Advances in molecular mechanisms of oral submucosal fibrogenic carcinogenesis

LI Bing-xia, WEN Qi-tao, WANG Tao✉, SUN Guan-yu, FU Xiao, SU Shou-da

1. School of Stomatology, Hainan Medical University, Haikou 571199, China

2. Department of Stomatology, Hainan General Hospital, Haikou 570311, China

Keywords:

ABSTRACT Oral submucosal fibrosis (OSF) is a chronic, progressive and insidious oral mucosal disease.Its development is highly associated with areca chewing and eventually evolves into oral squamous cell carcinoma (OSCC), which is currently recognized as a precancerous state by the International Center for Research on Cancer (IARC).Due to the extremely complex carcinogenesis process of OSF, the carcinogenesis mechanism is not clear, and most scholars believe that the disease is the result of multiple factors.Therefore, this paper mainly summarized the latest research on the molecular mechanism of OSF canceration, aiming at understanding the law of OSF canceration and providing theoretical reference for early detection, diagnosis and treatment of OSF canceration.

Oral submucous fibrosis (OSF) is an inflammatory reaction caused by excessive extracellular matrⅨ deposition, which leads to atrophy of oral mucosal epithelium, accumulation of collagen in the lamina propria of oral mucosa and reduction of blood vessels.The early pathological manifestations are edema of collagen fibers,vasodilation and congestion, visible subepithelial blisters and hyperkeratosis.In the late stage, dense fibers replace deep muscle tissue, collagen fibers are hyaline, blood vessels and fibroblasts are significantly reduced, with mild to moderate chronic inflammatory cell infiltration, and epithelial dysplasia in some cases.OSF can have a 7 % ～13 % chance of cancer, and with the development of social economy and the commercialization of areca, this probability will increase[1,2].Some epidemiological and case-control studies have shown that the disease has obvious regional characteristics.Southeast Asia and China ‘s Hainan, Hunan and Taiwan have higher prevalence rates.Residents in these areas have the habit of chewing betel nut, which is the main factor leading to OSF canceration[3,4].This article systematically reviews the research reports on OSF carcinogenesis in recent years at home and abroad, and reviews the molecular mechanisms of OSF carcinogenesis in terms of cell cycle changes, genetic susceptibility, epigenetic regulation, epithelialmesenchymal transition, hypoxia and angiogenesis.

1.Changes of cell cycle

Cell cycle is an accurate and orderly process, which is regulated by a variety of genes and proteins.When the expression of these genes and proteins is abnormal, cell proliferation is out of control, apoptosis is inhibited, and genetic instability factors are accumulated, which leads to tumorigenesis.Therefore, the change of cell cycle may be one of the mechanisms of OSF carcinogenesis.

p21 and p27 are tumor suppressors and important regulators of cell cycle checkpoints, which play an important role in genome integrity.In vitro studies have found that arecoline can enhance reactive oxygen species (ROS) in OSCC cells, down-regulate the expression of p21 and p27 in OSCC cells through the ROS / mTORC1 pathway, promote cell cycle G1 / S phase transition, and increase the probability of DNA replication errors, thereby increasing the risk of OSF carcinogenesis.

The p34cdc2-Cyclin B1 complex, the only complex formed by phosphokinase cell division cycle protein 2 (p34cdc2) and cyclin B1, is a key regulator of the G2 / M phase of the cell cycle.Vay et al.[5] found that the expression of p34cdc2-Cyclin B1 gradually increased in normal, OSF and OSF cancerous oral mucosa tissues,and its increased expression could phosphorylate the apoptosis inhibitor Survivin.Other studies have pointed out that the expression level of phosphorylation-Survivin (p-Survivin) was significantly higher in the OSF canceration to OSCC group than in the normal group and the OSF group, and the expression showed an upward trend during the carcinogenesis of OSF[6], suggesting that p-Survivin may promote cell division and proliferation by inhibiting apoptosis,which seriously affects the stability of mitosis and leads to the carcinogenesis of OSF.

The expression of proliferating cell nuclear antigen (PCNA) is closely related to cell division, which increases in G1 and S phases and decreases in G2 phase.PCNA is expressed in both OSF and OSCC.In OSF, it is mainly expressed in the epithelial basal layer and spinous layer, and in OSCC, it can be expressed in the whole epithelial layer[7].With the oral tissue from normal epithelium to OSF to OSCC, the distribution of PCNA gradually increased,confirming that cell cycle changes are associated with OSF carcinogenesis.

