PD2-1-8: Promotion of Squamous Differentiation and Tumorigenesis in Mouse Lung Expressing Human Keratin 14

Abstract

Squamous cell carcinoma accounts for 20% of all human lung cancers, and is strongly linked to cigarette smoking. Historically it has been the first lung cancer associated with distinct premalignant intraepithelial changes that evolve through basal cell hyperplasia, squamous metaplasia and increasing dysplasia to carcinoma in situ (CIS) and invasive carcinoma. However, it has been difficult to mimic this sequence in genetically engineered mice (GEM). Because lung squamous cell carcinoma, its progenitor cells (basal cells) and precursor lesions express high levels of human keratin 14 (hK14), we hypothesized that K14 is critical for squamous differentiation and carcinogenesis in the pulmonary epithelium that normally lacks squamous differentiation. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous features and K14 immunoreactivity, hK14 gene was constitutively expressed in mouse airways using Clara cell specific 10kDa protein (CC10) promoter. In order to investigate the effect of hK14 on chemically-induced lung tumorigenesis, mice were given tobacco-specific nitrosamine NNK (4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol), a major systemic carcinogen in cigarettes by intraperitoneal injections (100mg/kg body weight, 3 times). To further explore the role of hK14 in carcinogenesis, CC10-hK14 mice were crossed with the previously established CC10-SV40 TAg mice, a model for aggressive lung carcinomas. CC10-hK14 transgenic (TG) mice developed normally except at the age of 1 year they were 20% lighter (p= 0.014) than their wild type (WT) littermates. Light and electron microscopic examination of TG mice failed to show any evidence for overt squamous features in the airways such as keratinization, intercellular bridges or tonofilaments. However, by RT-PCR, quantitative-RT-PCR and immunohistochemistry (IHC), TG mouse lungs were positive for hK14, while wild type (WT) lungs did not show any expression. Notably, we also detected variable levels of the squamous differentiation markers involucrin, loricrin, small proline rich proteins and transglutaminase 1, an enzyme that mediates cornified cell envelope formation, a hallmark of squamous differentiation in TG lungs, while WT lungs were consistently negative. By 48 weeks the lungs of NNK-exposed TG mice almost tripled in weight (p=0.011), contained larger tumors and revealed a three-fold increase (p=0.002) in tumor multiplicity compared to the lungs of their WT littermates. No tumors were found in saline-exposed control mice. The bi-transgenic CC10-hK14-TAg mice became moribund or died of massive lung tumors on average at 18 weeks of age. Their lungs typically increased five-fold in weight and contained hK14 by RT-PCR, while the lungs of CC10-SV40 Tag mice had smaller tumors that were negative for hK14. While the tumors in TG mice were adenomas or adenocarcinomas, they expressed involucrin by IHC. Constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, which normally lacks K14 containing cells. Furthermore, it markedly enhances the progression of both chemically- and SV40 TAg- induced tumors. Our study is significant because it provides a novel in vivo model for a common premalignant pulmonary lesion in humans, characterized by hK14 expression and squamous differentiation. It will also be a potentially useful pre-clinical tool for chemoprevention research.