Inhibition of invasion and metastasis in oral cancer by targeting urokinase-type plasminogen activator receptor

There have been reports of strong correlations between poor prognosis in various cancers and concomitant expression of urokinase-type plasminogen activator (uPA) and its surface receptor (uPAR). We and others have previously shown that the uPA system plays a significant role in a subset of head and neck squamous cell carcinoma. In the present study, we found that uPAR is required for invasion and metastasis of highly malignant oral cancer cells (OSC-19). Treating OSC-19 cells with antisense oligonucleotides (AS) targeting uPAR resulted in a dramatic decrease of uPAR mRNA expression. Furthermore, pretreatment with AS or siRNA targeting uPAR inhibited progression of OSC-19 cells in experimental models. These results suggest that overexpression of uPAR increases the invasiveness and metastasis of OSC-19 cells, and that uPAR is a promising therapeutic target for regulation of progression of oral cancer.     

Single domain antibody against carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits proliferation,migration, invasion and angiogenesis of pancreatic cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC₅₀s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5 μM, 8 μM and 12 nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6.     

Inhibition of cancer cell invasion and metastasis by genistein

Genistein is a small, biologically active flavonoid that is found in high amounts in soy. This important compound possesses a wide variety of biological activities, but it is best known for its ability to inhibit cancer progression. In particular, genistein has emerged as an important inhibitor of cancer metastasis. Consumption of genistein in the diet has been linked to decreased rates of metastatic cancer in a number of population-based studies. Extensive investigations have been performed to determine the molecular mechanisms underlying genistein’s antimetastatic activity, with results indicating that this small molecule has significant inhibitory activity at nearly every step of the metastatic cascade. Reports have demonstrated that, at high concentrations, genistein can inhibit several proteins involved with primary tumor growth and apoptosis, including the cyclin class of cell cycle regulators and the Akt family of proteins. At lower concentrations that are similar to those achieved through dietary consumption, genistein can inhibit the prometastatic processes of cancer cell detachment, migration, and invasion through a variety of mechanisms, including the transforming growth factor (TGF)-β signaling pathway. Several in vitro findings have been corroborated in both in vivo animal studies and in early-phase human clinical trials, demonstrating that genistein can both inhibit human cancer metastasis and also modulate markers of metastatic potential in humans, respectively. Herein, we discuss the variety of mechanisms by which genistein regulates individual steps of the metastatic cascade and highlight the potential of this natural product as a promising therapeutic inhibitor of metastasis.     

Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers

Background  

Many breast, pancreatic, colonic and non-small-cell lung carcinoma lines express CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen, CEA), and antibodies to both can affect tumor cell growth in vitro and in vivo. Here, we compare both antigens as a function of histological phenotype in breast, pancreatic, lung, ovarian, and prostatic cancers, including patient-matched normal, primary tumor, and metastatic breast and colonic cancer specimens.     

Inhibition of invasion and metastasis of gastric cancer cells through snail targeting artificial microRNA interference

The zinc-finger factor Snai1 plays an important role in the down-regulation of E-cadherin expression and in the induction of epithelial-mesenchymal transition (EMT) during cancer progression. In gastric cancer tissues, we noted that Snail is abnormally high expressed and is remarkably associated with the lymph node metastasis. Using a plasmid containing newly synthesized artificial microRNA (amiRNA), we transfected gastric cancer cells to block Snail expression. Both Snail protein and mRNA levels were significantly decreased in stably transfected cells, while protein and mRNA expression of E-cadherin was up-regulated. In addition, migration and invasion potential were significantly decreased after knockdown of Snail.     