2.Genetic susceptibility

Genetic susceptibility mainly includes gene mutation, single nucleotide polymorphism (SNP) and loss of heterozygosity (LOH).

Alkaloids, polyphenols and their unique nitrosamines in areca nut have genotoxicity and mutagenicity, which can directly act on the oral mucosa and induce the occurrence of OSF, and then cancer.Ekanayaka et al.[8] observed that arecoline has mutagenicity in Ames test, and can induce sister chromatid exchange, chromosome aberration and micronucleus formation in several types of cells.

SNPs in the gene promoter region are associated with susceptibility to OSF and OSCC patients caused by chewing betel nut.Studies have found that there are high frequencies of high-risk alleles and genotypes of collagen, matrⅨ metalloproteinases (MMPs),transforming growth factor-β (TGF-β) and lysyloxidase (LOX) in OSF and OSCC, which change the transcriptional activity and the expression and function of corresponding proteins, and increase the risk of OSF carcinogenesis[9].

Ray et al.[10] found that LOH was present on all autosomes in OSCC specimens, and high-frequency LOH was positively correlated with the progression and recurrence of OSCC.At the same time, the LOH locus shared by OSF and OSCC contains a variety of tumor suppressor genes involved in maintaining the genetic integrity and defense ability of oral epithelium, such as PTEN, breast cancer genes 1,2 (BRAC1, BRAC2), fragile histidine triad (FHIT) and cytochrome P450 (CYP450).Once these tumor suppressor genes occur LOH, their DNA repair, cell adhesion and other functions will be affected, causing OSF to become cancerous.It is speculated that LOH may play an important role in the carcinogenesis of OSF and promote the carcinogenesis of OSF.

3.Epigenetic regulation

Epigenetic changes refer to the heritable changes of gene DNA sequence and function, resulting in phenotypic changes, including DNA modification, histone modification and non-coding RNA regulation.Because of its heritable and reversible characteristics, it has become an attractive candidate for the development of disease biomarkers, and has become an emerging target for the treatment of several human cancers.It also provides new insights into the molecular and cellular events related to OSF carcinogenesis.

Aberrant promoter methylation is one of the earliest molecular events in oral cancer.Dickkopf-1 (DKK1) is a gene that specifically inhibits the Wnt / β-catenin pathway and is abnormally expressed in various fibrotic diseases and tumors.The methylation level of DKK1 is lower in the normal group and higher in the OSF group and the OSF cancer group, suggesting that the hypermethylation of DKK1 may lead to the occurrence and canceration of OSF by silencing DKK1 gene or inhibiting the expression of DKK1 gene and abnormally activating the Wnt / β-catenin pathway[11].Therefore,the detection of human gene promoter CpG island methylation in primary tumor tissues and body fluids is a promising non-invasive screening and early cancer diagnosis method, which is expected to be applied to oral cancer screening.

Abnormal expression and dysfunction of non-coding RNA can promote the carcinogenesis of OSF.MicroRNAs (miRNAs) are often found to be located in fragile genomic locations and genomic regions associated with cancer risk, and have clear characteristics in the occurrence and development of cancer.As a tumor suppressor, the expression of miR-22 was inhibited in arecoline-treated OSCC cells,which was equivalent to the up-regulation of c-Myc oncogene, and c-Myc could also directly inhibit miR-22.In addition, p53 is a direct transcription factor of miR-22, which can induce miR-22 expression,but arecoline inhibits p53 expression, thereby indirectly inhibiting miR-22 expression[12], indicating that abnormal expression of miR-22 plays a key role in OSF carcinogenesis.The loss of control and dysfunction of long non-coding RNAs (lncRNAs) are associated with a variety of malignant tumors.Some studies have found that differentially expressed lncRNAs are involved in inflammation,Wnt and p53 signaling pathways by RNA sequencing of normal,OSF and OSCC oral mucosa tissues.These signaling pathways are involved in the inflammation and fibroelastic pathological changes of OSF and further carcinogenesis[13].Circular RNA (circRNA) is a special non-coding RNA.Wang et al.[14] conducted microarray analysis of circRNA and identified circEPSTI1 as a circRNA, which is in normal oral mucosa to OSF.

