Genetic regulation and potentially therapeutic application of cancer‐associated fibroblasts in oral cancer

Carcinoma‐associated fibroblast (CAF) is the most important host cell type in tumor microenvironment, which greatly contributes to tumor initiation, progression, and metastasis. Therefore, a large amount of data has emerged, showing the cancer‐promoting function of these cells via paracrine effects that escort tumor cells through all the steps of cancer development. CAF is a heterogeneous cell population that can arise from the differentiation of resting fibroblasts, epithelial cells, endothelial cells, and mesenchymal stem cells. This review summarizes the current knowledge of the role of CAFs in tumor progression, with a particular focus on the cellular and molecular features and recent advances in researches on the genetic status and microRNA regulation, and addresses the potential prognostic and therapeutic values for patients with oral cancer by targeting CAFs.     

N‐cadherin expression is correlated with metastasis of spindle cell carcinoma of head and neck region

J Oral Pathol Med (2011) 40 : 77–82 Spindle cell carcinoma (SpCC) is a biphasic tumor composed of conventional squamous cell carcinoma and a malignant spindle cell component. SpCC expresses both epithelial and mesenchymal markers by immunohistochemical analysis. There is mounting evidence for sarcomatoid transformation from the epithelial component, supporting the theory that SpCC is a monoclonal neoplasm originating from a stem cell giving rise to both components. The loss of E‐cadherin and the gain of N‐cadherin expression are known as the “cadherin switching”. Cadherin switching is a major hallmark of epithelial–mesenchymal transition (EMT). EMT is a crucial process in cancer progression providing cancer cells with the ability to escape from the primary focus, to invade stromal tissues, and to migrate to distant regions. Although E‐cadherin down‐regulation is well known in various cancers, there are a few studies on N‐cadherin expression in cancer. Here, therefore, we investigated N‐cadherin expression in the progression of head and neck SpCC. First, we examined cadherin switching in our established SpCC cell lines, SpCC‐1 and SpCC‐2. SpCC‐1 and SpCC‐2 cells were spindle in shape and showed cadherin switching. Moreover, we examined N‐cadherin expression in 15 SpCC cases by immunohistochemistry. Although N‐cadherin expression was not observed in non‐neoplastic squamous epithelium, high expression of N‐cadherin was observed in 10 of 15 SpCC cases. Interestingly, 6 of 7 SpCC cases with metastasis showed high expression of N‐cadherin. In conclusion, our findings suggest that N‐cadherin may play an important role in metastasis of SpCC in addition to the pathogenesis of SpCC of the head and neck.     

Areca nut extract upregulates vimentin by activating PI3K/AKT signaling in oral carcinoma

J Oral Pathol Med (2011) 40 : 160–166 Background: Areca nut is a group I carcinogen. Areca nut extract (ANE) is known to activate signaling pathways in oral epithelial cells. Activation of the serine/threonine protein kinase AKT/pKB (AKT) signaling pathway is known to be important during the neoplastic process. Vimentin is a mesenchymal intermediate filament and a regulator of tumor progression. This study investigated the impact of ANE on PI3K/AKT activation during vimentin expression. Materials and methods: Oral carcinoma cells were treated with ANE to explore the signaling changes underlying vimentin expression. Oral carcinoma tissues were subjected to immunohistochemical analysis to study the implications that vimentin expression has on patient survival. Results: After ANE treatment, the OECM‐1 and Fadu cells developed a fibroblastoid morphology and there was an increase in vimentin expression. The treatment also induced the phosphorylation of AKT and glycogen synthase kinase 3β in OECM‐1 cells. Blockage of phosphatidylinositol 3‐kinase (PI3K)/AKT signaling attenuated vimentin expression when it was induced by ANE. However, it did not affect ANE‐mediated extracellular signal‐regulated kinase (ERK) activation or cyclooxygenase 2 (COX‐2) upregulation. Oral carcinoma tissue samples were found to have significantly higher levels of vimentin and pAKT expression than their controls. Tumors exhibiting no vimentin expression and weak AKT phosphorylation were found to be associated with better survival than groups with high levels of expression. Conclusion: Our results imply that PI3K/AKT activation and vimentin expression are important pathogenic cascades in areca‐associated oral carcinogenesis.     

