Evidence that beta 1-6 branched Asn-linked oligosaccharides on metastatic tumor cells facilitate invasion of basement membranes

In previous studies we have shown that the ability of murine tumor cells to metastasize in situ is directly linked to expression of -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched complex-type Asn-linked oligosaccharides in tumor-cell glycoproteins. Here we demonstrate that cell-surface expression of beta 1-6 branched oligosaccharides in metastatic tumor cells is specifically associated with increased invasion of human amnion basement membranes in vitro. Compared to nonmetastatic SP1 murine mammary carcinoma cells, 2 metastatic sublines expressed higher levels of beta 1-6 branched oligosaccharides and were found to be invasive but poorly adhesive on the amnion basement membrane. Swainsonine, a non-toxic inhibitor of Asn-linked oligosaccharide processing which blocks the pathway prior to initiation of the beta 1-6 linked antenna, blocked metastatic tumor-cell invasion and increased adhesiveness. Swainsonine and the metalloprotease inhibitor O-phenanthroline inhibited invasion, apparently via independent mechanisms. O-phenanthroline did not affect tumor-cell adhesion to the amnion basement membrane and swainsonine did not block secretion of metalloproteases, beta-hexosaminadase or tissue plasminogen activator activity by the tumor cells. These results suggest that tumor-cell invasion of basement membranes requires both secretion of hydrolase activities and expression of beta 1-6 branched complex-type oligosaccharides at the tumor cell surface, such oligosaccharides being associated with reduced tumor-cell adhesion to extracellular matrix.     

Oncodevelopmental expression of--GlcNAc beta 1-6Man alpha 1-6Man beta 1--branched asparagine-linked oligosaccharides in murine tissues and human breast carcinomas

Increased--GlcNAc beta 1-6Man alpha 1-6Man beta--branching in asparagine-linked oligosaccharides has been observed in murine and human tumor cells and has recently been linked to enhanced metastatic potential in experimental tumor models. Leukoagglutinin (L-PHA) requires the beta 1-6-linked lactosamine antenna (beta 1-6 branch) for high affinity binding and was used in this study to quantitate these structures on glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal rodent tissues and cell lines were used to standardize the experimental conditions required to quantitate beta 1-6-branched oligosaccharide structures and the glycosyltransferase activity which initiates the synthesis of the antenna, beta 1-6 N-acetylglucosamine (GlcNAc)-transferase V (EC 2.4.1.155). Secondly, the levels of L-PHA-reactive oligosaccharide were compared in a series of benign and malignant human breast biopsies. Normal human breast tissue and benign lesions showed low expression but 50% of the primary malignancies examined showed significantly elevated L-PHA reactivity. GlcNAc transferase V activities in the human breast carcinomas and in normal murine tissues correlated with the levels of L-PHA reactive oligosaccharide in the tissues. GlcNAc transferase V showed similar ranges of activities, differing by approximately 5-fold between high and low expressing mouse tissues; fibroblasts with and without an activated H-ras oncogene; and low and high expressing human breast carcinomas. The results show that beta 1-6 branching in asparagine-linked oligosaccharides is dependent on tissue-specific regulation of GlcNAc transferase V activity. Secondly, a subset of human breast malignancies showed elevated levels of beta 1-6-branched oligosaccharides compared to benign samples, suggesting that further studies are warranted to determine whether the presence of these oligosaccharides is associated with metastatic disease and reduced patient survival time.     

Altered protein glycosylation of rat 3Y1 cells induced by activated c-myc gene

N-linked sugar chains of rat 3Y1 cells and tumorigenic cells derived by transfection with activated c-myc gene were quantitatively released as oligosaccharides from membrane preparations by hydrazinolysis. Structural analyses revealed that cells of both types contain bi-, tri- and tetra-antennary complex-type oligosaccharides as well as high-mannose-type oligosaccharides. However, the c-myc-transfected cells showed an increase in tri- and tetra-antennary oligosaccharides having the GlcNAc beta 1----4Man alpha 1----and/or the GlcNAc beta 1----6Man alpha 1----linkages with a decrease in biantennary oligosaccharides compared to control 3Y1 cells. The data suggest that c-myc gene has a potential role in the regulation of cellular protein glycosylation and that an elevated expression of c-myc gene in the cells leads to increased branch formation of outer chains in N-linked oligosaccharides concomitant with the acquisition of tumorigenicity.     