4.Epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) transforms polarized epithelial cells into mesenchymal phenotypes, which is characterized by the loss of cell-cell junctions and the acquisition of spindleshaped morphology to establish motor and invasive phenotypes.It plays a key role in inflammation, fibrosis and tumorigenesis.EMT can promote the accumulation of extracellular matrⅨ, which is an important step in the process of OSF and its carcinogenesis.The characteristics of EMT include the expression and loss of epithelial markers such as E-cadherin, cytokeratin, and increased abundance of mesenchymal markers such as N-cadherin, vimentin and integrin[15].

Chewing areca nut is considered to be the main cause of oral EMT and activation of buccal mucosal fibroblasts (BMF).Arecoline can reduce the expression of E-cadherin and increase the expression of N-cadherin and vimentin in BMF[16].Arecoline up-regulates the expression of E-cadherin transcriptional inhibitor Snail in a dosedependent manner, which in turn triggers the transdifferentiation of myofibroblasts, increases the expression of various fibrotic factors,such as type I collagen (Col-1) and alpha-smooth muscle actin(α-SMA), and increases the expression of interleukin-6 (IL-6).The elevated inflammatory cytokines lead to an imbalance between tissue repair and fibrosis formation in the oral cavity.The overexpression of Snail is related to the clinicopathological features of OSCC[17-19].IL-6 induces EMT in a variety of cancers through the Stat3 /Snail signaling pathway[20].Experiments have shown that IL-6 is a direct target of Snail and mediates the activation of myofibroblasts induced by Snail, indicating that there is a loop between Snail and IL-6 that positively regulates the transdifferentiation of areca-related myofibroblasts, suggesting that blocking Snail can be used as a powerful strategy for the treatment of OSF[21].It can be inferred that Snail is essential for maintaining the activity of myofibroblasts.The treatment targeting Snail may alleviate the carcinogenesis of OSF and down-regulate the chronic inflammation caused by it.In addition, arecoline can up-regulate the transcription of insulin-like growth factor-1 receptor (IGF-1R) and induce the phosphorylation of IGF-1R, thereby inducing the activation of E-cadherin transcription inhibitor zinc finger E protein ZEB1 and reducing the expression of E-cadherin[22].At the same time, it also deepens the understanding of EMT in the process of OSF carcinogenesis.

Integrin αvβ6 is involved in pathological fibrosis of multiple organs, and its expression is up-regulated in OSF, confirming that arecoline-dependent up-regulation of αvβ6 promotes the transformation of oral fibroblasts into myofibroblasts[22].The high expression rate of RAS gene in patients with OSF canceration may be one of the reasons for OSF canceration to OSCC.Some scholars have also pointed out that arecoline can up-regulate the expression of αvβ6 in keratinocytes, and then activate TGF-β1, TGF-β1 and RAS may be involved in regulating the EMT process of tumor cell invasion[19].

In recent years, the role of miRNAs in arecoline-induced EMT has been reported.The expression of miR-203 in OSF tissues was significantly lower than that in normal oral mucosa tissues.Its upregulation significantly inhibited cell proliferation, significantly upregulated the expression of cytokeratin 19 (CK19) and E-cadherin,and down-regulated the expression of N-cadherin and vimentin,thus reversing the EMT process[23].Other researchers have observed that arecoline can dose-dependently reduce the expression of miR-200b gene in human bone marrow mesenchymal stem cells.After overexpression of miR-200b in bone marrow mesenchymal stem cells, arecoline-induced myofibroblast activity was abolished.In addition, the increase of miR-200b inhibited the expression of α-SMA[24].

These studies suggest that betel nut leading to oral EMT may be a key link in the process of OSF carcinogenesis and may also be a potential target for preventing OSF carcinogenesis.

5.Hypoxia and angiogenesis

Hypoxia-inducible factor 1-alpha (HIF-1α) is considered to be associated with epithelial carcinogenesis[25].The stability of HIF-1α can promote the gene expression of tumorigenesis and angiogenesis, and obtain a more glycolytic phenotype, so that cancer cells do not rely on oxygen to produce ATP.The increase of neovascularization and glycolysis represents the adaptation to hypoxic microenvironment, which is related to tumor invasion and metastasis[26,27].

