CD23/FcεRII: molecular multi-tasking.

CD23 is the low-affinity receptor for immunoglobulin (Ig)E and plays important roles in the regulation of IgE responses. CD23 can be cleaved from cell surfaces to yield a range of soluble CD23 (sCD23) proteins that have pleiotropic cytokine-like activities. The regions of CD23 responsible for interaction with many of its known ligands, including IgE, CD21, major histocompatibility complex (MHC) class II and integrins, have been identified and help to explain the structure-function relationships within the CD23 protein. Translational studies of CD23 underline its credibility as a target for therapeutic intervention strategies and illustrate its involvement in mediating therapeutic effects of antibodies directed at other targets.

The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Transcription factors control invasion: AP-1 the first among equals.

Metastasis, the aggressive spread of a malignant tumor to distant organs, is a major cause of death in cancer patients. Despite this critical role in cancer outcomes, the molecular mechanisms that control this process are just beginning to be understood. Metastasis is largely dependent upon the ability of tumor cells to invade the barrier formed by the basement membrane and to migrate through neighboring tissues. This review will summarize the evidence that tumor cell invasion is the result of oncogene-mediated signal transduction pathways that control the expression of a specific set of genes that together mediate tumor cell invasion. We focus on the role of the transcription factor AP-1 to both induce the expression of genes that function as invasion effectors and repress other genes that function as invasion suppressors. This identifies AP-1 as a critical regulator of a complex program of gene expression that defines the invasive phenotype.

p120-catenin is required for the collective invasion of squamous cell carcinoma cells via a phosphorylation-independent mechanism.

Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival. p120-catenin has emerged as a key player in promoting E-cadherin stability and adherens junction integrity and has been proposed as a potential invasion suppressor by preventing release of cells from the constraints imposed by cadherin-mediated cell-cell adhesion. However, it has been proposed that tyrosine phosphorylation of p120 may contribute to cadherin-dependent junction disassembly during invasion. Here, we use small interfering RNA (siRNA) in A431 cells to show that knockdown of p120 promotes two-dimensional migration of cells. In contrast, p120 knockdown impairs epidermal growth factor-induced A431 invasion into three-dimensional matrix gels or in organotypic culture, whereas re-expression of siRNA-resistant p120, or a p120 isoform that cannot be phosphorylated on tyrosine, restores the collective mode of invasion employed by A431 cells in vitro. Thus, p120 promotes A431 cell invasion in a phosphorylation-independent manner. We show that the collective invasion of A431 cells depends on the presence of cadherin-mediated (P- and E-cadherin) cell-cell contacts, which are lost in cells where p120 expression is knocked down. Furthermore, membranous p120 is maintained in invasive squamous cell carcinomas in tumours suggesting that p120 may be important for the collective invasion of tumours cells in vivo.

Calpain 2 and Src dependence distinguishes mesenchymal and amoeboid modes of tumour cell invasion: a link to integrin function.

Cancer cells can invade three-dimensional matrices by distinct mechanisms, recently defined by their dependence on extracellular proteases, including matrix metalloproteinases. Upon treatment with protease inhibitors, some tumour cells undergo a 'mesenchymal to amoeboid' transition that allows invasion in the absence of pericellular proteolysis and matrix degradation. We show here that in HT1080 cells, this transition is associated with weakened integrin-dependent adhesion, consistently reduced cell surface expression of the alpha2beta1 integrin collagen receptor and impaired signalling downstream, as judged by reduced autophosphorylation of focal adhesion kinase (FAK). On examining cancer cells that use defined invasion strategies, we show that distinct from mesenchymal invasion, amoeboid invasion is independent of intracellular calpain 2 proteolytic activity that is usually needed for turnover of integrin-linked adhesions during two-dimensional planar migration. Moreover, an inhibitor of Rho/ROCK signalling, which specifically impairs amoeboid-like invasion, restores cell surface expression of alpha2beta1 integrin, downstream FAK autophosphorylation and calpain 2 sensitivity--features of mesenchymal invasion. These findings link weakened integrin function to a lack of requirement for calpain 2-mediated integrin adhesion turnover during amoeboid invasion. In keeping with the need for integrin adhesion turnover, mesenchymal invasion is uniquely sensitive to Src inhibitors. Thus, the need for a major pathway that controls integrin adhesion turnover defines and distinguishes cancer cell invasion strategies.

Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts.

