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Expression of the mucin MUC2, the structural component of the colonic mucus layer, is lowered in ulcerative colitis. Furthermore, interleukin (IL)-10 knockout (IL-10-/-) mice develop colitis and have reduced Muc2 levels. Our aim was to obtain insight into the role of Muc2 and IL-10 in epithelial protection. Muc2-IL-10 double-knockout (Muc2/IL-10(DKO)) mice were characterized and compared to Muc2 knockout (Muc2-/-), IL-10-/- and wild-type (WT) mice. Clinical symptoms, intestinal morphology and differences in epithelial-specific protein levels were analyzed. In addition, levels of the pro-inflammatory cytokines in colonic tissue and serum were determined. IL-10-/- mice were indistinguishable from WT mice throughout this experiment and showed no clinical or histological signs of colitis. Muc2/IL-10(DKO) and Muc2-/- mice showed significant growth retardation and clinical signs of colitis at 4 and 5 weeks, respectively. Muc2/IL-10(DKO) mice had a high mortality rate (50% survival/5 weeks) compared to the other types of mice (100% survival). Microscopic analysis of the colon of Muc2/IL-10(DKO) mice showed mucosal thickening, increased proliferation, superficial erosions and a diminished Muc4 expression. Furthermore, pro-inflammatory cytokines were significantly upregulated, both in tissue (mRNA) and systemically in Muc2/IL-10(DKO) mice. In conclusion, Muc2/IL-10(DKO) mice develop colitis, which is more severe in every aspect compared to Muc2-/- and IL-10-/- mice. These data indicate that (i) in case of Muc2 deficiency, the anti-inflammatory cytokine IL-10 can control epithelial damage, though to a limited extent and (ii) the mucus layer is most likely a key factor determining colitis.

Her2 is overexpressed in 20% to 30% of breast tumors and correlates with reduced disease-free and overall patient survival. Trastuzumab, a humanized monoclonal antibody directed against Her2, represents the first Her2-targeted therapy, which decreases the risk of relapse and prolongs patient survival. Resistance to trastuzumab, both inherent and treatment-acquired, represents a significant barrier to the effective treatment of Her2 (+) breast cancer. The Met receptor tyrosine kinase is aberrantly expressed in breast cancer and predicts poor patient prognosis. In this study, we find that Met is frequently expressed in Her2-overexpressing breast cancer cells, as well as Her2 (+) breast cancer. Importantly, Met contributes to trastuzumab resistance, as inhibition of Met sensitizes cells to trastuzumab-mediated growth inhibition, whereas Met activation protects cells against trastuzumab by abrogating p27 induction. Remarkably, Her2-overexpressing breast cancer cells rapidly up-regulate Met expression after trastuzumab treatment, promoting their own resistance. Our study suggests that a subset of Her2 (+) patients may benefit from combined inhibition of Her2 and Met. [Cancer Res 2008;68(5):1471–7]

Muc4 interacts with erbB2 and potentiates tumourigenesis and/or tumour growth. The expression of MUC4, the interaction of MUC4 with erbB2 and the status of erbB2 signalling in human gallbladder carcinomas were determined in order to gain a better understanding of the pathobiology. The expression levels of MUC4 protein and mRNA were increased in specimens of gallbladder carcinoma. Immunoprecipitation experiments showed an interaction between MUC4 and erbB2. This interaction was associated with the hyperphosphorylation of erbB2, MAPK and Akt and with the overexpression of cyclooxygenase-2. MUC4 was detected on the apical surface of cancerous epithelia and partially co-localised there with erbB2. Transfection experiments showed that MUC4 amplifies cell proliferation in the presence of heregulin through potentiating phosphorylation of erbB2 and its downstream signalling pathways. These findings suggest that MUC4 is up-regulated and interacts with erbB2 in human gallbladder carcinoma, and thereby support the potential implication of MUC4 in erbB2 activation.

Signaling through the EGFR or ErbB family of receptor tyrosine kinases must be precisely regulated to ensure the fidelity of tissue development and homeostasis, yet prevent tumor initiation and progression. The efficiency of receptor signaling in cells is tempered by a series of negative regulatory mechanisms that act directly on receptors to suppress their response to growth factor ligand. The past ten years have witnessed significant progress in the discovery of these pathways and the characterization of the mechanisms by which their loss in tumors might contribute to malignancy. These mechanisms include pathways that lead to receptor degradation, both in the absence and presence of activating ligand. The central components of such pathways are often E3 ubiquitin ligases, such as cbl or Nrdp1. Other mechanisms suppress the ability of receptors to respond to growth factor stimulation. Splice variants of ErbB receptor extracellular domains, proteins that contain leucine-rich repeats in their extracellular domains, and intracellular suppressor proteins such as RALT fall into this category. Here we review ErbB negative regulatory mechanisms, emphasizing what is known about the loss of these pathways in tumors, and highlighting the notion that pathway augmentation or restoration to tumor cells could ultimately be of therapeutic benefit to cancer patients.

As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.

