A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer

Jon Peñarando,Silvia Guil-Luna,César Díaz,Enrique Aranda,Rafael Mena,Laura M López-Sánchez,Marta Toledano,Carlos Villar,Victoria Gudiño,José Gómez-Barbadillo,Caroline Billard,Kevin Myant,Antonio Rodríguez-Ariza,Vanessa Hernández,Juan De La Haba-Rodríguez,Francisco Conde,Michela Raponi

doi:10.1186/s12915-017-0472-5

Abstract

BackgroundNitric oxide (NO) has been highlighted as an important agent in cancer-related events. Although the inducible nitric oxide synthase (iNOS) isoform has received most attention, recent studies in the literature indicate that the endothelial isoenzyme (eNOS) can also modulate different tumor processes including resistance, angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology and mesenchymal tumors is unknown.ResultsHere, we show that eNOS was significantly upregulated in VilCreERT2Apcfl/+ and VilCreERT2Apcfl/fl mouse intestinal tissue, with intense immunostaining in hyperproliferative crypts. Similarly, the more invasive VilCreERT2Apcfl/+Ptenfl/+ mouse model showed an overexpression of eNOS in intestinal tumors whereas this isoform was not expressed in normal tissue. However, none of the three models showed iNOS expression. Notably, when 40 human colorectal tumors were classified into different clinically relevant molecular subtypes, high eNOS expression was found in the poor relapse-free and overall survival mesenchymal subtype, whereas iNOS was absent. Furthermore, Apcfl/fl organoids overexpressed eNOS compared with wild-type organoids and NO depletion with the scavenger carboxy-PTIO (c-PTIO) decreased the proliferation and the expression of stem-cell markers, such as Lgr5, Troy, Vav3, and Slc14a1, in these intestinal organoids. Moreover, specific NO depletion also decreased the expression of CSC-related proteins in human colorectal cancer cells such as β-catenin and Bmi1, impairing the CSC phenotype. To rule out the contribution of iNOS in this effect, we established an iNOS-knockdown colorectal cancer cell line. NO-depleted cells showed a decreased capacity to form tumors and c-PTIO treatment in vivo showed an antitumoral effect in a xenograft mouse model.ConclusionOur data support that eNOS upregulation occurs after Apc loss, emerging as an unexpected potential new target in poor-prognosis mesenchymal colorectal tumors, where NO scavenging could represent an interesting therapeutic alternative to targeting the CSC subpopulation.

Highlights

Nitric oxide (NO) has been highlighted as an important agent in cancer-related events
Results endothelial isoenzyme (eNOS) is overexpressed in hyperproliferative regions and tumors of Apcfl/fl, Apcfl/+, and Apcfl/+ PTENfl/+ intestinal mouse tissue To explore the role of NO in the generation and maintenance of a cancer stem cell (CSC) subpopulation, we used the CSC-specific VilCreERT2 Apcfl/fl mouse model [25], where Apc-deficient cells maintain a crypt progenitor-like phenotype associated with the expansion of a stem Lgr5-positive cell population and early colorectal lesions [26]
Whereas intestinal tissue sections from normal mice showed no expression of eNOS, we found intense immunostaining for this NO synthase (NOS) isoform in epithelial cells from Apc-deficient crypts (Fig. 1a)

Summary

Introduction

Nitric oxide (NO) has been highlighted as an important agent in cancer-related events. In the CSC model, a small subpopulation of tumor cells possesses unlimited proliferative potential and chemoresistance, resulting in a tumor spreading and metastasis [4] This cellular subset with tumor-initiating properties has the capacity to evade conventional therapies, which contributes to the adverse survival rates, so new specific targets must be found [5]. The International CRC Subtyping Consortium analyzed different CRC data sets and have described four robust consensus molecular subtypes (CMSs) associated with clinical variables [11]: CMS1 (microsatellite instability), CMS2 (canonical), CMS3 (metabolic), and CMS4 (mesenchymal) These classifications differed in the number of tumor subtypes, they all agree in the identification of a stem-like mesenchymal subtype, which is associated with poor patient outcome in CRC [11], so new specific targets must be discovered to improve current treatments

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