BACKGROUND: Changes in cell shape and motility are important manifestations of oncogenic transformation, but the mechanisms underlying these changes and key effector molecules in the cytoskeleton remain unknown. The Fos oncogene induces expression of ezrin, the founder member of the ezrin/radixin/moesin (ERM) protein family, but not expression of the related ERM proteins, suggesting that ezrin has a distinct role in cell transformation. ERM proteins have been suggested to link the plasma membrane to the actin-based cytoskeleton and are substrates and anchoring sites for a variety of protein kinases. Here, we examined the role of ezrin in cellular transformation. RESULTS: Fos-mediated transformation of Rat-1 fibroblasts resulted in an increased expression and hyperphosphorylation of ezrin, and a concomitant increased association of ezrin with the cortical cytoskeleton. We tagged ezrin with green fluorescent protein and examined its distribution in normal and Fos-transformed fibroblasts: ezrin was concentrated at the leading edge of extending pseudopodia of Fos-transformed Rat-1 cells, and was mainly cytosolic in normal Rat-1 cells. Functional ablation of ezrin by micro-CALI (chromophore-assisted laser inactivation) blocked plasma-membrane ruffling and motility of Fos-transformed fibroblasts. Ablation of ezrin in normal Rat-1 cells caused a marked collapse of the leading edge of the cell. CONCLUSIONS: Ezrin plays an important role in pseudopodial extension in Fos-transformed Rat-1 fibroblasts, and maintains cell shape in normal Rat-1 cells. The increased expression, hyperphosphorylation and subcellular redistribution of ezrin upon fibroblast transformation coupled with its roles in cell shape and motility suggest a critical role for ezrin in oncogenic transformation.

Human squamous cell lung cancers express increased epidermal growth factor receptors.

Epidermal growth factor (EGF) promotes the growth of cultured benign and malignant cells. Recent studies have demonstrated that the amount of EGF receptor is elevated in squamous cell carcinoma cells in tissue culture when compared with normal epidermal cells. This study demonstrates that elevated levels of EGF receptor are detected in biopsy specimens of human squamous cell carcinomas of the lung with a murine monoclonal antibody, EGF-R1, which binds specifically to the receptor. The increased receptor ranged from 2.5- to 5-fold that of normal skin. These findings have been observed in 11 of 11 squamous carcinomas and two of two epidermoid head and neck cancers. Seven of eight adenocarcinomas, two of two small cell, and four of eight undifferentiated lung cancers had negligible amounts of EGF receptor. The EGF receptor antibody did not bind significantly to normal lung tissues and 35 nonepidermoid tumors. Therefore, EGF receptor may be an excellent marker for epidermoid malignancies.

AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program.

Multistage progression of Abelson virus-infected murine pre-B-cells to the tumorigenic state.

The tumorigenic potential of pre-B-cells at different stages of Abelson murine leukemia virus-induced transformation was determined. Cell lines with low growth potential in liquid culture were found (a) to have a dose-dependent growth requirement for conditioned medium obtained from bone marrow cultures, (b) to have low colony-forming ability in semisolid medium in the absence of conditioned medium, and (c) to be nontumorigenic when inoculated into syngeneic mice. Culture of the factor-dependent cells in vitro leads to the emergence of factor-independent variants, which eventually dominate the population by overgrowth. Cell lines that acquired a factor-independent phenotype were able to form colonies in semisolid medium and form tumors when inoculated into syngeneic mice. These results suggest that Abelson murine leukemia virus is sufficient to initiate transformation in the infected cell but that an additional genetic alteration is needed to confer tumorigenicity.

Quantitative estimation of epidermal growth factor receptor and c-erbB-2 in human breast cancer.

Epidermal growth factor receptor (EGFR) expression by human breast cancer has been shown to predict poor patient outcome, as has amplification of the c-erbB-2 proto-oncogene. We have developed a quantitative immunohistochemical method for measuring protein levels of both receptors and have applied this to a series of 123 breast primaries. We find EGFR expression is substantially lower than normal in nearly all breast cancers (97%). Quantification of p185erbB-2 indicates overexpression in 91% of the tumors. Two separate tumor populations are apparent with levels of c-erbB-2 expression ranging from 0.33 to 19 and 45 to 480 times normal, respectively. Within the lower population, p185erbB-2 expression is inversely related to EGFR expression (rank correlation, P < 0.0005). Using fluorescent in situ hybridization we show that tumors in the latter population have c-erbB-2 amplification and that amplification is restricted to this group. Our findings indicate that significant overexpression of p185erbB-2 occurs in the absence of amplification; these lower levels of expression may have functional significance. Fifty-three patients underwent in vivo bromodeoxyuridine labeling, allowing flow cytometric analysis of tumor cell cycle kinetics. EGFR expression correlates directly to the labeling index (P = 0.011) and indirectly to potential doubling time (P = 0.010), but not to the duration of the S-phase (P = 0.502). Conversely, p185erbB-2 expression does not relate to indices of proliferation. Our results have important implications for the use of both receptor types as therapeutic targets.

Isolation and characterisation of a uniquely regulated threonine, tyrosine phosphatase (TYP 1) which inactivates ERK2 and p54jnk.

