Abstract B15: Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression
Authors: Hanley Christopher J. , Mellone Massimiliano , Thomas Gareth J. 
Published: October 2016

Purpose: Smooth muscle actin (SMA)-positive myofibroblasts (cancer-associated fibroblasts), within the tumor microenvironment, promote many of the hallmarks of malignancy and correlate with poor prognosis in multiple cancer types. The purpose of this study was to investigate the mechanisms regulating myofibroblast differentiation in solid tumors in order to therapeutically target this cell population.

Experimental procedures:

Clinical Samples: Human tumor samples were analyzed by immunohistochemistry and RNA sequencing followed by bioinformatics analyses.

In vitro: Myofibroblast differentiation was analyzed in Human fetal foreskin fibroblasts (HFFF2), adult primary fibroblasts and cancer associated fibroblasts (CAFs) by Western blotting for α-SMA; immunofluorescence for α-SMA stress fiber formation; collagen gel contraction assays and analysis of gene expression by RNA sequencing/PCR. Intracellular Reactive Oxygen Species (ROS) generation was measured using FACS analysis of DCFH-DA fluorescence.

In vivo: To monitor tumor growth in vivo, a xenograft model of Head & Neck squamous cell carcinoma (HNSCC; 5PT) co-injected with HFFF2s in B6.129S7-Rag1tm1Mom/J mice; and an isograft model (K2) in C57BL/6-Tg(TRAMP)8247Ng/J mice was used. Lymph node (LN) metastases were assayed in the 5PT model using the LI-COR IRDye® 800CW 2-DG Optical Probe. Myofibroblast accumulation was assessed by immunohistochemistry for α-SMA and collagen deposition by Masson's trichrome staining.

Results: Using TGF-β1 treatment to model fibroblast-to-myofibroblast differentiation in vitro, we found that a delayed phase of intracellular ROS generation, beginning 24/48 hours post-treatment was required to induce differentiation in HFFF2s and adult primary fibroblasts.

RNA-Sequencing of fibroblasts treated with TGF-β1 showed that NOX4 was the most significantly up-regulated ROS-producing enzyme. NOX4 was also found to be up-regulated in the stroma of multiple solid tumors, co-localized with α-SMA, and correlating with a myofibroblast gene signature.

Genetic (si/shRNA) and pharmacological (GKT137831) inhibition of NOX4 abrogated multiple processes associated with myofibroblast differentiation, down-stream of TGF-β1: suppressing ROS generation; α-SMA expression; collagen gel contraction and expression of genes associated with ECM deposition (COL1A1, COL3A1, FN1 and CTGF). Furthermore, NOX4 inhibition reversed the myofibroblast phenotype in Cancer Associated Fibroblasts (CAFs) isolated from HNSCC and esophageal adenocarcinoma.

Genetic and pharmacological inhibition of NOX4 in fibroblasts significantly slowed tumor growth and myofibroblast accumulation in the 5PT and K2, in vivo, model systems. Notably, LN metastases in the 5PT model were significantly reduced following shRNA mediated NOX4 knockdown in HFFF2s.

Conclusions: NOX4 is a critical regulator of myofibroblast accumulation in multiple tumor types. Fibroblast-specific inhibition of this enzyme suppresses tumor growth and metastatic spread, suggesting that NOX4 is a promising therapeutic target for preventing myofibroblast differentiation within the tumor microenvironment.

Citation Format: Christopher J. Hanley, Massimiliano Mellone, Gareth J. Thomas. Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B15.