YAP1 Activation by Human Papillomavirus E7 Promotes Basal Cell Identity in Squamous Epithelia

Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly five percent of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector YAP1. The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 bind and degrade the tumor suppressor PTPN14. E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.

The Expression of Oct3/4A mRNA and Not Its Isoforms is Upregulated by the HPV16 E7 Oncoprotein

Oct3/4 a transcription factor is involved in maintaining the characteristics of cancer stem cells. Oct3/4 can be expressed differentially with respect to the progression of CC. In addition, Oct3/4 can give rise to three isoforms by alternative splicing of the mRNA Oct3/4A, Oct3/4B and Oct3/4B1. The aim of this study was to evaluate the mRNA expression from Oct3/4A, Oct3/4B and Oct3/4B1 in low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical cancer (CC) samples, and measure the effect of the HPV16 E7 oncoprotein on the mRNA expression from Oct3/4 isoforms in the C-33 A cell line. The expression levels of Oct3/4A, Oct3/4B and Oct3/4B1 mRNA were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in patients with LSILs, HSILs and CC. Additionally, C-33 A cells that expressed the HPV16 E7 oncoprotein were established to evaluate the effect of E7 on the expression of Oct3/4 mRNA isoforms. Oct3/4A (p=0.02), Oct3/4B (p=0. 001) and Oct3/4B1 (p<0. 0001) expression is significantly higher in patients with LSIL, HSIL and CC than in woman with non-IL. In the C-33 A cell line, the expression of Oct3/4A mRNA in the presence of the E7 oncoprotein increased compared to that in nontransfected C-33 A cells. Oct3/4B and Oct3/4B1 mRNA were expressed at similar levels among the different groups. These data indicate that only the mRNA of Oct3/4A is upregulated by the HPV16 E7 oncoprotein.

The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

Human papillomavirus type 16 circular RNA is barely detectable for the claimed transformation activity

The human papillomavirus type 16 (HPV16) E7 oncoprotein plays an essential role in cervical carcinogenesis and is encoded predominantly by a viral E6*I mRNA through alternative RNA splicing of a p97 promoter-transcribed bicistronic E6E7 pre-mRNA. Recently, Zhao et al. detected the HPV16 circular RNA circE7, which is generated aberrantly through backsplicing of the E6E7 pre-mRNA from two HPV16-positive cervical cancer cell lines, CaSki and SiHa. Based on their findings that HPV16 E7 was translated from circE7 and knockdown of circE7 in CaSki cells led to reduction of E7 oncoprotein, cell proliferation, and xenograft tumor formation, the authors claimed that circE7 is functionally important in cell transformation. We believe, however, that the reported circE7 function is overstated. We found that circE7 in CaSki cells is only 0.4 copy per cell and determine that the claimed circE7 function in the published report was resulted from off-targeting viral E7 linear mRNAs by their circE7 small interfering RNAs.

Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas

Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.

Non-Metabolic Functions of PKM2 Contribute to Cervical Cancer Cell Proliferation Induced by the HPV16 E7 Oncoprotein

Pyruvate kinase M2 (PKM2) mainly catalyzes glycolysis, but it also exerts non-glycolytic functions in several cancers. While it has been shown to interact with the human papillomavirus 16 (HPV16) E7 oncoprotein, the functional significance of PKM2 in HPV-associated cervical cancer has been elusive. Here, we show that HPV16 E7 increased the expression of PKM2 in cervical cancer cells. TCGA data analyses revealed a higher level of PKM2 in HPV+ than HPV− cervical cancers and a worse prognosis for patients with high PKM2 expression. Functionally, we demonstrate that shRNA-mediated PKM2 knockdown decreased the proliferation of HPV+ SiHa cervical cancer cells. PKM2 knockdown also inhibited the E7-induced proliferation of cervical cancer cells. ML265 activating the pyruvate kinase function of PKM2 inhibited cell cycle progression and colony formation. ML265 treatments decreased phosphorylation of PKM2 at the Y105 position that has been associated with non-glycolytic functions. On the contrary, HPV16 E7 increased the PKM2 phosphorylation. Our results indicate that E7 increases PKM2 expression and activates a non-glycolytic function of PKM2 to promote cervical cancer cell proliferation.