High expression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 and 8 in primary myelofibrosis

Primary myelofibrosis (PMF) is characterized by leukoerythroblastic anemia with circulating immature myeloid cells, including CD34+ cells, progressive splenomegaly and accumulation of connective tissue and neoangiogenesis in the bone marrow. Altered bone marrow stroma and cell adherence account for the egress of CD34+ cells from the bone marrow. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 has been implicated in cell adhesion, cellular invasiveness, angiogenesis, and inflammation, which are all key processes in the pathophysiology of PMF. Accordingly, CEACAMs may play an important role in these processes in patients with myelofibrosis as well. Using a genome-wide approach, several genes of the CEACAM family were found to be deregulated in patients with PMF and related myeloproliferative neoplasms (n = 69). In PMF patients, the CEACAM6 and 8 were significantly upregulated (fold change (FC) 12.5 and 14.0, respectively and FDR adjusted p values 7.71 × 10⁻⁷ and 1.48 × 10⁻⁵, respectively). The elevated expression of CEACAM genes may reflect clonal myeloid expansion and neutrophil activation being most exaggerated in patients with PMF. Alternatively, the highly elevated gene expression of CEACAM6 and 8 in PMF may be molecular markers of myelofibrotic transformation, implying enhanced proteolytic activity, egress of CD34+ cells into the circulation, and neoangiogenesis.     

Inhibition of experimental metastasis of human breast-carcinoma cells in athymic nude-mice by anti-alpha(5)beta(1) fibronectin receptor integrin antibodies

We have investigated the role of the human alpha(5) beta(1) fibronectin receptor integrin in experimental metastasis. Treatment of human MDA-MB-231 breast carcinoma cells with monoclonal antibodies specific for alpha(5) or beta(1) integrin subunits prior to injection into the tail veins of 7 to 9 week old athymic nude mice significantly decreased the median number of lung colonies that were formed. In contrast, treatment of the cells with monoclonal antibodies specific for the alpha(2) subunit had no significant effect. In vitro, the anti-alpha(5) and the anti-beta(1) monoclonal antibodies both strongly inhibited breast carcinoma cell adhesion to fibronectin, while only the anti-beta(1) monoclonal antibody inhibited adhesion to laminin. In a Boyden chamber invasion assay, the anti-beta(1) antibody almost completely inhibited invasion of the breast carcinoma cells through an artificial Matrigel basement membrane. The anti-alpha(5) monoclonal antibody inhibited in vitro invasion approximately 30%, only if fibroblast conditioned medium was present as a chemoattractant. Cell migration on fibronectin could be inhibited by both the anti-alpha(5) and the anti-beta(1) monoclonal antibody. These results indicate that the alpha(5) beta(1) integrin fibronectin receptor on MDA-MB-231 human breast carcinoma cells plays an important role in experimental hematogenous metastasis and may function in this process by a combination of mechanisms, including tumor cell attachment to fibronectin and fibronectin-directed extravasation of tumor cells into the target organ.     

Inhibition of lymphoma invasion and liver metastasis formation by pertussis toxin

We have examined whether pertussis toxin, an agent known to inhibit entry of normal lymphocytes into tissues, affects invasion and metastasis formation by malignant lymphoma and T-cell hybridoma cells. The toxin reduced invasion in vitro in hepatocyte cultures to 20% of control values. Inhibition was maximal after pretreatment for 2 h with approximately 100 ng/ml. The effect of pretreatment with 1 to 5 micrograms toxin/ml for 4 h persisted for at least 5 days, despite a more than 100-fold increase in cell number. The proliferation rate was not affected. Liver metastasis formation after tail vein injection of TAM2D2 T-cell hybridoma cells in syngeneic AKR mice, measured as liver weight, was reduced to 10 to 25% of controls after pretreatment of the cells for 4 h with 1 microgram pertussis toxin/ml. Metastasis to kidneys, ovaries, and lymph nodes was not, or less evidently, affected. With MB6A lymphosarcoma cells no effect was seen after treatment with 1 microgram/ml, but a significant reduction of the liver tumor burden to approximately 50% of controls was achieved by treatment with at least 5 micrograms toxin/ml. Spleen metastasis by MB6A cells was not affected. These results provide evidence for a similarity in invasion mechanisms of normal and malignant lymphoid cells, and they suggest that invasiveness is an important factor in the formation of lymphoma metastases, particularly in the liver.     

Inhibition of tumor growth, invasion and metastasis in papain-immunized mice

Papain-immunized mice possess serum antibodies which cross-react with cathepsin-B- and cathepsin-H-like endopeptidases isolated from B16 melanoma cells. The growth rate, invasion and metastasis of both the B16 melanoma and the Lewis lung carcinoma were inhibited in mice immunized with papain. These animals presented an increased mean survival time as compared to the tumor-bearing nonimmunized controls. Quantitative microscopy suggested that vasodilation and edema, associated with tumor invasion, are, at least partially, sustained by proteolytic enzymes, being strongly reduced when tumor cells were inoculated in papain-immunized mice.     

