Purpose We have previously demonstrated the presence of two cancer stem cell (CSC) subpopulations within metastatic head and neck cutaneous squamous cell carcinoma (mHNcSCC), expressing components of the renin-angiotensin system (RAS). The RAS promotes tumorigenesis. Cathepsins B, D and G are enzymes that provide bypass loops for the RAS. This study investigates the expression and localization of cathepsins B, D, and G in relation to CSC subpopulations within mHNcSCC.
Methods Immunohistochemical staining was performed to determine expression and localization of cathepsins B, D and G in 20 patient mHNcSCC tissue samples. Two representative mHNcSCC tissue samples underwent immunofluorescent staining of these cathepsins with embryonic stem cell markers OCT4 and SOX2 to determine the localization in relation to the CSCs, in two of the mHNcSCC samples. Western blotting and reverse transcription quantitative polymerase reaction (RT-qPCR) was performed on five mHNcSCC samples and four mHNcSCC-derived primary cell lines, to determine protein and transcript expression of these cathepsins, respectively. Enzyme activity assays determined whether these cathepsins were active.
Results Immunohistochemical staining demonstrated the presence of cathepsins B, D and G in all 20 mHNcSCC samples. Cathepsins B and D were localized to OCT4+ CSCs within the tumor nests (TNs) and peri-tumoral stroma (PTS). Cathepsin B was expressed by the SOX2+ CSCs in the TNs and PTS. Cathepsin G was localized to the chymase+ mast cells within the PTS. RT-qPCR demonstrated transcript expression of all three cathepsins, although cathepsin G was expressed at lower levels. Western blotting showed protein expression of cathepsins B and D. Enzyme activity assays showed that cathepsins B and D were active.
Conclusion Presence of cathepsins B and D on the CSCs and cathepsin G on the phenotypic mast cells suggest presence of bypass loops for the RAS which may be a potential novel therapeutic target in treatment of mHNcSCC.