Introduction: Head and neck squamous cell carcinoma (HNSCC) is a devastating and deadly disease, largely because it is diagnosed in late stage. Cure rates, currently at 50%, could increase to >80% with early detection. In this study, we evaluate soluble CD44 (solCD44) as an early detection tool for HNSCC by determining whether it reliably distinguishes HNSCC from benign disease of the upper aerodigestive tract. Methods: We carried out the solCD44 ELISA on oral rinses from 102 patients with HNSCC and 69 control patients with benign diseases of upper aerodigestive tract to determine the sensitivity and specificity of the test for differentiating HNSCC from benign disease. Furthermore, we did a pilot study using methylation-specific PCR primers on oral rinses from 11 HNSCC patients with low solCD44 levels and 10 benign disease controls. Results: Mean salivary solCD44 levels were 24.4 ± 32.0 ng/mL for HNSCC patients (range, 0.99-201 ng/mL) and 9.9 ± 16.1 ng/mL (range, 0.73-124 ng/mL) for the patients with benign disease (P < 0.0001). Depending on cutoff point and HNSCC site, sensitivity ranged from 62% to 70% and specificity ranged from 75% to 88%. Nine of 11 HNSCC and 0 of 10 controls with low solCD44 levels showed hypermethylation of the CD44 promoter. Conclusions: SolCD44 is elevated in the majority of HNSCC and distinguishes cancer from benign disease with high specificity. Whereas the solCD44 test lacks sensitivity by itself, methylation status of the CD44 gene seems to complement the solCD44 test. Our pilot data indicate that, together, these markers will detect HNSCC with very high sensitivity and specificity. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1348–55)

Cell behaviors are regulated by signaling pathways triggered by the activation of cell surface receptors. A key aspect of receptor signaling is the location of these receptors relative to their ligands and to other receptors, particularly in epithelia, whose cells are polarized by tight junction barriers into apical and basolateral membrane regions. In polarized epithelia, the co-receptor ErbB2 is often localized to the apical surface by its intramembrane ligand Muc4, thus segregating it from its partner ErbB3, which is sequestered at the lateral surface, co-localized with cadherin junctions. The ErbB2-ErbB3 receptor heterodimer, when activated, is a potent stimulator of cell proliferation; thus, the segregation mechanism helps maintain these cells in a differentiated state. Similarly, epidermal growth factor, the ligand for ErbB1, which is present in the apical fluid of some epithelia, is segregated from its receptor by the tight junction barrier. Loss of cell polarity and the tight junction barrier facilitates the interaction of ErbB2 with the hemidesmosome integrin α 6 β 4 . This integrin acts as a docking site for signaling pathways to promote cell proliferation and further disruption of cell junctions. The ultimate dissolution of tight junctions may result from activation of transforming growth factor–β receptors, one subunit of which is directly associated with the junction. This activation triggers degradation of critical tight junction components. These sequestration and segregation phenomena provide a model by which overexpression of the ErbB2 receptor kinase may trigger oncogenesis by initiating junction breakdown. Equally important, these mechanisms may act as a sensor for epithelial damage that can activate repair mechanisms.

Mucins are large, heavily O-glycosylated proteins expressed by epithelial tissues. The canonical function of membrane mucins is to provide protection to vulnerable epithelia by forming a steric barrier against assault, and by contributing to the formation of protective extracellular mucin gels. The aberrant overexpression of mucins is thought to contribute to tumor progression by allowing tumor cells to evade immune recognition, and by aiding in the breakdown of cell-cell and cell-matrix contacts to facilitate migration and metastasis. Recent evidence suggests that we should now modify our thinking about mucin function by considering their roles in signaling pathways leading to cellular growth control. Here we review the markedly divergent mechanisms by which membrane mucins, specifically MUC1 and MUC4, influence pathways contributing to cellular proliferation and survival. The cytoplasmic domain of MUC1 serves as a scaffold for the assembly of a variety of signaling proteins, while MUC4 influences the trafficking and localization of growth factor receptors, and hence their responses to external stimuli. We also discuss how tumor cells exploit these mechanisms to promote their own growth and metastasis.

The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase down-regulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.

Mucins are large, heavily O-glycosylated proteins expressed by epithelial tissues. The canonical function of membrane mucins is to provide protection to vulnerable epithelia by forming a steric barrier against assault, and by contributing to the formation of protective extracellular mucin gels. The aberrant overexpression of mucins is thought to contribute to tumor progression by allowing tumor cells to evade immune recognition, and by aiding in the breakdown of cell-cell and cell-matrix contacts to facilitate migration and metastasis. Recent evidence suggests that we should now modify our thinking about mucin function by considering their roles in signaling pathways leading to cellular growth control. Here we review the markedly divergent mechanisms by which membrane mucins, specifically MUC1 and MUC4, influence pathways contributing to cellular proliferation and survival. The cytoplasmic domain of MUC1 serves as a scaffold for the assembly of a variety of signaling proteins, while MUC4 influences the trafficking and localization of growth factor receptors, and hence their responses to external stimuli. We also discuss how tumor cells exploit these mechanisms to promote their own growth and metastasis.

