Many of the characteristics ascribed to cancer-associated fibroblasts (CAFs) are shared by activated, autophagic and senescent fibroblasts. Whilst most oral squamous cell carcinomas (OSCCs) are genetically unstable (GU-OSCC), genetically stable variants (GS-OSCC) have been described and, notably, CAF activation (myofibroblast differentiation) and senescence are characteristics particularly associated with GU-OSCCs. However, it is not known whether autophagy is disrupted in these cells or whether autophagy regulates the development of the myofibroblast and senescent phenotypes. In this study, we show that senescent CAFs from GU-OSCCs contained more autophagosomes than normal human oral fibroblasts (NHOFs) and CAFs from GS-OSCCs possibly due to autophagic impairment. Further, we show that deregulation of autophagy in normal fibroblasts, either by inhibition with autophagy inhibitor, SAR405, or activation with TGF-β1, induced fibroblast activation and senescence: In response to TGF-β1, autophagy was induced prior to the development of the activated and senescent phenotypes. Lastly, we show that both SAR405- and TGF-β1-treated NHOFs enhance OSCC cell migration but only TGF-β1-treated cells increase OSCC invasion through Matrigel, indicating that TGF-β1 has additional effects that are independent of fibroblast activation/senescence. These results suggest a functional role for autophagy in the development of myofibroblast and CAF phenotypes.
This study reviews the molecular landscape of oral potentially malignant disorders (OPMD). We examine the impact of tumour heterogeneity, the spectrum of driver mutations (TP53, CDKN2A, TERT, NOTCH1, AJUBA, PIK3CA, CASP8) and gene transcription on tumour progression. We comment on how some of these mutations impact cellular senescence, field cancerization and cancer stem cells. We propose that OPMD can be monitored more closely and more dynamically through the use of liquid biopsies using an appropriate biomarker of transformation. We describe new gene interactions through the use of a systems biology approach and we highlight some of the first studies to identify functional genes using CRISPR-Cas9 technology. We believe that this information has translational implications for the use of re-purposed existing drugs and/or new drug development. Further, we argue that the use of digital technology encompassing clinical and laboratory-based data will create relevant datasets for machine learning/artificial intelligence. We believe that therapeutic intervention at an early molecular premalignant stage should be an important preventative strategy to inhibit the development of oral squamous cell carcinoma and that this approach is applicable to other aerodigestive tract cancers.
The interrelationship between malignant epithelium and the underlying stroma is of fundamental importance in tumour development and progression. In the present study, we used cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), tumours that are characterized by the loss of genes such as TP53 and p16INK4A and with extensive loss of heterozygosity, together with CAFs from their more genetically stable (GS) counterparts that have wild-type TP53 and p16INK4A and minimal loss of heterozygosity (GS-OSCC). Using a systems biology approach to interpret the genome-wide transcriptional profile of the CAFs, we show that transforming growth factor-β (TGF-β) family members not only had biological relevance in silico but also distinguished GU-OSCC-derived CAFs from GS-OSCC CAFs and fibroblasts from normal oral mucosa. In view of the close association between TGF-β family members, we examined the expression of TGF-β1 and TGF-β2 in the different fibroblast subtypes and showed increased levels of active TGF-β1 and TGF-β2 in CAFs from GU-OSCC. CAFs from GU-OSCC, but not GS-OSCC or normal fibroblasts, induced epithelial-mesenchymal transition and down-regulated a broad spectrum of cell adhesion molecules resulting in epithelial dis-cohesion and invasion of target keratinocytes in vitro in a TGF-β-dependent manner. The results demonstrate that the TGF-β family of cytokines secreted by CAFs derived from genotype-specific oral cancer (GU-OSCC) promote, at least in part, the malignant phenotype by weakening intercellular epithelial adhesion.
Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.
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.
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and includes squamous cell carcinomas of the oropharynx and oral cavity. Patient prognosis has remained poor for decades and molecular targeted therapies are not in routine use. Here we showed that the overall expression of collagen subunit genes was higher in cancer-associated fibroblasts (CAFs) than normal fibroblasts. Focusing on collagen8A1 and collagen11A1, we showed that collagen is produced by both CAFs and tumour cells, indicating that HNSCCs are collagen-rich environments. We then focused on discoidin domain receptor 1 (DDR1), a collagen-activated receptor tyrosine kinase, and showed that it is over-expressed in HNSCC tissues. Further, we demonstrated that collagen promoted the proliferation and migration of HNSCC cells and attenuated the apoptotic response to cisplatin. Knockdown of DDR1 in HNSCC cells demonstrated that these tumour-promoting effects of collagen are mediated by DDR1. Our data suggest that specific inhibitors of DDR1 might provide novel therapeutic opportunities to treat HNSCC.