RESUMO
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.
Assuntos
Antígenos Transformantes de Poliomavirus , Antígenos Virais de Tumores , Poliomavírus das Células de Merkel , Células-Tronco Neoplásicas , Proteínas de Ligação a Retinoblastoma , Humanos , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Carcinoma de Célula de Merkel/genética , Diferenciação Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Poliomavírus das Células de Merkel/genética , Células-Tronco Neoplásicas/virologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/virologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias Cutâneas/virologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismoRESUMO
Hepatitis B viral (HBV) persistent infection plays a significant role in hepatocellular carcinoma (HCC) tumorigenesis. Many studies have revealed the pivotal roles of N6-methyladenosine (m6A) in multiple cancers, while the regulatory mechanism in stemness maintenance of HBV persistent infection-related HCC remains elusive. Here, we demonstrated that the level of m6A modification was downregulated by HBV in HBV-positive HCC, through enhanced stability of ALKBH5 mRNA. More specifically, we also identified that ALKBH5 mRNA was functionally required for the stemness maintenance and self-renewal in the HBV-positive HCC, but dispensable in HBV-negative HCC. Mechanistically, ALKBH5 demethylated the m6A modification in the 3' untranslated region of the oncogenic gene SNAI2 to prevent the recognition of YTHDF2 therewith stabilize SNAI2 transcripts, contributing to cancer stem cell traits in HBV-positive HCC. Moreover, the expression of SNAI2 reversed the suppression of stemness properties by knocking down ALKBH5. In addition, ALKBH5/SNAI2 axis accelerates tumor immune evasion through activated ligand of immune checkpoint CD155. Our study unveiled that the ALKBH5 induces m6A demethylation of the SNAI2 as a key regulator in HBV-related HCC, and identifies the function of ALKBH5/SNAI2/YTHDF2 axis in promoting the stem-like cells phenotype and immune escape during HBV infection. IMPLICATIONS: HBV promotes HCC stemness maintenance through elevate m6A modification of SNAI2 in an ALKBH5-YTHDF2-dependent manner and increases the expression of the ligand of immune checkpoint CD155.