The expression of HIF-1α in fibroblasts, epithelial cells and inflammatory cells increased in turn in normal, OSF and OSCC tissues[28].In addition, HIF-1α is up-regulated at both protein and gene levels in OSF tissues, and is associated with epithelial dysplasia.At the same time, arecoline in BMF can up-regulate the expression of HIF-1α gene in a dose-dependent manner[29,30].This indicates that changes in cell proliferation, maturation and metabolic adaptation increase the possibility of carcinogenesis.Carbonic anhydrase Ⅸ (CAⅨ) is a hypoxia-inducible enzyme that has been shown to be a direct target of HIF, which is responsible for regulating intracellular pH and participating in the formation of extracellular space.It is significantly up-regulated in cancer hypoxia and promotes tumor cell invasion[26].Some scholars have found that the total activity of CA and the expression level of CAⅨ gene in OSF are higher than those in normal BMF, and arecoline increases the expression of CAⅨ in normal BMF in a dose-dependent manner.The plasma CAⅨ level of patients who chew areca is higher than that of patients who do not chew areca.The plasma CAⅨ level of patients with oral cancer is statistically significantly correlated with clinical stage[31].

In the precancerous stage of several human cancers, angiogenesis switches may be activated.Therefore, tumor angiogenesis is not necessarily a feature of invasive tumors, but may be an early event in the development of cancer.A variety of target genes of HIF-1α, such as vascular endothelial growth factor (VEGF), angiogenin and fibroblast growth factor, regulate angiogenesis by promoting mitosis and migration activity of endothelial cells, and promote the proliferation and differentiation of irregular vascular endothelial cells [32].From OSF to OSCC to OSCC with OSF, the expression of HIF-1α showed an upward trend and was positively correlated with vascular density[33].In vivo studies have shown that the changes of OSF microvessels are mainly atrophy and reduction, and the increase of blood vessels in the later stage of OSF is considered as OSF carcinogenesis[34].It is concluded that the up-regulation of HIF-1α is an early event in the carcinogenesis of OSF.The expression of HIF-1α in OSCC tissues is accompanied by an increase in the number of blood vessels, but compared with the increase in the number of blood vessels, the number of fibroblasts increases more,that is, OSF carcinogenesis is due to fibroblasts.Increased fibrosis,more hypoxic conditions lead to increased expression of HIF-1α[25].

It can be seen that the effect of hypoxia on the progression of OSF carcinogenesis is mediated by a series of protein and genomic changes induced by hypoxia, which activate angiogenesis, anaerobic metabolism and other processes that enable tumor cells to survive or escape from hypoxic environments.

6.Other

In addition to the above mechanisms, in recent years, scholars have also studied the molecular mechanism of OSF carcinogenesis from cell senescence, tumor stem cells, trace elements, oral microbiome,immune microenvironment and other aspects.At the same time,new explorations have been made on the biomarkers and therapeutic targets of OSF carcinogenesis.These studies and explorations will help people to have a deep understanding of the molecular mechanism of OSF carcinogenesis and provide new strategies for its prevention and treatment.

In conclusion, OSF carcinogenesis is a complex and dynamic process, which mainly involves cell cycle changes, genetic susceptibility, epigenetic regulation, epithelial-mesenchymal transition, hypoxia and angiogenesis.From the occurrence and development of OSF to the carcinogenesis of OSF to OSCC, each mechanism interacts with each other and promotes the progression of OSF carcinogenesis.Therefore, further elucidating the molecular mechanism of OSF carcinogenesis induced by areca nut is of great significance for the prevention and treatment of areca nut chewing related diseases in the future.With the increasing economic and social burden caused by OSF and its carcinogenesis, clinicians and related researchers should strengthen primary prevention and carry out health education for patients with betel quid chewing habits.In addition, early detection, early diagnosis and early treatment of OSF are the key to preventing OSF carcinogenesis.We should continue to study the molecular mechanism of OSF carcinogenesis, determine the main molecular targeting methods, clarify the occurrence and carcinogenesis of OSF, and provide new ideas for the prevention and treatment of OSF carcinogenesis.

Authors’ contribution

Li Bingxia: Proposed research ideas, wrote papers and revised;Wang Tao, Wen Qitao: Check the full text, put forward suggestions for revision, and provide guidance; Sun Guanyu, Fu Xiao, Su Shouda: Assisted in collecting literature and sorting out documents.All authors declare no conflict of interest.