Asn-linked oligosaccharide processing and malignant potential

There is now considerable evidence that malignant transformation is accompanied by many changes in the oligosaccharide structures of glycoproteins and glycolipids. More recently, studies using tumour cell glycosylation mutants and inhibitors of Asn-linked oligosaccharide processing have suggested that the transformation-associated increase in GlcNAc beta 1-6Man alpha 1-6 Man beta- branching of complex-type oligosaccharides may enhance tumour cell metastasis. The synthesis of beta 1-6 branched oligosaccharides and related structures as well as their possible function in cell-cell interactions and tumour cell metastasis are discussed.     

N-glycoproteins bearing beta1-6 branched oligosaccharides from the A375 human melanoma cell line analysed by tandem mass spectrometry

Tumour-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of cancer cells. It is well known that the metastatic potential is associated with increased GlcNAc beta1-6 branching in N-glycans of tumour cells specifically recognized by a lectin from Phaseolus vulgaris leukoagglutinin (PHA-L). We identified proteins bearing GlcNAc beta1-6 branched N-glycans in the A375 human melanoma cell line by affinity chromatography separation on a PHA-L agarose column, followed by immunoidentification and tandem mass spectrometry (MS/MS) analysis. Amongst the proteins identified were integrin subunits alpha2, alpha3, alpha5 and beta1, as well as N-cadherin and lysosome-associated membrane proteins (LAMP-1 and LAMP-2). In addition, L1, Mac-2 binding protein (Mac-2-BP), activated leukocyte cell adhesion molecule/CD166 (ALCAM) and melanotransferrin were shown to react with PHA-L. Some of these proteins are connected mainly with nervous tissues or the immune system and play a crucial role in cell adhesion processes. The presence of GlcNAc beta1-6 branched oligosaccharides in these proteins may influence their adhesion properties, reducing adhesion of the cells to the extracellular matrix (ECM) and thus facilitating tumour cell invasion.     

Beta 1-6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia

Malignant transformation of murine and human cells is commonly associated with increased--GlcNAc beta 1-6Man alpha 1-6Man beta--branching in asparagine-linked oligosaccharides. Somatic mutations and drugs which block expression of the beta 1-6 branched oligosaccharides are potent inhibitors of tumor cell invasion and metastasis in animal models. This suggests that the oligosaccharides are required for metastasis to occur and therefore their increased presence in primary tumors may be diagnostic of metastatic disease. Although antibodies to the beta 1-6 branched portion of the oligosaccharides are not available, a plant lectin leukoagglutinin (L-PHA) has been shown to bind specifically to this structure. L-PHA lectin histochemistry was performed on paraffin sections of human breast and colon tissues. All breast carcinomas and epithelial hyperplasia with atypia showed significantly increased L-PHA staining compared to fibroadenomas and hyperplasia without atypia. In histological sections of colon, adenomas showed a small but significant increase in L-PHA staining compared to normal colonic epithelium, while carcinomas showed greatly increased reactivity. In addition, Dukes stage C tumors showed higher levels of L-PHA staining than stage A tumors. These results demonstrate that L-PHA-reactive beta 1-6 branched N-linked oligosaccharides are consistently increased in neoplasias of human breast and colon and that the level of L-PHA staining correlates with the pathological staging of the diseases.     

Asparagine-linked oligosaccharides associated with metastatic cancer

Malignant transformation and tumor progression are often accompanied by structural changes in the carbohydrate components of glycoproteins and glycolipids. Studies with tumor cell glycosylation mutants and specific inhibitors of glycosylation indicate that expression of sialylated and beta (1-6) branched asparagine (Asn)-linked oligosaccharides are required for tumor cell invasion and metastasis. The initiation of the beta (1-6) antenna appears to be oncodevelopmentally regulated and may be associated with the aberrant expression of blood group and embryonic carbohydrate sequences in Asn-linked oligosaccharides of tumors. Although the function of these oligosaccharides in the metastatic process remains unclear, there is some evidence that expression of these structures on certain cell-surface glycoproteins may reduce tumor cell adhesion to extracellular matrix. These observations suggest that low-toxicity drugs that inhibit the biosynthesis of these structures may be useful in the treatment of metastatic cancers.     

