Stromal laminin chain distribution in normal,hyperplastic and malignant oral mucosa: relation to myofibroblast occurrence and vessel formation

J Oral Pathol Med (2010) 39 : 290–298 Background: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains α2, α3, α4, α5 and γ2 in the stromal compartment/vascular structures in OSCC was analysed. Methods: Frozen tissue of OSCC (9× G1, 24× G2, 8× G3) and normal (2×)/hyperplastic (11×) oral mucosa was subjected to laminin chain and α‐smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. Results: Stromal laminin α2 chain significantly decreases and α3, α4, α5 and γ2 chains and also ASMA significantly increase with rising grade. The amount of stromal α3, α4 and γ2 chains significantly increased with rising ASMA positivity. There is a significant decrease in α3 chain positive vessels with neoplastic transformation. Conclusions: Mediated by myofibroblasts, OSCC development is associated with a stromal up‐regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning α3 and γ2 chain laminins during tumour angioneogenesis is suggested.     

SOX2通过PI3K/AKT通路调控口腔鳞癌细胞迁徙的机制研究

目的:研究过表达SOX2通过PI3K/AKT通路促进口腔鳞癌(oral squamous cell carcinoma,OSCC)细胞迁移及上皮间质转化(epithelial mesenchymal transition,EMT)的作用及机制.方法:收集OSCC组织及正常口腔黏膜组织,培养OSCC细胞株Tca83、SC...     

Expression of epithelial-mesenchymal transition markers at the invasive front of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common malignances. In epithelial-mesenchymal transition (EMT), epithelial cells switch to mesenchymal-like cells exhibiting high mobility. This migratory phenotype is significant during tumor invasion and metastasis.

Objective

: The aim of this study is to evaluate the expression of the EMT markers E-cadherin, N-cadherin and vimentin in OSCC.

Material and Methods

: Immunohistochemical detection of E-cadherin, N-cadherin and vimentin was performed on 20 OSCC samples. Differences in the expression of each protein at the invasive front (IF) and in the central/superficial areas (CSA) of the tumor were assessed. Differences in the expression of each protein at the IF of both histologically high- and low-invasive OSCCs were evaluated. Associations among expression of proteins at the IF were assessed. Correlations between the expression levels of each protein at the IF and the tumor stage and clinical nodal status were also evaluated.

Results

: Reduced expression of E-cadherin was detected in 15 samples (75%). E-cadherin expression was reduced at the IF when compared to the CSA and in high-invasive tumors when compared to low-invasive tumors. All samples were negative for N-cadherin, even though one sample showed an inconspicuous expression. Positive expression of vimentin was observed in 6 samples (30%). Nevertheless, there was no difference in vimentin expression between the IF and the CSA regions or between the low- and high-invasive tumors. Furthermore, no association was observed among protein expression levels at the IF. Finally, no correlations were observed between each protein’s expression levels and tumor stage or clinical nodal status.

Conclusions

: Reduced E-cadherin expression at the IF and its association with histological invasiveness suggest that this protein is a noteworthy EMT marker in OSCC. Although vimentin was also detected as an EMT marker, its expression was neither limited to the IF nor was it related to histological invasiveness.     

Cetuximab inhibits oral squamous cell carcinoma invasion and metastasis via degradation of epidermal growth factor receptor

Cetuximab (Erbitux, C225) is a chimeric monoclonal antibody that binds to the extracellular domain of epidermal growth factor receptor (EGFR), inhibiting tumor growth, invasion, angiogenesis and metastasis. However, the mechanisms underlying the effect of Cetuximab in human oral squamous cell carcinoma (OSCC) remain unclear. Here, we report that Cetuximab modulates EGFR protein stability through the ubiquitin/proteasome pathway, resulting in the inhibition of human OSCC growth. Cetuximab significantly inhibited the migration and invasion of human OSCC cells by blocking epithelial/mesenchymal transition (EMT) and the AKT and ERK pathways. Furthermore, Cetuximab‐inhibited cell growth by modulating the expression of integrin β5. Taken together, these results provide novel insights into the mechanism of Cetuximab action and suggest potential therapeutic strategies for OSCC.     