肿瘤相关成纤维细胞促进舌鳞癌细胞上皮间质转化

目的：检测舌鳞癌组织中肿瘤相关成纤维细胞是否诱导舌癌细胞的上皮间质转化。方法：通过体外原代培养舌鳞癌组织肿瘤相关成纤维细胞的培养上清作用于舌鳞癌细胞系Tca8113,通过检测细胞形态确认肿瘤细胞是否发生上皮间质转化。利用实时定量PCR及免疫印迹检测舌鳞癌细胞上皮标志及间质标志物表达。利用TGF-β/Smad信号通路阻断剂探究TGF-β在此过程的作用。结果：肿瘤相关成纤维细胞促进Tca8113的上皮间质转化。利用实时定量PCR检测及免疫印迹发现肿瘤相关成纤维细胞可以明显促进Tca8113细胞的Vimentin的表达。SB431542,TGF-β/Smad信号通路特异性阻断剂可明显阻断肿瘤相关成纤维细胞条件培养基对Tca8113上皮间质转化的诱导作用。结论：舌鳞癌组织中的肿瘤成纤维细胞可通过诱导舌鳞癌细胞的上皮间质转化促进舌鳞癌细胞的迁移及侵袭,可能在肿瘤发展过程发挥重要作用。     

Epithelial–mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro

Epithelial–mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase‐20 (MMP‐20), and kallikrein‐4 (KLK‐4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum‐free medium. Both the epithelial cells and fibroblasts were co‐cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in‐situ hybridization and RT‐PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP‐20 and KLK‐4 in epithelial cells of Malassez co‐cultured with periodontal ligament fibroblasts. in‐situ hybridization and RT‐PCR confirmed significant mRNA expression of these factors in co‐cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial–mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.     

Areca-treated fibroblasts enhance tumorigenesis of oral epithelial cells

Several hundred million Asians chew areca nut, which is strongly associated with oral carcinogenesis in people of this region. The impacts of areca nut extract on oral target cells are largely unclear. This study hypothesized an inductive role for areca-nut-exposed stromal cells in the progression of oral carcinomas in an at-risk population. Oral fibroblasts with chronic subtoxic areca nut extract treatment exhibited growth arrest and MMP-2 activation. The supernatant of arrested oral fibroblasts activated the AKT signaling pathway in oral carcinoma cells. The enhancement of proliferation, migration, and anchorage-independent growth of oral carcinoma cells elicited by such supernatant could be abrogated by blockers against MMP-2 or AKT. Subcutaneous co-injection of arrested oral fibroblasts into nude mice significantly enhanced the tumorigenicity of xenographic oral carcinoma cells. This study concludes that areca nut extract may impair oral fibroblasts and then modulate the progression of oral epithelial oncogenesis via their secreted molecules.     

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell‐carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell‐carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E‐selectin expression, which is mediated by eNOS production and p38 mitogen‐activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel‐induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.     

Lack of evidence for prognostic value of epidermal growth factor receptor intron‐1 CA repeats for oral carcinomas

Epidermal growth factor receptor (EGFR) expression is altered in several malignancies, including oral squamous cell carcinoma. A CA‐repeat polymorphism in intron‐1 (CA‐SSR‐1) of the EGFR gene is reported to influence EGFR expression and is associated with features of various solid tumors and outcomes of cancer patients. In the present study we evaluated the influence of length and zygosity of CA‐SSR‐1 on the survival of patients with oral squamous cell carcinoma. The length and zygosity of CA‐SSR‐1 was obtained through microsatellite analysis in 91 patients with oral cancer, who were treated in the Department of Oral and Maxillofacial Surgery of the University Medical Centre Hamburg Eppendorf, Germany, during the years 1998–2008. Follow up was conducted until 2016. Outcome measures were age, gender, tumor stage, occurrence of metastases, and date of recurrence or death. Statistical analysis was conducted using the chi‐square test and the log‐rank test. Neither length nor zygosity of the CA‐SSR‐1 in patients with oral squamous cell carcinoma was significantly correlated with sex, age, tumor size, tumor localization, lymph node involvement, metastasis status, disease‐free survival, or overall survival. Length and zygosity of the CA‐SSR‐1 polymorphism in EGFR is not able to serve as a prognostic biomarker in White European patients with oral squamous cell carcinoma.     