Beta1,6-branched oligosaccharides regulate melanin content and motility in macrophage-melanoma fusion hybrids

In previous studies, fusion of peritoneal macrophages or blood monocytes with mouse melanoma cells produced hybrids with upregulated expression of the glycosyltransferase beta1,6-N-acetylglucosaminyltransferase V (GnT-V) and its enzymatic product, beta1,6-branched oligosaccharides. This correlated with marked increases in motility, metastatic potential and, surprisingly, melanin content. This study was designed to establish direct roles for beta1,6-branched oligosaccharides in melanogenesis and motility. The levels of beta1,6-branched oligosaccharides were lowered by transfecting beta1,4-N-acetylglucosaminyltransferase III, a competitive inhibitor of GnT-V. beta1,4-N-acetylglucosaminyltransferase III transfection virtually eliminated melanin production and markedly decreased chemotactic motility. This implied that the metastatic and melanogenic phenotypes in hybrids were each upregulated by beta1,6-branched oligosaccharides. Although roles for beta1,6-branched oligosaccharides in motility and metastasis have been reported previously, this is the first study to directly implicate these structures in melanogenesis. Although drawn from experimental models, the findings might explain the well known hypermelanotic regions of human cutaneous malignant melanoma as hypermelanotic cutaneous malignant melanoma cells are rich in beta1,6-branched oligosaccharides. They might also explain why melanogenesis pathways differ between malignant and normal melanocytes as GnT-V is a myeloid-associated enzyme that is aberrantly expressed in melanoma cells but not in normal melanocytes.     

Relationships between heparanase activity and increasing metastatic potential of fibroblasts transfected with various oncogenes

We have examined the effects of transformation by activated H-ras and other transforming oncogenes on the activity of the enzyme, heparanase. Degradation of 3H-N-acetylated-partially N-desulfated heparan sulfate by cellular extracts of the transformants was assessed by gel permation chromatography. More extensive degradation was observed with 10T1/2 mouse embryo fibroblasts transfected with an activated H-ras oncogene. The cells having the highest metastatic potential (CIRAS-3) were shown to contain the greatest heparanase activity, giving 49% higher levels of activity than parental cells (P less than 0.0002). Furthermore, the enzyme activity produced by a series of H-ras transformed cell lines increased progressively with metastatic potential (non-parametric rank correlation coefficient r = 0.96). Transfection of NIH 3T3 fibroblasts with activated H-ras, v-src or v-fes oncogenes, which induced the metastatic phenotype, did not lead to large increases in heparanase activities. Also, inhibition of ras-induced malignancy by cotransfection of rat REF cells with the Ad2 E1a oncogene did not produce significant declines in heparanase activities. These results are consistent with the view that modifications in heparanase activity can play a role in the complex process of metastasis in some, but not all situations.     

N-acetylglucosaminyltransferase V activity in metastatic models of human hepatocellular carcinoma in nude mice

N-linked beta 1-6 branched oligosaccharides may contribute directly to the malignant phenotype including metastatic potential of tumour cells. Increased beta 1-6 branching was associated with an increased level of N-acetylglucosaminyltransferase V (GnT V). In this report, the tissues from two metastatic models of human hepatocellular carcinoma (HCC) in nude mice were obtained. GnT V activity and mRNA level were determined. Results showed that GnT V activity in highly metastatic LCI-D20 models (Liver Cancer Institute, passage time: 20 days) (413.1+/-86.4U) was much higher than that in low metastatic LCI-D35 model (passage time 35 days) (155.3+/-31.9U). Northern blot showed that the mRNA level of GnT V in two models had no change. During the selection of a highly metastatic LCI-D20 model, GnT V activity increased from 301.6+/-57.3U to 413.1+/-86.4U while the highly metastatic LCI-D20 model acquired higher metastatic ability after selection. When highly metastatic LCI-D20 model tissues were implanted subcutaneously (s.c.), the GnT V activity decreased dramatically from 413.1+/-86.4U to 94.9U. This is the first report that GnT V activity increased in HCC during metastasis in vivo.     

