Ectopic expression of a small cell lung cancer transcription factor, INSM1 impairs alveologenesis in lung development

For (tetO)₇CMV-INSM1 mice, a human INSM1 full-length cDNA (2.8 kb) was sub-cloned into a pBI-EGFP Tet vector containing the CMV promoter and tetracycline response element. The transgenic animal model was generated from Gene Targeting & Transgenic Facility, University of Connecticut Health Center (Farmington, CT). Two lines of transgenic mice bearing (tetO)₇CMV-INSM1 transgene were generated. The lung-specific Dox inducible CCSP-rtTA^-/tg transgenic line was obtained from Jackson laboratory. Bi-transgenic mice, named INSM1/rtTA, were generated by crossing (tetO)₇-CMV-INSM1 and CCSP-rtTA^-/tg. Wild type littermates lacking either rtTA or INSM1 allele were used as control. Transgenic mice were genotyped by PCR using genomic DNA from tail of fetal or postnatal mice. PCR primers for transgenes were: for CCSP-rtTA, forward primer 5’-ACTGCCCATTGCCCAAACAC-3’; reverse primer: 5’-AAAATCTTGCCAGCTTTCCCC-3’, for (tetO)₇CMV-INSM1, forward primer: 5’-CCTTGTACAACCGACAGCTC-3’, reverse primer: 5’-GAGTGAGCTGATACCGCTCG-3’. The PCR amplification was performed as follow: denatured at 95 °C for 3 mins followed by 35 cycles of amplification at 95 °C for 30s, 58 °C for 30s and extension at 72 °C for 30s. Animals were maintained in a pathogen-free vivarium in filtered cages according to the protocol approved by Institutional Animal Care and Use Committee from the Research Institute for Children, Children’s Hospital in New Orleans. All mice were maintained in C57BL/6 background. Dams bearing double transgenes were fed with Dox food (200 mg/kg; Bio Serv co., Frenchtown, NJ) for various time spans.

A human normal bronchial epithelial cell line, BEAS-2B was obtained from American Type Culture Collection. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high-glucose) with 10 % fetal calf serum (Atlanta Biological Inc., Norcross, Georgia), 1X Pen/Strep (10,000 IU penicillin and 10,000 ug/ml streptomycin) (Mediatech, Inc., Manassas, VA.) in a 5 % CO₂ incubator at 37 °C.

The 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS proliferation assay was carried out according to the manufacturer’s protocol (Promega Co., Madison, WI). The cells were seeded and infected with Ad-LacZ or Ad-INSM1 virus in a serum-free medium for 24 h and then cultured in Dulbecco’s modified Eagle’s medium (DMEM, high-glucose) with 10 % fetal calf serum. After culturing for 48 or 60 h, CellTiter 96® AQ_ueous One Solution Reagents were added into the culture medium and then incubated at 5 % CO₂, 37 °C for 4 h. The amount of soluble formazan produced by cellular reduction of MTS was measured for absorbance at 490 nm using a microplate spectrophotometer to calculate the cell viability.

Lung RNAs were extracted using TRIzol reagent (Life Technologies, San Francisco, CA) following the manufacturer’s instruction. RNA was treated with 2 units of DNase (Promega Co., Madison, WI) at 37 °C for 30 min to remove residual genomic DNA. Total lung RNA was used as template to synthesize cDNA by High Capacity RNA-to-cDNA™ Kit (Life Technologies, San Francisco, CA) following the manufacturer’s protocol. RNA was reverse transcribed and analyzed by PCR and/or real-time PCR for the expression of INSM1 and cyclin D1. The relative RNA concentration of the target gene was normalized to the concentration of the housekeeping gene, GAPDH. Primers for INSM1: INSM1-440aa 5’-ACGGAATTCTGCCACCTGTGCCCAGTGTGCGGAGAG-3’, INSM-510aa 5’-CACCTCGAGCTAGCAGGCCGGGCGCACGGGCACCTGCAG-3’ Primers for cyclin D1, forward 5’-TGCCTACAGCCCTGTTACCT-3’ and reverse 5’-ACTTTGCAGGACAGATCCCG-3’. Total RNA (20 ug) was separated on 1 % agarose/formaldehyde gel. The gel was transferred to a nitrocellulose membrane for 3 h in 20X SSC and then UV cross-linked. The membrane was pre-hybridized in Express Hybe Solution (Clontech, Mountain View, CA) for 1 h followed by hybridization in the same solution with ³²P-labeled INSM1 probe, washed, and exposed to autoradiography.

