Metastatic Hepatoblastoma Cells Exhibit a Distinct Cancer Stem Cell-like Phenotype Which Is Attenuated by PIM Inhibition

Background: Stem cell-like cancer cells (SCLCCs) are thought to be responsible for metastatic dissemination and drug resistance. We have previously established a metastatic human hepatoblastoma cell line that exhibited enhanced tumorigenicity and invasiveness, resistance to cisplatin, and increased PIM3 kinase expression, compared to the parent hepatoblastoma cell line. We sought to assess whether these metastatic cells exhibited an SCLCC phenotype and investigate the effect of PIM inhibition on that phenotype.

Methods: RNA sequencing of human hepatoblastoma (HuH6) and metastatic (HLM_3) cells was performed and differentially expressed genes were evaluated using Ingenuity Pathway Analysis. Flow cytometry evaluated cell surface expression of CD133, a marker of hepatoblastoma SCLCCs. Real-time qPCR assessed mRNA abundance of other embryonic stemness markers and gene expression was calculated using the ΔΔCt method. PIM inhibition was achieved using the small molecule PIM inhibitor AZD1208 and viability was assessed using an alamarBlue® assay. The Chou-Talalay method was used to evaluate the effect of combination therapy using AZD1208 and cisplatin, with combination indices (CI) less than 1 indicating synergism. Data were reported as mean ± standard error of the mean. Student’s t-test was used with p≤0.05 significant.

Results: Transcriptomic analysis revealed that HLM_3 cells upregulated genes involved in cancer cell stemness and pluripotency, epithelial-to-mesenchymal transition, and pro-survival, while downregulating cellular differentiation and apoptosis pathways. HLM_3 cells exhibited increased CD133 cell surface expression (57 ± 6 % vs. 31 ± 1.6 % in HuH6 cells, p≤0.01) and mRNA abundance of the stemness markers Oct4, Nanog, and Sox2 (2.6, 2.7, and 2.9 fold change compared to HuH6 cells, respectively, p≤0.05), indicating increased stemness. Treatment with AZD1208 at increasing concentrations (0 to 100 µM) for 72 hours decreased viability of HLM_3 cells (median lethal dose (LD50) of 46 ± 3 µM) and acted synergistically with cisplatin (CI of 0.25, 0.33, and 0.48 with the addition of 1, 5, and 10 µM of AZD1208 respectively, Figure 1A). Furthermore, PIM kinase inhibition with AZD1208 (0 to 10 µM) decreased CD133 surface expression and mRNA abundance of the stemness markers in HLM_3 cells (p≤0.05, Figure 1B-C), indicating a decrease in the SCLCC phenotype following PIM inhibition.

Conclusion: Metastatic hepatoblastoma cells exhibit an SCLCC phenotype compared to the parent hepatoblastoma cell line. PIM kinase inhibition decreased HLM_3 cell viability, attenuated the SCLCC phenotype of HLM_3 cells, and was synergistic with cisplatin with doses as low as 1 µM, highlighting the potential for PIM kinase inhibition as a useful tactic in the treatment of children with metastatic hepatoblastoma.