The Role of ZSCAN4 in Cancer Stemness

Abstract

Cancer stem cells (CSCs) represent a subset of cells within tumors that maintain the ability to self-renew, drive tumor heterogeneity, and contribute to therapeutic resistance and cancer recurrence. Over the past decade, embryonic factors have emerged as key regulators of cancer stemness and as therapeutic targets. Zscan4 is an early embryonic gene expressed in mouse embryonic and induced pluripotent stem cells where it plays critical roles in genomic stability, telomere maintenance, and pluripotency. Like other embryonic factors, ZSCAN4 is reactivated in cancer. In these studies, we define for the first time the role of ZSCAN4 in human CSCs of head and neck squamous cell carcinoma (HNSCC). We find that ZSCAN4 is enriched for in and marks the HNSCC CSC population. Induction of ZSCAN4 promotes the CSC phenotype, increases CSC factors and alters the epigenetic profile. Importantly, extreme limiting dilution analyses both in vitro and in vivo indicate that ZSCAN4 induction significantly increases the frequency of CSC. Consistently, ZSCAN4 depletion leads to loss of the CSC phenotype including CSC marker expression, the ability to form spheroids in non-adherent culture conditions, and hypersensitivity to genotoxic drugs. Furthermore, loss of ZSCAN4 severely impairs tumor growth in vivo. As our findings indicated that ZSCAN4 promotes the CSC phenotype, we next chose to study its regulation and turnover in cancer cells. Expression of Zscan4 is transient, and characterized by infrequent high expression peaks that are quickly down-regulated, suggesting its expression is tightly controlled. However, little is known about the protein degradation pathway responsible for regulating the human ZSCAN4 protein levels. We first determined the protein half-life of ZSCAN4 and elucidated the role of the ubiquitin proteasome system in ZSCAN4 degradation. Importantly, our data indicate an interaction between ZSCAN4 and the E3 ubiquitin ligase RNF20. RNF20 depletion stabilizes the ZSCAN4 protein half-life, suggesting that RNF20 negatively regulates ZSCAN4 stability. Due to the crucial cellular functions of ZSCAN4, these results have important implications in telomere regulation, stem cell biology, and cancer. Overall, our study suggests that ZSCAN4 plays a critical role in maintaining the undifferentiated state and survival of CSCs, indicating that ZSCAN4 is a potential therapeutic target in HNSCC