Expression of E-cadherin and vimentin in oral squamous cell carcinoma

The aim of the study is to determine the levels of E-cadherin, vimentin expression in tumor tissues from patients with oral squamous cell carcinoma (OSCC), and the relationship between the expression of E-cadherin, vimentin and epithelial-mesenchymal transition, in order to explore its values for predicting the invasion and metastasis of oral squamous cell carcinoma, short survival of patients in many types of cancer. E-cadherin and vimentin expression of 10 benign and 42 OSCC tumor tissues was examined by immunohistochemical staining. E-cadherin is positively expressed in normal oral mucosa epithelium, but vimentin expression is not found in normal oral mucosa epithelia; the E-cadherin and vimentin were expressed in 26 of 42 (61.9%) and 16 of 42 (38.1%), respectively. No statistically difference was found for E-cadherin and vimentin expression in patients with different age, gender and tumor location, E-cadherin and vimentin expression was significantly associated with lymph node metastasis and tissue location (P < 0.05); E-cadherin expression was also significantly associated with tumor stage (P < 0.05); there are significantly difference between infiltrative margin and central area in patients with oral squamous cell carcinoma for E-cadherin and vimentin positive expression (P < 0.05). E-cadherin and vimentin positive expression was associated with tumor metastasis of oral squamous cell carcinoma. Our study preliminarily confirmed that EMT phenomenon is existed during the development of oral squamous cell carcinoma. Co-evaluation of E-cadherin and vimentin might be a valuable tool for predicting OSCC patient outcome.     

Overexpression of FAM3C is associated with poor prognosis in oral squamous cell carcinoma

Expression of the family with sequence similarity 3 member C (FAM3C) is necessary for the epithelial-mesenchymal transition (EMT). However, the expression level and clinicopathological significance of FAM3C in oral squamous cell carcinoma (OSCC) has not been thoroughly elucidated to date. We performed immunohistochemical staining on human OSCC specimens with FAM3C, co-inhibitory immune checkpoints, EMT markers, and cancer stem cells (CSCs) markers to analyze the expression levels and clinicopathological features of FAM3C in OSCC. There were 210 primary OSCC specimens, 69 oral epithelial dysplasia and 42 normal oral mucosae in our human OSCC tissue microarrays cohort. We observed that FAM3C expression was upregulated in OSCC compared with normal mucosa and epithelial dysplasia (P?<? 0.001). Moreover, patients with higher FAM3C expression levels had a worse prognosis than those with lower expression levels (P < 0.05). Also, FAM3C expression was positively correlated with the immune checkpoints PD-L1, VISTA, and B7-H4, the EMT marker Slug and the CSC markers SOX2 and ALDH1. In conclusion, these findings suggested that overexpression of FAM3C in human OSCC may predict a poor prognosis for OSCC patients.     

SNAIL诱导MCF-7细胞上皮间质转化致阿霉素耐药性增加

 目的 观察SNAIL诱导后,MCF-7细胞上皮间质转化（EMT）的发生和P-糖蛋白(P-GP)的表达,探讨EMT致乳腺癌细胞耐药性增强的作用。方法 构建SNAIL真核表达载体,转染MCF-7细胞,用免疫荧光检测上皮标记物E-cadherin,间质标志物Vimentin的表达;MTT法评价细胞对阿霉素(ADM)的耐药性;流式细胞术检测细胞中P-GP和SNAIL的表达;Real-Time-PCR检测SNAIL、E-cadherin、Vimentin、MDR1 mRNA的表达。结果 MCF-7细胞转染SNAIL后E-cadherin表达较亲本细胞显著降低,Vimentin表达显著升高(P＜0.01),对ADM的耐药性及SNAIL、P-GP的表达也显著升高(P＜0.01),与Real-Time-PCR结果一致。结论 SNAIL诱导MCF-7细胞EMT后,增强了乳腺癌细胞中P-GP介导的对ADM的耐药。     

SNAIL induces epithelial-to-mesenchymal transition in a human pancreatic cancer cell line (BxPC3) and promotes distant metastasis and invasiveness in vivo

