Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomavirus 16 (HPV 16) causes cancer. Two viral oncoproteins of HPV 16, E6 and E7, are consistently expressed in these cancers. HPV 16 E6 and E7 proteins target p53 and Rb family members, respectively, for degradation thus inactivating the functiond of these tumor suppressor proteins. Tumor development requires the acquisition of a blood supply, a process known as angiogenesis. Tumor suppressors negatively regulate angigogenesis. Expression of HPV 16 E6 and E7 together in human foreskin keratinocytes (HFKs) increases the level of angiogenic inducers vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). Further, conditioned media from such cells are sufficient to alter endothelial cell behavior both in vitro and in vivo. To determine the individual contributions of HPV E6 and E7 to angiogenesis, translational termination linkers (TTLs) were inserted into the coding region of E6 or E7. Following retroviral transduction of the mutated cassette into HFKs, the ability of E7 in the context of the E6TTL mutation (E6TTLE7) and E6 in the context of the E7TTL mutation (E6E7TTL) to induce VEGF and IL-8 was compared to the LXSN control retrovirus. E7 and, to a lesser extent E6, increased the expression of VEGF and IL-8. Migration of human microvascular endothelial cells was not induced using conditioned media from either E6 or E7 expressing cells. Since the increased levels of VEGF and IL-8 induced by HPV 16E6ETTLE7 were not sufficient to alter endothelial cell behavior, immunological depletion experiments were used to determine whether either angiogenic factor was required for HPV 16E6 and E7 together to induce HMVEC migration. Only VEGF was required. Preliminary data suggest that the ability of HPV 16 E7 to induce angiogenic factors is dependent upon degradation of a specific Rb family member.

Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.

Indiana University-Purdue University Indianapolis (IUPUI) / Cervical cancer is caused by the persistent infection of human papilloma virus (HPV) in the cervix epithelium. Although effective preventative care is available, the widespread nature of infection and the variety of HPV strains unprotected by HPV vaccines necessitate a better understanding of the disease for development of new therapies. A major tumor suppressing mechanism is the inhibition of cell division by tissue structure; however, the underlining molecular circuitry for this regulation remains unclear. Recently, the Yap transcriptional co-activator has emerged as a key growth promoter that mediates contact growth arrest and limits organ size. Thus, we aimed to uncover upstream signals that connect tissue organization to Yap regulation in the inhibition of cervical cancer. Two events that disrupt tissue structure were examined including the loss of the tumor suppressor LKB1 and the expression of the viral oncogene HPV16-E6. We identified that Yap mediates cell growth regulation downstream of both LKB1 and E6. Restoration of LKB1 expression in HeLa cervical cancer cells, which lack this tumor suppressor, or shRNA knockdown of LKB1 in NTERT immortalized normal human dermal keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion, and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). Interestingly, LKB1’s suppression of Yap activity required neither the canonical Yap kinases, Lats1/2, nor metabolic downstream targets of LKB1, AMPK and mTORC1. Instead, the scaffolding protein NF2 was required for LKB1 to induce a specific actin cytoskeleton structure that associates with Yap suppression. Meanwhile, HPV16-E6 promoted Yap activation in all stages of keratinocyte differentiation. E6 activated the Rap1 small GTPase, which in turn promoted Yap activity. Since Rap1 does not mediate differentiation inhibition caused by E6, E6 may play a role in promoting cell growth through Rap1-Yap activation rather than preventing growth arrest through the disruption of differentiation. Altogether, the LKB1-NF2-Yap and E6-Rap1-Yap pathways represent two examples of a novel phenomenon, whereby the structure of a cell directly influences its gene expression and proliferation.