Cell lysates were extracted with the lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1 mM DTT, 0.2 mM PMSF, 1 ug/ml aproptinin, 1 ug/ml leupeptin, 1 mM Na₃VO₄ and 1 mM NaF), separated on 10 % SDS-PAGE gel, and transferred onto the nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was blocked with 5 % BSA in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1 % Tween-20), probed with specific primary antibody at 4 °C overnight, and bound with HRP-conjugated secondary antibody (Bio-Rad) at room temperature for 1 h. The membrane was developed with a chemi-luminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA), and the blot was autographed onto a X-ray film (Fuji Photo Film Co., Japan).

Human small cell lung carcinoma tissue array (LC802b) was used in this study. Each specimen collected from any clinic was consented to by both hospital and individual. Discrete legal consent form was obtained and the rights to hold research uses for any purpose or further commercialized uses were waived (US Biomax, Rockville, MD). An IRB exemption (#8885) was obtained from Louisiana State University Health Sciences Center, New Orleans. Mouse tissue sections were prepared from fetal (E17.5), neonatal (newborn and postnatal days 7, PN7), and 3-week old mice (PN21) fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C. Tissue sections were stained by hemotoxylin and eosin (H&E staining) for the histopathology study. For immunohistochemistry, sections were blocked with 5 % BSA in PBS and incubated with either anti-CCSP, synatophysin, or cyclin D1 antibody (Cell Signaling Technology, Beverly, MA) at 4 °C overnight. Then, the slides were incubated with secondary antibodies conjugated with HRP for 1 h at room temperature and developed using a diamino-benzidine (DAB) histochemistry kit (Life Technologies, San Francisco, CA). For immunohistochemical staining of INSM1, anti-INSM1 antibody (Santa Cruz biotechnology, Inc., CA) with a MACH 3 biotin free polymer detection kit was used on the human tissue array slide and Mouse-on-Mouse HRP-polymer bundle kit were used on mouse tissue sections (Biocare Medical, Concord, CA).

For preparing the mouse lung samples, we euthanized mice with overdosed ketamine/xylazine, open the chest cavity, removed the sternum and ribs to expose the heart. We inserted the butterfly needle into the right ventricle and nicked at the right atrium to build a direct route to pulmonary circulation for lung perfusion and fixation with cold PBS and formalin. After perfusion, we injected 2 ml of 10 % formalin into trachea to fix and inflate the lung. The dissected lungs were measured by weight. The preserved lung samples were subjected to histological analysis. Lung tissue sections were stained with hematoxylin and eosin. Sections were visually scanned for position-matching regions compared with controls. At least four representative lobes were selected from each animal. A Kodak MI image system was used to measure terminal air space area. We drew the outline of the alveoli and used software to measure the area size at 200X magnification. For each sample, we measured 4 fields and each field at least 20 alveoli. The air spaces were distinguished from tissue based on intensity and the number of pixels acquired for each air space when converted to square micrometers.

The cells were seeded and infected with Ad-LacZ virus or Ad-INSM1 virus for 48 or 96 h. Cells were collected and fixed with cold 70 % ethanol at 4 °C overnight. The fixed cells were washed twice with PBS and then the cells were incubated in PI staining solution (PBS with 0.2 mg/mL DNase-free RNase, 0.1 % Triton X-100, and 1 mg/mL propidium iodine) at room temperature for 30 min before analysis on the flow cytometer.

