Lmo4 in the lung epithelium, we generated Shh-cre;Lmo4 fl/fl mice in which Lmo4 was excised from E9.5 in the epithelium of the lung primordia. Since the cre allele was knocked into the Shh locus resulting in loss of one Shh allele, Shh-cre;Lmo4 fl/+ animals were used as controls. Shh-cre;Lmo4 fl/fl pups were born at normal mendelian ratio and survived to adulthood without any lung abnormalities compared to controls (Additional file 1: Figure S1B and data not shown), although complete excision of Lmo4 was observed in the epithelium of Shh-cre;Lmo4 fl/fl embryos (Additional file 1: Figure S1B). These results demonstrate that although Lmo4 is expressed in the embryonic lung, its role in the epithelium is not essential for normal lung morphogenesis.

Loss of Lmo4 affects lung repair after H1N1-induced lung injury
Given that Lmo4 is expressed in CD104-positive cells that have recently been described to participate in the regeneration of the epithelium after flu-mediated lung injury [22], we investigated whether Lmo4 was required for adult lung progenitor cell function. We first observed that under steady state conditions, the number of CD104 + cells was reduced in Shh-cre;Lmo4 fl/fl compared to Shh-cre;Lmo4 fl/+ control animals (Fig. 2a). However, when plated in colony formation assay on matrigel, the remaining CD104 + cells present in Shh-cre;Lmo4 fl/fl lungs had the same colony formation capacity as control animals (data not shown). These results suggested that loss of Lmo4 does not affect CD104 + progenitor cells colony forming capacity in vitro.
To further explore the role of Lmo4 in vivo we infected Shh-cre;Lmo4 fl/fl and control mice with H1N1 flu virus. Intra-nasal administration of murine adapted  [20]. In the damaged areas, proliferation is maximal at 11 days post-infection and is driven by CD104-positive keratin 5-positive distal basal cells [20][21][22]. Interestingly, Lmo4 is expressed in these regions of active proliferation with keratin 5-positive cells expressing Lmo4 (Fig. 2b). Co-staining for Lmo4 and p63, another marker of basal cells, was also observed (Fig. 2b). Shh-cre;Lmo4 fl/fl mice showed an increased sensitivity to H1N1 infection with only 68 % of animals surviving to 11 days post infection compared to 89 % in the control group (Fig. 2c, log rank test p = 0.028, n ≥ 27). In both genotypes, animals that survived to 11 days recovered and returned to normal health within a few days. Interestingly, however, loss of Lmo4 was associated with a significant reduction in the percentage of keratin 5 expressing cells and Ki67-positive proliferating cells after H1N1 infection (Fig. 2d). Reduced animal recovery (68 %) and lower proliferation in Lmo4-depleted animals that survive the injury, suggest that Lmo4 is required for the proliferation of lung epithelial cells for the repair of the injured lung but is not essential for progenitor cells activity after flu-mediated injury.

Loss of Lmo4 affects lung repair after naphthalene-induced lung injury
Lmo4 expression in club cells in adult airways suggested that it might play an important role in repair after airway injury [37]. Treatment of mice with naphthalene damages the club cells lining the bronchiole with maximal injury of the airways observed two days following naphthalene treatment [26,38]. Repair of the epithelium is driven by a population of variant club cells that are resistant to naphthalene-induced injury with newly regenerated epithelium observed one week after treatment [38]. In wild-type mice, although the majority of club cells are depleted two days after naphthalene injection, Lmo4 was detected in cells lining the airways at this time point (Fig. 3a). Interestingly, staining for the proliferation marker Ki67 revealed the presence of proliferative cells in the layer of flattened Lmo4-positive cells lining the basement membrane of the bronchiole four days following injury (Fig. 3a). Six days following injury, the number of proliferative cells increased and these cells co-expressed Lmo4 (Fig. 3a), suggesting that Lmo4 may play a role in lung epithelial cell proliferation after naphthalene injury. To explore the role of Lmo4 in airway repair following naphthalene injury, Shh-cre;Lmo4 fl/fl mice were treated with naphthalene and the lungs collected to analyse proliferation and airway repair. Lmo4 loss resulted in delayed repair with a reduction in the number of CC10-positive club cells five days post-naphthalene administration compared to control animals (Fig. 3b). BrdU immunostaining further showed a reduction in the number of proliferative cells in Shh-cre;Lmo4 fl/fl lungs compared to control ( Fig. 3c and d, Wilcoxon rank sum test p = 0.0002). However, the animals survived the injury and eventually, the airways of Shh-cre;Lmo4 fl/fl mice returned to normal (data not shown). These results were confirmed using Scgb1a1-cre ER ;Lmo4 fl/fl mice in which deletion of Lmo4 occurs specifically in club cells after tamoxifen administration [30] (Additional file 2: Figure S2A). Scgb1a1-cre ER ;Lmo4 fl/fl mice were first treated with corn oil or tamoxifen followed by naphthalene 2 weeks later. Analysis of BrdU incorporation in the lung five days after injury showed a significant reduction in the number of BrdU-positive cells in the airways of tamoxifen-treated mice (Fig. 3e, Wilcoxon rank sum test p = 0.039), consistent with the data observed in Shh-cre;Lmo4 fl/fl mice. These results suggest that Lmo4 promotes proliferation of epithelial cells after naphthaleneinduced injury but is dispensable for complete repair of the airways.

