Effects of suppressing notch signaling on phenotypes of HPV positive cervical cancer cell lines

Abstract

Notch encodes a protein family of transmembrane receptors that regulates differentiation, proliferation and apoptosis in various cell types. Upon receptor-ligand interaction. Notch are cleaved by a presenilin-dependent γ-secretase. This active form of Notch forms a complex with other proteins in the nucleus and functions as transcription activator. MAMLs are scaffold proteins essential for forming a stable transcriptional activation complex containing Notch in the nucleus. In humans, aberrant Notch1 expression was identified as a causative factor in the development of T-cell acute lymphoblastic leukemia and lymphoma, establishing it as protooncogene. Several reports indicated that Notch signaling is associated with transformation of HPV-positive epithelial cells but the exact roles of Notch signaling in HPV mediated cervical cancer is still controversial. Disruption of Notch1 HPV16 integrations may contribute to malignancy of cervical cancer. In contrast, other studies suggested that Notch signaling acted as a tumor suppressor in HPV-mediated transformation. In this study, we used γ-secretase inhibitor (GSI) or dominant negative MAML1 (DN-MAML1) to suppress Notch signaling pathway and examined the effects of Notch signaling in HPV-positive cervical cancer cell lines (HeLa, CaSki, SiHa). All cell lines expressed Notch1, and only CaSki constitutively expressed cleaved Notch1 (Vall 744). After GSI treatment for 4 days, complete abrogation of the cleaved Notch1 and increased level of total Notch1 were observed in CaSki. On the other hand, expression of Hes1, MAML1 and TP53 were increased. During this period, GSI treatment did not affect cell viability and cell cycle progression in all cell lines tested. Surprisingly, GSI treatment resulted in increased cell proliferation in CaSki. Upon DN-MAML1 retroviral transduction, Notch1 and Hels1 mRNA expression were decreased. IN addition, DN-MAML1 also induced proliferation of CaSki, resulting in formation of smaller colonies, but difference in cell cycle progression was not detected. These results indicated that suppression of Notch signaling by γ-secretase inhibitor or DN-MAML1 led to cell proliferation in HPV-positive cervical cancer cell lines.