TGF‐β1 regulates the invasive and metastatic potential of mucoepidermoid carcinoma cells

J Oral Pathol Med (2011) 40 : 762–768 Background: Patients with mucoepidermoid carcinoma exhibit poor long‐term prognosis because of the lack of therapeutic strategies that effectively block tumor progression. We have previously characterized the Ms cells as a highly metastatic mucoepidermoid carcinoma cell line that expresses high levels of transforming growth factor β1 (TGF‐β1). Here, we studied the effect of suppressing TGF‐β1 by RNA silencing on the invasive and metastatic potential of mucoepidermoid carcinoma. Methods: Cell motility, substratum adhesion, and transmembrane invasion were estimated by migration, matrigel adhesion, and matrigel invasion assay. Matrix metalloproteinase (MMP)‐2 and MMP‐9 activity were determined using gelatin gel zymography. Balb/c nu/nu nude mice lung metastatic model was used to test the metastatic ability of the Ms cells. Lung metastatic tumors were experimentally induced by mice tail vein inoculation of cancer cells. Results: TGF‐β1 silencing inhibits cell motility, substratum adhesion, and transmembrane invasion. In vivo, a significant decrease in lung metastasis was observed when mice received tail vein injections of TGF‐β1‐silenced mucoepidermoid carcinoma cells, as compared to controls. Conclusion: These results unveil a critical role for TGF‐β1 in the progression of mucoepidermoid carcinomas and suggest that patients with this malignancy may benefit from therapeutic inhibition of the effectors of the TGF‐β1 pathway.     

Curcumin suppresses the proliferation and tumorigenicity of Cal27 by modulating cancer‐associated fibroblasts of TSCC

Cancer‐associated fibroblasts (CAFs) are “activated” fibroblasts in the tumor microenvironment (TME) and play a vital role in all steps of cancer development. Increasing evidence focusing on the function of CAFs suggests that CAFs are candidate therapeutic targets and that drugs targeting the modification of CAFs would suppress tumor progression and be beneficial to tumor treatment and prevention. In the present study, we found that curcumin reversed the phenotype of CAFs to that of peri‐tumor fibroblast (PTF)‐like cells by downregulating the expression of α‐SMA (a special marker for CAFs) and inhibiting the secretion of pro‐carcinogenic cytokines, including transforming growth factor‐β1 (TGF‐β1), matrix metalloproteinases 2 (MMP2), and stromal cell‐derived factor‐1 (SDF‐1). We further demonstrated that the conditioned medium (CM) derived from CAFs promoted the proliferation of Cal27, and this effect was confirmed by the xenograft model. More importantly, we found that curcumin blocked the CAF‐mediated enhancement of Cal27 proliferation in vitro and in vivo. In conclusion, our data suggest that curcumin reverses cell phenotype from CAF to PTF‐like cells and suppresses the CAF‐mediated proliferation and tumorigenicity of Cal27 by inhibiting TSCC CAFs.     

Myofibroblasts in oral potentially malignant disorders: Is it related to malignant transformation?

In oral cancer, acquisition of α‐smooth muscle actin (α‐SMA)‐positive fibroblasts, known as myofibroblasts or carcinoma‐associated fibroblasts (CAF), is an important event for progression and metastasis. However, the contribution of myofibroblasts in oral potentially malignant disorders (OPMD) remains controversial. This systematic review provides evidence that immunodetection of myofibroblasts may identify oral submucous fibrosis (OSMF) with high risk of malignant transformation, but does not represent an auxiliary tool to predict the malignant potential of leukoplakia and erythroplakia, the most common OPMD.     