MUC4 is a heterodimeric membrane mucin, composed of two tightly linked subunits and implicated in the protection of wet-surfaced epithelia. Although human MUC4 and its rat analogue Muc4/sialomucin complex have been extensively studied in the adult human and in the adult and embryonic rat, respectively, there has been little attention paid to date to the human embryo. Based on studies with our monoclonal antibody 1G8 and commercial tissue arrays, we describe some unexpected features of the expression of MUC4 in human embryonic epithelia. In the human small intestine and colon, MUC4 appears at an earlier relative stage of development, compared to gestation time, than in the rat. Interestingly, MUC4 also appears in the embryo in the skin, then disappears late in gestation, consistent with its absence in adult skin. These results are consistent with an important protective role for MUC4 in the human embryo that is different from that in the rat or in the adult human.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease that is only cured 50% of the time. A better understanding of the molecular mechanisms involved in HNSCC progression may lead to earlier detection and improved cure rates. CD44 is a ubiquitous transmembrane glycoprotein comprising a family of alternatively spliced isoforms involved in cell migration and cell proliferation. CD44 isoforms containing the variant 3 (v3) exon include a growth factor binding site and may be involved in tumor progression. To characterize CD44v3-containing isoforms expression in HNSCC we purified RNA from four HNSCC cell lines and performed RT-PCR using junction primer strategies followed by gel elecrophoresis. Cloning and sequencing of HNSCC cell line PCR products revealed two isoforms. One of these, CD44v3-10, has been previously described. The other isoform, CD44v3, has not been characterized in HNSCC tissues. To further study this isoform, we purified RNA from 19 HNSCC tissues, 7 normal margin tissues and 5 true normal tissues. Following reverse-transcription, we performed quantitative PCR using junction primers specific for CD44v3. Results show that HNSCC tumor tissues expressed mean CD44v3 levels that were elevated 4.5 times more than true normal tissues (p0.01). Mean CD44v3 values for HNSCC tumors were 0.43 +/- 0.44 while mean levels for true normal tissues were 0.10 +/- 0.11. Levels in tumor tissue did not vary significantly with tumor characteristics such as site, stage, prior treatment, or nodal status. In addition, to characterize the role of this molecule plays in tumor progression, we overexpressed CD44v3 in a HNSCC cell line. Our results indicate that although higher levels of CD44v3 did not affect the rate of proliferation, a significant increase in migration was observed. CD44v3 may provide a target for future diagnostic and therapeutic interventions for HNSCC.

Mucins provide a protective barrier for epithelial surfaces, and their overexpression in tumors has been implicated in malignancy. We have previously demonstrated that Muc4, a transmembrane mucin that promotes tumor growth and metastasis, physically interacts with the ErbB2 receptor tyrosine kinase and augments receptor tyrosine phosphorylation in response to the neuregulin-1 beta (NRG1 beta) growth factor. In the present study we demonstrate that Muc4 expression in A375 human melanoma cells, as well as MCF7 and T47D human breast cancer cells, enhances NRG1 beta signaling through the phosphatidylinositol 3-kinase pathway. In examining the mechanism underlying Muc4-potetiated ErbB2 signaling, we found that Muc4 expression markedly augments NRG1 beta binding to A375 cells without altering the total quantity of receptors expressed by the cells. Cell-surface protein biotinylation experiments and immunofluorescence studies suggest that Muc4 induces the relocalization of the ErbB2 and ErbB3 receptors from intracellular compartments to the plasma membrane. Moreover, Muc4 interferes with the accumulation of surface receptors within internal compartments following NRG1 beta treatment by suppressing the efficiency of receptor internalization. These observations suggest that transmembrane mucins can modulate receptor tyrosine kinase signaling by influencing receptor localization and trafficking and contribute to our understanding of the mechanisms by which mucins contribute to tumor growth and progression.

Cleavage of Muc4/SMC precursor into two subunits is an essential processing step for maturation and occurs within a GD-PH sequence. Recent evidence indicates that cleavage of the precursor of gel-forming mucin MUC2 within the same tetrapeptide sequence occurs by a non-enzymatic, autocatalytic cleavage at low pH, and in cells in the late secretory pathway. Here we provide evidence that the cleavage step of Muc4/SMC processing occurs by a proteolytic mechanism. First, processing of Muc4/SMC precursor to ASGP-2 was inhibited in the presence of the mechanism-based serine protease inhibitor, Pefabloc SC, under conditions that did not block synthesis of other proteins. This inhibition led to an increased level of the precursor. Second, neutralization of the acidic environment of the late secretory pathway with NH4Cl did not inhibit cleavage of Muc4/SMC precursor. These results indicate that the two mucins can be processed by cleavage at the same peptide site by different mechanisms. J. Cell. Biochem. 97: 1267–1274, 2006. © 2005 Wiley-Liss, Inc.

Mammary function is dependent on its three-dimensional organization, which is established and maintained by cell adhesive junctions linked through the membrane to the cell cytoskeleton. These junctions serve not only as structural elements, but also function as initiators and integrators of cell signals. In this review we discuss three types of glycoproteins whose interactions impinge on the function of mammary cell-cell junctions, cadherins, ErbB receptor tyrosine kinases and membrane mucins, as a microcosm of events regulating mammary cell behaviors. Actions of these components are integrated by the critical signaling element beta-catenin. When functioning properly, these glycoproteins, beta-catenin and associated signaling pathways mesh into a highly structured program for development and function of the gland. However, disruption or dysfunction of these glycoproteins or the signaling elements can lead to disorganization of the epithelia and ultimately to neoplasia.