Role of Transforming Growth Factor‐beta1 in Cyclosporine‐Induced Epithelial‐to‐Mesenchymal Transition in Gingival Epithelium

Background: It has been proposed that cyclosporin A (CsA) may induce epithelial‐to‐mesenchymal transition (EMT) in gingiva. The aims of the present study are to confirm the notion that EMT occurs in human gingival epithelial (hGE) cells after CsA treatment and to investigate the role of transforming growth factor beta1 (TGF‐β1) on this CsA‐induced EMT. Methods: The effects of CsA, with and without TGF‐β1 inhibitor, on the morphologic changes of primary culture of hGE cells were examined in vitro. The changes of protein and messenger RNA (mRNA) expressions of two EMT markers (E‐cadherin and alpha‐smooth muscle actin) in the hGE cells after CsA treatment with and without TGF‐β1 inhibitor were evaluated with immunocytochemistry and real‐time polymerase chain reaction. Results: The epithelial cells became spindle‐like, elongated, and disassociated from neighboring cells and lost their original cobblestone monolayer pattern when CsA was added. However, the epithelial cells stayed in their original cobblestone morphology with treatment of TGF‐β1 inhibitor on top of the CsA treatment. When CsA was given, the protein and mRNA expressions of E‐cadherin and α‐SMA were significantly altered, and these alterations were significantly reversed with pretreatment of TGF‐β1 inhibitor. Conclusions: CsA could induce Type 2 EMT in gingiva by changing the morphology of epithelial cells and altering the EMT markers/effectors. The CsA‐induced gingival EMT is dependent or at least partially dependent on TGF‐β1.     

Expression of fibroblast growth factor receptor like 1 protein in oral squamous cell carcinoma and its influence on tumor cell proliferation and migration

目的 检测成纤维细胞生长因子受体样蛋白1（FGFRL1）在口腔鳞状细胞癌（OSCC）中的表达情况,并探究其在OSCC细胞增殖和迁移中的作用。方法 利用Western blot检测FGFRL1蛋白在OSCC组织、癌旁正常组织、OSCC细胞株及正常上皮细胞中的表达;通过FGFRL1小干扰RNA（siRNA）干扰HN4细胞,影响FGFRL1蛋白的表达,利用CCK-8及Ki67实验检测FGFRL1对肿瘤细胞增殖能力的影响;细胞划痕及Transwell实验检测FGFRL1对肿瘤细胞迁移能力的影响;利用Western blot检测其对上皮间充质转化（EMT）相关指标蛋白的影响。结果 FGFRL1蛋白在OSCC组织中的表达量高于癌旁正常组织（t=2.820,P=0.047 8）;FGFRL1蛋白在OSCC细胞系中的表达量高于在HOK细胞中的表达量。实时荧光定量聚合酶链反应（qRT-PCR）结果显示,FGFRL1 RNA在HOK细胞中的表达量低于在OSCC细胞系中的表达量。将FGFRL1 siRNA转染的HN4细胞作为实验组,NC siRNA处理的HN4细胞作为对照组。Ki67免疫荧光试验结果显示,实验组与对照组在48 h（P=0.478 1）及72 h（P=0.334 2）细胞增殖活力差异无统计学意义。细胞划痕实验结果显示,在12 h（P=0.022 8）、24 h（P=0.005 1）及36 h（P=0.009 5）实验组细胞划痕面积百分比均比对照组小。Transwell实验结果显示,实验组在16 h（P=0.008 7）及24 h（P=0.008 6）细胞迁移数较对照组少。FGFRL1 siRNA干扰使得HN4细胞神经性钙黏附素蛋白和波形蛋白的表达量下降,上皮钙黏附素蛋白的表达量上升。结论 FGFRL1蛋白在OSCC组织中的表达量高于癌旁正常组织,在OSCC细胞株中的表达量高于正常上皮细胞。FGFRL1基因沉默对肿瘤细胞增殖无影响,但对肿瘤细胞EMT和细胞迁移具有一定的抑制作用。     

Transforming growth factor‐β‐regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF‐β), as a novel biomarker for correct prediction and early detection of OSCC‐associated bone invasion. TGF‐β knockdown and treatment with a TGF‐β‐neutralizing antibody decreased the level of fractalkine in the culture media of HSC‐2 and YD10B OSCC cells. Treatment with a fractalkine‐neutralizing antibody reduced TGF‐β‐stimulated invasion by HSC‐2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild‐type or TGF‐β knockdown OSCC cells in mouse calvaria. TGF‐β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor‐bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF‐β and mediates TGF‐β‐induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC‐induced bone destruction.     

