ABSTRACT
AIM: 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), which promotes tumorigenesis. Cathepsins B, D and G are enzymes that constitute bypass loops for the RAS. This study investigated the expression and localization of cathepsins B, D, and G in relation to CSC subpopulations within mHNcSCC. METHODS: Immunohistochemical staining was performed on mHNcSCC tissue samples from 20 patients to determine the expression and localization of cathepsins B, D, and G. Immunofluorescence staining was performed on two of these mHNcSCC tissue samples by co-staining of cathepsins B and D with OCT4 and SOX2, and cathepsin G with mast cell markers tryptase and chymase. Western blotting and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were performed on five mHNcSCC samples and four mHNcSCC-derived primary cell lines, to determine protein and transcript expression of these three cathepsins, respectively. Enzyme activity assays were performed on mHNcSCC tissue samples to determine whether these cathepsins were active. RESULTS: Immunohistochemical staining demonstrated the presence of cathepsins B, D and G in in all 20 mHNcSCC tissue samples. Immunofluorescence staining showed that cathepsins B and D were localized to the CSCs both within the tumor nests and peri-tumoral stroma (PTS) and cathepsin G was localized to the phenotypic mast cells within the PTS. Western blotting demonstrated protein expression of cathepsin B and D, and RT-qPCR demonstrated transcript expression of all three cathepsins. Enzyme activity assays showed that cathepsin B and D to be active. CONCLUSION: The presence of cathepsins B and D on the CSCs and cathepsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for mHNcSCC.
ABSTRACT
We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling.
Subject(s)
Biomarkers, Tumor/metabolism , Cathepsin B/metabolism , Cathepsin D/metabolism , Head and Neck Neoplasms/pathology , Melanoma/secondary , Neoplastic Stem Cells/pathology , Skin Neoplasms/secondary , Adult , Aged , Aged, 80 and over , Apoptosis , Biomarkers, Tumor/genetics , Cathepsin B/genetics , Cathepsin D/genetics , Cell Proliferation , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Lymphatic Metastasis , Male , Melanoma/genetics , Melanoma/metabolism , Middle Aged , Neoplastic Stem Cells/metabolism , Prognosis , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Tumor Cells, CulturedABSTRACT
BACKGROUND: There is accumulating evidence of the presence of embryonic stem cell (ESC)-like cells in benign tumors. AIM: This study aimed to identify ESC-like cells in Schwannoma using the induced-pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4 and c-MYC. METHODS: Immunohistochemical (IHC) staining (n = 20) and RT-qPCR (n = 6) were performed on Schwannoma tissue samples (STS) to investigate protein and mRNA expression of these iPSC markers, respectively. Immunofluorescence (IF) staining was performed to investigate co-localization of the iPSC markers with CD34, α-SMA and CD133. RESULTS: IHC staining and RT-qPCR demonstrated protein and mRNA expression of all five iPSC markers, respectively. IF staining showed expression of SOX2, KLF4 and c-MYC on the tumor cells and the endothelium of the tumor microvessels which also expressed OCT4, while NANOG was exclusively expressed on the endothelium of the tumor microvessels. The OCT4+/CD34+ endothelium expressed CD133. CONCLUSION: We have identified a putative OCT4+/SOX2+/NANOG+/KLF4+/c-MYC+/CD133+ ESC-like subpopulation on the endothelium of tumor microvessels and an OCT4-/SOX2+/NANOG-/KLF4+/c-MYC+/CD133+ ESC-like subpopulation, within Schwannoma.