SNAIL, a potent repressor of E-cadherin expression, plays a key role in inducing epithelial-to-mesenchymal transition (EMT) in epithelial cells. During EMT, epithelial cells lose cell polarity and adhesion, and undergo drastic morphological changes acquiring highly migratory abilities. Although there is increasing evidence that EMT is involved in the progression of some human cancers, its significance in the progression of pancreatic cancer remains elusive. In Panc-1, a well-known human pancreatic cancer cell line in which EMT is triggered by TGF-β1 treatment, SNAIL and vimentin are highly expressed, whereas E-cadherin expression is scant. In contrast, another human pancreatic cancer cell line, BxPC3, in which SNAIL expression is not detected, has high levels of E-cadherin expression and does not undergo EMT upon TGF-β1 treatment. After transfecting the SNAIL gene into BxPC3, however, the cells undergo EMT with remarkable alterations in cell morphology and molecular expression patterns without the addition of any growth factors. Furthermore, in an orthotopic transplantation model using SCID mice, SNAIL-transfected BxPC3 displayed highly metastatic and invasive activities. In the immunohistochemical analysis of the tumor derived from the SNAIL-expressing BxPC3, alterations suggestive of EMT were observed in the invasive tumor front. SNAIL enabled BxPC3 to undergo EMT, endowing it with a highly malignant potential in vivo. These results indicate that SNAIL-mediated EMT may be relevant in the progression of pancreatic cancer, and SNAIL could be a molecular target for a pancreatic cancer intervention.     

Combined therapy with cisplatin and 5-AZA-2CdR modifies methylation and expression of DNA repair genes in oral squamous cell carcinoma

The methylation and expression of DNA repair system genes has been studied in several tumor types. These genes have been associated with resistance to chemotherapy treatments by epigenetic regulation. Studies have yet to show the effects of combined therapy using an epigenetic drug (5-aza-2CdR) and cisplatin (CDDP) on DNA repair genes in oral squamous cell carcinoma (OSCC). This study proposed to investigate the effects of CDDP in combination with 5-aza-2CdR on the methylation of MGMT and MLH1 genes in oral cancer cells. Oral squamous cell carcinoma cell lineages (SCC-9, SCC-15, and SCC-25) were submitted to 72 hours of treatment: 0.1 μM CDDP (or 4.44 μM SCC-9), 0.1 μM and 0.3 μM 5-aza-2CdR (or 1 μM and 3 μM SCC-9), and the drugs in combination. Cell viability was assessed by MTT, DNA methylation of MGMT and MLH1 genes by Methylation Sensitivity High-Resolution Melting (MS-HRM), and the relative expression of the genes by RT-qPCR. The results show that all treatments reduced cell viability; however, in SCC-15 and SCC-9 (IC₅₀ value), 5-aza-2CdR promotes cell sensitization to cytotoxic effect of cisplatin. The MGMT promoter region was 100% demethylated in the SCC-15 and SCC-25 cells but partially (50%) methylated in SCC-9 before drug treatment. Treatment with IC₅₀ CDDP value kept the methylation status and decreased MGMT expression in SCC-9; MGMT gene in SCC-15 and SCC-25 cells became downregulated after treatment with 5-aza-2CdR. MLH1 was demethylated, but the treatments with low-doses and combined drugs decreased the expression in SCC-9 and SCC-25; however high doses of 5-aza-2CdR and drug combination with IC₅₀ value CDDP increased expression of MLH1 in SCC-9. The data presented suggest that epigenetic drugs associated with chemotherapy have clinical translational potential as a therapy strategy to avoid or reverse cancer resistance, requiring further investigation.     

Differential expression of fatty acid synthase (FAS) and ErbB2 in nonmalignant and malignant oral keratinocytes

