Katerina Strati

Human papillomavirus E7 binds Oct4 and regulates its activity in HPV-associated cervical cancers

Octamer binding transcription factor-4 (Oct4), is highly expressed in stem cells and has indispensable roles in pluripotency and cellular reprogramming. In contrast to other factors used for cellular reprogramming, a role for Oct4 outside embryonic stem cells has been elusive and highly controversial. Emerging evidence implicates Oct4 in the carcinogenic process, but the mechanism through which Oct4 may be functioning in cancers is not fully appreciated. Here, we provide evidence that Oct4 is expressed in human cervical cancer and this expression correlates with the presence of the human papillomavirus (HPV) oncogenes E6 and E7. Surprisingly, the viral oncogenes can complement exogenously provided Oct4 in reprogramming assays, providing functional validation for their ability to activate Oct4 transcription in Mouse Embryonic Fibroblasts (MEFs). To interrogate potential roles of Oct4 in cervical cancers we knocked-down Oct4 in HPV(+) (HeLa & CaSki) and HPV(-) (C33A) cervical cancer cell lines and found that Oct4 knockdown attenuated clonogenesis, only in the HPV(+) cells. More unexpectedly, cell proliferation and migration, were differentially affected in HPV(+) and HPV(-) cell lines. We provide evidence that Oct4 interacts with HPV E7 specifically at the CR3 region of the E7 protein and that introduction of the HPV oncogenes in C33A cells and human immortalised keratinocytes generates Oct4-associated transcriptional and phenotypic patterns, which mimic those seen in HPV(+) cells. We propose that a physical interaction of Oct4 with E7 regulates its activity in HPV(+) cervical cancers in a manner not seen in other cancer types.

Decitabine Treatment Induces a Viral Mimicry Response in Cervical Cancer Cells and Further Sensitizes Cells to Chemotherapy

Purpose: To investigate the anti-cancer, chemosensitizing and/or immunomodulating effects of decitabine (DAC) to be used as a potential therapeutic agent for the treatment of cervical cancer (CC). Methods: Cervical cancer cell lines were treated with low doses of DAC treatment used as a single agent or in combination with chemotherapy. End-point in vitro assays were developed as indicators of the anti-cancer and/or immunomodulating effects of DAC treatment in CC cells. These assays include cell viability, cell cycle analysis, apoptosis, induction of a viral-mimicry response pathway, expression of MHC-class I and PD-L1 and chemosensitivity. Results: High and low doses of DAC treatment induced reduction in cell viability in HeLa (HPV18+), CaSki (HPV16+) and C33A (HPV−) cells. Specifically, a time-dependent reduction in cell viability of HeLa and CaSki cells was observed accompanied by robust cell cycle arrest at G2/M phase and alterations in the cell cycle distribution. Decrease in cell viability was also observed in a non-transformed immortal keratinocyte (HaCat) suggesting a non-cancer specific target effect. DAC treatment also triggered a viral mimicry response through long-term induction of cytoplasmic double-stranded RNA (dsRNA) and activation of downstream IFN-related genes in both HPV+ and HPV− cells. In addition, DAC treatment increased the number of CC cells expressing MHC-class I and PD-L1. Furthermore, DAC significantly increased the proportion of early and late apoptotic CC cells quantified using FACS. Our combination treatments showed that low dose DAC treatment sensitizes cells to chemotherapy. Conclusions: Low doses of DAC treatment promotes robust induction of a viral mimicry response, immunomodulating and chemosensitizing effects in CC, indicating its promising therapeutic role in CC in vitro.

A paradigm for post‐embryonic Oct4 re‐expression: E7‐induced hydroxymethylation regulates Oct4 expression in cervical cancer

Abstract The Octamer‐binding transcription factor‐4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post‐embryonic re‐expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)‐related than HPV‐unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4‐transcriptional output and Oct4‐related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4‐protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl‐binding proteins (MBD2 and MBD3), are determinants of Oct4‐driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the O ct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re‐expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re‐expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.