Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma

Aim We have previously demonstrated the putative presence of two cancer stem cell (CSC) subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC), which express components of the renin–angiotensin system (RAS). In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC. Methods 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC. Results IHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G. Conclusion The presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

Methods: 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC.
results: IHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase + phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G.
conclusion: The presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.
Keywords: oral tongue, squamous cell carcinoma, cancer, cathepsin, renin-angiotensin system, cancer stem cells, oral cavity, head and neck inTrODUcTiOn Oral cavity cancers are the eighth most common cancer worldwide (1) accounting for 2% of cancer mortality (2). Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity cancer (3). Risk factors for OTSCC include tobacco and alcohol consumption which act synergistically to promote carcinogenesis (1). Current mainstay treatment for OTSCC is surgery often with post-operative radiotherapy, and sometimes chemotherapy (4). The 5-year survival rate of 50-55% for OTSCC (3) has remained largely unchanged over the past 40 years (5).
Cancer stem cells (CSCs), demonstrated in many types of cancers, have been proposed to be the origin of cancer including OTSCC (6). Increased tumor size, local invasion, local recurrence, and regional metastasis have been associated with overexpression of CSC markers (7).
We have recently characterized two CSC subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC) with an OCT4 − subpopulation within the tumor nests (TNs) that also expresses EMA; and an OCT4 + subpopulation within the peri-tumoral stroma that does not express EMA (6).
The renin-angiotensin system (RAS) is a hormonal system classically associated with blood pressure and body fluid regulation. Recent literature has demonstrated its role in cancer growth and metastasis (8) by promoting angiogenesis and cell proliferation (9,10). Angiotensinogen (ANG) undergoes conversion to angiotensin I (ATI) by renin, the active form of pro-renin (8,11). Pro-renin receptor (PRR) is the receptor for both pro-renin and renin (12). ATI is then converted to angiotensin II (ATII) by the action of angiotensin converting enzyme (ACE) (8). Vasoactive ATII acts on angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2) (8).
We have recently demonstrated the presence of two putative CSC subpopulations in MDOTSCC: one within the TNs and the other within the peri-tumoral stroma (6). We have also demonstrated the expression of components of the RAS by these CSCs (13). The CSC subpopulation within the TNs expresses PRR, ATIIR1, and ATIIR2, while the CSC subpopulation within the peri-tumoral stroma expresses PRR, ACE, ATIIR1, and ATIIR2 (13).
Cathepsins B, D, and G are proteases that provide putative bypass loops for the RAS. Cathepsin B, a cysteine protease, is a pro-renin processing enzyme and converts inactive pro-renin to active renin. Cathepsin D, an aspartyl protease, is functionally homologous to renin. Cathepsin G, a serine protease, can directly produce ATII from ANG and ATI, and is functionally homologous to ACE (14). We have previously demonstrated an ESC-like population within infantile hemangioma (15) that expresses components of the RAS (14,15) and also cathepsins B, D, and G, suggesting the existence of bypass loops (14).
In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations within MDOTSCC. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to confirm mRNA transcription. Protein expression and localization of each cathepsin was determined by 3,3-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining. Enzymatic activity assays were performed to demonstrate the activity of these cathepsins.

Tissue samples
Moderately differentiated oral tongue squamous cell carcinoma samples from four male and five female patients, aged 30-73 (mean, 58.2) years, were sourced from the Gillies McIndoe Research Institute Tissue Bank for this study, which was approved by the Central Regional Health and Disability Ethics Committee (ref. no 12/CEN/74). Written consent was obtained from all participants.

histochemical and ihc staining
Hematoxylin and eosin (H&E) staining was used to confirm the presence and appropriate histological grading on 4 μm thick formalin-fixed paraffin-embedded of MDOTSCC sections from nine patients by an anatomical pathologist (HDB). These MDOTSCC sections were then used for DAB IHC staining, as previously described (6,13) Immunofluorescent (IF) IHC staining was performed to determine co-expression of two proteins on two samples of MDOTSCC from the original cohort of nine patients used for DAB IHC staining. Vectafluor Excel anti-mouse 488 (readyto-use; cat# VEDK2488, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Life Technologies, Carlsbad, CA, USA) were utilized to detect the combinations. All IF IHC-stained slides were mounted in Vecta Shield Hardset mounting medium with 4′,6′-diamino-2-phenylindole (Vector Laboratories).
Positive control tissues used for the primary antibodies were human placenta for cathepsin B; human breast cancer for cathepsin D; mouse bone marrow for cathepsin G; and human seminoma for OCT4 and SALL4. A negative MDOTSCC control sample was prepared for DAB IHC staining by using an IgG isotype control (ready-to-use; cat# IR600, Dako, Santa Clara, CA, USA). For IF IHC staining, a negative control was performed using a section of MDOTSCC tissue with the combined use of primary isotype mouse (ready-to-use; cat# IR750, Dako, Copenhagen, Denmark) and rabbit (read-to-use; cat# IR600, Dako) antibodies.
All antibodies were diluted with Bond primary antibody diluent (cat# AR9352, Leica), and DAB and IF IHC staining was carried out on the Leica Bond Rx autostainer, as previously described (16).

