Characterization of the cancer stemness effect of gene MAEL in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, predominantly accounting for over 80% of cases. Due to the delayed diagnosis and limited treatment options for patients, HCC ranks as a priority leading cause of cancer-related death worldwide in a recent investigation. Thus, there is an unmet need to better understanding the mechanism of HCC progression, which may shed a light onlead to the the development of novel therapeutic strategies. The cancer stem cell (CSC) correlates with inferior survival outcomes and resistance to treatment in many solid tumors, including HCC. Limited studies reported the role of maelstrom (MAEL), the cancer/testis associated antigen in regulating cancer stemness-related genes and protein expression. However, the function of MAEL in CSCs remains elusive. Therefore, this thesis focuses on revealing the molecular mechanism for CSCs property maintaining by MAEL. TCGA transcriptome sequencing data of 371 primary HCC samples and 50 non-tumor samples identified that MAEL was significantly up-regulated in HCC. In HCC, over-expression of MAEL was associated with poor overall survival, male gender, . The knockout of MAEL by CRISPR-Cas9 significantly inhibited HCC cell proliferation, colony formation, self-renewal, and metastasis abilities in vitro and compromised HCC tumorigenicity and tumor initiating ability in vivo. MAEL knockout dramatically impeded HCC tumorigenicity and tumor-initiating ability in vivo. In CD133+ cells, MAEL exhibited tumor initiation ability compared to CD133- cells. We demonstrated that MAEL knockout increased Sorafenib-induced apoptosis in vitro.

To understand the underlying mechanism, through RNA-Seq of MAEL knockout and wildtype cells, it has been identified MAEL enriched in signaling pathways regulating stem cell pluripotency by KEGG pathways enrichment. RNA sequencing data and investigations by other researchers provided the clues for this line of research. RNA-Seq revealed that MAEL could promote PTGS2 transcription by messenger RNA (mRNA)level. PTGS2, which further activated protein kinase B (PKB/Akt) phosphorylation, resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 translocation by Akt phosphorylation, which in turn increased interleukin 8 (IL-8) transcription. Western blot indicated that knockout of MAEL resulted in suppression of stemness-associated gene Sox2, Klf4 and C-Myc expression being suppressed.

Interestingly, it demonstrated Sorafenib response in MAEL short hairpin RNA (shRNA) knockdown models relative to wild-type models. This finding added a new dimension to our understanding of the functional mechanism of MAEL, suggesting that MAEL-associated stemness in HCC may contribute to Sorafenib treatment resistance. These findings suggest that MAEL may be considered as a novel prognostic marker and therapeutic target for HCC in the future.