The retinoblastoma protein contributes to maintaining the stability of HPV E7 in cervical cancer cells

High-risk human papillomaviruses (HR HPV)-16 and -18, and other closely related subtypes, are associated with at least 90% of human cervical cancers. Cervical cancers and derived cell lines continuously express high levels of the HPV oncoprotein E7, known to degrade the tumor suppressor retinoblastoma protein (pRB). This E7-pRB interaction is important for the maintenance and progression of malignancy. In the case of HPV E6, substrate recognition has been reported to play an important role in stabilizing the viral oncoprotein; however, such regulation of E7 has so far not been investigated. Using biochemical, immunostaining, and clonogenic assays, we describe an intriguing role for pRB in the stabilization of E7 oncoprotein in HPV-positive cervical-cancer-derived cell lines. The knockdown of pRB expression by RNA interference results in a significant decrease in the levels of E7 protein in CaSki, SiHa, HeLa, and C-4 I cells. We show that pRB knockdown regulates HPV E7 at the transcription and protein levels, and significantly reduces the half-life of E7 protein by at least twofold in SiHa and HeLa cells. We also demonstrate that the destabilization of E7 caused by pRB knockdown results in significant inhibition of cell proliferation and colony formation of HPV-16 and -18 E7-positive SiHa and HeLa cells. Furthermore, the expression of wild-type pRB in pRB-depleted cells significantly restored E7 levels. Therefore, we propose that pRB, in addition to being a degradation target for HPV E7, is crucial for its stabilization.IMPORTANCEThe human papillomavirus (HPV) viral proteins E6 and E7 cooperatively contribute to tumorigenesis by disrupting cellular targets. These oncoproteins are degraded via the proteasome pathway; however, they are continuously expressed in cervical cancer cell lines. The retinoblastoma protein, pRB, is a degradation target of high-risk (HR) HPV E7 oncoprotein. Several studies have shown that the binding of E7 to pRB is important for its E7-mediated inactivation and demonstrated how pRB protein levels respond to the presence and absence of E7. However, the modulatory role of pRB on E7 protein levels has so far not been reported. Here, we report a novel regulatory relationship between E7 and pRB. We found that the continuous expression of pRB is critical for E7 stabilization. We demonstrate that this pRB-related E7 destabilization occurs in part through enhanced protein turnover. Thus, our findings provide new insights into the importance of the E7-pRB interaction in driving tumorigenesis.