The recent discovery of the vaccinia virus protein phosphatase VH1, and its mammalian counterparts has highlighted a novel subfamily of protein tyrosine phosphatases that exhibit dual specificity toward phosphotyrosine- and phosphoserine/threonine-residues. We have identified further members of this subfamily. The characterisation of one clone in particular, which we have named threonine-tyrosine phosphatase 1 (TYP 1), encodes a protein homologous to CL100, but differs dramatically in its regulation. TYP 1 is not expressed in human fibroblasts unlike other CL100-like genes. Furthermore, northern analysis has demonstrated that following mitogenic stimulation of squamous cells, induction of TYP 1 mRNA reaches its maximal levels after four hours, in contrast to the immediate early CL100-like genes. Both TYP 1 and CL100 mRNAs are induced upon TGF-beta treatment of squamous cell lines sensitive to the growth factors antiproliferative effects. When TYP 1 is transfected into COS-1 cells, the gene product inhibits both ERK2 and p54 MAP kinase subfamilies. In addition, we show that purified TYP 1 protein efficiently inactivates recombinant ERK2 in vitro by the concomitant dephosphorylation of both its phosphothreonine and -tyrosine residues. TYP 1 encodes a nuclear protein, which when expressed in COS cells is stabilised by EGF treatment.

Fos-transformation activates genes associated with invasion.

Fos oncoproteins transform cells by functioning as transcription factors. Over-expression of c-fos results in minimal morphological transformation while the two viral isolates, FBJ and FBR v-fos, result in full morphological transformation. Fos-transformed cells are serum dependent for proliferation but not for morphological transformation. To identify Fos target genes which might be involved in morphological transformation we screened a cDNA library constructed from RNA isolated from serum starved FBR-transformed cells with cDNA probes prepared from both FBR-transformed cells and untransformed parental fibroblasts, 208F. We identified 10 genes which are differentially expressed between FBR and 208F cells. One is a novel gene. Nine are upregulated in c-fos- and FBJ-transformed cells and also in mutant c-Ha-Ras-transformed 208Fs. All nine of the upregulated genes have been associated previously with invasion or metastasis. We demonstrate that the FBR-transformed cells are invasive in an in vitro assay and that their ability to invade is enhanced by platelet derived growth factor. We conclude that the fos oncogenes target genes involved in morphological transformation, and invasion.

A role for epidermal growth factor receptor, c-Src and focal adhesion kinase in an in vitro model for the progression of colon cancer.

We have examined the function of the epidermal growth factor (EGF) receptor, c-Src and focal adhesion kinase (FAK) in the progression of colon cancer using an in vitro progression model. A non-tumorigenic cell line was derived from a premalignant colonic adenoma (PC/AA) from which a clonogenic variant was established (AA/C1). Following sequential treatment with sodium butyrate and the carcinogen N-methyl-N'-nitro-N-nitro-soguanidine an anchorage-independent line was isolated which, with time in culture, became tumorigenic when injected into athymic nude mice (AA/C1/SB10). We have shown that both EGF receptor and FAK protein levels were elevated in the carcinoma cells as compared to the adenoma cells, while the expression and activity of c-Src were unaltered during the adenoma to carcinoma transition. EGF induced the movement of the carcinoma cells into a reconstituted basement membrane which was not seen with the premalignant adenoma cells. This increased motility was accompanied by an EGF-induced increase in c-Src kinase activity, relocalisation of c-Src to the cell periphery and phosphorylation of FAK in the carcinoma cells but not in the adenoma cells. This suggests that c-Src plays a role in the biological behaviour of colonic carcinoma cells induced by migratory factors such as EGF, perhaps acting in conjunction with FAK to regulate focal adhesion turnover and tumour cell motility. Furthermore, although c-Src has been implicated in colonic tumour progression, we demonstrate here that in the adenoma to carcinoma in vitro model c-Src is not the driving force for this progression but co-operates with other molecules in carcinoma development.

Isolation of novel, transcriptionally active AP-1 binding sites: implications for cellular transformation.

