Cancer Stem Cells in Moderately Differentiated Lip Squamous Cell Carcinoma Express Components of the Renin–Angiotensin System

Aim We investigated the expression of the renin–angiotensin system (RAS) by cancer stem cell (CSC) subpopulations we have identified in moderately differentiated lip squamous cell carcinoma (MDLSCC). Method Ten MDLSCC samples underwent 3,3-diaminobenzidine (DAB) and immunofluorescent immunohistochemical (IHC) staining for (pro)renin receptor (PRR), angiotensin-converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and receptor 2 (ATIIR2). NanoString analysis and Western blotting (WB) were performed on six MDLSCC samples for gene and protein expression, respectively. Results IHC staining showed expression of PRR, ATIIR1, and ATIIR2 on cells within the tumor nests (TNs) and the stroma. ACE was localized to the microvessels within the stroma. WB detected PRR, ACE, and ATIIR2. NanoString analysis confirmed gene expression of PRR, ACE, and ATIIR1. Conclusion Components of the RAS: PRR, ATIIR1, and ATIIR2 are expressed on two CSC subpopulations in MDLSCC, one within the TNs and the other within the stroma. The endothelium of the microvessels within the stroma expresses ACE.

inTrODUcTiOn Oral cavity cancer is the sixth most common cancer globally with the majority being squamous cell carcinoma (SCC) (1,2). Oral cavity SCC (OCSCC) affects the lip, oral tongue, floor of mouth, hard palate, buccal mucosa, maxillary and mandibular alveolus, and the retromolar trigone (1,3). The incidence of lip cancer is highest among white populations in Canada and Australia (2).
The cancer stem cell (CSC) concept proposes that a population of cells within a tumor are tumorigenic (11,12). CSCs have been proposed as the origin of cancer cells and account for tumor resistance to therapeutic agents and give rise to metastases (11,13). Although there are no distinctive markers that differentiate normal stem cells from CSCs, several stem cell markers have been used to identify CSCs in OCSCC (14,15).
The renin-angiotensin system (RAS) is a major hormonal system that regulates blood pressure and fluid balance through its effects in the kidney, cardiovascular, and central nervous systems (19,20). Angiotensinogen is cleaved by renin to form angiotensin I (ATI) via the (pro)renin receptor (PRR) (19). Angiotensin-converting enzyme (ACE) then cleaves ATI to angiotensin II (ATII). ATII is the main effector of the RAS and mediates its effects through angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2) (19,20).
Expression of several components of the RAS have been demonstrated in abnormal tissues such as mammary hyperplasia and ductal carcinoma in situ (21) and cancer cells in human breast, lung, pancreas, prostate, skin cancer, and glioblastoma multiforme (GBM) (21)(22)(23). These components have also been correlated with inflammation, tumor growth, and angiogenesis (21,24). Although the RAS has been implicated in carcinogenesis (20,25), there are little data demonstrating the presence of the RAS in lip SCC.
This study investigated the expression of components of the RAS: PRR, ACE, ATIIR1, ATIIR2, in the CSC subpopulations within MDLSCC that we had previously characterized (16). Positive human control tissues used for primary antibodies for IHC staining were placenta for PRR and ERG; liver for ATIIR1, kidney for ACE and ATIIR2, skin for SOX2, and seminoma for OCT4 and SALL4. A negative control by omitting the primary antibody on a MDLSCC sample was also prepared (data not shown).

Tissue samples
All antibodies were diluted with Bond primary antibody diluent (cat# AR9352, 143 Leica) and all DAB and IF IHC staining was carried out on the Leica Bond Rx autostainer as previously described (26).

nanostring gene analysis
Six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining were utilized for isolation of   (Figure 1A, brown). ACE was present within the endothelium of the microvessels within the stroma between the TNs (Figure 1B, brown) but not the cells within the TNs. Weak to moderate cytoplasmic and nuclear staining of ATIIR1 was observed in cells within the TNs and cells within the stroma (Figure 1C, brown). There was strong perinuclear and cytoplasmic staining of ATIIR2 in cells within the TNs, the stroma and skeletal muscle (Figure 1D, brown). A summary of staining patterns across all 10 samples is given in Table S1 in Supplementary Material. Positive human control tissues demonstrated the expected staining pattern in placenta for PRR ( Figure S1A in Supplementary Material) and ERG (data not shown), liver for ACE ( Figure  S1B in Supplementary Material) and ATIIR1 ( Figure S1C in Supplementary Material, brown) and kidney for ATIIR2 ( Figure  S1D in Supplementary Material, brown). The omission of the primary antibody in a MDLSCC sample provided a control for the secondary antibody (data not shown).