nanostring mrna analysis
Six snap-frozen samples of MDOTSCC from the original cohort of nine patients used for DAB IHC staining were used for isolation of total mRNA for NanoString nCounter™ Gene Expression Assay (Nanostring Technologies, Seattle, WA, USA), as previously described (6,13). Probes for the genes encoding for cathepsin B (NM_001908.2), cathepsin D (NM_001909.3), cathepsin G (NM_001911.2), and the housekeeping gene PGK1 (NM_000291.3) were used in the analysis. Raw data were analyzed by nSolver™ software (NanoString Technologies). Results were normalized against the housekeeping gene, graphed using Excel (Microsoft Office 2013), and subjected to t-tests for related samples, to compare the relative abundance of each cathepsin.   (Figure 1A, brown) by cells predominantly within the TNs (Figure 1A, brown, arrows) and cells within the peri-tumoral stroma (Figure 1A, brown, arrowheads). Granular cytoplasmic staining of cathepsin D (Figure 1B, brown) was localized to cells within the TNs (Figure 1B, brown, arrows) and some cells within the peri-tumoral stroma (Figure 1B,  brown, arrowheads). Cathepsin G was expressed in the cytoplasm of only some cells within the peri-tumoral stroma (Figure 1C, brown, arrowheads).
Positive controls for cathepsins B ( Figure S1A in Supplementary Material, brown), D ( Figure S1B in Supplementary Material, brown), and G ( Figure S1C in Supplementary Material, brown) demonstrated expected staining patterns in human placenta (19) and breast cancer (20), and mouse bone marrow (21), respectively. The negative control showed minimal staining ( Figure S1D in Supplementary Material, brown).

iF ihc staining
To localize cathepsins B, D, and G in relation to the CSC subpopulations, IF IHC staining was performed on two representative MDOTSCC samples from the original cohort of nine patients used for DAB IHC staining.
To further characterize the cathepsin D + (Figure 2G, red) and cathepsin G + (Figure 2H, red) cells, we performed co-staining with tryptase, as we have reported in infantile hemangioma (14). This confirmed that tryptase (Figures 2G,H, green) was not expressed by the cathepsin D + (Figure 2G, red) cells, but was expressed by the cathepsin G + (Figure 2H, red) cells.
Images illustrating the individual stains demonstrated in Figure 2 are presented in Figure S2 in Supplementary Material. Minimal staining was present on the negative control ( Figure  S2Q in Supplementary Material), confirming the specificity of the primary antibodies used.

nanostring mrna analysis
NanoString mRNA analysis for cathepsins B, D, and G was normalized against the housekeeping gene, PGK1, confirming transcriptional activation for both cathepsins B and D in all six MDOTSCC samples, whereas cathepsin G was detected at low levels in four of the six samples studied (Figure 3).
Statistical analysis of the gene transcripts confirmed significantly greater presence of cathepsin B than cathepsin D (t = 3.073, p < 0.05), and that both cathepsins B and D were significantly more abundant than cathepsin G (t = 4.701 and t = 4.885, respectively, p < 0.01).
colorimetric In Situ hybridization Colorimetric in situ hybridization confirmed the presence of mRNA for cathepsin B (Figure 4A, pink, arrows), cathepsin D ( Figure 4B, pink, arrows), and cathepsin G (Figure 4C, pink, arrows) in cells within all six MDOTSCC samples examined.
Appropriate staining was seen on positive controls for cathepsin B ( Figure S3A in Supplementary Material, pink, arrows), cathepsin D ( Figure S3B in Supplementary Material, pink, arrows), and cathepsin G ( Figure S3C in Supplementary Material, pink, arrows), indicating the presence of mRNA transcripts in the controls. The negative control performed on each run showed the absence of mRNA transcripts for any marker ( Figure S3D in Supplementary Material).