The aim of this study was to investigate fatty acid synthase (FAS) and ErbB2 expression in nonmalignant oral epithelium and oral or head and neck squamous cell carcinomas (OSCC/HNSCC). Morphologically normal, hyperkeratotic, and dysplastic oral epithelium as well as well-differentiated and poorly differentiated OSCC were immunohistochemically evaluated for FAS, ErbB2, and Ki-67. These proteins were also analyzed in a tissue microarray with 55 HNSCC. SCC-9 cells were used to study FAS and ErbB2 during differentiation. FAS expression was higher in hyperkeratosis, dysplasias, and OSCC than in normal epithelium. Well-differentiated OSCC/HNSCC were more positive for FAS than the poorly differentiated tumors. ErbB2 was observed at the surface of nonmalignant and well-differentiated OSCC/HNSCC keratinocytes and in the cytoplasm of poorly differentiated cells. Ki-67 index was progressively higher from normal oral epithelium to OSCC, inversely correlated with cell surface ErbB2, and positively correlated with intracytoplasmic ErbB2. Finally, SCC-9 cell cultures were enriched in membrane ErbB2-positive cells after differentiation by anchorage deprivation. In conclusion, FAS is overexpressed in OSCC/HNSCC and hyperkeratotic oral epithelium and ErbB2 is found at the cell surface of differentiating keratinocytes and in the cytoplasm of poorly differentiated tumor cells. Ki-67 index is higher in epithelial dysplasias and OSCC than in morphologically normal oral epithelium.     

Loss of ELF3 immunoexpression is useful for detecting oral squamous cell carcinoma but not for distinguishing between grades of epithelial dysplasia

Early diagnosis and targeted therapy are crucial to mitigating the morbidity and mortality of oral squamous cell carcinoma. Among the potentially malignant oral disorders, epithelial dysplasia has known association with malignant transformation, but defensible gradation of dysplasia severity presents unmet challenges. Published microarray data has denoted dysregulation of CLSP, ELF3, IFI44, USP18, and CXCL13 genes in potentially malignant oral disorders. The present study investigated the diagnostic potential of these gene products to grade oral epithelial dysplasia severity. Archived biopsies from independent patient cohorts comprised “training” (n = 107) and “test” (n = 278) sample sets. Immunoreactivity for candidate markers was determined in the “training” set of normal oral mucosa (NOM), mild dysplasia (MD), moderate to severe dysplasia, and oral squamous cell carcinoma (OSCC). The diagnostic potential of ELF3 immunoscoring to improve detection and severity gradation of epithelial dysplasia was assessed with the “test” set. A reciprocal relationship between disease severity and immunoreactivity score for CLSP and ELF3 was observed (MD/NOM to OSCC: P < .08, Mann-Whitney U test), whereas elevated IFI44 immunostaining was present for OSCC compared to MD/NOM (P < .08, Mann-Whitney U test). Loss of ELF3 immunostaining effectively distinguished OSCC from non-malignant tissues (sensitivity = 0.81; specificity = 0.56; area under the curve [AUC] = 0.68) but did not distinguish dysplasia from NOM (sensitivity = 0.55; specificity = 0.40; AUC = 0.47) or moderate to severe dysplasia from MD (sensitivity = 0.63; specificity = 0.51; AUC = 0.57). The results confirm via immunohistochemistry the relevance of published CLSP, ELF3, and IFI44 (but not USP18 or CXCL13) gene expression data to potentially malignant oral lesion severity. Loss of ELF3 immunostaining discriminated OSCC from dysplasia but was unreliable for grading dysplasia severity.     

Expression of programmed cell death-ligand 1 in oral squamous cell carcinoma and oral leukoplakia is associated with disease progress and CD8+ tumor-infiltrating lymphocytes

ObjectiveIn recent years, monoclonal antibodies targeting programmed cell death-ligand 1 (PD-L1) have become a promising cancer immunotherapy. However, the role of PD-L1 in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), including oral leukoplakia (OLK), remains controversial. The aim of the present study was to investigate the expression level of PD-L1 in OSCC and OPMDs, and examine its relationship with CD8 expression and different clinicopathological features.MethodExpression of PD-L1 and CD8 were conducted in 41 OSCC, 21 OLK, and 25 normal mucosa samples by immunohistochemistry. Then, the density of PD-L1 expression was measured, and its correlation with CD8 expression and different clinicopathological features was analyzed.ResultsPD-L1 protein was detected in 97.6% of OSCC, 61.9% of OLK, and 0% of normal tissues. PD-L1 was highly expressed in human OSCC tissue (P < 0.0001), when compared to both OLK and control tissues. PD-L1 positivity was significantly associated with CD8 density (P < 0.0001, r = 0.8491). The PD-L1 high expression OSCC group displayed a trend for improved overall survival (OS) and disease-free survival (DFS) compared to the low expression group, although the differences were not significant. Moreover, the expression level of PD-L1 in OSCC was positively correlated with the pathological grade (P < 0.0001), but it was independent of age, gender, smoking, drinking, tumor size, lymph node status, or recurrence (P > 0.05). Also, there was a significant upregulation of PD-L1 expression observed in the OLK group compared to the control group (P < 0.0001). PD-L1 positivity in OLK patients was associated with gender and smoking habits (P < 0.05), but it did not correlate with age, drinking, or dysplasia (P > 0.05).ConclusionThe upregulation of PD-L1 may be associated with disease progress and CD8+ tumor-infiltrating lymphocytes in oral premalignant and malignant lesions.     