iF ihc staining
Double IF IHC staining showed that PRR (Figure 2A, red) was expressed by the SALL4 + cells (Figure 2A, green) within the TNs and the stroma. PRR (Figure 2B, red) was also detected in the OCT4 + CSC subpopulation (Figure 2B, green) and a separate OCT − CSC subpopulation (Figure 2B, red) within the stroma. Expression of ACE (Figure 2C, green) was observed  in endothelium of the microvessels that also expressed SOX2 (Figure 2C, red). Expression of ACE was localized exclusively to the endothelium of the microvessels that expressed the endothelial marker ERG (Figure 2D, red). ATIIR1 (Figure 2E, green) was expressed by cells within the TNs as well as the microvessels that also expressed SOX2 (Figure 2E, red). Perinuclear expression of ATIIR2 (Figures 2F,G, red) was demonstrated in cells within the TNs that expressed SALL4 (Figure 2F, green) as well as cells within the stroma, regardless of whether they were OCT4 + (Figure 2G, green) or OCT4 − (Figure 2G, red). Separated IF IHC images of Figure 2 are shown in Figure S2 in Supplementary Material.

Western Blotting
Western blotting confirmed the presence of PRR at an expected molecular weight of 35 kDa (Figure 3A) within the extracts of all six MDLSCC samples examined. ACE (Figure 3B) was detected in five of the six MDLSCC samples analyzed, at the expected molecular weight of 195 kDa. ATIIRI was detected at 41 kDa in five samples, however, its proximity to the control ruled out significance (data not shown). ATIIR2 was detected in four of the six MDLSCC samples at approximately 50 kDa (Figure 3C), which is larger than the native form of the protein and may be due to glycosylation. β-actin confirmed approximately equal total protein loading in all samples (Figures 3A-C).

nanostring gene analysis
NanoString gene analysis for PRR, ACE, ATIIR1, and ATIIR2 performed on six MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining, confirmed the presence of PRR and ACE mRNA in all six samples, and ATIIR1 mRNA in one sample. ATIIR2 mRNA was below detectable levels in all six samples (Figure 4).

DiscUssiOn
The RAS is a complex endocrine system that is a therapeutic target for hypertension and has been associated with the metabolic system (28), inflammation (29,30), and carcinogenesis (22,25). Although physiologically the liver is the primary source of circulating AGN, it is also expressed by other cell types including adipocytes, mesangial cells, and proximal tubule epithelial cells of the kidney, and neurons and glial cells in the brain (28). Components of the RAS were identified by IHC staining in all 10 MDLSCC samples examined in our study. Interestingly, ACE was localized to the endothelium of the microvessels located within the stroma, between the TNs. The presence of ACE on the endothelium of the microvessels within the stroma that also expresses the ESC marker SOX2 is intriguing. This is consistent with recent reports of a primitive endothelial phenotype in close proximity to colorectal tumors (31) suggesting its role in regulating epithelial to mesenchymal transition by influencing angiogenesis (20,22). However, this remains the topic of further investigation.
Expression of PRR was seen in the SALL4 + /OCT4 − CSCs within the TNs and the SALL4 + /OCT4 + CSCs within the stroma. Expression of ATIIR1 was present in SOX2 + CSCs within the TNs and the endothelium of the microvessesls within the stroma. ATIIR2 was expressed by the SALL4 + /OCT4 − CSCs within the TNs, as well as the SALL4 + /OCT4 + CSCs within the stroma. The expression of PRR, ACE, and ATIIR2 was supported by WB. While the presence of mRNA for PRR and ACE was confirmed in all six samples, ATIIR1 was detected by NanoString gene analysis in only one sample and ATIIR2 was below detectable levels in all samples. This could be attributed to degradation of mRNA or lower rate of mRNA transcription compared to protein translation. Although this study demonstrates novel findings of the expression of components of the RAS by the CSC subpopulations in MDLSCC, its descriptive nature necessitates further work into the functional role of the RAS, including a larger sample size.
The hallmarks of cancer (12,32) are the distinct capabilities of cells to proliferate, survive, and disseminate, and the regulation of these features by the RAS suggests its direct effect on angiogenesis (25,33).
Recent studies have demonstrated the presence of components of RAS in breast cancer (22,34). To the best of our knowledge, this is the first report demonstrating the expression of components of the RAS by the CSC subpopulations within MDLSCC. The localization of the RAS to the CSC subpopulations within MDLSCC adds to the similar finding in oral tongue (35) and buccal mucosal (27) SCC and GBM (36). This would suggest that CSCs may be a novel therapeutic target for this cancer by using drugs that modulate the RAS.
Previous studies have demonstrated an association of the use of ACE inhibitors and angiotensin II receptor 1 blockers with reduced risk of development of skin SCC (37), and β-blockers administration which reduce renin levels (38), is associated with improved survival of patients with malignant melanoma (39). Although there is currently no literature on the potential role RAS modulators on lip SCC, propranolol has been shown to reduce viability, induce apoptosis, and inhibit production of the proangiogenic protein VEGF in head and neck SCC cell lines (40). Further work is needed to determine the validity of targeting CSCs by modulating the RAS in MDLSCC and other types of cancer as a novel therapeutic approach.

eThics sTaTeMenT
This study was approved by the Central Regional Health and Disability Ethics Committee.