Plasminogen activator/plasmin system suppresses cell-cell adhesion of oral squamous cell carcinoma cells via proteolysis of E-cadherin

The participation of plasminogen activator/plasmin system in the expression and function of E-cadherin was examined in oral squamous cell carcinoma (SCC) cells. Treatment of SCC cells with plasminogen reduced the Ca2+-dependent cell aggregation. SCC cells expressed E-cadherin at the cell membrane, and released a small amount of soluble E-cadherin at 80 kDa in the culture medium. Addition of plasminogen to SCC cells led to a decrease in the amount of E-cadherin of the cell membrane and the enhancement of the shedding of E-cadherin ectodomain. Plasmin directly cleaved E-cadherin of SCC cells and enhanced the motility of SCC cells. These results suggested that plasminogen activator/plasmin system might directly mediate the proteolytic processing of E-cadherin in oral SCC cells and that might facilitate the progression of oral SCC by downregulation of E-cadherin-mediated cell-cell adhesion.     

E-cadherin increases squamous cell carcinoma antigen expression through phosphatidylinositol-3 kinase-Akt pathway in squamous cell carcinoma cell lines

Squamous cell carcinoma antigen (SCCA) has been used for the management of squamous cell carcinoma, especially for evaluating therapeutic effects and monitoring recurrence. It has been reported that SCCA has several biological activities and influences behavior of cancer cells. E-cadherin is a cell adhesion molecule and plays important roles in the process of cancer invasion and metastasis. Our previous studies have shown that blockage of E-cadherin action by anti-E-cadherin antibody treatment suppresses SCCA production in squamous cell carcinoma cells. This finding strongly suggests that E-cadherin regulates SCCA expression. The present study was, therefore, undertaken to investigate the correlation between E-cadherin and SCCA2. For this purpose, E-cadherin cDNA was transfected into squamous cell carcinoma cell lines, SiHa and SKG IIIa. Overexpression of E-cadherin increased SCCA2 expression together with cell aggregation. We also examined the involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathway, which is one of major signaling pathways from E-cadherin. E-cadherin transfection increased phosphorylated Akt expression concomitantly with the increase in SCCA2 expression, and the increased SCCA2 expression was inhibited by a PI3K inhibitor. In conclusion, SCCA2 is up-regulated by E-cadherin through PI3K-Akt pathway, suggesting that SCCA2, as well as E-cadherin, may be involved in the regulation of cancer behavior.     

Induction of alpha2-antiplasmin inhibits E-cadherin processing mediated by the plasminogen activator/plasmin system, leading to suppression of progression of oral squamous cell carcinoma via upregulation of cell-cell adhesion

The plasminogen activator/plasmin system is one of the main protease systems involved in tumor cell invasion and metastasis. Our previous study has shown that plasmin degrades E-cadherin and promotes cell dissemination by downregulation of E-cadherin-mediated cell-cell adhesion in oral squamous cell carcinoma (SCC) cells. To examine the effect of downregulation of the plasminogen activator/plasmin system by alpha2-antiplasmin (alpha2-AP) on cell-cell adhesion mediated by E-cadherin in oral SCC cells, the oral SCC cell line SCCKN was stably transfected with alpha2-AP cDNA. Induction of alpha2-AP expression led to the inhibition of the proteolysis of E-cadherin by plasminogen activator/plasmin in SCC cells, resulting in the enhancement of the cell aggregation and the suppression of the cell motility. Moreover, alpha2-AP also reduced the ability of SCC cells to invade type I collagen gel, and suppressed tumorigenicity in vivo. These results suggested that downregulation of the plasminogen activator/ plasmin system by alpha2-AP might be a potent therapeutic approach to prevent the progression of oral SCC.     