Objective: Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease which is cured only 50% of the time. If diagnosed early, survival rates could reach 80%, but there is currently no practical method for early detection. CD44 comprises a family of isoforms that, in certain tumors, are alternatively spliced and overexpressed in tissues and circulation. Here we examine salivary soluble CD44 (solCD44) expression in HNSCC patients and normal controls to determine its potential as a screening tool. Method: We did a solCD44 ELISA on saliva from 26 HNSCC patients, 10 normal volunteers, conditioned media (CM) of 4 HNSCC cell lines, and 1 CD44-negative cell line (COS-7). Western blot was done on CM from 2 HNSCC cell lines (UMSS11B and FaDu), COS-7, 3 HNSCC, and 2 normal saliva specimens to verify ELISA antibody specificity. SolCD44 levels were significantly elevated in HNSCC patients compared with normal controls (7.85 ng/mL for HNSCC patients and 1.09 ng/mL for normal controls, P < 0.001). Results: The test detected 79% of mucosally invasive HNSCC using preliminary cutoff points. SolCD44 levels did not vary significantly with tumor size, stage, recurrence, history of radiation treatment, or tobacco and alcohol risk factors. A 65 to 75 kDa band, corresponding to solCD44, was detected in all of the HNSCC cell line CM and saliva whereas normal samples showed a fainter band or were undetectable. Conclusion: In this preliminary analysis, the salivary solCD44 ELISA seems to effectively detect HNSCC at all stages. Further study is indicated because early detection is clearly important in this disease.

Few molecules have been more closely linked to cancerthan the protein-tyrosine kinases. Tyrosine phosphorylationand the tyrosine kinase enzymes, which create phosphoty-rosine residues in their substrate proteins, were discoveredduring studies of the oncogenic factors in tumour viruses.The identification of v-src as the transforming factor of theRous sarcoma virus and the recognition of its activity as aprotein-tyrosine kinase conceptualized the oncogene theoryof tumourigenesis, creating a model, which has dominatedmuch of cancer research over the past two decades. Theimportance of the work on viral oncogenes in the earlyresearchon tyrosine phosphorylation is shown by an account-ing of landmark events in this area (Table 1) (Hunter, 1998).The discovery of a cellular counterpart (c-src) of the viralsrc (v-src) tyrosine kinase indicated a much broader role forthese enzymes and suggested a major role for c-src and tyro-sine phosphorylation in mediating normal cell behaviours.For example, c-src has been implicated in such diverse cellfunctions as platelet aggregation, cell cycle control, andcell motility. The diversity of cellular functions of tyrosinekinases was further indicated by early observations that boththe epidermal growth factor (EGF) receptor, important inepithelial cell growth, and the insulin receptor, a key com-ponent in metabolic regulation, are tyrosine kinases. Anothertyrosine kinase receptor, called

Muc4/sialomucin complex (SMC), a large heterodimeric mucin composed of an extracellular mucin subunit ASGP-1 and a transmembrane subunit ASGP-2, is present at the rat ocular surface localized mainly to the most superficial layers of the epithelia. To investigate corneal homeostasis and the functions of Muc4/SMC at the ocular surface, we developed a corneal epithelial cell culture system from corneal explants, from which migrating cells formed an epithelial sheet resembling the native epithelium with regard to microanatomy, expression of characteristic markers, cell migration, and Muc4/SMC expression. Cells migrating from the explants expressed smooth muscle actin. Proliferation was detected only on the edge of epithelial sheet in the immature epithelium and throughout the sheet in confluent cultures. Microscopy revealed that the epithelial sheet was formed from four to six layers of cells expressing keratin 3 and Muc4/SMC in forms identical to those expressed at ocular surface in vivo. Electron microscopy showed cells in various morphological states in the process of releasing from the surface of the multilayer (desquamating). Surprisingly, few of these cells showed evidence of apoptosis, either by morphological or DNA fragmentation analyses. These results suggest a new model for desquamation from stratified epithelia, in which desquamation and apoptosis are independent and sequential processes. Desquamating cells also exhibit a high level of Muc4/SMC. Since Muc4/SMC has been shown to be a potent anti-adhesive and a repressor of apoptosis, we propose that it plays a role in the non-apoptotic desquamation process.

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.