Expression of extracellular matrix metalloproteinase inducer and enhancement of the production of matrix metalloproteinase-1 in tongue squamous cell carcinoma

Recent studies have found that in addition to promoting cellular invasion, overexpression of metalloproteinase -1 (MMP-1) is associated with the initial stages of cancer development. Extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein, has been reported to be highly expressed in tumor cells and induce production of MMPs from peritumor fibroblasts (PTFs) adjacent to the tumor cells. The expression of EMMPRIN in tongue squamous cell carcinoma (SCC) was investigated in this study. It was found that EMMPRIN was expressed at the cell membrane throughout the entire lesion in tongue SCC. Immunofluorescence staining localized EMMPRIN to the cell membrane in a highly invasive tongue SCC cell line (Tca 8113). EMMPRIN mRNA was expressed at a high level in Tca 8113, whereas MMP-1 mRNA was expressed in PTF but harder to be detected in Tca 8113. Co-culture of Tca 8113 with PTF stimulated production of MMP-1. EMMPRIN was highly expressed in tongue SCC, and could induce local production of MMP-1. These data indicate that EMMPRIN might play an important role in tongue SCC progression and invasion.     

Inhibition of autophagy augments chemotherapy in human salivary adenoid cystic carcinoma

Although cisplatin (DDP)‐based adjuvant chemotherapy is widely used in the treatment of salivary adenoid cystic carcinoma (SACC), SACCs have developed resistance to cisplatin, resulting in chemotherapy failure. Autophagy serves as a critical adaptive response, which was increased in tumor cells in chemotherapy. However, the function of autophagy is not clear in SACC. In this study, apoptosis induced by DDP in SACC high metastatic cell line (ACC‐M) was revealed using MTT assay, flow cytometry, and caspase‐3 immunoblotting. The autophagy activation induced by DDP treatment was measured by transmission electron microscopy, green fluorescent protein–light chain 3 plasmid transfection LC3 immunoblotting and p62 immunoblotting. 3‐methyladenine (3‐MA) or small interference RNA targeting beclin 1 (beclin 1 siRNA) inhibited autophagy and significantly enhanced DDP‐induced apoptosis. ACC‐M xenografts in nude mice further verified the synergistic effect of DDP and 3‐MA. In conclusion, autophagy activation was caused to protect cancer cells from DDP‐induced apoptosis and autophagy inhibition could be a promising strategy for adjuvant chemotherapy in SACC.     

Immunomodulatory aspects in the progression and treatment of oral malignancy

Inflammation substantially affects the risk of oral malignancy. Pro-inflammatory cytokine, interferon (IFN)-γ, confers anti-tumor activity using several different mechanisms. Conversely, higher expression of interleukin (IL)-17 is associated with worse prognosis. Monocyte chemotactic protein (MCP)-1 correlates positively with poor long-term survival of head and neck squamous cell carcinoma (HNSCC) patients. IL-1α affects cancer associated fibroblasts and macrophages, and promote several malignant phenotypes including immune suppression. Some anti-inflammatory cytokines, including IL-10 and transforming growth factor (TGF)-β, relate to pro-tumoral activities.Among immune checkpoint modulators, programmed death (PD-)1 and PD-ligand (L)1 facilitate oral squamous cell carcinoma (OSCC) cell evasion from immune surveillance, and the expression status of these has a prognostic value.OSCCs contain tumor associated macrophages (TAMs) as major stromal cells of their tumor microenvironment. Among the two distinctive states, M2 macrophages support tumor invasion, metastasis and immune suppression. Crosstalk between TAMs and OSCC or cancer-associated fibroblasts (CAF) plays an important role in the progression of OSCC.Clinical trials with blocking antibodies against IL-1α or melanoma-associated antigens have been reported as therapeutic approaches against OSCCs. The most promising approach activating antitumor immunity is the blockade of PD-1/PD-L1 axis. Manipulating the polarization of pro-tumorigenic macrophages has been reported as a novel therapeutic approach.     