Rack1 promotes epithelial cell-cell adhesion by regulating E-cadherin endocytosis

E-cadherin and its cytoplasmic partners, catenins, mediate epithelial cell-cell adhesion. Disruption of this adhesion allows cancer cells to invade and metastasize. Aberrant activation of the Src tyrosine kinase disrupts cell-cell contacts through an E-cadherin/catenin-dependent mechanism. Previously we showed that Rack1 regulates the growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and in the intrinsic apoptotic and Akt cell survival pathways. Here we show that Rack1, partly by inhibiting Src, promotes cell-cell adhesion and reduces the invasive potential of colon cancer cells. Rack1 stabilizes E-cadherin and catenins at cell-cell contacts by inhibiting the Src phosphorylation of E-cadherin, the ubiquitination of E-cadherin by the E3 ligase Hakai and the endocytosis of E-cadherin. Upon depletion and restoration of extracellular calcium, Rack1 facilitates the re-assembly of E-cadherin-containing cell-cell contacts. Rack1 also blocks HGF-induced endocytosis of E-cadherin, disruption of cell-cell contacts and cell scatter. Our results uncover a novel function of Rack1 in maintaining the junctional homeostasis of intestinal epithelial cells by regulation of the Src- and growth factor-induced endocytosis of E-cadherin.     

Tumor-associated serpin, squamous cell carcinoma antigen stimulates matrix metalloproteinase-9 production in cervical squamous cell carcinoma cell lines

Squamous cell carcinoma antigen (SCCA) is clinically used as a tumor marker for patients with squamous cell carcinoma of various organs. SCCA1 and its highly homologous molecule, SCCA2, belong to the serine proteinase inhibitors (serpins) family, suggesting that these proteins may be involved in the malignant behavior of squamous cell carcinoma cells. The aim of this study is to functionally characterize these tumor-associated serpins regarding the potential to influence the production of matrix metalloproteinases (MMPs), which play a key role in tumor cell invasion and metastasis. Cervical squamous cell carcinoma cell lines, CaSki cells and SKG-IIIa cells were incubated with SCCA1 or SCCA2 and MMP production was analyzed by gelatin zymography. Both SCCA1 and SCCA2 significantly increased production of proMMP-9, but not proMMP-2. These stimulatory effects were still observed when cells were treated with SCCA mutants lacking the proteinase inhibitory activity of serpins. Furthermore, treatments with various forms of SCCAs, which are generated by interacting with their target proteinases, diminished the stimulatory effect of SCCAs, implying the importance of the conformational structure of SCCAs in the stimulatory effects of SCCAs on proMMP-9 production. In addition, in vitro invasion assay showed that SCCA1 and SCCA2 significantly promoted the activity of cell invasion. It is concluded that SCCAs can alter the invasive phenotype of cervical squamous cell carcinoma cells, probably by stimulating proMMP-9 production, and that intact conformational structure of SCCAs, but not proteinase inhibitory activity of serpins, is required for its stimulatory activity on proMMP-9 production.     

Chemopreventive alteration of the cell-cell adhesion in head and neck squamous cell cancer

Approximately 310,000 new cases of oral and pharynx cancer account for a major cause of neoplasm related morbidity and mortality world-wide. Unfortunately, the survival rate has not improved significantly in the last decade. The vast majority of head and neck cancer is squamous cell carcinoma. The major adhesion-proteins involved in the development and maintenance of all solid tissue are the Cadherins. Cadherins are the transmembrane components of the adherent junction with interaction with plakoglobin and beta-catenin. Downregulation of Cadherins and catenins is frequently observed in many types of human cancer. Sulindac sulfone is one of the new therapeutic apoptotic agents that show promise in the treatment of cancer. In this study, we incubated sulindac sulfone with a head and neck cancer cell line and investigated the outcome of E-Cadherin. Immunohistochemical and Western blot analyses were then performed, with different concentrations of sulindac sulfone (100, 200, 400, 600, and 800 microMol) for 48 h. At 400 microMol of sulindac sulfone a decrease of 21% was observed; at 600 microMol, 44% decrease of beta-catenin concentration was seen, and incubation with 800 microMol resulted in 73% reduction of secreted beta-catenin. Incubation with sulindac sulfone seemed to stop proliferation; however, with respect to the controls, there was no increased reduction of the total protein. Sulindac sulfone resulted in an increase of E-Cadherin content in the head and neck squamous cell cancer cell line after 48 h of incubation; however, the reactivity was restricted to the adherent junctions. At increasing concentrations of sulindac sulfone, intercellular E-Cadherin immunostaining intensifyied. ELISA also depicted significant rising levels of E-Cadherin. Sulindac sulfone contributes to the inactivation of cGMP phospho-diesterase. Thus, the accumulation of cellular cGMP and protein kinase G is induced. The following degradation of the phosphorylated beta-catenin and the dissociation from the Cadherin-catenin complex releases E-Cadherin. This may also contribute to growth inhibition and co-ordinate with apoptosis induction. It is not really clear as to, which pathway results in the elevation of the E-Cadherin proteins. However, in epithelial cancer cells, the Cadherin-catenin complex serves as a target for the chemopreventive agent, sulindac sulfone.     

