1. Incidence of Pulmonary Fibrosis in Lungs of CUX1 Transgenic Mice Scott Helgeson 1, Ethan Harris 2, Betty Herndon 1, Gregory Van Der Heuvel 3, Richard Baybutt 2, Agostino Molteni 1 1 UMKC School of Medicine, 2 Wheaton College, 3 Western Michigan University BACKGROUND AND INTRODUCTION CUX1 (CUT-like homeobox 1) genes are present in all metazoans. In humans, the CUX1 gene is a transcriptional repressor and has been shown to play a significant role in cell cycle progression (specifically G1-S phase), cell differentiation, and morphogenesis. CUX1 expression is responsible for the inhibition of cyclin kinase inhibitors p21 and p27, and the repression of these CKIs is associated with increased cell proliferation. Recent studies show a correlation between CUX1 overexpression and increased tumor growth in multiple cell cancer lines. (1,2,3) In our and other laboratories it has been previously reported that these mice develop severe polycystic kidneys, reduced spermatogenesis, liver fibrosis, and hepatomegaly (4,5), and a preliminary study also showed bronchiectasis and pulmonary inflammation (6). In this study, we investigated the impact of CUX1 overexpression on the fibrotic pulmonary process, in view of previous publications related to some fibrotic development in the lungs (6). METHODS Wild-type and transgenic mice constitutively expressing CUX1 were maintained and bred at Wheaton College To ensure genotype accuracy, tail biopsies of mice were regularly sent to Transnetyx for genotyping Mice were sacrificed by cervical dislocation following anesthetization with isofluorane as recommended by the Panel on Euthanasia of the American Veterinary Association Lungs were harvested following euthanasia and fixed with formalin until histological analysis could be performed Tissue was cut into 4 micrometers slices and stained with H&E and trichrome The histopathological damage was evaluated with H&E stained slides for inflammation, bronchiectasis, emphysema, vasculitis, and bronchitis Trichrome slides were evaluated by ImageJ analysis for assessment of the presence of collagen About 10 photographs were taken at 100 and 400 magnification for assessment of vascular thickening, severity of bronchiectasis, septal inflammation, and the presence of fibrosis RESULTS 1.Histological staining (HandE) of the lungs of CUX1 mice showed extensive inflammation of the septa with a few patchy areas of emphysema and severe bronchiectasis, particularly involving the medium and large sized caliber bronchi (fig 1B) 2.Mild septal inflammation, little bronchiectasis, and some collagen deposition was also evident in the wild type mice (fig 1A) 3.Trichrome staining of CUX1 mice lungs showed evidence of pulmonary fibrosis with collagen deposition throughout the organs with the highest concentration in the adventitia of peribronchial arteries and bronchial basal membranes (fig 2 C,D). ImageJ software indicated marked increase in collagen in the basal membrane of the bronchi, especially when bronchiectasis was present and the intensity of the collagen measured by ImageJ was 17.06% in the lungs of CUX1 mice versus 8.73% in the wild type (p<0.05) (fig 3) 4.Bronchial basal membranes were thickened. The bronchial epithelium presented areas of pluristratification alternating with areas of necrosis 5.The peribronchial arteries had large adventitia with scattered fibroblasts (fig 4) and thickening of the media with reduced lumen patency [64% in CUX1 versus 76% in wild type (p=0.015) and reduced media/adventitia ratio 42% vs 64% (p=0.018)] (fig 5) CONCLUSIONS Transgenic mice constitutively expressing CUX1 developed significantly higher amounts of inflammation, bronchiectasis, and fibrosis compared to wild type mice In addition, extensive collagen was found in the adventitia of the pulmonary vessels and airways This suggests that overexpression of CUX1 promotes lung pathology in addition to other organ hyperplasia in a similar manner demonstrated in previous studies of CUX1 overexpression in the kidney and liver of these transgenic mice We suggest that CUX1 primarily exerts an inhibitive effect on CKI’s p21 and p27 which are key to CKI’s involvement in arresting G1-S phase transition Further investigation is necessary to determine the specific mechanism through which CUX1 overexpression leads to the lung changes described Would some patients with polycystic kidney disease also have aberrant repression of p21 and p27 and develop pulmonary fibrosis? Would this pulmonary fibrotic damage eventually lead to neoplasia in the affected organ? TRICHROME SLIDES Fig 2 Representative sections of lung peribronchial arteries and bronchi of WTF (A,B) and CUX1 (C,D) mice – Trichrome 400x Fig 1 Representative sections of lungs of WTF (A) and CUX1 (B) mice – H and E 100x H AND E SLIDES Fig 4 Lumen Patency Fig 5 Media/adventitia ratio Fig 3 Image J Lung Fibrosis AB A B C D REFERENCES 1.Ramdzam, Z.M., Nepveu, A. CUX1, a haploinsufficient tumour suppressor gene overexpressed in advanced cancers. Nature Reviews Cancer 14, 673-682 (2014). 2.Zen, W.R. et al. Loss of heterozygosity and reduced expression of the CUTL1 gene in uterine leiomyomas. Oncogen 14, 2355-2635 (1997). 3.Zeng, W.R. et al. Refined mapping of the region of loss of heterozygosity on the long arm of chromosome 7 in human breast cancer defines the location of a second tumor suppressor gene at 7q22 in the region of the CUTL1 gene. Oncogene 18, 2015-2021 (1999). 4.Vanden Heuvel, G.B. et al. Hepatomegaly in transgenic mice expressing the homeobox gene CUT-1. Mol. Carcinog. 43, 18-30 (2005). 5.Cadieux, C. et al. Polycystic kidneys caused by sustained expression of Cux1 isoform p75. J. Biol. Chem. 283, 13817-13824 (2008). 6.Hariss, H. et al. Pathological and fibrotic changes in the lungs of CUX1 transgenic mice. 18 th International Colloquim on Lung and Respiratory Airways Fibrosis, Mont Tremblant, Quebec, Canada, Sept. 20-24, 2014. Acknowledgement: Funding: Wheaton College, Wheaton IL and Goldmacher Foundation (Dr. Gary Salzman) (University of Missouri – Kansas City School of Medicine, Kansas City, MO) Staining: Pathology Department, Truman Medical Center Poster Assistance: Mr. J. Moran, Office of Marketing and Communications UMKC-SOM