Objectives/Hypothesis: Expression of the membrane mucin MUC4 has been associated with a variety of malignancies, including squamous cell carcinoma of the upper aerodigestive tract. MUC4 modulates cell signaling pathways as an intramembrane ligand of ErbB2. The hypotheses of the study were that MUC4 expression would correlate with ErbB2 expression and that MUC4 expression would correlate with clinical outcomes in squamous cell carcinoma of the upper aerodigestive tract. Study Design: Retrospective chart review was combined with immunohistochemical analysis of paraffin-embedded tumor specimens from patients treated with initial definitive surgical resection at an academic tertiary care medical center. Methods: MUC4 and ErbB2 receptor expression was localized by immunohistochemical studies using archival formalin-fixed and paraffin-embedded tissue. A limited number of fresh-frozen tissues were further analyzed by Western blot. Clinical outcomes and histopathological parameters were determined by retrospective chart review and correlated with immunohistochemical findings. Results: One hundred fifty-four patients were analyzed with a median follow-up of 12 months among 54 patients who died and 49 months among 100 surviving patients. Membrane expression of MUC4 and ErbB2 was seen in 12% and 13% of tumors, respectively. MUC4 expression was not correlated with pathological grade. A significant correlation was found between MUC4 expression and ErbB2 expression. Multivariate survival analyses revealed that patients whose tumors exhibited MUC4 membrane expression had statistically significant improvement in survival and longer time to recurrence compared with patients whose tumors did not express MUC4 as defined by immunohistochemical staining patterns. No correlations between ErbB2 expression and survival or recurrence were observed. Conclusion: Patients with tumors that retain MUC4 expression exhibit improved survival and decreased recurrence in squamous cell carcinoma of the upper aerodigestive tract. Correlations between MUC4 expression patterns and ErbB2 expression are also observed, suggesting that MUC4-ErbB2 mediated cell signaling pathways may provide insights into this clinical result.

Neuregulins play a critical role in the developing heart, nervous, and mammary systems. Neuregulin-1-induced cardiac, neuronal, and mammary differentiation is based on a cell-cell communication model, where the ligand neuregulin-1 is produced and secreted by one cell type, which does not express its receptors erbB3 and erbB4 and acts on neighboring cell types that do express these receptors. We proposed that neuregulin-1 affects fetal lung maturation through a similar mechanism. Immunostaining showed neuregulin-1 in fetal lung that increased in fibroblasts at the onset of surfactant synthesis. Neuregulin-1 beta was found to be secreted by the fetal lung fibroblast and stimulated type II cell surfactant synthesis. Both fetal lung fibroblast-conditioned media and neuregulin-1 stimulated erbB2 receptor phosphorylation in type II cells. The effects of neuregulin-1 and of fibroblast-conditioned media on both surfactant synthesis and type II cell erbB2 phosphorylation were specifically blocked by antibody to neuregulin-1. Thus, neuregulin-1 beta may control fetal lung maturation through mesenchymal-epithelial interactions in a paracrine mechanism similar to that described for the developing heart, brain, and mammary systems.

The lacrimal gland is an important source of components for the ocular tear fluid. Though mucins are not generally considered a product of the lacrimal gland, our results clearly show Muc4/SMC is produced by the gland in soluble and membrane forms. The secreted, soluble form is likely produced for the soluble phase of the ocular tear film. Analyses of ErbB2 and the Muc4/SMC-ErbB2 complex in the lacrimal gland suggest a second function for Muc4/SMC, a role in cell regulation through ErbB signaling. The nature of those signals and the cell functions they regulate will be subjects for future investigations.

The membrane mucin Muc4, also called sialomucin complex (SMC), is a heterodimeric complex of two subunits, ASGP-1 and ASGP-2, derived from a single gene. It is produced by multiple epithelia in both membrane and soluble forms and serves as a protective agent for the epithelia. The membrane form of Muc4 acts as a steric barrier to the apical cell surface of epithelial or tumor cells. An important example is the uterus of the rat, in which Muc4 expression is downregulated for blastocyst implantation. The soluble form facilitates the protection and lubrication of epithelia by mucous gels composed of gel-forming mucins, as in the airway, where Muc4 is proposed to participate in mucociliary transport as a constituent of the periciliary fluid. The soluble form is also found in body fluids, such as milk, tears, and saliva. The transmembrane subunit ASGP-2 acts as an intramembrane ligand and activator for the receptor tyrosine kinase ErbB2. Formation of this ligand-receptor complex is proposed to repress apopotosis in epithelial and cancer cells in which the ligand-receptor complex is formed, providing a second type of cell protective mechanism. Muc4 expression is regulated in epithelial tissues in a cell- and tissue-specific manner during epithelial differentiation. In stratified epithelia, it is predominantly in the most superficial, differentiated layers, often coincident with ErbB2. Dysregulation of Muc4 expression may contribute to cell and tissue dysfunction, such as the proposed contribution of Muc4 to mammary tumor progression. These observations clearly show that Muc4 has multiple roles in epithelia, which may provide insights into aberrant behaviors of these tissues and their derivative carcinomas.

Activation of the epidermal growth factor (EGF) receptor by its ligand, EGF, rapidly enhances receptor internalization and degradation, which desensitizes receptor signaling. In contrast, we have shown previously that exposure to oxidative stress in the form of hydrogen peroxide (H(2)O(2)) activated the EGF receptor but that the levels of activated receptors did not decline, which resulted in prolonged receptor signaling. This study provides mechanistic insights into these different modes of EGF receptor activation. Here we demonstrate that the pattern of receptor tyrosine phosphorylation induced by H(2)O(2) differs from that induced by its ligand, EGF. Importantly, H(2)O(2) generates a receptor with negligible phosphorylation at tyrosine 1045, the major docking site for the ubiquitin ligase c-Cbl. As a result, H(2)O(2)-activated receptors fail to recruit c-Cbl and do not undergo ubiquitination and endocytosis. In summary, H(2)O(2) stimulation results in an activated receptor uncoupled from normal down-regulation, a process that may contribute to oxidant-mediated tumorigenesis.

Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled–coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.

We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) α-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.

The lacrimal gland is an important source of components for the ocular tear fluid. Though mucins are not generally considered a product of the lacrimal gland, our results clearly show Muc4/SMC is produced by the gland in soluble and membrane forms. The secreted, soluble form is likely produced for the soluble phase of the ocular tear film. Analyses of ErbB2 and the Muc4/SMC-ErbB2 complex in the lacrimal gland suggest a second function for Muc4/SMC, a role in cell regulation through ErbB signaling. The nature of those signals and the cell functions they regulate will be subjects for future investigations.

The membrane mucin Muc4, also called sialomucin complex (SMC), is a heterodimeric complex of two subunits, ASGP-1 and ASGP-2, derived from a single gene. It is produced by multiple epithelia in both membrane and soluble forms and serves as a protective agent for the epithelia. The membrane form of Muc4 acts as a steric barrier to the apical cell surface of epithelial or tumor cells. An important example is the uterus of the rat, in which Muc4 expression is downregulated for blastocyst implantation. The soluble form facilitates the protection and lubrication of epithelia by mucous gels composed of gel-forming mucins, as in the airway, where Muc4 is proposed to participate in mucociliary transport as a constituent of the periciliary fluid. The soluble form is also found in body fluids, such as milk, tears, and saliva. The transmembrane subunit ASGP-2 acts as an intramembrane ligand and activator for the receptor tyrosine kinase ErbB2. Formation of this ligand-receptor complex is proposed to repress apopotosis in epithelial and cancer cells in which the ligand-receptor complex is formed, providing a second type of cell protective mechanism. Muc4 expression is regulated in epithelial tissues in a cell- and tissue-specific manner during epithelial differentiation. In stratified epithelia, it is predominantly in the most superficial, differentiated layers, often coincident with ErbB2. Dysregulation of Muc4 expression may contribute to cell and tissue dysfunction, such as the proposed contribution of Muc4 to mammary tumor progression. These observations clearly show that Muc4 has multiple roles in epithelia, which may provide insights into aberrant behaviors of these tissues and their derivative carcinomas.

According to a 2012 study by the U.S. Center for Disease Control and Prevention (CDC), African-American women had a 41% higher rate of breast cancer mortality during 2005-2009 than did Caucasian women. Although overall cancer mortality rates have declined significantly in the U.S. since the early 1990s, the black-white mortality gap has been steadily growing. The underlying causes of this racial disparity are likely diverse, and include factors such as lower quality of care and reduced access to mammographic screening. However, more aggressive tumor biology may also contribute significantly to racial disparity in breast cancer outcomes. Recently, a low frequency single nucleotide polymorphism (SNP) of the HER2 receptor tyrosine kinase, W452C, which occurs predominantly in African-American women (about 10% frequency), was significantly correlated with breast cancer. This study reported that tumors from patients harboring W452C do not amplify the erbB2 (HER2) gene or overexpress the protein, suggesting that this variant may contribute to breast cancer development through a mechanism distinct from the amplification common to HER2-positive breast cancer patients. Understanding the structural and functional differences associated with W452C will allow us to identify new strategies for customized breast cancer treatments for African American women. To address the role of the W452C SNP in breast cancer we applied a cell-free in vitro reconstitution system that uses nanolipoprotein particles (NLPs) to solubilize and support functional membrane proteins. Cell-free produced wild type HER2 and W452C were tested for tyrosine phosphorylation as well as specific binding to therapeutic anti-HER2 monoclonal antibodies trastuzumab and pertuzumab. Our results showed comparable tyrosine phosphorylation levels for both wild type and W452C HER2, suggesting that the variant itself might not alone be a driver of cancer. We observed a decreased binding affinity of trastuzumab for W452C compared to wild type HER2, suggesting that W452-positive patients might not respond to trastuzumab treatment. On the other hand, we found that W452C has a higher affinity for pertuzumab. Overall, our studies suggest that the W452C variant may be differentially sensitive to clinically pertinent therapeutic agents. Further characterization of this variant will be important for the development of more effective and precise therapeutic interventions. This in turn could lead to targeted measures that help address racial disparity problems in breast cancer mortality. Citation Format: Wei He, Matthew Saldana, Tiffany Scharadin, Steven Hoang-Phou, Denise Trans, Dennis Chang, Kermit Carraway, Paul Henderson, Matthew A. Coleman. In vitro-based biochemical studies of erbb2 gene variation to address racial disparities in breast cancer mortality. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3333. doi:10.1158/1538-7445.AM2014-3333

