AbstractAn in vitro premalignant transformation model was established to investigate molecular changes during early lung carcinogenesis. hTERT/Cdk4-immortalized human bronchial epithelial cells (HBECs), harboring few genetic alterations, were exposed to tobacco smoke carcinogens (benzo[a]pyrene, cigarette smoke condensate, or N-methylnitrosourea) for 15 weeks. Transformed cell lines, defined by their ability to form colonies in soft agar, were used as models for molecular investigations, associated with changes in morphology and phenotype. This study shows for the first time that hTERT/Cdk4-immortalized HBECs have the ability to bioactivate the model-PAH, B[a]P sufficiently for transformation. Transformed HBECs provided suitable models for further investigations of molecular changes involved in bioactivation and transformation. Transformation was found to be largely associated with changes in cellular morphology from an epithelial to a mesenchymal-like shape. Transformed cells also gained an increased migration capability. These phenotypic changes may characterize an activation of the regulatory, developmental program termed epithelial-to-mesenchymal transition (EMT). This program is important during embryogenesis, but is also activated during cancer progression. Gene expression analyses revealed that the transformed cells also had reduced expression of the E-cadherin-gene (CDH1), and an increased expression of the N-cadherin-gene (CDH2). This cadherin switch is considered a molecular hallmark of EMT. These results indicate that EMT may also be activated during premalignant transformation. Steroid receptor signaling pathways have been hypothesized to be involved in lung carcinogenesis, possibly through an interaction with carcinogen metabolism. In this study, increased expression of androgen receptor, estrogen receptor β, and partly also estrogen receptor α was shown to be associated with transformation. This supports the hypothesis of a possible role of steroid receptor pathways during lung carcinogenesis. The forkhead box A (FOXA) transcription factors are involved in regulating steroid receptor activity and may form a link between steroid receptors and carcinogen metabolism. Little is known about these transcription factors in lung cancer. This study shows that the expression of FOXA1 and FOXA2 was significantly changed during premalignant transformation. FOXA2 has been found to act as a suppressor of EMT in human lung cancer and in this study we observed both a downregulation of FOXA2 and the E-cadherin-gene (CDH1). In conclusion, steroid receptor pathways may be involved in carcinogen induced in vitro transformation of human lung cells, possibly through an interaction with carcinogen metabolism, but also through regulating EMT.