Inhibition of invasion,gelatinase activity,tumor take and metastasis of malignant cells by N-acetylcysteine

The thiol N-acetylcysteine (NAC) is currently considered one of the most promising cancer chemopreventive agents by virtue of its multiple and coordinated mechanisms affecting the process of chemical carcinogenesis. Recent studies have shown that an unpaired cysteine residue in the propeptide plays a key role in inactivation of latent metastasis-associated metalloproteinases: the present study was designed to assess whether NAC could also affect tumor take, invasion and metastasis of malignant cells. As assessed by zymographic analysis, NAC completely inhibited the gelatinolytic activity of type-IV collagenases in the cells tested (gelatinases A and B). Moreover, NAC was efficient in inhibiting the chemotactic and invasive activities of tumor cells of human (A2058 melanoma) and murine origin (K1735 and B16-F10 melanoma cells as well as C87 Lewis lung carcinoma cells) in Boyden-chamber assays, which are predictive of the invasive and metastatic properties. Reduced glutathione (GSH) had a similar, although less effective activity. The number of lung metastases decreased sharply when B16-F10 murine melanoma cells, injected i.v. into nude mice, were pre-treated with NAC and resuspended in medium supplemented with 10 mM NAC. In other experiments NAC was given in drinking water, starting 48-72 hr before subcutaneous inoculation of either B16-F10 cells or of their highly metastatic variant B16-BL6, or intramuscular injection of LLC cells. In all experiments NAC treatment decreased the weight of the locally formed primary tumor and produced a dose-related delay in tumor formation. Spontaneous metastasis formation by B16-F10 and B16-BL6 tumors was slightly yet significantly reduced by oral administration of NAC. However, this was not observed for Lewis lung tumors. These data indicate that NAC affects the process of tumor-cell invasion and metastasis, probably due to inhibition of gelatinases by its sulfhydryl group, with the possible contribution of other mechanisms, including the potent antioxidant activity of this thiol. © 1995 Wiley-Liss, Inc.     

Inhibition of tumor invasion and metastasis by a novel lysophosphatidic acid (cyclic LPA).

Fetal calf serum (FCS) and 1-oleoyl lysophosphatidic acid (LPA) were previously found to be potent inducers of invasion (transcellular migration) in an in vitro system. A novel LPA, composed of cyclic phosphate and cyclopropane-containing hexadecanoic acid (PHYLPA), first isolated from myxoamoebae of Physarum polycephalum, and its synthetic derivatives (cLPA) were tested for their ability to inhibit tumor cell invasion and metastasis. Amoung these, Pal-cLPA, which has a palmitoyl moiety, was most potent in inhibiting invasion, with 93.8% inhibition at the concentration of 25 microM. Invasion in vitro by mouse melanoma cells (B16), human pancreatic adenocarcinoma cells (PSN-1), human lung cancer cells (OC-10) and human fibrosarcoma cells (HT-1080) was also inhibited by Pal-cLPA. The stimulation of MMI cells with LPA triggered F-actin formation, which was impaired by the addition of Pal-cLPA at invasion-inhibitory concentration. Pal-cLPA induced a rapid increase in adenosine 3',5'-cyclic monophosphate (cAMP) concentration in MMI cells. The addition of dibutyryl cAMP significantly abrogated LPA-induced invasion by MM1 cells and actin polymerization in the cells. The inhibition of MM1 cell invasion by Pal-cLPA may be ascribed to an increased level of cAMP. Pal-cLPA also suppressed invasion in vitro by MM1 cells induced by FCS dose dependently, without affecting proliferation. It also suppressed the pulmonary metastasis of B16 mouse melanoma cells injected into the tail vein of C57BL/6 mice. Thus, Pal-cLPA is effective in inhibiting invasion and metastasis of a variety of tumor cells.     

