Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

Abstract

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.

Reduced S-nitrosylation of TGFβ1 elevates its binding affinity towards the receptor and promotes fibrogenic signaling in the breast

“…Such NO-associated tissue stiffening is largely attributed to the downmodulation of SNO-mediated inhibition of ECM-remodeling enzymes, including tissue transglutaminases and matrix metalloproteinases (27, 47). In addition, our study reports that a major fibrogenic protein, TGFβ1, is another contributor to tissue stiffening induced by reduction of SNO (11, 12). While in healthy cells a subset of proteins are constitutively S-nitrosylated to remain under control, in disease states, on the other hand, SNO levels could be dysregulated, contributing to disruption of homeostasis (30).…”

“…We previously reported that pharmacological inhibition of basal nitric oxide (NO) production in wild-type mice induced precancerous progression of mammary glands accompanied by highly desmoplastic stroma. These glands also showed elevated TGFβ activity and fibrogenic signaling (11, 12). To explore the mechanism of this phenomenon, we modulated NO levels in vitro using cell lines of MCF10A human breast cancer progression series comprising normal MCF10A and malignant CA1d cells (29).…”

“…Our previous and present studies strongly suggest that TGFβ1 is highly S-nitrosylated for latency in normal breast cells. However, as the basal NO production declines possibly due to the increase in oxidative stress, TGFβ1 becomes denitrosylated and derepressed, contributing to the induction of desmoplasia and precancerous progression (11, 12, 27). Roles of TGFβ1 in precancerous progression, however, remain controversial.…”

“…To determine the mechanism of precancerous progression of the breast, we previously inhibited nitric oxide (NO) in healthy mammary glands of wild-type mice (11,12). NO is a bioactive signaling molecule produced throughout the body, and its aberrant production is linked to different risk factors of breast cancer (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…We found that the basal levels of NO plummeted along with breast cancer progression. Importantly, we saw that pharmacological deprivation of NO in healthy mammary glands induced precancerous progression accompanied by desmoplasia and TGFβ upregulation (11, 12). These findings suggested that NO plays critical roles in suppressing TGFβ activity and that its deficit could lead to precancerous progression of the breast.…”