REGULATORY DIVERSITY OF CANCER STEM CELL MARKER EXPRESSIONS UNDERSCORES INTERTUMORAL HETEROGENEITY WITH THERAPEUTIC IMPLICATION IN HEPATOCELLULAR CARCINOMA

Abstract

<p>Introduction: Expression of different functioning liver cancer stem cell (LCSC) markers in the <br>cancer cells of hepatocellular carcinoma (HCC) contributes to various subpopulations and may <br>have implications in drug treatment response. However, information of their consensus signaling <br>pathways and upstream upregulation is scarce. <br>Methods: We performed a comprehensive, unbiased examination of LCSC markers in same set <br>of patients’ HCC samples by flow cytometry, fluorescence-activated cell-sorting, and subsequent <br>transcriptomic analyses. Relevant assays (sphere formation assay, luciferase reporter assays, <br>quantitative-PCR, multi-color immunofluorescence) were performed using knockdown and <br>overexpression approaches to identify the upstream transcriptional regulation and downstream <br>consensus signaling genes on the concerned LCSC markers. <br>Results: We observed heterogeneous sub-populations denoted by different well-established <br>LCSC markers in different combinations in HCC samples. Among the 6 LCSC markers examined <br>(CD24, CD13, EpCAM, CD47, CD133, CD44), it was mainly CD24, CD13 and EpCAM contributing <br>to the heterogeneity, while the remaining ones had either ubiquitous or negligible expression. To <br>identify their signaling pathways, RNA-sequencing of HCC cells positive for individual markers <br>was performed and showed enriched gene activation regarding cell cycle phases, cytoskeleton <br>organization and kinase activity. On the other hand, there was also repression on innate immune <br>system. Presence of HCC cells with concomitant expression of multiple LCSC markers was also <br>detected. Among the cells showing CD24, CD13 and EpCAM triple expression, the concerned <br>LCSC markers showed concomitant upstream regulation by a combination of transcription factors <br>(ELF1, ELF4, CTCF, SP1 and FOXA1) to drive the expressions of these markers. Furthermore, upon <br>sorting the marker-high and marker-low HCC cells according to the three LCSC markers and <br>subsequent transcriptome sequencing on the sorted cells, we found diverse signaling pathways <br>with only a low degree of overlapping among these different individual LCSC markers in promoting <br>cancer stemness. <br>Conclusion: Well-established LCSC markers had specific signaling pathways. However, the <br>apparent lack of consensus pathways implicates the importance of combination drug treatment <br>against the LCSC populations in HCC treatment. More importantly, we demonstrated the upstream <br>regulatory diversity of concomitant LCSC marker signaling. The data may provide proof-of-<br>concept support to implicating a more effective therapeutic outcome by pinpointing the upstream <br>transcription regulatory machineries in a collective manner.<br></p>