cell counting and statistical analyses
Cell counting for cathepsins B, D, and G ( Figure 5) in all nine samples demonstrated significantly more cells staining positively for cathepsin B within the TNs, than those within the peri-tumoral stroma (91% vs. 77%, t = 10.281, p < 0.000). Analysis using χ 2 method showed that there were significantly more cathepsin B + cells than cathepsin D + cells (χ 2 = 409.9.261, p < 0.0001), and that there was significantly more cathepsin D + cells relative to the cathepsin G + cells (χ 2 = 3356.0, p < 0.0001). Consequently, we conclude that there was significantly more cathepsin B than cathepsin G.

enzymatic activity assays
To determine the functionality of the cathepsins B, D, and G within MDOTSCC, we performed enzymatic activity assays, which confirmed the functional activity for cathepsin B ( Figure 6A) and cathepsin D (Figure 6B), but not cathepsin G ( Figure 6C) within all three MDOTSCC samples, as compared with the expected activity for the positive and negative controls (Figures 6A-C).

DiscUssiOn
We have recently demonstrated expression of components of the RAS by the two CSC subpopulations within MDOTSCC (13). The novel finding of the expression and localization of cathepsins B, D, and G to these CSC subpopulations provides further insights into the biology of this cancer.
It is intriguing that cathepsin B is present in both CSC subpopulations within the TNs and the peri-tumoral stroma. However, cathepsin D is localized only to the CSC subpopulation within the TNs, while cathepsin G is localized exclusively to the tryptase + phenotypic mast cells within the peri-tumoral stroma, similar to the finding in infantile hemangioma (14).
The expression of cathepsin B has been recently reported in oral cavity SCC (22). Yang et al. (22) have demonstrated that increased expression of cathepsin B is correlated with lymph node metastasis, higher tumor grade, and significantly poorer overall survival.
The finding that only four of the six MDOTSCC samples displayed relative low abundance of cathepsin G by NanoString mRNA analysis may be due to either rapid degradation of mRNA for this protein or sampling bias. However, the low transcriptional abundance may reflect the relatively low numbers cells that stained positively for cathepsin G by IHC staining.
Cathepsins B, D, and G exist in precursor forms, with the mature form of cathepsin B being 29 kDa, and its precursor form being 39 kDa (23). Its conversion requires a single cleavage of the precursor protease by either cathepsin B or in acidic conditions (23,24). Cathepsin D has two precursor forms-the 51 kDa pre-pro-enzyme is cleaved, removing the signal peptide to form   the 48 kDa pro-enzyme (25). Active cathepsin D is formed by removal of the 44 amino acid pro-domain of the pro-enzyme (25). Cathepsin D can be cleaved to form a single or two-chain form (26). It is exciting to speculate that both cathepsin B and cathepsin D undergo "intra-tumoral" post-translational modification following synthesis, with the latter being confirmed by enzyme activity assays we have performed.
The precursor of cathepsin G is a 32.5 kDa protein which includes a signal peptide and a pro-dipeptide at the N terminus (27)(28)(29). This is cleaved by another protease, cathepsin C, to form the active 28.5 kDa cathepsin G (27,29). Although we have confirmed transcriptional activation for cathepsin G and demonstrated localization of this protein to cells by IHC staining within the MDOTSCC samples, we used in this study, it was not possible to determine whether cathepsin G would undergo posttranslational modification into the active form, based on the activity data. However, given the relatively low number of cathepsin G + cells, such activity may not be detectable by the activity kit used in this study. Furthermore, it is interesting that cathepsin G is expressed by mast cells, consistent with previous reports (14). However, its precise role in MDOTSCC carcinogenesis remains to be determined.
Recent literature suggests that the RAS plays a crucial role in cancer growth and metastasis (8,9), specifically cell proliferation (10). We have demonstrated expression of cathepsins B and D by the two CSC subpopulations, and the expression of cathepsin G by the phenotypic mast cells within MDOTSCC that we have recently identified. This suggests the putative presence of bypass loops for the RAS in MDOTSCC (30).
This report suggests CSCs as a potential therapeutic target for MDOTSCC, through modulation of cathepsin B and D, and potentially G, in addition to modulation of the classical RAS. A larger study is needed to validate these findings. limitations 1. Further study with a bigger sample size would be needed to confirm the observation of this study.

Further study including well and poorly differentiated
OTSCC, in addition to moderately differentiated lesions may improve understanding of this aggressive cancer. 3. Functional study using in vitro and in vivo models of MDOTSCC would be needed to further validate the results of this study.