MicroRNA-335 inhibits invasion and metastasis of colorectal cancer by targeting ZEB2

To determine whether chemotherapy treatment at least 6 months prior to the detection of hepatic steatosis is associated with advanced hepatic fibrosis. Demographics, comorbid conditions, and laboratory data for cancer patients with hepatic steatosis were reviewed. The primary end point of this study was a low probability of fibrosis as calculated by the AST-to-platelet ratio index (APRI)—a surrogate for the absence of histologic bridging fibrosis and/or cirrhosis. Of 279 patients, 117 (41.9 %) were treated with chemotherapy and 197 (66.3 %) had a low probability of fibrosis by APRI. A smaller proportion of patients treated with chemotherapy had a low probability of hepatic fibrosis compared with untreated patients (64.1 vs. 75.3 %, p = 0.04). On multivariable analysis, chemotherapy treatment was a negative predictive factor for a low probability of fibrosis (OR 0.366 [95 % CI 0.184–0.708], p < 0.01). Among chemotherapy-treated patients, 75 (64.1 %) had a low probability of fibrosis. There were no differences in chemotherapy duration (mean 7.8 vs. 7.5 cycles) and interval from last dose to steatosis diagnosis (24.3 vs. 21.4 months) between patients with and without a low probability of fibrosis. A smaller proportion of patients treated with irinotecan or 5-fluorouracil had a low probability of fibrosis (37.3 vs. 66.7 %, p = 0.04). On multivariable analysis, irinotecan or 5-fluorouracil treatment was a negative predictive factor for low probability of fibrosis (OR 0.277 [95 % CI 0.091–0.779], p = 0.02). Prior chemotherapy treatment, especially with 5-fluorouracil or irinotecan, is a negative predictor for the absence of advanced hepatic fibrosis among patients with steatosis.     

Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor

Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumors. In this study, ATF-Fc, an antibody-like molecule comprising the amino-terminal fragment of human uPA (ATF) linked to the Fc fragment of human IgG1 via a flexible linker was developed. Its antitumor activities were evaluated in vitro and in vivo. The results showed that ATF-Fc had obvious cytotoxic effect on several types of tumor cells, which is dependent on cellular expression of uPAR and its Fc fragment. Treatment with ATF-Fc caused a significant suppression on tumor growth and metastasis of xenograft human tumors (MCF-7 breast cancer and BGC-823 gastric cancer) in athymic nude mice. Furthermore, we demonstrated that ATF-Fc had an anti-angiogenesis activity both in vitro and in vivo. In conclusion, we provided a novel therapeutic antibody-like molecule in the management of a variety of solid tumors by disrupting the uPA/uPAR interaction.     

Overexpression of CEACAM6 promotes migration and invasion of oestrogen-deprived breast cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast and lung and is associated with tumourigenesis, tumour cell adhesion, invasion and metastasis. In this study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERalpha)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation, and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6-15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced phosphorylated Akt and c-Src expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen-deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.     

The E-cadherin cell-cell adhesion complex and lung cancer invasion, metastasis, and prognosis

BACKGROUND: Lung cancer is the most common cause of cancer deaths in the western world. Progress in treatment results has been limited, and the prognosis is poor with a 5-year survival less than 15%. Based on new developments in molecular biology, our knowledge about lung carcinogenesis and mechanisms for invasion and metastasis has expanded and may in the future lead to more specific targeted therapies and better prognosis. The E-cadherin-catenin complex is critical for intercellular adhesiveness and maintenance of normal and malignant tissue architecture. Reduced expression of this complex in malignant disease is associated with tumour invasion, metastasis, and unfavorable prognosis. METHODS: This review is based on search in the Medline database from 1991 to 2001. We have reviewed the relevance of the E-cadherin-catenin adhesion complex in malignancy in general and lung cancer in particular. Furthermore, its role as target for specific therapy is discussed. RESULTS: Available data indicate that alterations of proteins involved in the E-cadherin-catenin complex are early incidents in cancer development. Reduced or altered expression of one or more of the components in this complex is associated with extended invasive and progressive behavior of cancer cells. Consistently, the E-cadherin-catenin complex appears to be increasingly delicate with regard to cancer prognosis. beta-Catenin, one of the components of the adhesion complex, also plays a significant role in cell signal transduction, gene activation, apoptosis inhibition, and increased cellular proliferation and migration. CONCLUSION: Inactivation of the E-cadherin-catenin adhesion complex, induced by genetic and epigenetic events, plays a significant role in multistage carcinogenesis, and seems to be associated with dedifferentiation, local invasion, regional metastasis, and reduced survival in lung cancer.     

