The HPV-18 E7 CKII phospho acceptor site is required for maintaining the transformed phenotype of cervical tumour-derived cells

Abstract

The Human Papillomavirus E7 oncoprotein plays an essential role in the development and maintenance of malignancy, which it achieves through targeting a number of critical cell control pathways. An important element in the ability of E7 to contribute towards cell transformation is the presence of a Casein Kinase II phospho-acceptor site within the CR2 domain of the protein. Phosphorylation is believed to enhance E7 interaction with a number of different cellular target proteins, and thereby increase the ability of E7 to enhance cell proliferation and induce malignancy. However, there is little information on how important this site in E7 is, once the tumour cells have become fully transformed. In this study, we have performed genome editing of the HPV-18 E7 CKII recognition site in C4-1 cervical tumour-derived cells. We first show that mutation of HPV18 E7 S32/S34 to A32/A34 abolishes CKII phosphorylation of E7, and subsequently we have isolated C4-1 clones containing these mutations in E7. The cells continue to proliferate, but are somewhat more slow-growing than wild type cells, reach lower saturation densities, and are also more susceptible to low nutrient conditions. These cells are severely defective in matrigel invasion assays, partly due to downregulation of matrix metalloproteases (MMPs). Mechanistically, we find that phosphorylation of E7 plays a direct role in the ability of E7 to activate AKT signaling, which in turn is required for optimal levels of MMP secretion. These results demonstrate that the E7 CKII phospho-acceptor site thus continues to play an important role for E7’s activity in cells derived from cervical cancers, and suggests that blocking this activity of E7 could be expected to have therapeutic potential.

Human papillomavirus E6 and E7: What remains?

“…Once the cervical cells become malignant, both E6 and E7 are required to maintain the phenotype and promote cell survival [ 64 , 65 ], but it is still not clear which, of all the pathways targeted by the two oncoproteins, are essential in doing this. However, the CKII phospho-acceptor site in HPV-18 E7 does seem to be required [ 66 ]. CKII phosphorylation of E7 is needed for the induction of cell transformation [ 67 ], and increases E7's affinity for several targets [ [68] , [69] , [70] ]; cervical cancer cells with gene-edited abolition of E7's CKII recognition site are severely compromised in proliferation in low-nutrient conditions and in invasion ability [ 66 ].…”

“…However, the CKII phospho-acceptor site in HPV-18 E7 does seem to be required [ 66 ]. CKII phosphorylation of E7 is needed for the induction of cell transformation [ 67 ], and increases E7's affinity for several targets [ [68] , [69] , [70] ]; cervical cancer cells with gene-edited abolition of E7's CKII recognition site are severely compromised in proliferation in low-nutrient conditions and in invasion ability [ 66 ]. These data suggest the potential of therapeutically targeting this CKII phospho-acceptor site, and, CKII inhibitors appear promising in clinical trials [ 71 , 72 ].…”

“…The contribution of insulin-activated transcription has recently come to the fore; stimulation of the insulin receptor leads to transactivation of a number of genes via recruitment of p300 (a target of E7 - [ 47 ]) and TIP60 (an E6 target - [ 45 ]) leading to chromatin modification and upregulated transcription of genes involved in glucose metabolism [ 128 ]. The role of nutrients in the tumour and cancer environment is very relevant: for example, mutation of the E7 CKII site has no deleterious effect in a nutrient-rich environment, and the effects are only seen when cancer cells are starved of nutrient [ 66 ]. In addition, HPV-18 E6's PBM-dependent binding to Sorting Nexin 27 (SNX27) modulates SNX27's interaction with its cargoes, including the GLUT1 glucose transporter, and leads to increased cellular glucose uptake [ 129 ].…”

CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function

“…As well as regulating E1-E2 functions, CK2 can also regulate the function of E7 proteins. Phosphorylation of a CK2 consensus sequence on E7 is important for E7 degradation of p130 and the promotion of S-phase in differentiated keratinocytes (97), and a HPV18 E7 CK2 target residue is required for maintaining the transformed phenotype of cervical cancer cells (98). Overall, this critical role of CK2 during multiple stages of viral life cycle supports initiatives to investigate the anti-viral and therapeutic effects of CK2 inhibition on HPV infections and disease.…”

miR‑4417 targets lncRNA PSMG3‑AS1 to suppress cell invasion and migration in cervical squamous cell carcinoma

“…Human papillomavirus (HPV) infection is a major risk factor for cervical cancer ( 3 ). With the increased understanding of the molecular mechanisms of HPV infection ( 4 , 5 ), as well as the popularization of HPV screening and vaccination ( 6 , 7 ), the incidence and mortality rates of cervical cancer have significantly dropped over the past decades ( 6 , 7 ). However, the HPV screening rate in developing countries, such as China, remains low, and most patients with cervical cancer are diagnosed at advanced stages and have a poor survival ( 8 ).…”