Loss of Lmo4 delays initiation of hyperplasia in K-Ras LSL-G12D/+ mice sensitized with naphthalene
Administration of naphthalene following K-Ras G12D activation results in earlier tumor onset and increased tumor burden due to activation of progenitor cells in response to injury [27]. Given that Lmo4 is involved in the regulation of the proliferation of epithelial cells after naphthalene injury, we assessed whether loss of Lmo4 would affect initiation of tumor growth in naphthalene-mediated sensitization of K-Ras G12D -driven tumors. We used K-Ras LSL-G12D model in which cre-mediated excision of the "STOP" cassette by intranasal administration of cre-expressing adenoviruses leads to activation of the oncogenic K-Ras G12D in the lung [28,39]. Upon Ad5-CMV-Cre infection, these mice initially develop adenomatous hyperplasia, which progresses to adenomas and adenocarcinomas [28]. K-Ras LSL-G12D/+ ;Lmo4 fl/fl , K-Ras LSL-G12D/+ ;Lmo4 fl/+ and K-Ras LSL-G12D/+ ;Lmo4 +/+ mice were infected with Ad5-CMV-cre resulting in activation of K-Ras G12D and excision of Lmo4 alleles in lung cells, followed one week later by naphthalene administration. Lungs were collected for analysis six days after naphthalene injection. At this time-point, areas of hyperplastic epithelium were observed around the airways of control animals but not in K-Ras LSL-G12D/+ ;Lmo4 fl/fl mice (Fig. 4a). Consistently, there was a marked decrease in the number of BrdU-positive cells in K-Ras LSL-G12D/+ ;Lmo4 fl/fl compared to K-Ras LSL-G12D/+ mice ( Fig. 4a and b, one-way ANOVA p = 0.03, n ≥ 3). However, when lungs were collected 11 weeks following naphthalene injury (12 weeks following Ad5-CMV-cre administration), the number of proliferating cells in the hyperplastic airways was indistinguishable between the genotypes (Fig. 4c and d, Wilcoxon rank sum p = 0.5895). Lung weight (data not shown) and tumor area in the parenchyme (Additional file 2: Figure S2B) were also similar between the groups. These results indicate that loss of Lmo4 delays initiation of hyperplasia after naphthalene injury but this effect is rapidly overcome by signaling pathways activated by K-Ras G12D .

Loss of Lmo4 does not affect tumor progression in K-Ras G12D -driven mouse model of lung cancer
We then assessed whether Lmo4 participated in tumor growth in the K-Ras LSL-G12D model in the absence of naphthalene sensitization. K-Ras LSL-G12D/+ , K-Ras LSL-G12D/+ ; Lmo4 fl/+ and K-Ras LSL-G12D/+ ;Lmo4 fl/fl mice were infected intra-nasally with Ad5-CMV-cre resulting in activation of  (Fig. 5a, log rank test p = 0.06). Histological examination and analysis of lung weight upon collection at ethical endpoint showed no significant difference in tumor burden in Lmo4-depleted lungs compared to control mice ( Fig. 5b and c, one-way ANOVA p = 0.33, n ≥ 13), although Lmo4 expression was reduced in the tumors as assessed by Western Blot (Additional file 2: Figure S2C). All tumors presented histological features of adenocarcinomas and detailed morphological analysis did not show any significant difference in tumor grade, number of mitosis, presence of atypical adenomatous hyperplasia or bronchial hyperplasia between any of the genotypes (data not shown). Immunostaining for TTF-1, a marker of adenocarcinoma, and Sox2, a marker present in early lesions originating from CC10positive cells and lost in advanced adenocarcinoma [40], revealed no difference between K-Ras LSL-G12D/+ ;Lmo4 fl/fl and K-Ras LSL-G12D/+ mice (Fig. 5d).
To evaluate whether loss of Lmo4 could affect tumour initiation, we collected mice of each genotype 12 weeks after Ad5-CMV-cre administration. Given that Lmo4 is essentially expressed in the airways, we quantified the number of BrdU-positive cells in hyperplastic airways and found no difference between Lmo4-depleted and control lungs ( Fig. 5e and f, Wilcoxon rank sum p = 0.2773). Analysis of the lung weight (data not shown) or tumor area (Additional file 2: Figure S2D) did not show any significant difference between the cohorts. These results indicate that Lmo4 is not essential for tumor cell proliferation and overall tumor growth in the K-Ras G12Dinduced mouse model of lung cancer.