Migration induced by epidermal and hepatocyte growth factors in oral squamous carcinoma cells in vitro: role of MEK/ERK, p38 and PI-3 kinase/Akt

J Oral Pathol Med (2012) 41 : 547–558 Background: Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti‐tumour signal inhibitors on the migratory activity. Methods: Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time‐lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. Results: In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose‐dependent activation of cell migration. Addition of the EGFR‐specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF‐induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI‐3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF‐induced migration was particularly sensitive to PI‐3 K‐inhibition, while in C12 cells, both HGF‐ and EGF‐induced migration were highly sensitive to p38‐blockade. Conclusion: The results demonstrate that the MEK/ERK, p38 and PI‐3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.     

胚胎干细胞上清液诱导Tca8113细胞发生上皮-间充质转化的实验研究

目的:采用胚胎干细胞培养上清液诱导口腔鳞癌细胞发生上皮-间充质转化,初步探讨其意义.方法:应用129小鼠胚胎干细胞培养上清液诱导培养舌鳞癌Tca8113细胞.分别在1周和2周后,采用免疫荧光和免疫细胞化学检测诱导前、后Tca8113细胞中Oct4、角蛋白(CK)和波形丝蛋白(vimentin)的表达情况.半定量反转录PCR检测snail、slug、E-cadherin和CD44、Bmi-1、hTERT表达随诱导时间的变化情况.结果:Tca8113细胞经条件培养基诱导后,细胞形状逐渐由铺路石样变为长梭形;Oct4集中表达在细胞核中,vimentin转变为阳性表达,CK表达无明显变化;上皮-间充质转化标记snail mRNA表达上调,E-cadherin下调,slug无明显变化.同时,癌干细胞标记CD44、Bmi-1和hTERT mRNA表达上调.结论:口腔鳞癌细胞经胚胎干细胞培养上清液诱导后,可发生上皮-间充质转化,从而可能具备部分干细胞特性.     

Increased expression of Smad proteins,and in particular Smad3, in oral lichen planus compared to normal oral mucosa

J Oral Pathol Med (2010) 39 : 639–644 Backgound: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organisation (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. EMT occurs during embryogenesis and wound healing but also in some human diseases such as cancer and fibrosis. A factor known to induce EMT is transforming growth factor‐β (TGF‐β), which uses the Smad proteins as mediators for its signalling. TGF‐β is also often over‐expressed in squamous cell carcinoma of the head and neck (SCCHN). Methods: In the present study we mapped expression of Smad proteins in OLP lesions by immunohistochemistry, and compared to expression in normal and sensitive oral mucosa. The latter group of patients had developed SCCHN after shorter or longer periods of diffuse oral symptoms. The aim was to see if there were any signs of EMT related changes in the OLP lesions, as judged by changes in the TGF‐β pathway. Conclusion: Changes in the TGF‐β pathway related to EMT are seen in the very earliest stages of oral malignancy and become more severe as lesions progress.     

