There is now compelling evidence that the tumour stroma plays an important role in the pathogenesis of cancers of epithelial origin. The pre-eminent cell type of the stroma is carcinoma-associated fibroblasts. These cells demonstrate remarkable heterogeneity with activation and senescence being common stress responses. In this review, we summarise the part that these cells play in cancer, particularly oral cancer, and present evidence to show that activation and senescence reflect a unified programme of fibroblast differentiation. We report advances concerning the senescent fibroblast metabolome, mechanisms of gene regulation in these cells and ways in which epithelial cell adhesion is dysregulated by the fibroblast secretome. We suggest that the identification of fibroblast stress responses may be a valuable diagnostic tool in the determination of tumour behaviour and patient outcome. Further, the fact that stromal fibroblasts are a genetically stable diploid cell population suggests that they may be ideal therapeutic targets and early work in this context is encouraging.
Senescent cancer-associated fibroblasts (CAF) develop a senescence-associated secretory phenotype (SASP) that is believed to contribute to cancer progression. The mechanisms underlying SASP development are, however, poorly understood. Here we examined the functional role of microRNA in the development of the SASP in normal fibroblasts and CAF. We identified a microRNA, miR-335, up-regulated in the senescent normal fibroblasts and CAF and able to modulate the secretion of SASP factors and induce cancer cell motility in co-cultures, at least in part by suppressing the expression of phosphatase and tensin homologue (PTEN). Additionally, elevated levels of cyclo-oxygenase 2 (PTGS2; COX-2) and prostaglandin E2 (PGE2) secretion were observed in senescent fibroblasts, and inhibition of COX-2 by celecoxib reduced the expression of miR-335, restored PTEN expression and decreased the pro-tumourigenic effects of the SASP. Collectively these data demonstrate the existence of a novel miRNA/PTEN-regulated pathway modulating the inflammasome in senescent fibroblasts.
The dissemination of cancer cells to local and distant sites depends on a complex and poorly understood interplay between malignant cells and the cellular and non-cellular components surrounding them, collectively termed the tumour microenvironment. One of the most abundant cell types of the tumour microenvironment is the fibroblast, which becomes corrupted by locally derived cues such as TGF-β1 and acquires an altered, heterogeneous phenotype (cancer-associated fibroblasts, CAF) supportive of tumour cell invasion and metastasis. Efforts to develop new treatments targeting the tumour mesenchyme are hampered by a poor understanding of the mechanisms underlying the development of CAF. Here, we examine the contribution of microRNA to the development of experimentally-derived CAF and correlate this with changes observed in CAF derived from tumours. Exposure of primary normal human fibroblasts to TGF-β1 resulted in the acquisition of a myofibroblastic CAF-like phenotype. This was associated with increased expression of miR-145, a miRNA predicted in silico to target multiple components of the TGF-β signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-β1-induced myofibroblastic differentiation and reverted CAF towards a normal fibroblast phenotype. We conclude that miR-145 is a key regulator of the CAF phenotype, acting in a negative feedback loop to dampen acquisition of myofibroblastic traits, a key feature of CAF associated with poor disease outcome.
Cancer-associated fibroblasts (CAF) remain a poorly characterized, heterogeneous cell population. Here we characterized two previously described tumor-promoting CAF sub-types, smooth muscle actin (SMA)-positive myofibroblasts and senescent fibroblasts, identifying a novel link between the two. Analysis of CAF cultured ex vivo, showed that senescent CAF are predominantly SMA-positive; this was confirmed by immunochemistry in head & neck (HNSCC) and esophageal (EAC) cancers. In vitro, we found that fibroblasts induced to senesce develop molecular, ultrastructural and contractile features typical of myofibroblasts and this is dependent on canonical TGF-β signaling. Similar to TGF-β1-generated myofibroblasts, these cells secrete soluble factors that promote tumor cell motility. However, RNA-sequencing revealed significant transcriptomic differences between the two SMA-positive CAF groups, particularly in genes associated with extracellular matrix (ECM) deposition and organization, which differentially promote tumor cell invasion. Notably, second harmonic generation imaging and bioinformatic analysis of SMA-positive human HNSCC and EAC showed that collagen fiber organization correlates with poor prognosis, indicating that heterogeneity within the SMA-positive CAF population differentially impacts on survival. These results show that non-fibrogenic, SMA-positive myofibroblasts can be directly generated through induction of fibroblast senescence and suggest that senescence and myofibroblast differentiation are closely linked processes.
HPV-independent head and neck squamous cell carcinoma (HNSCC) is a common cancer globally. The overall response rate to anti-PD1 checkpoint inhibitors (CPIs) in HNSCC is ~16%. One major factor influencing the effectiveness of CPI is the level of tumor infiltrating T cells (TILs). Converting TILlow tumors to TILhigh tumors is thus critical to improve clinical outcome. Here we describe a novel DNA vaccines to facilitate the T-cell infiltration and control tumor growth. We evaluated the expression of target antigens and their respective immunogenicity in HNSCC patients. The efficacy of DNA vaccines targeting two novel antigens were evaluated with or without CPI using a syngeneic model. Most HNSCC patients (43/44) co-expressed MAGED4B and FJX1 and their respective tetramer-specific T cells were in the range of 0.06-0.12%. In a preclinical model, antigen-specific T cells were induced by DNA vaccines and increased T cell infiltration into the tumor, but not MDSC or regulatory T cells. The vaccines inhibited tumor growth and improved the outcome alone and upon combination with anti-PD1 and resulted in tumor clearance in approximately 75% of mice. Pre-existence of MAGED4B and FJX1-reactive T cells in HNSCC patients suggests that these widely expressed antigens are highly immunogenic and could be further expanded by vaccination. The DNA vaccines targeting these antigens induced robust T cell responses and with the anti-PD1 antibody conferring excellent tumor control. This opens up an opportunity for combination immunotherapy that might benefit a wider population of HNSCC patients in an antigen-specific manner.