170. TGF? SIGNALING IN AN IN VITRO SEMINOMA MODEL

Abstract

Testicular cancer, the second most common malignancy in young men, has a 95% cure rate but can result in infertility or subfertility. Its incidence has increased significantly in recent decades (1). This cancer is thought to arise during embryogenesis, based on the persistence of embryonic germ cell markers such as Blimp1 (2), Oct3/4 (3) and Nanog (3) in adult seminoma cells. TCam2 cells are a recently characterised in vitro seminoma model (4).  We show by Q-PCR and immunofluorescence that they also express these early germ cell markers. TGFβ signaling plays a key role during germ cell development, and is implicated in the development of testicular cancers (5, 6). To investigate this further, we first determined whether the pathway is active in TCam2 cells. By Q-PCR we demonstrate expression of the TGFβ downstream transcription factors Smad 2, 3 and 4, and Activin type I and II receptors. Importantly, ActRIIA, which is undetectable in adult testicular germ cells, but readily detected in human foetal germ cells (7) and clinical seminoma samples (6), is readily detectable at both the mRNA and protein level in TCam2 cells. Furthermore, 24 hour treatment with Activin (5 and 50ng/ml) or BMP4 (5 and 50ng/ml) induces a 3-4 fold increase in ActRIIA mRNA levels, but not ActRIA, ActRIB or ActRIIB. Strikingly, in TCam2 cells BMP4 and to a lesser extent retinoic acid, but not activin, support survival and proliferation of TCam2 cells in the absence of serum. This is consistent with known roles of BMP4 and retinoic acid in enhancing murine foetal germ cell proliferation/self-renewal and survival (8, 9), and activin inhibition of foetal murine germ cell proliferation (10). This study is the first to demonstrate a functional response in seminoma cells consistent with their foetal germ cell-like identity and forms the basis for future mechanistic analyses of the role of TGFβ signaling in human testicular cancer.