Increased AP-1 DNA-binding activity, in the context of TRE-binding, is not a consequence of Fos transformation. In this report we investigate the possibility of a change in binding site preference by vFosAP-1 compared with AP-1 from an untransformed cell. Fos binding sites were immunoselected from random sequence oligonucleotides using a pan Fos anti-serum with nuclear protein from quiescent FBRp75v-fos-transformed (FBR) and normal (208F) rat fibroblasts. The selected oligonucleotides were aligned by computer and a consensus described for the sequences bound by AP-1 from the two cell lines. The vFos binding site is shown to be a consensus TRE, whereas the sequence ACCACATC is described as the cellular Fos protein family consensus. We demonstrate that sequences differing from the TRE consensus can bind AP-1 and direct transcription. AP-1 DNA-binding activity differs between normal and transformed cells with several of the selected oligonucleotides. These sequences also demonstrate differential transcriptional activation between normal and transformed cells. In particular, the 208F consensus has no transcriptional activity in FBR cells. Further, EGF differentially influences the transcriptional activity of the oligonucleotides in 208F and FBR cells. Our results suggest that AP-1 may change its preferred binding site depending on the proteins available at any given time, the sequences flanking a non-consensus TRE or even the environment in which the cell exists. These differences in binding site preference and transcriptional activation may result in the increased transforming ability of the v-fos oncogene compared with the c-fos proto-oncogene and may extend the potential target genes beyond those with an AP-1 consensus binding site.

Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells.

We have previously shown that the transcription factor AP-1 regulates the expression of genes which allow neoplastically transformed rat fibroblasts to become invasive. Searches for further AP-1 target genes led to the identification of a gene encoding a novel rat kelch family member, named kelch related protein 1 (Krp1). Kelch family members are characterized by a series of repeats at their carboxyl terminus and a BTB/POZ domain near their amino terminus. Rat Krp1 has a primarily cytoplasmic localization, and a small fraction appears to accumulate and co-localize with F-actin at membrane ruffle-like structures in the tips of pseudopodia. Overexpression of Krp1 in transformed rat fibroblasts led to the formation of dramatically elongated pseudopodia, while expression of truncated Krp1 polypeptides resulted in a reduction in the length of pseudopodia. We propose that the transformation-specific expression of Krp1 is required for pseudopod elongation, which are structures that are required for cell motility and invasion.

Regulation of a multigenic invasion programme by the transcription factor, AP-1: re-expression of a down-regulated gene, TSC-36, inhibits invasion.

The transcription factor AP-1 (activator protein-1) is required for transformation by many oncogenes, which function upstream of it in the growth factor-ras signal transduction pathway. Previously, we proposed that one role of AP-1 in transformation is to regulate the expression of a multigenic invasion programme. As a test of this proposal we sought to identify AP-1 regulated genes based upon their differential expression in 208F rat fibroblasts transformed by FBR-v-fos (FBR), and to determine if they functioned in the invasion programme. Subtracted cDNA libraries specific for up- or down-regulated genes in FBRs compared to 208Fs were constructed and analysed. Northern analysis revealed that the cDNAs in both libraries represented differentially expressed genes. Nucleic acid sequence analysis of randomly selected cDNA clones from each library coupled with searches of nucleic acid and amino acid sequence databases determined that many of the cDNAs represented proteins that function in various aspects of the invasion process. Functional analysis of one the down-regulated genes, TSC-36/follistatin-related protein (TSC-36/Frp), which has not previously been associated with invasion, demonstrated that its expression in FBRs inhibited in vitro invasion. These results support the proposal that AP-1 in transformed cells regulates a multigenic invasion programme.

Transcriptional regulation of cell invasion: AP-1 regulation of a multigenic invasion programme.

The focus of this review will be on the regulation of the multigenic invasion programme by activator protein-1 (AP-1). Investigation of AP-1-regulated gene expression in transformed cells can be used to identify the genes in the multigenic invasion programme and to validate them as targets for diagnosis or therapy.

Localization of EGF receptors in frozen tissue sections by antibody and biotinylated EGF-based techniques.

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EGF-based technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF was detected. The remaining eight tumors were negative by both techniques. The discordant cases may reflect a lower level of sensitivity of the ligand-binding technique or, alternatively, abnormal receptors may have been expressed in these tissues. EGF receptors could be detected in rat liver with biotinylated EGF but not with the antibody, indicating the usefulness of the ligand-based technique in cross-species studies.

Quantitative radioimmunohistochemical measurements of p185(erbB-2) in frozen tissue sections.

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.

Anti-immunoglobulin and anti-CD40 stimulation induces CD25 expression by resting human tonsillar B lymphocytes.

In this report, the effect of ligation of a number of B-cell surface molecules upon expression of CD25, the 55-kDa inducible component of the IL-2 receptor complex found on T and B lymphocytes, is reported. IL-4 is the only cytokine apparently capable of promoting CD25 expression in human high-density quiescent tonsillar B cells; neither IL-10 nor IL-13 could induce CD25 expression. Cross-linking of the antigen receptors or CD40 with antibody elicited CD25 expression in a dose-dependent manner. Stimulation with anti-CD40 promoted CD25 expression in approximately 25% of B cells, while anti-Ig caused 80% or more of cells to become CD25+. In experiments where the stimuli were used in combination, some additive effects upon CD25 expression were noted, but no obvious synergistic effects could be detected.