Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice

Abstract

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreER^tam), which is under the control of the cytokeratin 14 promoter (K14-CreER^tam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-ras^G12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.

Keywords: Ang-(1-7); K-ras; mTor; oral cancer; papilloma; tumor.

Figure 1

The classical and beneficial arms of RAS. Angiotensinogen is produced by the liver, then hydrolyzed by renin to form angiotensin I (AngI). Next, Ang I is hydrolyzed by angiotensin-converting enzyme (ACE) to produce the octapeptide angiotensin II (AngII). AngII can stimulate two receptors, AngII type 1 receptor (AT1) and AngII type 2 receptor (AT2). ACE2 catalyzes Ang II to generate the heptapeptide angiotensin-(1-7) (Ang-(1-7)), which can also be a product of ACE activity cleaving angiotensin-(1-9) (Ang-(1-9)). Ang (1-7) counteracts the effects of AngII by interacting with the receptors Mas (Mas) and/or MrgD.

Figure 2

Levels of gene expression of members of the beneficial arm of the renin-angiotensin system in head and neck squamous cell carcinoma (HNSCC). Data from the TCGA HNSCC study (21 datasets, n = 604 samples, hence 566 with mRNA analyses) were used to investigate the expression of ACE2 (A), Mas (B), and MrgD (C), along with distinct anatomic locations of HNSCC. Analyses were performed using the UCSC Xena browser.

Figure 3

(A) Scheme of experimental groups and timeline used in the study. A representative example of K14-CreER^tam/LSL-K-ras ^G12D/+ mice treated with vehicle (B) after Tam induction as indicated in the upper scheme. (C) Histology of an oral squamous cell papilloma from a K14-CreER^tam/LSL-K-ras ^G12D/+ mouse upon Tam induction and vehicle treatment for 30 days (n = 4 animals). The hyperplastic epithelium covering thin stromal finger-like projections that grow out of the oral squamous epithelium. Two representative examples of K14-CreERtam/LSL-K-ras G12D/+ mice treated with angiotensin-(1-7) (D,E) and the histology (F,G) of small oral squamous papilloma from those representative K14-CreERtam/LSL-K-rasG12D/+ mice upon Tam induction and angiotensin-(1-7) treatment during 30 days (n = 6 animals). Original magnification: ×4.

Figure 4

(A–C): Proliferative status of the oral papilloma from the K14-CreER^tam/LSL-K-ras ^G12D/+ mice treated with vehicle or angiotensin-(1-7). (A) p-H3 expression in a control mouse. The immunostaining is widely extended in the basal layers (inset). (B) p-H3 expression is reduced after angiotensin-(1-7) treatment (detail depicted in the lower inset). (C) p-H3 positive cells were counted and quantified as a percentage of total cells. Angiotensin-(1-7) treated tumors showed a significant reduction of p-H3 labeled cells. Student t-test (n = 4) * p < 0.05. (D–G) pS6 immunostaining in representatives papilloma developed by K14-CreER^tam/LSL-K-ras ^G12D/+ mice treated with vehicle (D,F) or angiotensin-(1-7) (E,G). Level of pS6 in the tumor of a mouse treated with the vehicle was more prominent and showed stronger signal intensity than the angiotensin-(1-7) treatment (E,G). Original magnifications: A,B ×10; D,E ×20; and F,G ×40.