ANO1在口腔鳞癌中的表达及临床分析

 目的 探讨ANO1基因及蛋白在口腔鳞癌中的表达及其临床意义。方法 应用免疫组化SP法及Northern blot检测81例口腔鳞癌组织及相应正常组织的ANO1基因及蛋白的表达进行检测,并结合临床病理资料和基因蛋白表达特征对比作差异显著性检验及相关分析。利用western blot检测ANO1在多株鳞癌细胞株的表达。结果 ANO1在口腔鳞癌组织中的阳性表达明显高于正常组织,有显著性差异( P <0.05);有淋巴结转移的口腔鳞癌组织ANO1阳性表达高于无转移的口腔鳞癌组织。有显著性差异( P <0.05);随着口腔鳞癌临床分期的升高,ANO1的阳性表达率升高(P＜0.05);而ANO1的阳性表达率与病理分级,年龄和性别暂无关(P＞0.05)。Hep-2的内源性ANO1表达最低, 而SCC-25细胞株的内源性ANO1表达最高。 结论 ANO1可能在口腔鳞癌的发生和进展过程中起到重要作用。     

Over-expression of integrin-linked kinase correlates with aberrant expression of Snail, E-cadherin and N-cadherin in oral squamous cell carcinoma: implications in tumor progression and metastasis

Integrin-linked kinase (ILK), an intracellular protein with serine/threonine protein kinase activities, plays a key role in integrin mediated cell-excellular matrix interactions, regulating cell proliferation, apoptosis, differentiation, and migration. ILK has been implicated in the development and progression in several malignancies. However, the role of ILK and ILK-mediated epithelial?Cmensenchymal transition (EMT) in the progression of oral squamous cell carcinoma (OSCC) has not been well understood. Here, by immunohistochemistry, we studied the expression of ILK, Snail, E-cadherin and N-cadherin in 98 primary OSCC specimens and analyzed their correlations with clinicopathologic profiles and clinical outcome. We also investigated the expression of ILK in 42 corresponding lymph node metastases. Positive expression of ILK protein was detected in 87.8?% of the primary tumors and 100?% of metastatic lesions. Increased ILK expression was correlated strongly with enhanced tumor invasion, higher tumor grade, advanced clinical stage, positive lymph node status and increased risk of recurrence. Higher ILK expression was also observed in lymph node metastases in comparison with the corresponding primary tumor. Moreover, up-regulation of Snail and N-cadherin and down-regulation of E-cadherin correlated significantly with both ILK over-expression and tumor invasion. Patients with higher ILK expression exhibited shorter disease-free survival while those with absent E-cadherin expression exhibited shorter overall and disease-free survival. Taken together, our results suggest that ILK may have an important role in progression and metastasis of OSCC, possibly through EMT involving up-regulation of Snail and consequent aberrant expression of E-cadherin and N-cadherin. ILK should be considered as a critical prognostic indicator for patients with OSCC.     

