Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma

Carcinoembryonic antigen (CEA), a widely used tumor marker, is attached by a glycosylphosphatidylinositol (GPI) anchor motif to the cell membrane. Recent study suggested that membrane-bound CEA might be cleaved by glycosylphosphatidylinositol-phospholipase D (GPI-PLD). We studied the effect of GPI-PLD on the cleavage of CEA to elucidate the implication for metastatic potential in colorectal carcinoma cells. CEA amount of conditioned medium was changed by suramin and phenanthroline (activator and inhibitor of GPI-PLD) only in SW620 and SW837 which expressed both CEA and GPI-PLD mRNA. Suramin treatment also augmented migratory activity and decreased cell surface CEA expression in SW620 and SW837. Furthermore, GPI-PLD knockdown cells using GPI-PLD-specific siRNA in SW620 and SW837 showed decreased CEA secretion from cell membrane and the migration activity, increased membrane-bound CEA amount. Splenic injection of SW620 and SW837 induced marked hepatic metastases in nude mice. These results suggest that membrane-bound CEA is cleaved by GPI-PLD and that this cleavage enhances the metastatic potential in colorectal carcinoma cells.     

Induction of carcinoembryonic antigen and its crossreactive gene products in human colonic adenomas and carcinomas by transforming growth factor-beta

The inducing effect of transforming growth factor-beta (TGF-B) on carcinoembryonic antigen (CEA) secretion and the cellular expression of CEA and CEA crossreactive glycoproteins (CEA-GLY) was examined from a panel of human colonic cell lines with different phenotypic classification. This panel included carcinomas with dissimilar differentiation characteristics and metastatic behavior, and premalignant adenomas derived from colonic polyps. A great degree of heterogeneity was observed in the endogenous levels of CEA secretion and the cellular expression of CEA and CEA-GLY species. The response profiles of the different cell lines to TGF-B treatment were also found to be heterogenous. However, TGF-B was able to induce CEA secretion and up-modulated the cellular expression of CEA and CEA-GLY from a majority of the cells tested. More importantly, TGF-B was able to exert these effects on carcinoma cells that secrete or express minimal or nondetectable amounts of these glycoproteins. These biologic modifying effects of TGF-B may have potential in augmenting the efficacy of CEA as a colon cancer marker, and in antibody-directed radioimaging and therapeutics. Further investigation in vivo in an experimental animal model system is warranted.     

Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential

Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10(-/-) mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.     

In vitro derivation and characterization of a colorectal cancer stem cell subpopulation

In the present publication we describe for the first time the derivation of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells’ primary role in the development of malignant neoplasms.     

Cell Specific CD44 Expression in Breast Cancer Requires the Interaction of AP-1 and NFκB with a Novel cis-Element

Breast cancers contain a heterogeneous population of cells with a small percentage that possess properties similar to those found in stem cells. One of the widely accepted markers of breast cancer stem cells (BCSCs) is the cell surface marker CD44. As a glycoprotein, CD44 is involved in many cellular processes including cell adhesion, migration and proliferation, making it pro-oncogenic by nature. CD44 expression is highly up-regulated in BCSCs, and has been implicated in tumorigenesis and metastasis. However, the genetic mechanism that leads to a high level of CD44 expression in breast cancer cells and BCSCs is not well understood. Here, we identify a novel cis-element of the CD44 directs gene expression in breast cancer cells in a cell type specific manner. We have further identified key trans-acting factor binding sites and nuclear factors AP-1 and NFκB that are involved in the regulation of cell-specific CD44 expression. These findings provide new insight into the complex regulatory mechanism of CD44 expression, which may help identify more effective therapeutic targets against the breast cancer stem cells and metastatic tumors.     

Carcinoembryonic antigen gene and carcinoembryonic antigen expression in the liver metastasis of colorectal carcinoma.

