Tie2 activity in cancer associated myofibroblasts serves as novel target against reprogramming of cancer cells to embryonic-like cell state and associated poor prognosis in oral carcinoma patients.

Abstract

Background: Myofibroblastic cancer-associated fibroblasts (CAF) in tumor stroma serves as an independent poor prognostic indicator, supporting higher stemness in oral cancer; however, the underlying biology is not fully comprehended. Here, we have explored the crucial role of Tunica Interna Endothelial Cell Kinase (Tie2/TEK) signaling in transition and maintenance of myofibroblastic phenotype of CAFs, and as possible link with the poor prognosis of head and neck squamous cell carcinoma (HNSCC) patients.

Methods: Bulk and single cell RNA-sequencing (scRNAseq) methods and in-depth bioinformatic analysis were applied for CAF and cancer cells co-culture for studying molecular relationships. In vitro 3D-spheroid-forming ability, expression of stemness markers, in vivo tumor formation ability in zebrafish embryo and syngeneic mouse allografts formation was conducted to test stemness, upon targeting CAF-specific Tie2 activity by gene silencing or with small molecule inhibitor. Immunohistochemistry analysis was performed to locate the distribution of Tie2 and αSMA in primary tumors of oral carcinoma. Prognosis in HNSCC patient cohort from The Cancer Genome Atlas (TCGA) study was analysed based on single sample gene set enrichment score (ssGSEA) and Kaplan-Meier analysis.

Results: Autocrine or exogenous TGFβ-induction in CAF led to the recruitment of histone deacetylase 2 (HDAC2) on the promoter of Tie2-antagonist, Angiopoietin-2 (ANGPT2), resulting in its downregulation, leading to phosphorylation of Tie2 (Y992) and subsequent activation of SRC (Y418). This led to SRC/ROCK mediated αSMA-positive stress-fiber formation with gain of myofibroblast phenotype. The CAF-specific Tie2-signaling was responsible for producing embryonic-like cell state in co-cultured cancer cells; with enhanced tumor initiating ability. Tie2 activity in CAF exerted the dynamic gene expression reprogramming, with the upregulation of 'cell migration' and downregulation of 'protein biosynthesis' related gene-regulatory-network modules in malignant cells. The AUCell scores calculated for gene signatures derived from these modules showed significant concordance in independently reported scRNAseq studies of HNSCC tumors and significant association with poor prognosis in HNSCC patient cohort.

Conclusions: CAF-specific Tie2 activity may serve as direct stromal-target against cancer cell plasticity leading to poor prognosis of oral cancer patients. Overall, our work has provided wider applicability of Tie2-specific functions in tumor biology, along with its known role in endothelial cell-specific function.