Cathepsins B, D, and G Are Expressed in Metastatic Head and Neck Cutaneous Squamous Cell Carcinoma.

Felix Humphries,Nicholas Bockett,Swee T Tan,Erin Paterson,Josie Patel,Paul F Davis,Bridget Chang-Mcdonald

doi:10.3389/fonc.2021.690460

Abstract

AimWe 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.MethodsImmunohistochemical 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.ResultsImmunohistochemical 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.ConclusionThe 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.

Highlights

Cutaneous squamous cell carcinoma is the second most common skin cancer, affecting most commonly the head and neck region [1]
This study investigated the expression of cathepsins B, D and G and their localization in relation to the cancer stem cells (CSCs) subpopulations we have recently identified in Metastatic HNcSCC (mHNcSCC) [25]
To confirm co-expression of two proteins, immunofluorescence staining was performed on two of the 20 mHNcSCC tissue samples, using the same primary antibodies with the same concentrations as used for immunohistochemical staining, and co-staining with OCT4 (1:30; cat#309M-16, Abcam) or SOX2 (1:500; PA1-094, Thermo Fisher Scientific) which was used as a surrogate marker for the CSC subpopulations we have recently identified in mHNcSCC [25], and mast cell markers tryptase (1:300; cat#nCL-MCTRYP428, Leica) and chymase (1:1500, cat#PA528317, Invitrogen)

Summary

Introduction

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, affecting most commonly the head and neck region [1]. 14% of head and neck cSCC (HNcSCC) are considered high-risk lesions [4] and up to 5% develop metastasis to the regional nodes [7]. Metastatic HNcSCC (mHNcSCC) is an aggressive disease with a 5-year survival of 34-44% despite intensive treatment that typically involves surgery and adjuvant radiotherapy [9, 10]. This poor prognosis has been attributed to the presence of cancer stem cells (CSCs) [11]

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