Regulation of IGFBP-3 dependent apoptosis by HPV-16 E7

Human papillomaviruses (HPVs) of the high-risk type (e.g., HPV16) cause cancers in humans, including cervical and other anogenital cancers. E7 is the major transforming oncoprotein of the high-risk HPVs. On the basis of the current concept, several growth-suppressing pathways must be independently subverted by E7 in order to immortalize primary human epithelial cells. In accordance with this hypothesis, E7 has the ability to interact and hence functionally inactivate several cellular growth regulatory proteins. One of these E7 target proteins is insulin- like growth factor binding protein-3 (IGFBP-3), which belongs to the IGF-axis of growth regulatory proteins and occurs as secreted and intracellular, nuclear protein. It is suggested that both differentially localized IGFBP-3 forms can induce apoptotic cell death; however, the underlying mechanisms are poorly understood. IGFBP-3 mediated apoptosis can be inhibited by E7, which directly binds to IGFBP-3 and triggers its proteolytic cleavage. In order to better understand the molecular mechanisms for E7-induced cell immortalization, we will focus on the regulation of IGFBP-3-dependent apoptosis by E7. For this reason we will try to distinguish between the proapoptotic effects by means of secreted and intracellular IGFBP-3, to do this we shall delete the secretorial signal sequence within IGFBP-3 to generate a non-secretable and thereby intracellular form of IGFBP-3 (IGFBP-3DS). If ectopically expressed IGFBP-3DS does induce apoptotic cell death, we will determine the structural domains within IGFBP-3 that are necessary for this activity. Furthermore, we shall use an IGFBP-3 mutant that is unable to enter the nucleus in order to clarify whether nuclear localization of IGFBP-3 plays a role for IGFBP-3-dependent apoptosis. Next, we shall study the influence of E7 on the proapoptotic activity of IGFBP-3DS. We further intend to address the molecular mechanism underlying the proteolytic degradation of IGFBP-3. To this end, we shall analyze whether IGFBP-3 is polyubiquitinated under certain conditions, and whether this modification is necessary for its proteasome-dependent degradation. If IGFBP-3 does prove to be a target for the ubiquitin-proteasome pathway, we will map out the sequence requirements within IGFBP-3 necessary and sufficient for its degradation. Finally, we shall clarify whether multiubiquitination plays a role in E7 induced IGFBP-3-proteolysis.

Human papillomaviruses (HPVs) of the high-risk type (e.g., HPV16) can cause cervical cancer in humans. E7 is the major transforming oncoprotein of these viruses. On the basis of the current concept, several growth- suppressing pathways must be independently subverted by E7 in order to immortalize primary human epithelial cells. In accordance with this hypothesis, E7 has the ability to interact and hence functionally inactivate several cellular growth regulatory proteins. One of these E7 target proteins is insulin-like growth factor binding protein-3 (IGFBP-3), which belongs to the IGF-axis of growth regulatory proteins and occurs as secreted and nuclear protein. It is suggested that both differentially localized IGFBP-3 forms can induce apoptotic cell death; however, the regulation of nuclear IGFBP-3 is poorly understood. IGFBP-3 mediated apoptosis can be inhibited by E7, which directly binds to IGFBP-3 and triggers its proteolytic cleavage; however, the underlying mechanisms are also not well understood. In this project we made several important scientific advances contributing to the better understanding of the molecular mechanisms underlying the control of IGFBP-3 by cellular mechanisms and by E7. We demonstrated for the first time, that the stability of nuclear IGFBP-3 is regulated by polyubiquitination and, subsequently proteasome-dependent proteolysis and this contributes to the regulation of apoptosis induced by nuclear IGFBP-3. We show that E7 interacts with nuclear IGFBP-3 and accelerates its ubiquitin/proteasome- dependent proteolysis, leading to a dramatic reduction of the IGFBP-3 half live and that contributes to the regulation of apoptosis mediated by nuclear IGFBP-3. Finally, by the development of highly specific anti-HPV-16 E7 antibodies we could show for the first time in cervical biopsies that the levels of the HPV-16 E7 oncoprotein strongly increase in cervical carcinogenesis, underlining the important role of the E7 oncoprotein for cervical cancer. We compared the E7 levels with those of IGFBP-3 in cervical biopsies and found in 46 from 46 analyzed cervical carcinomas, that the intracellular IGFBP-3 levels inversely correlate with the E7 levels. While we found that IGFBP-3 was expressed in basal cells of the normal cervical squamous epithelium, IGFBP-3 was undetectable in the HPV-16 E7 expressing cervical cancers. These findings suggest that E7 probably also induces the proteolysis of IGFBP-3 in vivo.