High expression of shMDG1 gene is associated with low metastatic potential of tumor cells

Metastasis is the primary cause of mortality associated with cancer. Molecular mechanisms leading to metastatic spread are poorly studied. To get a better understanding of this process, we compared the gene expression pattern of two isogenic cell lines, HET-SR and HET-SR1 (Rous Sarcoma Virus-transformed embryo hamster fibroblasts) with different metastatic activity using the differential display technique. A novel cDNA of hamster gene shMDG1 (Syrian hamster homologue of microvascular differentiation gene 1), which had 94% homology with rat MDG1 gene, was identified. Expression of shMDG1 was increased in low metastatic HET-SR cell line in comparison to high metastatic HET-SR1. Sequence analysis of the ORF of shMDG1 gene showed that it belongs to the DnaJ/heat-shock proteins of 40 kDa (HSP40) chaperones family, considered to function as a cochaperone of HSP70 family. In order to confirm involvement of shMDG1 in metastasis, we injected parental and shMDG1 overexpressed cells into animals. We showed that overexpression of the shMDG1 gene significantly diminished the metastatic activity of both HET-SR and HET-SR1 cells. The shMDG1-induced repression of metastasis was not connected with alterations in cell proliferation and motility in vitro, but correlated well with a decrease in content of the Asn-linked beta1-6 branched oligosaccharides on cell surface.     

Multiple effects on drug-sensitivity, genome stability and malignant potential by combinations of h-ras, C-myc and mutant p53 gene overexpression

Activation of specific oncogenes and inactivation of tumor suppressor genes play major roles in mechanisms leading to neoplastic transformation. The potential involvement of these genes in determining genome stability is an important issue. To examine the relationships between altered oncogene expression and the effects on genome stability, we have investigated the drug sensitivity properties of mouse 10T1/2 fibroblasts transfected with combinations of H-ras, c-myc and the proline 193 mutant form of p53. The relative colony forming efficiencies of these cells were investigated in the absence or presence of various concentrations of the chemotherapeutic agents, methotrexate, N-(phosphonacetyl)-L-aspartate (PALA) or hydroxyurea. The effects of altered oncogene expression were found to be drug and locus specific, and to lead to increased drug resistance (e.g. H-ras transfectants were significantly resistant to methotrexate or PALA), decreased drug resistance (e.g. H-ras/-myc transfectants were significantly less resistant to PALA or hydroxyurea than H-ras transfected cells), or to no significant change in drug sensitivity (e.g. H-ras transfected cells were not significantly different in sensitivity to hydroxyurea than non-transfected cells). Gene amplification was an important but not the only mechanism for drug resistance. Cells that were transfected with p53 (H-ras/p53 or H-ras/c-myc/p53) exhibited the greatest drug resistance properties with all three chemotherapeutic agents, in keeping with the important role of p53 in DNA repair and DNA amplification mechanisms. Although both H-ras/p53 and H-ras/c-myc/p53 groups exhibited very similar genome stability characteristics as determined by drug sensitivity results, they were significantly different in their abilities to produce transformed foci in vitro and lung metastases in vivo. The H-ras/c-myc/p53 transfected cells formed significantly higher numbers of transformed foci and exhibited a greater malignant potential. These results are consistent with observations that H-ras expression directly correlates with malignant potential, and that H-ras/c-myc/p53 transfected cells have higher H-ras expression than H-ras/p53 transfected cells. Alterations in genomic integrity through changes in onocogene expression play important roles in mechanisms determining drug sensitivity; in addition to genome destabilization, other events are critically involved in regulating transformed and malignant characteristics.     