Carcinoembryonic antigen cell adhesion molecule 1 inhibits the antitumor effect of neutrophils in tongue squamous cell carcinoma

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), a transmembrane glycoprotein, has multiple functions. In tongue squamous cell carcinoma (TSCC), CEACAM1 overexpression is correlated with neutrophil infiltration, and both are associated with poor clinical outcomes. However, the mechanism underlying CEACAM1's effect on neutrophil function in TSCC remains unclear. We cocultured tongue carcinoma cells overexpressing CEACAM1‐4L, CEACAM1‐4S and differentiated HL‐60 cells. This significantly upregulated the expression of MMP‐9, interleukin 8, and VEGF‐A in the differentiated HL‐60 cells and downregulated the expression of TNF‐α, relative to vector and blank control groups (P < 0.05). Additionally, CEACAM1 overexpression in tongue carcinoma cells weakened the cytotoxicity of differentiated HL‐60 cells in the coculture system (P < 0.05). Thus, CEACAM1 expression in TSCC may induce an antitumor to protumor transformation of neutrophils. We performed qRT‐PCR and ELISA to evaluate the underlying mechanism, and found that CEACAM1 expression in tongue carcinoma cells upregulated transforming growth factor β1 (TGF‐β1) expression, while blocking of TGF‐β1 inhibited the neutrophils’ changes in the coculture system. Immunohistochemical analysis of clinical specimens revealed strong expression of TGF‐β1 protein in TSCC. TGF‐β1 expression was positively correlated with CEACAM1 expression, lymph node metastasis, and tumor recurrence. Double immunofluorescence results revealed colocalization of CEACAM1 and TGF‐β1 protein in TSCC. A xenograft nude mouse model revealed that CEACAM1 overexpression in TSCC promoted tumor formation and growth, and was associated with more neutrophils infiltration. Our results indicate that CEACAM1 overexpression in TSCC may induce transformation of neutrophils from antitumor to protumor type via TGF‐β1, which may further promote tumor progression.     

宫颈鳞状细胞癌患者血清鳞状细胞癌抗原变化的临床意义

目的　研究子宫颈鳞状细胞癌患者血清中鳞状细胞癌抗原水平与病理分级、临床分期及对治疗反应之间的关系。方法　利用雅培公司提供的 IMX全自动快速粒子酶免疫分析系统 ,对 2 5例正常献血员和 83例经病理学诊断的宫颈鳞状细胞癌患者 ,进行了血清中鳞状细胞癌抗原血清检测并分析其与病理分级、临床分期之间关系。其中 80例患者放射治疗前后血清鳞状细胞癌抗原水平变化情况进行了自身比较。结果　中晚期宫颈鳞状细胞癌患者血清鳞状细胞癌抗原水平与病理分级、临床分期之间无相关性。放射治疗前后患者血清鳞状细胞癌抗原水平有明显变化。结论　对子宫颈鳞状细胞癌患者 ,在治疗前后检测血清鳞状细胞癌抗原水平 ,可以作为对放射治疗疗效判断的参考指标之一。     

Role of complex cadherins in cell-cell adhesion evaluated by spheroid formation in renal cell carcinoma cell lines

We have previously shown that renal cell carcinoma (RCC) cell lines expressed a complex set of cadherins, e.g. E-cadherin, N-cadherin and cadherin-6. It is also reported that E-cadherin and cadherin-6 have a predictive value for estimating a patient's prognosis in RCC. However, E-cadherin is infrequently expressed in RCC as compared with N-cadherin and cadherin-6. In the present study, therefore, we performed a functional analysis of these cadherins as a cell adhesion molecule using spheroid culturing and spheroid-blocking assay. In E-cadherin expressers, compact spheroid formation was observed, and it was inhibited by anti-E-cadherin antibody. In contrast, in E-cadherin-absent lines, cadherin-6 apparently played a role to form relatively loose spheroids and this spheroid formation was inhibited by anti-cadherin-6 antibody. In both spheroids, the anti-N-cadherin antibody could not inhibit their formation, suggesting that N-cadherin was not an essential molecule for spheroid formation in the cell lines expressing complex cadherins. The anti-N-cadherin antibody inhibited spheroid formation only in the cell line that expressed N-cadherin alone. Using western blot analysis, immunohistochemistry and immunoprecipitation, these cadherins were linked to catenins to make a functional adhesion molecule. In conclusion, E-cadherin and cadherin-6 are shown to act in cell-cell adhesion whereas N-cadherin might play a somewhat different role from cell-cell adhesion in RCC cell lines.     

Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma

Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (scoreor=2 (mean+/-s.d.: 2%+/-2.4 vs 7.5%+/-10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G(351) to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.     

E-钙黏蛋白在喉鳞状细胞癌中的表达及其意义

目的探讨E-钙黏蛋白(E-Cadherin,E-Cad)在喉鳞状细胞癌侵袭转移中的作用,旨在初步明确其在喉鳞状细胞癌淋巴结转移判断中的作用。方法应用免疫组化S-P法检测66例喉鳞状细胞癌标本中E-Cad的表达情况,以其癌旁正常黏膜作为对照,并分析比较E-Cad表达与喉鳞状细胞癌患者年龄、病理分级、临床分期、临床分型、淋巴结转移等方面的关系,探讨其临床意义。结果E-Cad在喉鳞状细胞癌组织中阳性表达率为3.0%,在癌旁正常黏膜中75.8%,两者比较差异有显著性(P<0.05)。E-Cad阴性表达率与喉鳞状细胞癌患者的年龄、临床分期、临床分型以及淋巴结转移有显著性相关(P<0.05);而与病理分级方面无显著性相关(P>0.05)。结论E-Cad可能是喉鳞状细胞癌侵袭转移机制中的重要肿瘤标志物,它对于喉鳞状细胞癌淋巴结转移的判断具有重要意义。     

ILK及E-钙黏蛋白的表达在宫颈鳞癌发病中的意义

目的 研究整合素连接激酶(ILK)、E-钙黏蛋白(E-cadherin)在宫颈上皮内瘤变(CIN)及宫颈鳞癌组织中的表达情况,分析其与临床病理资料的关系。方法 采用免疫组化方法检测ILK及E-cadherin在54例CIN、38例宫颈鳞癌及20例正常宫颈组织中的表达。结果 ILK在正常宫颈、CINⅠ、CINⅡ~Ⅲ及宫颈鳞癌中的阳性表达率分别为25.0%、68.8%、78.9%、97.3%。ILK在CIN及宫颈鳞癌中的阳性表达率显著高于正常宫颈(P＜0.05)。E-cadherin在正常宫颈、CINⅠ、CINⅡ~Ⅲ及宫颈鳞癌中的阳性表达率分别为100.0%、75.0%、55.3%、36.8%。E-cadherin在CIN及宫颈鳞癌中的阳性表达率显著低于正常宫颈(P＜0.05)。在宫颈鳞癌组织中ILK的表达与E-cadherin呈负相关(r=-0.531,P＜0.01)。E-cadherin阳性表达还与宫颈鳞癌临床分期、分化程度、淋巴结转移有关(P＜0.05)。结论 ILK、E-cadherin可作为区分正常宫颈组织与CIN及宫颈鳞癌的标记物,E-钙黏蛋白还与宫颈癌恶性生物学行为有关。     

Establishment of an esophageal squamous cell carcinoma tumor line and development of serum squamous cell carcinoma-related antigen in athymic nude mice

A serially transplantable tumor line (IMEs-1) derived from human esophageal squamous cell carcinoma (SCC) of a 71-year-old female was established in nude mice. The histology of IMEs-1 closely resembled that of the original tumor and tumor doubling time was 3.2 days. Production of SCC-related antigen (SCCRA) and carcinoembryonic antigen (CEA) in the serum of tumor-bearing mice was observed. To evaluate the usefulness of SCCRA for esophageal cancer, the relationship between the serum levels of SCCRA and tumor burden in athymic nude mice bearing IMEs-1 was studied. The mean serum levels of SCCRA, as determined by radioimmunoassay, gradually rose in direct proportion to the tumor growth gauged by tumor volume. However, the serum levels of CEA did not increase until the last few weeks of the assay. These results suggest the possibility of using SCCRA as a tumor marker for esophageal SCC. In addition, this nude mouse assay system will provide a theoretical model to study the usefulness of SCCRA.     