人癌胚抗原cDNA的克隆与真核表达质粒的构建

[目的] 将人癌胚抗原(CEA)cDNA克隆到真核表达载体pIRES中，构建真核表达质粒pIRES-CEA，以期在哺乳动物细胞中高效、稳定表达，并为下一步连接上热休克蛋白基因(HSP70)从而构建CEA-HSP融合核酸疫苗作准备。[方法] 从CEA阳性的大肠癌组织中提取总RNA，经RTPCR方法获取人癌胚抗原cDNA，用内切酶XhaⅠ和NotⅠ分别酶切质粒pIRES和CEA cDNA，通过T4连接酶将带有黏性末端的CEA-cDNA插入到DIRES质粒的XbaⅠ和NotⅠ酶切位点上。[结果] 通过PCR扩增获得了CEA基因，并将CEA-cDNA正向克隆到载体pIRES中。[结论] 已经成功构建了真核表达质粒pIRES-CEA，并为下一步连接上热休克蛋白基因（HSP70）从而构建CEA-HSP融合核酸疫苗作准备。     

前列腺癌中雌激素受体,癌胚抗原的测定研究

应用免疫组化方法对20例前列腺癌组织切片进行ER、CEA定性和定位观察,结果表明前列腺癌ER阳性率为45％(9/20),CEA阳性年为35％(7/20),ER、CEA的表达与前列腺癌的分化状态无明显关系。认为前列腺癌内ER的测定对选择内分泌治疗有参考价值。     

MiR-125a suppresses tumor growth,invasion and metastasis in cervical cancer by targeting STAT3

MiR-125a has been characterized as a tumor suppressor in several cancers. However, the role of miR-125a in cervical cancer is unknown. In this study, we found the expression of miR-125a was downregulated in cervical cancer patients, and negatively correlated with the tumor size, FIGO stage, and preoperative metastasis. Kaplan-Meier analysis showed that miR-125a expression predicted favorable outcome for cervical cancer patients. Dual luciferase assays identified the STAT3 gene as a novel direct target of miR-125a. Functional studies showed that miR-125a overexpression significantly suppressed the growth, invasion and epithelial-mesenchymal transition (EMT) of cervical cancer cells both in vitro and in vivo via decreasing STAT3 expression. Moreover, miR-125a conferred to G2/M cell cycle arrest, accompanied by inhibition of several G2/M checkpoint proteins. Mechanistically, inactivation of miR-125a during cervical carcinogenesis was caused by HPV suppression of p53 expression. Clinically, STAT3, the expression of which, predicted poorer outcome, was inversely correlated with miR-125a in cervical cancer. These data highlight the importance of miR-125a in the cell proliferation and progression of cervical cancer, and indicate that miR-125a may be a useful therapeutic target for cervical cancer.     

Potential ability of morphine to inhibit the adhesion,invasion and metastasis of metastatic colon 26-L5 carcinoma cells

Morphine is frequently used for cancer patient's terminal medical care to relieve cancer pain. In the present study, we examined the inhibitory effect of morphine on experimental lung metastasis and invasion of colon 26-L5 cells. Morphine was found to significantly reduce the number of tumor colonies and the weight of the tumor-containing lung. Morphine inhibited the adhesion and migration of colon 26-L5 cells to extracellular matrix components and invasion into reconstituted basement membrane Matrigel, without affecting the cell proliferation in vitro. Notably, naloxone, an antagonist of morphine, abrogated morphine-induced inhibition of tumor cell adhesion, but did not affect the inhibitory effect on the production of matrix metalloproteinases (MMPs) from tumor cells. These results suggest that morphine inhibited the adhesive and invasive properties of tumor cells by different inhibitory mechanisms that involved the mediation of an opioid receptor.