Enhanced expression of podoplanin in ameloblastomas

J Oral Pathol Med (2010) 39 : 103–109 Objective: Podoplanin, a mucin‐type transmembrane glycoprotein, is specifically expressed by lymphatic but not blood vascular endothelial cells, and is also widely expressed in various specialized cell types throughout the body. Recent studies have demonstrated that it mediates a pathway leading to collective cell migration and invasion in vivo and in vitro. In the present study, we carried out an immunohistochemical investigation of podoplanin to clarify whether it is expressed in human ameloblastomas (AMs), which are characterized by locally aggressive behavior with a high rate of recurrence. In addition, we examined the localization of the epithelial marker E‐cadherin and the mesenchymal marker vimentin to clarify whether AMs show epithelial–mesenchymal transition (EMT). Methods: Paraffin‐embedded tissue specimens of 38 AMs were examined immunohistochemically using antibodies against podoplanin, E‐cadherin, and vimentin. Results: Immunohistochemical reactivity for podoplanin was detected in the cell membrane and cytoplasm of most odontogenic tumor epithelial cells in AMs. Podoplanin was expressed strongly in peripheral columnar cells and slightly in central stellate reticulum‐like cells. E‐cadherin was expressed in central stellate reticulum‐like cells and showed decreased expression in peripheral columnar cells. Immunoreactivity for E‐cadherin was weak or negative in keratinizing cells of acanthomatous AMs, suggesting terminal differentiation of the tumor cells. Immunohistochemical reactivity for vimentin was found in stromal cells, but partial or no reaction was observed in neoplastic cells. Conclusion: Expression of podoplanin in AMs is considered to be associated with neoplastic odontogenic tissues; this molecule might play a role in the collective cell migration of tumor nests in AMs. The pattern of expression of E‐cadherin and vimentin suggests that invasion in AMs occurs in the absence of EMT. The migration and invasion mediated by podoplanin in AMs could be related to cytoskeletal reorganization.     

Activation of inflammasomes in dendritic cells and macrophages by Mycoplasma salivarium

Interleukin‐1β (IL‐1β) plays crucial roles in the pathogenesis of periodontal disease. It is produced after the processing of pro‐IL‐1β by caspase‐1, which is activated by the inflammasome‐a multiprotein complex comprising nucleotide‐binding domain leucine‐rich repeat‐containing receptor (NLR), the adaptor protein apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC), and procaspase‐1. Mycoplasma salivarium preferentially inhabits the gingival sulcus and the incidence and number of organisms in the oral cavity increase significantly with the progression of periodontal disease. To initially clarify the association of this organism with periodontal diseases, this study determined whether it induces IL‐1β production by innate immune cells such as dendritic cells or macrophages by using Mycoplasma pneumoniae as a positive control. Both live and heat‐killed M. salivarium and M. pneumoniae cells induced IL‐1β production by XS106 murine dendritic cells as well as pyroptosis. The activities were significantly downregulated by silencing of caspase‐1. Bone‐marrow‐derived macrophage (BMMs) from wild‐type and NLR‐containing protein 3 (NLRP3)‐, ASC‐, and caspase‐1‐deficient mice were examined for IL‐1β production in response to these mycoplasmas. Live M. salivarium and M. pneumoniae cells almost completely lost the ability to induce IL‐1β production by BMMs from ASC‐ and caspase‐1–deficient mice. Their activities toward BMMs from NLRP3‐deficient mice were significantly but not completely attenuated. These results suggest that live M. salivarium and M. pneumoniae cells can activate several types of inflammasomes including the NLRP3 inflammasome. Both M. salivarium and M. pneumoniae cells can activate THP‐1 human monocytic cells to induce IL‐1β production. Hence, the present finding that M. salivarium induces IL‐1β production by dendritic cells and macrophages may suggest the association of this organism with periodontal diseases.     

Role of oral microbiome in oral oncogenesis,tumor progression,and metastasis

Squamous cell carcinoma is the most common malignant tumor of the oral cavity and its adjacent sites, which endangers the physical and mental health of patients and has a complex etiology. Chronic infection is considered to be a risk factor in cancer development. Evidence suggests that periodontal pathogens, such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola, are associated with oral squamous cell carcinoma (OSCC). They can stimulate tumorigenesis by promoting epithelial cells proliferation while inhibiting apoptosis and regulating the inflammatory microenvironment. Candida albicans promotes OSCC progression and metastasis through multiple mechanisms. Moreover, oral human papillomavirus (HPV) can induce oropharyngeal squamous cell carcinoma (OPSCC). There is evidence that HPV16 can integrate with host cells’ DNA and activate oncogenes. Additionally, oral dysbiosis and synergistic effects in the oral microbial communities can promote cancer development. In this review, we will discuss the biological characteristics of oral microbiome associated with OSCC and OPSCC and then highlight the mechanisms by which oral microbiome is involved in oral oncogenesis, tumor progression, and metastasis. These findings may have positive implications for early diagnosis and treatment of oral cancer.