lncRNA PCAT1表达下调抑制口腔鳞状细胞癌细胞的增殖、生长、侵袭、迁移及上皮-间充质转化

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA) PCAT1对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞的增殖、生长、侵袭及迁移的影响,并探讨其可能机制。方法:采用Lipofectamine 200将PCAT1 siRNA转染入OSCC细胞分别利用RT-qPCR和Western blot检测相关基因的mRNA及蛋白表达;分别利用CCK-8实验和集落形成实验检测OSCC细胞的增殖及生长能力;利用细胞侵袭实验和细胞迁移实验检测OSCC细胞的侵袭及迁移能力。结果:PCAT1在OSCC组织和细胞中的表达与癌旁正常组织和正常口腔细胞黏膜角质细胞相比显著上调(P 0. 05)。转染PCAT1 siRNA可以显著降低PCAT1在Tca8113和TSCCa细胞中的表达(P 0. 05)。PCAT1的低表达可以显著抑制Tca8133和TSCCa细胞的增殖、生长、侵袭及迁移(P 0. 05)。PCAT1在Tca8113和TSCCa细胞中的低表达可以抑制ZEB1、N-cadherin和vimentin的mRNA及蛋白表达,同时增加E-cadherin的mRNA及蛋白表达(P 0. 05)。结论:沉默PCAT1能够抑制OSCC细胞的增殖、生长及转移,该作用可能与调控上皮-间充质转化有关。     

Possible involvement of ΔNp63 downregulation in the invasion and metastasis of oral squamous cell carcinoma via induction of a mesenchymal phenotype

Epithelial-to-mesenchymal transition (EMT), an essential developmental program, is involved in tumor progression. ΔNp63, a homolog of p53, is associated with the EMT program, but the detailed mechanism remains to be elucidated. In this study, we investigated the role of ΔNp63 in EMT during progression of oral squamous cell carcinoma (OSCC). Five OSCC cell lines and specimens from 78 patients with OSCC were used. The expressions of ΔNp63, p63α, p63β and epithelial markers (cytokeratins 5 and 14) was detected in the OSCC cells, but not in SQUU-B cells (high metastatic potential). E-cadherin was expressed in all OSCC cells. Mesenchymal markers were strongly expressed in the SQUU-B cells. Knockdown of endogenous ΔNp63 in HSC-2 cells induced morphological changes to the spindle shape, decreased the expression of epithelial markers, increased the expression of mesenchymal markers, increased migration and reduced proliferation. By contrast, SQUU-B cells overexpressing ΔNp63β showed changed their morphology from stromal cell-like to epithelial cells. However, E-cadherin expression was not affected by ΔNp63 knockdown or overexpression. Immunohistochemical staining revealed that cancer cells expressing vimentin were found at the invasive front in the OSCC specimens. The intensity of ΔNp63 expression was also decreased in these cells. Interestingly, the vimentin positivity or decreased intensity of ΔNp63 was positively associated with metastases and poor prognosis in the OSCC patients. These results indicated that ΔNp63 downregulation in cancer cells induces a mesenchymal phenotype that is related to tumor progression of OSCC.     

Correlated analysis of semi-quantitative immunohistochemical features of E-cadherin,VEGF and CD105 in assessing malignant potentiality of oral submucous fibrosis

Oral submucous fibrosis, a potentially premalignant condition for oral squamous cell carcinoma, manifests both non-dysplastic and dysplastic grades. Early and specific identification of its malignant potentiality suffers from diagnostic limitations that may be addressed by correlated molecular pathology attributes having histopathological backdrop. Present study correlates expressional alteration in prime epithelial marker E-cadherin, with neo-angiogenic molecules viz. VEGF and CD105 for elucidation of malignant potentiality in different stages of oral submucous fibrosis. Sixty-eight incision biopsies from normal oral mucosa (n = 10), non-dysplastic (n = 18) and different dysplastic grades (n = 40) of oral submucous fibrosis were semi-quantitatively analyzed for immunohistochemical expressions of E-cadherin (membranous and cytoplasmic), VEGF and CD105 which were further statistically correlated. The loss of membranous E-cadherin with increase in cytoplasmic accumulation in differentiative layers of epithelium through the progression of dysplasia was noted along with up-regulation in VEGF expressions. The number of CD105⁺ blood vessels and their major axis also showed significant increase from non-dysplasia toward higher grades of dysplasia. The positive correlation between deregulated expression of epithelial cell–cell adhesion molecule and increase in neo-angiogenic attributes of oral submucous fibrosis with increase in dysplastic grades indicated elucidatory potential of molecular expression features in assessment of malignant potentiality in oral submucous fibrosis.     

