J Liver Cancer.  2025 Mar;25(1):123-133. 10.17998/jlc.2025.03.08.

Identification of new biomarkers of hepatic cancer stem cells through proteomic profiling

• Choi SH
• Lee HY
• Yun SH
• Jang SJ
• Kim SU
• Park JY
• Ahn SH
• Kim DY

• KMID: 2567070
• DOI: http://doi.org/10.17998/jlc.2025.03.08

Abstract

Backgrounds/Aims
In hepatocellular carcinoma (HCC), which exhibits high mortality and recurrence rates globally, the traits of cancer stem cells (CSCs) that significantly influence recurrence and metastasis are not well understood. CSCs are self-renewing cell types identified in most liquid and solid cancers, contributing to tumor initiation, growth, resistance, recurrence, and metastasis following chemo-radiotherapy or trans-arterial chemoembolization therapy.
Methods
CSCs are classified based on the expression of cell surface markers such as CD133, which varies depending on the tumor type. Proteomic analysis of liver cancer cell lines with cancer stem cell potential and HCC cancer cell lines lacking stem cell propensity was conducted to compare and analyze specific expression patterns.
Results
Proteomic profiling and enrichment analysis revealed higher expression of the calcium-binding protein S100 family in CD133+ Huh7 cells than in CD133- or wild-type cells. Furthermore, elevated expression of S100 family members was confirmed in an actual CD133+ liver cancer cell line via protein-protein network analysis and quantitative polymerase chain reaction (qPCR).
Conclusion
The S100 family members are not only new markers of cancer stem cells but will also assist in identifying new treatment strategies for CSC metastasis and tumor advancement.

Keyword

Carcinoma, hepatocellular; Neoplastic stem cells; Proteomic analysis; CD133-PROM1; S100 proteins

Figure

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  Figure 1. Identification of CD133+ and CD133- Huh7 cell lines. The CD133+ cell line was isolated from the HCC cell line Huh7, and a CD133- cell line lacking cancer stem cell propensity was also isolated. To confirm whether the isolated CD133+ Huh7 cell line had cancer stem cell line propensity, the expression of representative cancer stem cell markers CD44, CD24, EpCAM, and CD90 was assessed. WT, wild type; EpCAM, epithelial cell adhesion molecule.

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  Figure 2. Proteomic profiling of CD133+ and CD133- Huh7 cell lines. The proteomic profiling analysis of CD133+ Huh7 was compared with wild-type and CD133- type. (A) CD133+ HCC cell lines showed distinct expression patterns compared to wild-type or CD133- type, while wild-type and CD133- cells showed similar expression patterns. (B) Proteomic profiling revealed various genetic changes, identifying four types of expression changes. The most significant expression difference was observed in biological regulation (approximately 61%). (C) Among the three categories (biological response, metabolic process, and cellular components) with major expression changes, we identified significant changes in biological regulation (approximately 64%), amine metabolic processes (approximately 40%), and structural development (approximately 58%). This profiling was conducted in duplicate analyses with high statistical significance. HCC, hepatocellular carcinoma.

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  Figure 3. Enrichment network of CD133+ Huh7 and CD133- Huh7. (A) Labelling-free quantification (LFQ) of proteins compared mass spectral peak intensities between CD133+ and CD133- and confirmed that CD133- and wild-type had similar intensities, while CD133+ type had higher peak intensities that were different from that of CD133- and wild-type. (B) Analysis of the CD133+-specific protein network confirmed the network composition of various biological regulation-related proteins such as S100P.

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  Figure 4. Comparison of the S100 family of CD133+ Huh7 over CD133- Huh7. Based on the protein network results with a high correlation to CD133+, we compared the actual expression levels and found that the S100 family members were more highly expressed in CD133+ cells than in the wild-type cells, whereas their expression was lower in CD133- cells than in wild-type cells. Conversely, ANXA1 was barely expressed in CD133+, while its expression increased in CD133- cells, compared to wild-type, although not significantly. WT, wild type.

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Reference

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