口腔鳞癌及癌前病变中TGFβ的表达

目的：研究转化生长因子β（TGFβ）在口腔鳞癌及新癌病变中的表达特点。方法：免疫组织化学染色。结果：TGFβ的阳性表达见于癌前上皮分化良好的区域，而上皮的增殖区则为阴性表达。随着上皮由单纯性增生－轻度异常增生－中度异常增生－重度异常增生－磷癌的转变，TGFβ1的表达逐渐减少以及消失。结论TGFβ与口腔鳞癌的发生有着密切的关系。TGFβ1自分泌的减少有利于癌前、上皮的癌变及鳞癌的形成。     

Organotypic culture of normal,dysplastic and squamous cell carcinoma‐derived oral cell lines reveals loss of spatial regulation of CD44 and p75NTR in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre‐cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell‐associated markers in dermis‐based organotypic cultures of oral epithelial cells from normal tissue (OKF6‐TERT2), mild dysplasia (DOK), severe dysplasia (POE‐9n) and OSCC (PE/CA P J15). Expression of CD44, p75^NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75^NTR was evident for organotypic cultures of normal (OKF6‐TERT2) and dysplasia (DOK and POE‐9n) but was lacking for OSCC (PE/CA PJ15)‐derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75^NTR, CD24 antigens and ALDH activity (ALDEFLUOR^® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis‐based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC‐derived cells.     

微小RNA663b对口腔鳞状细胞癌细胞迁移、侵袭和上皮间充质转化的影响

目的　探讨微小RNA663b（miR-663b）对口腔鳞状细胞癌（OSCC）细胞迁移、侵袭和上皮间质转化（EMT）的影响。方法　通过基因表达数据库（GEO）使用R Studio进行OSCC组织与癌旁正常组织的差异表达分析。实时荧光定量聚合酶链反应（qRT-PCR）检测miR-663b在组织和细胞中的表达。检测miR-663b敲除质粒的HN30细胞转染效率，Transwell法检测迁移、侵袭能力。生物信息学方法预测miR-663b的靶向mRNA，双荧光素酶实验验证结合情况。Western blot检测EMT相关标志物的表达。结果　miR-663b在OSCC组织中表达上调，在HN30、CAL27、SCC-9细胞中表达较HOEC细胞高（P<0.05）。敲除miR-663b可以抑制HN30细胞的迁移和侵袭（P<0.05），抑制EMT的发生。生信预测SH3BP2与miR-663b靶向结合，SH3BP2低表达的患者预后较差（P<0.05）。双荧光素酶实验证明miR-663b可以与SHBP2靶向结合（P<0.05）。敲除miR-663b的OSCC细胞中SH3BP2的表达增加，EMT的发生受到抑制。结论　敲除miR-663b可靶向调节SH3BP2抑制OSCC细胞的迁移、侵袭和EMT。     

Mechanism of long-chain noncoding RNA PCGEM1 in the regulation of the invasion and metastasis of oral squamous carcinoma cells via transforming growth factor β2/Smad2 signaling pathway

目的 探讨长链非编码RNA（lncRNA）PCGEM1通过转化生长因子β2（TGF β2）/Smad2信号通路调控口腔鳞状细胞癌（OSCC）侵袭和转移的机制。方法 将60例OSCC患者癌组织及距离癌组织超过2 cm处的正常组织纳入研究,实时定量聚合酶链反应（qRT-PCR）检测miR-148a、lncRNA PCGEM1在OSCC组织及正常组织、人正常口腔黏膜上皮细胞（OMEC）及人源OSCC细胞株KB、BcaCD885、SCC-4、CAL27、SCC-15中的表达情况;分析lncRNA PCGEM1和miR-148a表达与患者临床病理信息之间的关系。构建lncRNA PCGEM1沉默细胞系KB-siPCGEM1及阴性对照（KB-NC）,并以KB作为空白对照组,采用MTT、Transwell和划痕实验检测lncRNA PCGEM1对KB细胞增殖、侵袭和迁移能力的影响;使用生物信息学网站starBase预测lncRNA PCGEM1可以互补结合的微小RNA（miRNA）,再根据www.microRNA.org网站预测相应miRNA可靶向结合的基因;免疫印迹（Western blotting）检测TGF β2/Smad2信号通路蛋白表达情况。结果 qRT-PCR结果显示,OSCC组织中lncRNA PCGEM1、miR-148a的表达水平高于正常组织（P<0.05）;lncRNA PCGEM1和miR-148a在不同TNM分期、淋巴结转移和组织分化程度的患者癌组织中表达差异均有统计学意义（P<0.05）;与空白对照组和KB-NC组相比,KB-siPCGEM1组细胞OD_{492 nm}值下降,细胞侵袭数量及迁移率降低,差异均具有统计学意义（P<0.05）;生物信息学预测结果显示,lncRNA PCGEM1可与miR-148a互补结合,miR-148a与TGFβ2存在靶向结合位点;qRT-PCR和Western blotting检测结果显示,KB-siPCGEM1组中miR148a、TGFβ2及p-Smad 2的表达明显低于空白对照组与KB-NC组（P<0.05）,空白对照组和KB-NC组的差异无统计学意义（P>0.05）。结论 lncRNA PCGEM1在OSCC中高表达,高表达lncRNA PCGEM1可能通过上调miR-148a水平,强化TGF β2/Smad2信号通路,从而促进OSCC的进展。     