Antipain-induced suppression of oncogene expression in H-ras-transformed NIH3T3 cells

Antipain (AP; 50 micrograms/ml) inhibits transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. To determine whether AP effects on transformation are associated with alterations in oncogene expression, NIH3T3 cells were cotransfected with an activated H-ras oncogene and the selectable marker gene aph, and gene expression was quantified. Fifty percent of geneticin-resistant colonies which were exposed to AP failed to express the transformed phenotype as determined by their inability to grow in soft agar. Northern blot analysis of the transformed and nontransformed colonies revealed that suppression of H-ras transformation by AP was associated with a decrease in expression of the exogenously transfected H-ras gene by approximately 4-fold. Expression of the endogenous oncogene c-myc was decreased by approximately 2.5-fold, to levels seen in untransfected cells. AP-treated colonies that retained the transformed phenotype had levels of oncogene expression that were similar to untreated ras-transformed colonies. Southern blot analysis revealed no effects of AP on incorporation or copy number of the H-ras gene.     

Upregulation of alpha and beta integrin subunits in metastatic macrophage-melanoma fusion hybrids

Fusion of cancer cells with migratory bone-marrow-derived cells such as macrophages can produce cancer cells with increased metastatic potential. To study this, we fused mouse macrophages with weakly metastatic mouse melanoma cells and generated a panel of hybrid clones. About half of these showed increased metastatic potential in mice. These hybrids expressed traits and molecules that were known indicators of tumor progression in melanoma (chemotaxis toward fibronectin, melanogenesis, autophagy, cMet, MCR1, SPARC, cell surface LAMP-1, GnT-V and β1,6-branched oligosaccharides). Here, we investigated integrin subunit expression in selected hybrids. Integrins, especially those that are substrates for the glycosyltransferase GnT-V and carriers of β1,6-branched oligosaccharides, play an important role in cell migration. We report increased expression of the integrin subunits α3, α5, α6, αv, β1, and β3 in metastatic hybrids compared with parental melanoma cells and a weakly metastatic hybrid. Notably, each of these subunits is also a substrate for GnT-V. Integrin subunit expression was further increased by inducers of cyclic AMP. Expression of these integrin subunits is a characteristic of macrophages and also associated with progression in melanoma and other cancers. In summary, our studies of macrophage-melanoma hybrids show that several α and β integrin subunits are upregulated in the metastatic lines. This adds further support for the theory that generation of a metastatic phenotype may be initiated through a single event: fusion of migratory bone marrow-derived cells with cancer cells.     

Molecular characterization of P2B/LAMP-1, a major protein target of a metastasis-associated oligosaccharide structure

Sialylated and GlcNAc beta 1-6Man alpha 1-6Man beta 1 (beta 1-6 branched) complex-type oligosaccharides linked to asparagine residues of membrane glycoproteins in metastatic murine tumor cells have been associated with efficient tumor cell metastasis. A large proportion of these oligosaccharide structures, in several unrelated malignant cell lines, have been shown to be associated with a glycoprotein termed P2B, with a molecular weight of 130,000. This glycoprotein has recently been purified from the metastatic MDAY-D2 cell line and shown to be biochemically similar to a lysosomal associated membrane glycoprotein (LAMP-1). We report here the details of a 2147 nucleotide complementary DNA encoding the entire murine P2B polypeptide which was immunoselected from a lambda gt11 expression library and sequenced. The sequence is similar to a complementary DNA coding for mouse LAMP-1 with the exception of a 5' untranslated region, a leader signal-sequence, and various insertions, deletions, and substitutions in the 3' untranslated domain. An open reading frame of 405 amino acids encodes a mature polypeptide of 382 residues with a predicted molecular weight of 42,000. P2B/LAMP-1 possesses 20 asparagine-linked glycosylation sites separated into equal halves by a central, putative hinge region and is anchored by a carboxy, membrane-spanning, domain. Topological considerations dictate that cell surface expression of P2B/LAMP-1 exposes the bulk of the glycoprotein into the extracellular compartment. Immunofluorescent staining of fibroblast cells indicated that P2B/LAMP-1 was associated with lysosomal membranes and, to a lesser degree, select surfaces of plasma membrane. An amino acid comparison of the murine sequence with its recently cloned rat, human, and chicken counterparts shows a conservation of 17 of 20 asparagine-linked glycosylation consensus sites, eight of eight cysteine residues, and other selected protein domains. The interspecies conservation of these domains suggests that they are important for the structure and function of the P2B/LAMP-1 glycoprotein. Northern analysis revealed that P2B/LAMP-1 is widely expressed in normal murine tissues and tumor cell lines. However, in two experimental models of metastasis, where changes in branching of oligosaccharides on P2B/LAMP-1 have been shown to occur, comparable levels of P2B/LAMP-1 mRNA were found in both metastatic and nonmetastatic cell lines.     