The presence of the carcinoembryonic antigen (CEA) gene and CEA expression in the liver was tested to identify their possible roles in the liver metastasis of colorectal carcinoma. The CEA gene in the liver was identified by amplifying the CEA-specific N-terminal domain exon with digoxigenin-dUTP labeling in 16 colorectal carcinomas with liver metastases. Next, CEA expression was tested by immunostaining using the anti-CEA monoclonal antibody (T84.66, ATCC). Liver tissues from 13 stomach cancer patients and 12 colorectal cancer patients without liver metastasis were also tested as control groups. Three grades (<25%, 25-50%, and 50%< or =) were given according to the proportion of positive cells. The CEA gene was amplified in the metastatic tumor cells of the liver (2.6 +/- 0.2, mean grade +/- SEM) and their surrounding hepatocytes (1.5 +/- 0.2) in all cases. CEA expression was found in all metastatic tumor cells and 14 cases of the surrounding hepatocytes. Among the control groups, the CEA gene of the hepatocytes was found in 9 cases each of the colorectal and the stomach cancers that did not exhibit CEA expression. The level of serum CEA was related with the numbers and volume of liver metastases, but not with CEA expression in tumor cells and surrounding hepatocytes. The CEA gene in the metastatic tumor cells, not in the hepatocytes, was closely associated with CEA expression in the surrounding hepatocytes (p<0.01). Although the precise mechanism of CEA gene regulation in hepatocytes remains to be proven, the CEA gene in the metastatic tumor of the liver seems to affect CEA expression in the surrounding hepatocytes facilitating liver metastasis in colorectal carcinoma.     

RNA干扰抑制癌胚抗原表达对结肠癌细胞增殖的影响

癌胚抗原（CEA）作为肿瘤标志物,在多种恶性肿瘤中高表达.为探索CEA在肿瘤发生发展过程中的生物学作用,本研究以结肠癌细胞株8307为研究对象,应用RNAi技术抑制CEA在8307细胞中的表达,观察其对8307细胞生长的影响.依据siRNA设计原则,设计合成靶向CEA的shRNA并克隆到pSilencer2.1-U6 neo载体中,成功构建了CEA shRNA表达载体,通过脂质体介导转染8307细胞,经G418筛选出抑制CEA表达的稳定细胞.RT-PCR、Western印迹分别检测CEA mRNA及蛋白表达水平,MTT及集落形成实验检测细胞增殖活性.实验结果显示,构建的CEA shRNA表达载体明显抑制CEA mRNA及蛋白水平的表达.MTT实验中,CEA表达下调的8307细胞生长活性降低,与对照组相比,抑制率达39%（P<0.05）,细胞集落形成能力也显著降低（P<0.05）.上述结果初步证明,构建的CEA shRNA表达载体能明显降低CEA的表达,并抑制结肠癌细胞增殖活性.     

Molecular profile, tissue distribution and prognostic evaluation of a human melanoma-carcinoma antigen recognized by the murine monoclonal antibody B1.1

Using the murine monoclonal antibody (MoAb) B1.1 we have analyzed the immunochemical profile and the tissue distribution of a human melanoma associated antigen (MAA) carrying an epitope shared by the 180 kd CEA. Results of this study have demonstrated that the epitope expressed by the MAA is carried by a distinct set of molecules of 110-140 kd. Similarly to the 180 kd CEA molecules synthesized by carcinomas, the expression of the melanoma associated CEA like components (MA-CEA) is upregulated by IFN-alpha. The tissue distribution of MA-CEA is not restricted to malignant primary and metastatic melanocytic lesions but is found also at low levels in 64% of benign nevi. No circulating CEA was found in patients bearing widespread metastatic disease of MA-CEA positive lesions. Preliminary clinical evaluation of stage I melanoma patients bearing MoAb B1.1 positive lesions has not shown a significative prognostic association of this phenotypic marker with clinical course of the disease.     