口腔鳞癌中S100A4蛋白和E-cad的表达及意义

目的 探讨口腔鳞状细胞癌（oral squarnous cell carcinoma,OSCC）中S100A4蛋白和上皮钙粘蛋白（E—cadherin,E—cad）的表达及意义。方法 采用免疫组化SP法检测61例OSCC组织中S100A4、E—cad表达情况,分析二者的表达与临床病理特征之间的关系。结果 S100A4蛋白的表达与组织学分级无关（P〉0．05）,与淋巴结转移呈正相关（P〈0．05）;E-cad的表达与组织学分级呈正相关（P〈0．05）,与淋巴结转移呈负相关（P〈0．05）;S100A4蛋白和E—cad的表达呈负相关（P〈0．05）。结论 E-cad对OSCC的分化起重要作用;E-cad、S100A4蛋白和OSCC的侵袭和转移密切相关;S100A4蛋白和E-cad的表达与口腔鳞癌的进展密切相关,是判断口腔鳞癌生物学行为、预测转移趋势的有价值的指标。     

Clinicopathological significance and prognostic implication of nuclear factor-κB activation in colorectal cancer

ObjectiveThe aim of the present study was to evaluate the clinicopathological significance of phosphorylated nuclear factor-κB (pNF-κB) expression, and its impact on epithelial–mesenchymal transition and angiogenesis in colorectal cancer (CRC).MethodsWe carried out immunohistochemistry of pNF-κB on 261 human CRC tissues, and evaluated nuclear expression, regardless of cytoplasmic expression. We also investigated the correlation between pNF-κB expression and clinicopathological characteristics, survival, and epithelial–mesenchymal transition and angiogenesis-related markers in CRC.ResultspNF-κB was expressed in the nuclei of 164 of the 261 CRC tissues (62.8%). Furthermore, pNF-κB was significantly correlated with frequent perineural invasion, lymph node metastasis, and higher pTNM stage. However, there was no significant correlation between pNF-κB expression and other clinicopathological parameters. Among the epithelial–mesenchymal transition markers examined, SNAIL expression was significantly correlated with pNF-κB expression (P = 0.001) but E-cadherin expression was not. CRC with pNF-κB expression had significantly higher SIRT1 expression levels and hypoxia-inducible factor-1α expression levels than CRC without pNF-κB expression (P < 0.001 and P < 0.001, respectively). However, there was no correlation between the expression levels of pNF-κB and VEGF. pNF-κB expression was significantly correlated with worse overall and recurrence-free survival rates (P < 0.001 and P < 0.001, respectively).ConclusionpNF-κB expression was significantly correlated with aggressive tumor behaviors and worse survival rates. Furthermore, pNF-κB expression may affect tumor invasion and progression through SNAIL-related epithelial–mesenchymal transition and SIRT1- and hypoxia-inducible factor-1α-induced angiogenesis.     

汉黄芩素对人口腔鳞状细胞癌细胞生长和侵袭的影响

目的:探讨汉黄芩素对人口腔鳞状细胞癌SCC-4细胞生长和侵袭的影响及其作用机制。方法:使用不同浓度(0、20、40、60、80和100 mg/L)的汉黄芩素处理SCC-4细胞不同时间,分别用MTT法检测细胞活力,流式细胞术及Annexin V/PI双染法检测细胞凋亡,Transwell小室检测细胞侵袭能力,Western blot法检测Wnt/β-catenin信号通路的活化。结果:汉黄芩素呈剂量及时间依赖性地抑制细胞生长,同时诱导细胞大量凋亡,抑制细胞的侵袭。汉黄芩素明显抑制了细胞中β-catenin的活化,同时其下游靶分子细胞周期蛋白D1(cyclin D1)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)和MMP-9的表达水平降低,而抗凋亡蛋白Bcl-2的表达增加。用Wnt/β-catenin通路激动剂Li Cl处理后,汉黄芩素抑制的Wnt/β-catenin通路分子活化明显增强,同时细胞生长能力明显增强,而凋亡能力降低,还明显减弱了汉黄芩素对细胞侵袭能力的抑制作用。结论:汉黄芩素主要通过抑制Wnt/β-catenin信号通路来调控口腔鳞状细胞癌细胞的生长及侵袭进程,具有一定的抗口腔鳞状细胞癌发展的作用,可成为临床上口腔鳞状细胞癌防治的潜在药物。