LMO4 is expressed in human lung squamous cell carcinoma
Gene expression profiling analysis of lung tumors showed that LMO4 is highly expressed in advanced adenocarcinomas, which prompted us to analyse its role in K-Ras G12D mouse model of adenocarcinoma [15]. As we did not observe any effect of Lmo4 loss on tumor-free survival in this model, we decided to further determine LMO4 expression in the different subtypes of NSCLC in early stage disease. We performed immunostaining for LMO4 in NSCLC tissue-microarray (TMA) of lung cancer samples obtained from patients undergoing lung cancer surgery (stage I-II disease) comprising 34 squamous cell carcinomas, 40 adenocarcinomas and ten otherwiseclassified NSCLCs including large cell carcinomas. Scoring of LMO4 expression showed that LMO4 was significantly more expressed in squamous cell carcinomas compared to other tumor types ( Fig. 6a and b, Wilcoxon rank sum test, p < 0.001). Consistently, analysis of The Cancer Genome Atlas (TCGA) data showed an increase in LMO4 expression in lung squamous cell carcinomas (n = 224) compared to adenocarcinomas (n = 125) (Fig. 6c, one-way ANOVA p < 0.0001). These results suggest that LMO4 may play a more prominent role in the pathology of early stage squamous cell carcinoma than in adenocarcinomas. Depletion of Lmo4 in a mouse model of lung squamous cell carcinoma would enable to further explore this hypothesis.