Small cell lung cancer tumors are derived from pulmonary NE cells (PNECs), therefore their antigenic profile coincides with that of NE cells. In this study, we used immunohistochemical staining to examine 35 cases of different clinical stages of small cell lung cancer and 5 normal lung tissues for INSM1 expression. All the small cell lung cancer tissues were strongly positive for INSM1. INSM1 signal was not detected on normal adjacent tissues from lung cancer patients or normal lung tissues (Fig. 1). The expression pattern of INSM1 in NE lung cancer is consistent with the previous Northern blot analysis that revealed INSM1 mRNA is highly expressed in nearly 100 % of small cell lung carcinomas (SCLC) cell lines but not in normal adult lung tissues [6, 7]. Here, we showed that the INSM1 protein is highly over-expressed in 35 SCLC tumor tissues confirming that INSM1 is a specific and sensitive NE marker of small cell lung cancer. However, the functional role of INSM1 in NE lung cancer or normal lung in PNEC development is still unclear.

In order to determine the effect of INSM1 in normal lung development, we used a conditional lung-specific INSM1 transgenic mouse model. Transgenic Tet-on-INSM1 responder mice were bred with the lung-specific, club cell secretory protein (CCSP) promoter-rtTA activator mice to generate bi-transgenic progeny carrying both alleles, CCSP-rtTA and Tet-on-INSM1 (Fig. 2a). In this bi-transgenic model, INSM1 expression is induced by binding the tetracycline analogue Dox to rtTA, which in turn activates the Tet-on-CMV promoter and the transcription of INSM1 gene. Dox containing food was fed from the initial mating day to the weaning day (postnatal day 21, PN21) to ensure the full effect of INSM1 expression during lung development. Lung samples were collected at embryonic day (E) 17.5, newborn (PN0), and 3-week wean day (PN21). INSM1 was selectively expressed in a subset of respiratory epithelial cells, bronchial, and type II epithelial cells of lung tissues. The ectopic over-expression of INSM1 was spatially and temporally under the control of the lung specific CCSP-promoter and Dox. Two Tet-on-INSM1 transgenic lines (named 14-4-5 and 14-2-2) were generated and included in this study. Previous studies indicated that the CCSP-promoter directs rtTA transgene expression as early as post-conception day 14, E14 [9]. To ensure the fidelity of our transgenic system in regulating INSM1 expression, we treated the bi-transgenic (INSM1/rtTA), single transgenic (INSM1 or rtTA), and wild type mice with Dox food from the beginning of conception day. At each time point, E17.5, newborn (PN0), postnatal day 7 (PN7), and postnatal day 21 (PN21), the lung tissues were collected and subjected to Northern blot analyses, RT-PCR, real-time PCR, and immunohitochemical staining for INSM1 expression (Fig. 2). The results revealed that Dox food induced respiratory epithelium-specific INSM1 over-expression in the bi-transgenic mice as compared to single transgene, or wild-type littermates (Fig. 2b and e). INSM1 over-expression was detected in both lines of bi-transgenic lung tissues under Dox induction. Without Dox induction there was no INSM1 signal in bi-transgenic lung tissues (Fig 2c). The INSM1 expression level varies in different bi-transgenic animals (Fig. 2d). Since the over-expression of INSM1 is under the control of CCSP-promoter, the immunohistochemical staining showed that INSM1 over-expression was co-localized with CCSP expressing cells (Fig 2f). We performed the immunohistochemical staining of transgenic mouse tissues with anti-CCSP, anti-INSM1, or anti-synaptophysin antibody using the consecutive tissue sections from PN21 Bi-Tg, and PN7 CTRL lungs (Fig. 2e, f, g). We observed that a few bronchiolar epithelial cells with synaptophysin signal are CCSP-positive on the CTRL mouse lung section (Fig. 2g). On PN21 Bi-Tg lung section, INSM1-positive cells are also CCSP positive (Fig. 2f). We detected ASCL-1 and INSM1 expression on the consecutive tissue sections from E17.5 wild type and Bi-Tg lungs (Fig. 2h). The ASCL-1 positive cells are INSM1-positive. Synaptophysin and ASCL-1 are well known NE cell markers and were used as PNEC markers. The bi-transgenic lung was subjected to a time course study (Fig. 3a). The INSM1 transcript is expressed in all four time points, from E17.5, PN0, PN7, to PN21. Among the lung tissues that we collected from both lines of animals, the expression of transgenic INSM1 was significantly increased with age at the RNA level (Fig. 3b). The immunohistochemical staining of INSM1 showed that the over-expression of INSM1 on bronchial epithelial cells of bi-transgenic mice is consistently positive from embryonic stage (E17.5) until PN21. However, INSM1 expression in normal lung is only scarcely detected in E17.5 fetal lung (Fig. 3c). Although over-expression of INSM1 mRNA was increased by age, the protein levels were not significantly increased in a time-dependent manner.

