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.

SDF-1 and PDGF enhance alphavbeta5-mediated ERK activation and adhesion-independent growth of human pre-B cell lines.

CD23 acts through the alphavbeta5 integrin to promote growth of human pre-B cell lines in an adhesion-independent manner. alphavbeta5 is expressed on normal B-cell precursors in the bone marrow. Soluble CD23 (sCD23), short CD23-derived peptides containing the arg-lys-cys (RKC) motif recognized by alphavbeta5 and anti-alphavbeta5 monoclonal antibodies (MAbs) all sustain growth of pre-B cell lines. The chemokine stromal cell-derived factor-1 (SDF-1) regulates key processes during B-cell development. SDF-1 enhanced the growth-sustaining effect driven by ligation of alphavbeta5 with anti-alphavbeta5 MAb 15F-11, sCD23 or CD23-derived RKC-containing peptides. This effect was restricted to B-cell precursors and was specific to SDF-1. The enhancement in growth was associated with the activation of extracellular signal-regulated kinase (ERK) and both these responses were attenuated by the MEK inhibitor U0126. Finally, platelet-derived growth factor also enhanced both alphavbeta5-mediated cell growth and ERK activation. The data suggest that adhesion-independent growth-promoting signals delivered to B-cell precursors through the alphavbeta5 integrin can be modulated by cross-talk with receptors linked to both G-protein and tyrosine kinase-coupled signalling pathways.

TGF-beta-mediated activation of RhoA signalling is required for efficient (V12)HaRas and (V600E)BRAF transformation.

Transforming growth factor beta-1 (TGF-beta) acts as both a tumour suppressor and a tumour promoter in a context-dependent manner. The tumour-promoting activities of TGF-beta are likely to result from a combination of Smad and non-Smad signalling pathways but remain poorly understood. Here we show that TGF-beta-mediated activation of RhoA is dependent on the kinase activity of ALK5 and that continuous ALK5 activity maintains basal RhoA-ROCK signalling, cell morphology and actin dynamics in serum-starved rodent fibroblasts independently of Smad2, Smad3 and Smad4. In immortalized human diploid fibroblasts, we show that oncogenic rewiring by transduction of (V12)HaRas instigates regulation of RhoA-ROCK signalling through an autocrine TGF-beta1-ALK5 pathway. Furthermore, we show that ALK5-mediated activation of RhoA is required for efficient (V12)HaRas, V-Raf and (V600E)BRAF transformation and (V12)HaRas-mediated anchorage-independent growth. These findings identify a new pro-oncogenic activity of TGF-beta and indicate that tumours harbouring (V12)HaRas and (V600E)BRAF mutations may be susceptible to TGF-beta signalling inhibitors.

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.

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.

Low expression of HER2 protein in breast cancer is biologically significant.

HER2 status is routinely tested using immunohistochemistry or FISH following the licensing of a therapeutic agent targeting HER2. However, neither of these methods provides quantitative information relating to the 70-80% of patients with levels of expression lower than the assay detection thresholds. In this study, radioimmunohistochemistry was used to detect quantitative HER2 protein expression in 178 breast cancers. Survival analysis was performed, as were correlations with known prognostic variables and with overexpression of other HER family members. It is demonstrated that the populations expressing very high and very low levels of HER2 are each associated with increased risk of cancer-specific death on survival analysis (p = 0.0043). The group with low levels of HER2 was more likely to be of higher grade, EGFR-positive and ER/HER3/HER4-negative. HER2-positive cases were frequently ER-negative/HER3-positive, whilst cases with normal HER2 expression were often ER-positive/HER4-positive. The aggressive nature of the tumour group with low HER2 expression may be explained by actions of other HER family members, particularly EGFR, but whether these or other factors have a negative regulatory effect on HER2 expression remains to be determined.

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.

The CD23a and CD23b proximal promoters display different sensitivities to exogenous stimuli in B lymphocytes.