A human squamous cell carcinoma cell line.

An epithelial cell line COLO 16 has been established from a human squamous carcinoma, characterized and maintained for over two years. The cells produce a parathyroid-like hormone and carcinoembryonic antigen. The line is definitely not a "HeLa contaminant." The cell line is available to other investigators.     

The role of the cell-cell adhesion molecule E-cadherin in large bowel tumour cell invasion and metastasis.

It has been suggested that the selective loss of E-cadherin expression can generate invasiveness in human carcinoma cells and might be a predictor of metastasis. Frozen sections of samples from 44 patients, 43 with suspected large bowel cancer and one with a liver recurrence were examined for E-cadherin expression using the antibody 6F9 specific for the human E-cadherin molecule. Twelve of the 40 patients with carcinoma already had lymph node involvement at the time of surgery. Samples from the primary carcinomas of only nine of these 12 patients showed reduced E-cadherin expression. However, the one lymph node with metastatic spread examined did show reduced E-cadherin expression. Four of the 40 carcinoma patients had liver involvement at the time of surgery. The primary carcinoma samples from only three of these four patients showed reduced E-cadherin expression. In addition only two out of the three liver metastases examined showed reduced expression. The primary carcinoma samples from seven patients with no evidence of tumour spread also exhibited reduced expression. Overall, analysis of the data suggests that there is no absolute correlation between reduced E-cadherin expression and tumour spread in carcinomas of the large bowel.     

上皮钙黏着蛋白对人炎性乳腺癌细胞系生物学特性的影响

目的 研究上皮钙黏着蛋白（E-cadherin）对人炎性乳腺癌细胞系SUM149的生长、侵袭等生物学特性的影响。方法 应用脂质体法基因转染技术,将编码E-cadherin基因显性负调控突变体（H-2k^d-E-cadherin）质粒导入SUM149中,应用RT-PCR、流式细胞分析法筛选、鉴定出E-cadherin基因显性负调控突变体高表达的阳性克隆。观察转染前后人炎性乳腺癌细胞系SUM149生长、侵袭能力等特性的变化以及相关分子的改变。结果 RT-PCR及Western印迹法结果均显示,与对照组（未转染及空质粒组）相比,转染后高表达小鼠H-2k^d的阳性克隆细胞的内源性上皮钙黏着蛋白表达明显下调,其细胞生长速度增殖无明显变化,而体外侵袭能力分析显示其侵袭能力明显下降,进一步研究发现,基质金属蛋白酶MMP-1、MMP-9在mRNA水平明显下调;明胶酶谱分析也显示,MMP-9明显下调。结论 在E-cadherin高表达的人炎性乳腺癌细胞系SUM149中,其表达的下调可明显抑制其侵袭能力。     

Prognostic value of human papillomavirus and squamous cell carcinoma antigen in head and neck squamous cell carcinoma

To clarify the synergistic influence of human papillomavirus (HPV) status and squamous cell carcinoma antigen (SCCA) mRNA expression on head and neck squamous cell carcinoma (HNSCC) prognosis, HPV DNA presence and SCCA1 and SCCA2 mRNA expression were determined by PCR and quantitative real‐time RT‐PCR, respectively, in 121 patients with primary HNSCC who were receiving curative treatment. HPV DNA was detected in 28.1% (34/121) of HNSCC cases, and only high‐risk types (HPV‐16, HPV‐33, HPV‐35 and HPV‐58) were observed. Positive HPV status showed a significantly better prognosis than negative HPV status (P = 0.022). An elevated SCCA2/SCCA1 mRNA ratio was an independent predictor of disease recurrence (P = 0.004). In addition, HPV‐negative patients with a high SCCA2/SCCA1 ratio (>0.27) had a significantly lower recurrence‐free survival rate than HPV‐negative patients with a low SCCA2/SCCA1 ratio (P < 0.011). Our findings revealed that both HPV status and the SCCA2/SCCA1 mRNA ratio are independently associated with prognosis in HNSCC. Patients with both a HPV‐negative status and a high SCCA2/SCCA1 ratio might need intensified treatment and rigorous follow up after treatment because of the high risk of recurrence.