E-cadherin和Vimentin在OSCC上皮-间质转化中的作用及表达

目的:探讨E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)在口腔鳞状细胞癌(OSCC)上皮-间质转化中的作用及表达。方法:选取76例口腔鳞状细胞癌(OSCC)患者和40例健康者作为对照组,利用RT-PCR检测OSCC和正常口腔黏膜组织中TGF-β1、E-cadherin和Vimentin表达。结果:OSCC患者组织中TGF-β1 mRNA和Vimentin mRNA相对表达量均高于对照组,而E-cadherin mRNA相对表达量低于对照组,差异均有统计学意义(P<0.05);TGF-β1、E-cadherin和Vimentin在OSCC组织中表达均与分化程度有关,分化程度越高,TGF-β1 mRNA和Vimentin mRNA相对表达量越高,而E-cadherin mRNA相对表达量则越低;Pearson相关分析显示,OSCC中TGF-β1 mRNA相对表达量与E-cadherin mRNA相对表达量呈负相关(r=-0.322,P=0.004),而与Vimentin mRNA相对表达量呈正相关(r=0.661,P=0.000)。结论:TGF-β1和Vimentin 在OSCC组织中表达增强,而E-cadherin则表达降低,提示EMT过程在OSCC发生及进展过程中发挥重要作用。     

Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 124–130 Background: Loco‐regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl‐2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl‐2 expression in lymphatic endothelial cells. Methods: Endothelial cells were selectively retrieved from paraffin‐embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT‐PCR was used to evaluate Bcl‐2 expression in tumor‐associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)‐C on the expression of Bcl‐2 in primary human lymphatic endothelial cells. Results:  We observed that Bcl‐2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage‐matched tumors without metastasis. VEGF‐C induced Bcl‐2 expression in lymphatic endothelial cells via VEGFR‐3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF‐C and induce Bcl‐2 in lymphatic endothelial cells. Conclusions: Collectively, this work unveiled a mechanism for the induction of Bcl‐2 in lymphatic endothelial cells and suggested that endothelial cell Bcl‐2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.     

Effects of HB-EGF and epiregulin on wound healing of gingival cells in vitro

Oral Diseases (2011) 17 , 785–793 Objective: Gingival wound healing is important to periodontal disease and surgery. This in vitro study was conducted to assess the manner in which heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) and epiregulin cooperatively participate in the wound‐healing process in the gingival epithelial and fibroblast cells of the oral mucosa. Material and Methods: Gingival epithelium and fibroblast were separated from gingival tissue biopsies and prepared to primary cultures. The changes in the mRNA expression were evaluated via real‐time PCR. The effects on cell proliferation, migration, and repopulation were evaluated in vitro. Results: The different regulation of expressions of HB‐EGF, epiregulin, and epidermal growth factor receptors was observed over time and with different gingival cell types. HB‐EGF exerted a cell migration‐inducing effect on both epithelial and fibroblast cells, whereas epiregulin did not. Both growth factors functioned as mitogens for epithelial cell proliferation, but not for fibroblast proliferation. HB‐EGF strongly promoted epithelial cell repopulation and mildly promoted fibroblast repopulation, whereas epiregulin promoted only fibroblast repopulation. Conclusion: These results indicated that both growth factors might function importantly in the wound‐healing process of human gingival tissue via the different regulation of the expression, cell migration, proliferation, and repopulation.