The effect of H-ras oncogene transfection on response of mink lung epithelial cells to growth factors and cytotoxic drugs

Mink lung epithelial cells were transfected with c-myc and activated H-ras genes. The transfected sublines formed colonies in soft agar and were tumorigenic when injected subcutaneously into athymic nude mice. DNA synthesis was measured in each of the cell lines by 3H-thymidine incorporation and in the parent line there was dose related stimulation of DNA synthesis by epidermal growth factor (EGF) and inhibition by transforming growth factor-beta (TGF-beta). The c-myc transfected line had a reduced inhibitory response to TGF-beta and an exaggerated stimulatory response to EGF whereas the activated H-ras1 transfected line did not respond to TGF-beta or EGF. The activated H-ras1 transfected line was significantly more resistant to doxorubicin (ID50, 4.4 nM) and vincristine (ID50, 4.9 nM) than the parent mink lung epithelial cell line (ID50, 2.7 nM and 2.4 nM respectively). It would appear that oncogene transfection can alter the sensitivity of mink lung epithelial cells to both exogenous growth factors and cytotoxic drugs.     

Functional interaction of p53 with HPV18 E6, c-myc and H-ras in 3T3 cells.

Wild-type (wt) p53 has been suggested to be the product of a tumor-suppressor gene. Recently, it has been shown that the E6 oncoproteins of human papillomavirus (HPV) types 16 and 18, like the SV40 large T antigen, are physically associated with wt p53. We have investigated the functional interaction of wt p53 with the viral oncogene products of HPV16 and 18 and with cellular oncogenes by transfection of NIH3T3 cells with p53 wt alone or with several oncogene(s). We found that over-expression of HPV18 E6, c-myc or activated H-ras, like SV40 large T, can partially overcome the growth-inhibitory effect of wt p53 in NIH3T3 cells, while HPV16 E6 and E7, HPV18 E7, k-fgf, c-fos and mutant (mt) p53 do not. Further studies indicate that HPV18 E6 and c-myc can overcome the antiproliferative effect, but not the antitransforming effect, of wt p53, while activated H-ras can overcome both the antiproliferative and antitransforming effects of wt p53. These data show evidence of a functional interaction between HPV18 E6 and wt p53, and suggest that the cooperation of HPV E6 and cellular oncogenes c-myc and H-ras, which are activated in several cases of human cervical cancers, may be necessary to overcome completely the anti-oncogenic function of p53 in the development of these tumors.     

Glycoproteins associated with metastatic potential of cancer

Carbohydrate moieties of glycoproteins have been implicated to be involved in cellular adhesion. Therefore, certain carbohydrate structures in glycoproteins are expected to be associated with metastatic behaviour of cancer cells; such carbohydrate structure can be used as an indicator for the treatment of cancer patients based on the predicted metastatic potential of the cancer cells. The following results recently reported is interesting from the above view point. In certain cancer cells of the mouse, beta 1----6 branching of asparagine-linked oligosaccharides, which can be detected by the reactivity with PHA-L lectin, associates with increased metastatic potential. In human bladder carcinomas, reactivity with Lotus tetragonolobus agglutinin (LTA) correlates with increased metastatic potential. LTA appears to react with Lex structure [Gal beta 1----4(Fuc alpha 1----3)GlcNAc] in the cancer cells. Expression of sialilated dimeric Lex increases in the metastatic nests of human colon carcinomas. On the other hand, sialyl antigen MGl has the tendency to be expressed in human gastric adenocarcinoma of low metastatic potential. MGl is defined by a monoclonal antibody raised against Ricinus communis agglutinin receptors isolated from human gastric adenocarcinoma xenografted in nude mice. Continued efforts using monoclonal antibodies and lectins may yield arrays of carbohydrate markers of clinical value to predict the metastatic potential.