Discussion
Identifying factors regulating lung progenitor cell activity following lung injury is instrumental to deciphering processes driving lung repair. With their distinct properties to damage specific cell subtypes and to activate different progenitor cells, H1N1 infection and naphthalene treatment constitute complementary approaches to evaluate the role of particular proteins in the regulation of lung repair. Our results show that Lmo4 participates in the regulation of lung epithelial cell proliferation in these two models of injury. Lmo4 is dispensable for epithelial repair and animal survival but its loss delays recovery and increases sensitivity to H1N1-mediated injury. We also show that Lmo4 participates in the onset of lung cell proliferation induced by naphthalene in the context of oncogenic K-Ras G12D , further suggesting that Lmo4 plays an important role in lung epithelial cell proliferation.
Lmo4 is expressed in the developing lung with its expression becoming restricted to the airway lineages during development. This pattern of expression of Lmo4 in the embryonic lung is reminiscent of the expression profile of the transcription factor Sox2 where Sox2 is expressed only in the proximal airways and is absent in the distal tips of the branching epithelium that expresses Sox9 [41,42]. Similar to Sox2, the expression pattern of Lmo4 becomes restricted to club cells and ciliated cells in the adult lung and is absent in the alveoli [42]. Given that half of Lmo4 -/mice present breathing difficulties and die shortly after birth [11] we investigated the role of Lmo4 during lung morphogenesis by specifically ablating its expression in the lung epithelium from E9.5. No lung defects were observed in the conditional knock-out animals and their survival to adulthood was similar to their littermate controls. We observed that Lmo4 is expressed in both the epithelium and the mesenchyme until E13.5 before becoming restricted to the epithelium. Deletion of Lmo4 specifically in the mesenchyme would be required to evaluate whether Lmo4 plays a critical role in the mesenchyme during early lung morphogenesis.
Lmo4 has been implicated in the proliferation of progenitor cells in normal neural crest development where knockdown of Lmo4 leads to loss of neural crest precursor formation at the neural plate border [5] prompting us to evaluate the role of Lmo4 in lung progenitor cell activity. Repair of the adult lung is mediated by different progenitor cells depending on the type of injury inflicted. Following naphthalene administration, a rare population of variant club cells that do not express Cyp2f2 is maintained and responsible for the repopulation of the cells lining the airways [26,30]. On the other hand, recent lineage tracing experiments showed that a rare population of keratin-5 positive basal cells present in the distal lung expand after flu injury to repair the lung [21] indicating that distinct progenitor cell populations are involved in the repair processes following naphthalene or H1N1 injury. Our results show that Lmo4 promotes epithelial cell proliferation in both models of lung injury. However, in vitro colony formation assay did not show any significant difference in the progenitor activity of Lmo4 conditional knockout cells compared to control cells, suggesting that Lmo4 may participate in cell proliferation but is not essential for lung progenitor cell function. Consistently, animals with Lmo4-depleted lungs manage to eventually recover from injury. Knock-down of Lmo4 has been shown to potentiate TGFβ signaling in HEK293 cells [10]. We previously showed that TGFβ inhibited lung epithelial cell colony formation capacity in vitro while SB431542, an inhibitor of TGFβ signaling, increased colony forming capacity in vitro [43]. Loss of Lmo4 in Shhcre;Lmo4 fl/fl mice may therefore increase TGFβ signaling resulting in reduced epithelial cell proliferation. Other factors have been implicated in the proliferation of lung progenitor cells such as Bmi1 which regulates bronchioalveolar stem cells (BASCs) expansion following naphthalene injury [44], p38α and PTEN which are involved in the expansion of BASCs [45,46]. Stabilisation of β-catenin has also been shown to accelerate repair and increase CC10 expression three days following naphthalene injury [47]. Factors regulating distal basal cell expansion after H1N1 injury are still undefined. Lmo4 most likely cooperates with other regulatory factors to enable progenitor cells expansion and repair of the injured lung.
Recent studies have investigated the cell of origin of the different subtypes of lung cancer. Induction of oncogene expression or loss of tumor suppressor genes in specific cellular compartments using adenoviral delivery or genetically modified mouse models showed that neuroendocrine cells were the most likely cell of origin of small cell lung cancer [48] while both CC10-positive club cells and alveolar type II cells are involved in the initiation of tumor growth in K-Ras LSL-G12D/+or K-Ras LSL-G12D/+ ;p53 +/--induced adenocarcinoma [40,49]. In these models, expansion of CC10-positive cells after naphthalene injury has been described [40]. We observed that Lmo4 is expressed in club cells and is present in remaining proliferating cells lining the airways after naphthalene injury. Interestingly, Lmo4 deletion reduced cell proliferation after naphthalene injury in K-Ras LSL-G12D/+ mice, suggesting that upon K-Ras G12D expression, Lmo4 is required for optimal proliferation of CC10-positive cells resistant to naphthalene injury. However this initial effect on cell proliferation is not observed 12 weeks following injury suggesting that Lmo4 is redundant for long-term Kras G12D -driven tumorigenesis. Using CMV-cre adenoviruses to activate K-Ras G12D expression and deplete Lmo4 expression, we targeted all cell types in the lung, including alveolar type II cells that are the predominant cell of origin in K-Ras G12D -induced adenocarcinoma [40,49] but do not express Lmo4. The lack of difference in overall survival between K-Ras LSL-G12D/+ ;Lmo4 fl/fl and K-Ras LSL-G12D/+ mice may therefore reflect the initiation of adenocarcinoma formation in alveolar type II cells.
Deletion of Lmo4 and activation of K-Ras G12D exclusively in CC10-positive cell would enable to resolve this hypothesis. However, given that the role of Lmo4 in progenitor cells is redundant, it is also likely that in the context of oncogenic transformation, Lmo4 is important in the early phase of transformation but is rapidly taken over by other factors controling transformed cell proliferation.
LMO4 is overexpressed in 50 % of breast cancer and 83 % of pancreatic adenocarcinoma [3,50]. In lung cancer, gene expression analysis showed that LMO4 is overexpressed in SCLC and advanced adenocarcinoma [15] but its expression has not been investigated in early stage disease in NSCLC. The association between LMO4 expression and patient outcome is still unclear. High LMO4 expression is associated with worse outcome in breast cancer [3] while in pancreatic adenocarcinoma, low LMO4 expression is associated with poor outcome [50]. In head and neck squamous cell carcinomas, LMO4 has been reported to be overexpressed at the invasive front of the tumor but its expression did not correlate with patient outcome [13,51]. In a cohort of 84 NSCLC resected specimen from patients with stage I and II disease, we found that LMO4 expression was significantly higher in squamous cell carcinoma compared to adenocarcinoma. The size of the cohort in our current study did not enable us to evaluate correlation with patient outcome. Further investigations will be necessary to evaluate whether LMO4 is a predictor of outcome in lung squamous cell carcinoma and whether it plays a role in squamous cell carcinoma tumor progression. Lung squamous cell carcinoma is mostly observed in smokers. Our results show that loss of Lmo4 delays repair in acute models of lung damage such as naphthalene and H1N1 injury. It would be of interest to explore whether Lmo4 plays a prominent role in the repair of the lung in long-term injury models observed after exposure to oxidant such as cigarette smoke or ozone [52]. A role for Lmo4 in the regulation of cells proliferation in long-term injury may provide information regarding its role in the pathology of lung squamous cell carcinoma.

Conclusions
Factors regulating lung repair following chemical or viral damage are still largely unknown. Here we show that the transcriptional regulator Lmo4 promotes lung epithelial cell proliferation and tissue repair following injury. Additionally, Lmo4 was found to modulate the early proliferative response observed after activation of oncogenic K-Ras G12D expression in the context of naphthalene sensitization, although loss of Lmo4 did not affect overall tumor-free survival. This work provides novel insights into factors regulating lung repair following tissue damage caused by chemicals and pathogens present in the environment.