The single human CD23 gene encodes two protein products differing by six or seven amino acids in the extreme N-terminal cytoplasmic domain. The patterns of expression of CD23a and CD23b transcripts differs as a function of cell type and cell stimulation, with expression of CD23a being largely restricted to B cells and CD23b synthesis being inducible in a variety of haematopoietic cells by a range of exogenous stimuli. In this study, short defined sequences of the CD23a and CD23b proximal promoter regions were used to drive expression of exogenous reporter genes in transiently-transfected B cells exposed to a range of cellular stimuli. The CD23a promoter was activated only by IL-4, whereas the CD23b promoter was stimulated not only by IL-4, but also by stimulation with anti-mu, and anti-CD40. Deletion mutant analysis illustrated that of the two putative STAT6 binding sites present in the CD23a proximal promoter, deletion of the first site abrogated IL-4-driven transcriptional activation. Conversely, deletion of both STAT6 binding sites in the CD23b promoter was required before IL-4 sensitivity was lost. When the same CD23b promoter mutants were studied in the context of anti-CD40 and anti-mu stimulation of transfected cells, deletion of the NF-kappaB site abrogated anti-CD40-driven transcriptional activation, but not anti-mu-mediated effects which required additional deletion of putative AP1 sites lying close to the CD23b initiator methionine codon. The data of this report are consistent with the interpretation that the upstream regions of the CD23a and CD23b isoform coding sequences show distinct sensitivities to agents which induce CD23 protein expression at the plasma membrane, and that transcriptional activation by discrete stimuli reflects activation of particular transcriptional regulatory factors.

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.

A functional role for interleukin (IL)-4-driven cyclic amp accumulation in human b lymphocytes.

Interleukin-4 (IL-4) regulates the expression of the 55-kDa alpha-subunit (CD25) of the IL-2 receptor complex in human B lymphocytes. This report suggests that the cAMP/protein kinase A (PKA) component of the IL-4 receptor signalling programme in human tonsillar B cells has a functionally important role in regulating expression of the CD25 gene by attenuating activity of a protein binding to a potent negative regulatory element (NRE) in the CD25 promoter; this effect can be mimicked by agents that elevate cAMP and blocked by inhibitors of PKA but not protein kinase C (PKC). In a B-cell line that fails to elevate cAMP, attenuate NRE-binding protein (NRE-BP) activity or express CD25 following IL-4 treatment, stimulation of cAMP accumulation by forskolin facilitates IL-4-mediated induction of both the endogenous gene and an exogenous reporter gene under the control of a minimal promoter/enhancer fragment of the CD25 gene.

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.

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.

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.

Inhibition of apoptosis in a human pre-B-cell line by CD23 is mediated via a novel receptor.

Human CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B-acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.

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.

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.

Negative regulation of the interleukin (IL)-2 receptor alpha chain (CD25) promoter region in human B lymphocytes.

Studies of the CD25 gene promoter region in T cells have revealed the presence of a 31-bp region, including an 11-bp negative regulatory element (NRE), which profoundly suppresses CD25 expression. This report illustrates that the same region acts as a negative regulator of CD25 expression in human B lymphocytes. Human B cells contain DNA-binding protein activities which bind specifically to an oligonucleotide equivalent to the 11-bp core region of the NRE, and stimulation of tonsillar B cells or Daudi Burkitt's lymphoma B cells with interleukin 4 (IL-4) results in loss of binding activity for oligonucleotides containing the NRE; in contrast, IL-4 enhanced binding activity for the NRE in the Jurkat T cell line. Transient transfection analyses using deletion mutants lacking both the 11-bp core NRE and both the NRE and an adjacent putative retinoic acid response element (RARE) motif illustrated that the NRE element is a functional suppressor of CD25 transcription in B cells. Thus, deletion of the NRE element increased the basal level of CD25 promoter activity and also conferred IL-4 inducibility on reporter gene expression in transiently transfected tonsillar B lymphocytes.

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.

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.