In prostate cancer, the androgen receptor (AR) is a major regulator of gene transcription. We previously demonstrated that the E3 ubiquitin ligase Nrdp1 is transcriptionally regulated by the AR [Chen, L., Cancer Res, 2010]. The effect of the AR on ErbB3 is mediated by Nrdp1, which regulates the degradation of ErbB3. Therefore, we investigated the function of the various domains of Nrdp1 on ErbB3 in prostate cancer. ErbB3 is a major factor in cell growth and proliferation so finding ways to regulate it would be beneficial for patient survival. We found that the Nrdp1 protein had two splice variants, a full length 36kDa protein and a C-terminal 28kDa protein. In addition, the full-length protein remained in the cytoplasm of LNCaP cells, which are androgen dependent, while the 28kDa protein went into the nucleus of C4-2 cells, an androgen independent subline of LNCaP cells. To further investigate the subcellular difference and role of these two splice variants on ErbB3, we used 4 flag tagged vectors that contain either full-length Nrdp1 (Nrdp1), an N-terminal Nrdp1 containing amino acids 1-169 (Zn), a C-terminal Nrdp1 containing amino acids 169-317 (32) , and an Nrdp1 lacking the coiled coil domain (ΔCC). We found that the ΔCC vector caused an increase of cell proliferation in LNCaP cells, an androgen dependent cell line, with no to little change in cell proliferation in its androgen independent sub cell lines, LNCaP R273H (LNCaP stably transfected with a mutated p53), C4-2, and C4-2B, and PC3 and PC3 WT-AR cells. Despite this, we found that the ΔCC protein caused a decrease of ErbB3 protein in all cell lines. We also performed subcellular fractionation on these cell lines transfected with the Nrdp1 flag vectors. All of the Nrdp1 proteins were found in the cytoplasm of the cell lines. In addition, the ΔCC protein was also found in the nuclear fraction of LNCaP R273H, C4-2, and C4-2B, while in LNCaP cells the Zn protein was also found in the nuclear fraction and in PC3 cells the 32 protein was also found in the nuclear fraction. In LNCaP cells, ErbB3 was found in the nucleus when transfected with Nrdp1 protein, Zn protein, and 32 protein, but found in the cytoplasm when transfected with the ΔCC protein. However, in LNCaP R273H, C4-2, and C4-2B cell lines, ErbB3 was found in the nucleus when transfected with all of the Nrdp1 vectors. Based on available data, we concluded that the coiled coil domain is important for ErbB3 protein regulation, but it had opposing effects in castration sensitive and resistant cells. In castration sensitive LNCaP cells, the coiled coil domain of Nrdp1 likely suppresses ErbB3-mediated cell proliferation by preventing ErbB3 translocation from the nucleus to the cytoplasm. However, in castration resistant cells, the coiled coil domain prevents the degradation of ErbB3 by Nrdp1. Further studies are required to determine the mechanism of action of the Nrdp1 coiled coil domain in these two phenotypes. Citation Format: Rosalinda M. Savoy, Salma Siddiqui, William H. Fry, Kermit L. Carraway, Paramita Ghosh. Changes in Nrdp1 regulation of ErbB3 in androgen-dependent vs. independent prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4438. doi:10.1158/1538-7445.AM2014-4438

ErbB2 has been implicated in numerous functions, including normal and aberrant development of a variety of tissues. Although no soluble ligand has been identified for ErbB2, we have recently shown that ASGP-2, the transmembrane subunit of the cell surface glycoprotein Muc4 (also called sialomucin complex, SMC), can act as an intramembrane ligand for ErbB2 and modulate its activity. Muc4/SMC is abundantly expressed at the apical surface of most epithelia of the rat female reproductive tract. Since Muc4/SMC can interact with ErbB2 when they are expressed in the same cell and membrane, we investigated whether these two proteins are co-expressed and co-localized in tissues of the female reproductive tract. Using an anti-ErbB2 antibody from Dako, we found moderate staining at the basolateral surface of the oviduct and also around the cell membrane of the most superficial and medial layers of the stratified epithelia of the vagina. In contrast, Neomarkers neu Ab1 antibody intensely stained the apical surface of the epithelium of the oviduct and the medial and basal layers of the stratified epithelia of the vagina, substantially overlapping the distribution of Muc4/SMC. Furthermore, Muc4/SMC and ErbB2 association in different tissues of the female reproductive tract was demonstrated by co-immunoprecipitation analysis. Interestingly, phosphorylated ErbB2 detected by anti-phospho-ErbB2 is primarily present at the apical surface of the oviduct. Thus, our results show that differentially localized forms of ErbB2 are recognized by different antibodies and raise interesting questions about the nature of the different forms of ErbB2, the mechanism for differential localization, and possible functions of ErbB2 in the female reproductive tract. They also raise a cautionary note about the use of different ErbB2 antibodies for expression and localization studies. © 2001 Wiley-Liss, Inc.

Muc4 is a heterodimeric glycoprotein complex consisting of a peripheral mucin subunit tightly but noncovalently linked to a transmembrane subunit. Muc4 is overexpressed on a number of human breast tumors. Overexpression of Muc4 has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance and promote metastasis. In addition, Muc4 has been shown to act as a ligand for ErbB2/HER2, the target of the therapeutic antibody Herceptin. Using A375 melanoma and MCF7 breast adenocarcinoma cells stably transfected with tetracycline regulatable Muc4, we have investigated whether its overexpression can repress antibody binding to cell surface-expressed ErbB2.