Potential for Cripto-1 in defining stem cell-like characteristics in human malignant melanoma

The diagnosis of melanoma is becoming ever more frequent. Although surgical excision of early lesions is associated with relatively significant high cure rates, treatment modalities are largely unsuccessful for advanced disease. Characteristics such as cellular heterogeneity and plasticity, expression of certain molecules such as the multidrug resistance protein-1 (MDR1) or the aberrant expression of embryonic signaling molecules and morphogens like Nodal, important for self renewal and pluripotency, suggest that a stem cell-like population may reside in aggressive melanomas. This perspective focuses on preliminary findings obtained in our laboratory which indicate that the expression of the Nodal co-receptor, Cripto-1, in a subset of malignant melanoma cells may be exploited to identify possible melanoma stem cells (MSC). In fact, the use of anti-Cripto-1 antibodies to cell sort Cripto-1-positive cells in the metastatic melanoma cell line C8161 has identified a slow growing, sphere forming subpopulation that expresses increased levels of Oct4, Nanog and MDR1. If current in vivo studies confirm the self renewal and tumorigenic characteristics of these cells, the expression of Cripto-1 may represent a useful marker to identify cancer stem cells in melanoma, and possibly other aggressive tumors as well.     

Stable, high-level expression of a carcinoembryonic antigen-encoding cDNA after transfection and amplification with the dominant and selectable asparagine synthetase marker

The introduction and expression of cloned genes in a wide variety of animal cells requires the convenient use of dominant selectable markers. Very few of these markers can be amplified in copy number, a necessary feature if variable and high-level expression of the gene of interest is required. We describe the successful dominant transfection and amplification of a vector containing the Escherichia coli asparagine synthetase (AS)-encoding gene, asnA, transfected into a variety of human and rodent cell lines. An unlinked co-transfected expression vector containing the CEA cDNA, encoding human carcinoembryonic antigen, can be co-amplified with the asnA marker leading to extremely high levels of CEA synthesis. In addition, we show that the expression of both the asnA marker and the co-transfected CEA construct are stable in normal and amplified transfectants after prolonged culture in the absence of selective pressure.     

ABCG2: A potential marker of stem cells and novel target in stem cell and cancer therapy

ABCG2 is a member of the ATP binding cassette (ABC) transporters, which can pump a wide variety of endogenous and exogenous compounds out of cells. Widely expressed in stem cells, ABCG2 is also found to confer the side population phenotype and is recognized as a universal marker of stem cells. Although the precise physiological role of ABCG2 in stem cells is still unclear, existing data strongly suggest that ABCG2 plays an important role in promoting stem cell proliferation and the maintenance of the stem cell phenotype. In addition, ABCG2 is also found to be expressed in a number of cancer cells and appears to be a marker of cancer stem cells. Moreover, ABCG2 expression in tumors may contribute to their formation and progression. Thus, ABCG2 has potential applications in stem cell and tumor therapy.     

Diagnostic utility of GLUT-1 expression in the cytologic evaluation of serous fluids

OBJECTIVE: To evaluate the extent to which adenocarcinomas in body cavity fluids express GLUT-1 in comparison to currently available markers for adenocarcinomas. STUDY DESIGN: Archival paraffin-embedded cell blocks of serous fluids from 25 cases of benign effusions containing reactive mesothelial cells and 39 cases of malignant effusions with metastatic adenocarcinoma (11 ovarian, 11 pulmonary, 9 gastrointestinal and 8 breast) were retrieved from the surgical pathology files. All cases were stained with antibodies for GLUT-1, Ber-Ep4, B72.3 and CEA. Positive staining was defined as distinct linear membrane staining for GLUT-1 and Ber-EP4, cytoplasmic staining for CEA, and cytoplasmic or membrane staining for B72.3. Strong staining in at least 10% of the tumor cells was required in order to consider the case positive for the particular marker. RESULTS: GLUT-1 was expressed in 72% (28 of 39) of cases of malignant effusions: 100% (11 of 11) from the ovary, 91% (10 of 11) from the lung, 67% (6 of 9) from the gastrointestinal tract and 12% (1 of 8) from the breast. None (0 of 25) of the benign effusions expressed GLUT-1. Malignant effusions expressed CEA in 74% (29 of 39), Ber-Ep4 in 85% (33 of 39), and B72.3 in 62% (24 of 39). Benign effusions expressed CEA in 3 cases and B72.3 in 2 cases. CONCLUSION: GLUT-1 is a useful marker that can be applied to cytologic specimens. It can be used as a reliable component of an antibody panel to distinguish reactive mesothelial cells from metastatic adenocarcinoma in particular adenocarcinomas of body cavity effusions, in particular adenocarcinomas of ovarian and pulmonary origin.     