We examined the lungs for any gross morphological abnormalities. Samples from PN21 mice revealed that the lung size of bi-transgenic mice is 40 % larger than the control littermates (Fig. 4a and b). However, the size is not significantly different in the lungs collected from E17.5 or PN0 among the bi-transgenic, single transgenic, and wild type mice (data not shown). Similarly, histological analysis of the pulmonary structure at E17.5 shows no defect in lung morphology (Fig. 4c and d). In contrast, the histopathological data revealed that the alveolar space in the bi-transgenic mice were significantly larger than those in the single transgenic or wild-type littermates at PN21 (Fig. 4e). After quantification with Kodak MI SE software and statistical analysis, the average alveolar space of the bi-transgenic mice is five times larger than control (Fig 4f). This observation indicates that ectopic expression of INSM1 disrupts alveolar septation that causes air space enlargement. It is likely that the INSM1 expression interrupts the last alveolar stage development in the bi-transgenic animal lung.

Immunohistochemical staining of control or bi-transgenic lung for cyclin D1 revealed that bi-transgenic lung has a weaker signal in the nuclei of bronchial epithelial cells and respiratory bronchiole epithelial cells (Fig. 5a-f). The results indicate that over-expression of INSM1 reduces cyclin D1 expression in club cells and CCSP-promoter active bronchial epithelial cells suggesting the reduction of cyclin D1 expression could cause cell cycle arrest and decreased cell proliferation.

Down regulation of cyclin D1 causes cell cycle arrest and interferes with bronchiolar epithelial cell proliferation. We used Ad-INSM1 or Ad-LacZ to infect a normal bronchial epithelial cell line, BEAS-2B. The results showed that over-expression of INSM1 caused the decrement of cyclin D1 (Fig. 5g). In vitro data of INSM1 suppressing cyclin D1 expression is consistent with the observation in our bi-transgenic mouse model where reduced cyclin D1 expression was seen in bronchial epithelial cells and club cells. To further support our hypothesis on the functional effects of INSM1 on bronchial epithelial cell growth, we over-expressed INSM1 in BEAS cells for 24 h in serum-free medium followed by serum stimulation at various time points and analyzed the cell growth with a MTS assay and flow cytometric analysis with propidium iodide staining. The MTS assay showed that INSM1 caused cell death starting at 48 h, as compared to the vehicle treated group. A statistically significant decrement of cell viability was found at 60 h (O.D₄₉₀ = 1.091 v.s 1.58) and 96 h (O.D₄₉₀ = 1.61 v.s 2.21) (Fig. 6a). The Ad-INSM1 infected cells showed reduced cell proliferation, as 32.86 % of the total population was found in the G₀/G₁ region as compared to 16.9 % of control Ad-LacZ infected cells (Fig. 6b). In addition, the G₂/M population of cells in the Ad-INSM1 infected sample decreased to 40.98 % as compared to the Ad-LacZ control group, 54.18 %. This result suggests that over-expression of INSM1 caused cell cycle arrest at the G₀/G₁ phase probably through the reduction of cyclin D1 expression.

Human small cell lung carcinoma tissue array (LC802b) was used in this study. Each specimen collected from any clinic was consented to by both hospital and individual. Discrete legal consent form was obtained and the rights to hold research uses for any purpose or further commercialized uses were waived (US Biomax, Rockville, MD). An IRB exemption (#8885) was obtained from Louisiana State University Health Sciences Center, New Orleans.

Animals were maintained in a pathogen-free vivarium in filtered cages according to the protocol approved by Institutional Animal Care and Use Committee from the Research Institute for Children, Children’s Hospital in New Orleans.

Not applicable.

Data and materials will be available for public upon request.