Hormone receptor status, erbB2 expression and cancer stem cell characteristics of circulating tumor cells in breast cancer patients

The most important predictor for disease-free and overall survival of breast cancer patients is the presence of axillary lymph node metastasis. For surveillance during recurrence-free follow-up or in metastatic disease no marker is available at the moment. Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. Indeed, only CTC that exhibit specific molecular characteristics including stem cell characteristics, could be able to create new metastasis. Hormone therapy or anti-erbB2 therapies are prescribed according to the hormone (ERα/PR expression) and erbB2 status of the initial tumor. Nonetheless, it appears that the CTC, and consequently the metastatic cells, may have a very different hormone and erbB2 status. An optimal individualized treatment could then be obtained by characterizing ERα and erbB2 status in the CTC and comparing it to the primary tumor.     

Altered expression of sialyl-Tn, Lewis antigens and carcinoembryonic antigen between primary and metastatic lesions of uterine cervical cancers.

Immunohistochemical examination was performed of serial sections of 24 normal human adult cervical tissues and 53 human cervical carcinomas including 36 cases with lymph node metastasis. For this investigation, monoclonal antibodies directed to Lewis-X, Lewis-Y, sialyl-dimeric Lewis-X (SDLX), sialyl-Tn (STn) and carcinoembryonic antigen (CEA) were used. STn and CEA antigens were expressed very weakly in the normal cervical epithelium but strongly in the cancer cells, indicating the antigens to be oncogenic antigens of cervical squamous cell carcinoma. No significant difference in immunoreactivity was observed between primary and metastatic lesions of carcinoma or between primary lesions with and without metastasis. However, the expression patterns of STn and Lewis-Y antigens were quite different between primary lesions and metastatic lesions. In primary lesions the cancer cell nests tended to be stained centrally, but in metastatic lesions the cancer cell nests tended to be stained peripherally. This finding may reflect an important role of these carbohydrate chains in the process of metastasis of cervical squamous cell carcinoma to regional lymph nodes.     

Altered expression of sialyl-Tn,Lewis antigens and carcinoembryonic antigen between primary and metastatic lesions of uterine cervical cancers

Summary Immunohistochemical examination was performed of serial sections of 24 normal human adult cervical tissues and 53 human cervical carcinomas including 36 cases with lymph node metastasis. For this investigation, monoclonal antibodies directed to Lewis-X, Lewis-Y, sialyl-dimeric Lewis-X (SDLX), sialyl-Tn (STn) and carcinoembryonic antigen (CEA) were used. STn and CEA antigens were expressed very weakly in the normal cervical epithelium but strongly in the cancer cells, indicating the antigens to be oncogenic antigens of cervical squamous cell carcinoma. No significant difference in immunoreactivity was observed between primary and metastatic lesions of carcinoma or between primary lesions with and without metastasis. However, the expression patterns of STn and Lewis-Y antigens were quite different between primary lesions and metastatic lesions. In primary lesions the cancer cell nests tended to be stained centrally, but in metastatic lesions the cancer cell nests tended to be stained peripherally. This finding may reflect an important role of these carbohydrate chains in the process of metastasis of cervical squamous cell carcinoma to regional lymph nodes.     

SVCT2 Is Expressed by Cerebellar Precursor Cells,Which Differentiate into Neurons in Response to Ascorbic Acid

Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.