Sialomucin complex (SMC) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 and a transmembrane subunit ASGP-2, which is highly overexpressed on the surface of ascites 13762 rat mammary adenocarcinoma cells. ASGP-2 appears to be a ligand for the growth factor receptor Erb(beta)-2. In normal rat mammary gland the levels of both SMC and Erb(beta)-2 are sharply increased during pregnancy, In normal cultured mammary epithelial cells (MEC) SMC is post- transcriptionally regulated by Matrigel and TGF(beta). SMC expression in the 13762 mammary adenocarcinoma cells is unaffected by Matrigel or TGF%. In contrast, ErbB2 is maximally expressed when MEC are embedded in Matrigel. Although SMC and Erb(beta)-2 can be found in a complex in whole lactating rat mammary tissue, the two proteins are more readily co- immunoprecipitated from freshly isolated MEC, whose polarity is disrupted as well as in the tumor, which also has its polarity disrupted. Finally, in tumor cell lines that overexpress SMC, antibody binding to Erb(beta)-2 is significantly reduced, while the overall levels of Erb(beta)-2 in the cells are not reduced. Thus, overexpression of SMC and formation of the SMC/Erb(beta)-2 complex may contnbute to increased metastatic potential and decreased responsiveness of some breast cancers to anti-Erb(beta)-2 therapeutic agents.

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

The epidermal growth factor (EGF)-like family of growth factors elicits cellular responses by stimulating the dimerization, autophosphorylation, and tyrosine kinase activities of the ErbB family of receptor tyrosine kinases. Although several different EGF-like ligands are capable of binding to a single ErbB family member, it is generally thought that the biological and biochemical responses of a single receptor dimer to different ligands are indistinguishable. To test whether an ErbB receptor dimer is capable of discriminating among ligands we have examined the effect of four EGF-like growth factors on signaling through the ErbB4 receptor homodimer in CEM/HER4 cells, a transfected human T cell line ectopically expressing ErbB4 in an ErbB-null background. Despite stimulating similar levels of gross receptor tyrosine phosphorylation, the EGF-like growth factors betacellulin, neuregulin-1beta, neuregulin-2beta, and neuregulin-3 exhibited different biological potencies in a cellular growth assay. Moreover, the different ligands induced different patterns of recruitment of intracellular signaling proteins to the activated receptor and induced differential usage of intracellular kinase signaling cascades. Finally, two-dimensional phosphopeptide mapping of ligand-stimulated ErbB4 revealed that the different growth factors induce different patterns of receptor tyrosine phosphorylation. These results indicate that ErbB4 activation by growth factors is not generic and suggest that individual ErbB receptors can discriminate between different EGF-like ligands within the context of a single receptor dimer. More generally, our observations significantly modify our understanding of signaling through receptor tyrosine kinases and point to a number of possible models for ligand-mediated signal diversification.

Overexpression of the membrane mucin MUC4/Sialomucin complex (SMC) has been observed during malignant progression of mammary tumors in both humans and rats, suggesting that deregulation of MUC4/SMC expression might facilitate development of these malignancies. As previously reported, overexpression of SMC results in suppression of both cell adhesion and immune killing of tumor cells. SMC also acts as a ligand for ErbB2/Neu, modulating phosphorylation of the receptor tyrosine kinase in the presence and absence of heregulin. The present studies investigated the effect of Muc4/SMC up-regulation on primary tumor growth using a tetracycline-inducible SMC expression system in a xenotransplanted tumor model. SMC up-regulation provoked rapid growth of transfected A375 melanoma in nude mice. Up-regulation of SMC, however, did not significantly increase proliferation of A375 cells in vitro. Instead, a strong suppression of apoptosis was observed in situ in SMC-overexpressing tumors. These data suggest that Muc4/SMC expression promotes tumor growth in vivo at least in part via suppression of tumor cell apoptosis. Importantly, reduction of apoptosis was also observed in vitro, indicating that anti-apoptotic effect of SMC is independent of tumor-host interactions. These findings strongly suggest that SMC up-regulation alters intracellular signaling to favor cell survival, providing for the first time evidence for the regulation of programmed cell death by a gene of the MUC family.

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

The neuregulins comprise a subfamily of epidermal growth factor (EGF)-like growth factors that elicit diverse cellular responses by activating members of the ErbB family of receptor tyrosine kinases. Although neuregulin-1 and neuregulin-2 are both binding ligands for the ErbB3 and ErbB4 receptors, they exhibit distinct biological activities depending on cellular context. In MDA-MB-468 human mammary tumor cells, neuregulin-2beta (NRG2beta) inhibits cell growth, whereas neuregulin-1beta (NRG1beta) does not. In these cells, NRG2beta appears to preferentially act through the EGF receptor, stimulating receptor tyrosine phosphorylation and the recruitment of phospholipase C-gamma, Cbl, SHP2, and Shc to that receptor. NRG1beta preferentially acts through ErbB3 in these cells by stimulating the tyrosine phosphorylation and recruitment of phosphatidylinositol 3-kinase and Shc to that receptor. In MDA-MB-453 cells, both NRG1beta and NRG2beta stimulate the tyrosine phosphorylation of the ErbB2 and ErbB3 receptors to similar extents, but only NRG1beta potently stimulates morphological changes consistent with their differentiation. The profiles of SH2 domain-containing proteins that are efficiently recruited to activated receptors differ for the two factors. These observations indicate that despite their overlapping receptor specificity, the neuregulins exhibit distinct biological and biochemical properties. Since both of these cell lines express only two of the known ErbB receptors, our results imply that EGF-like ligands might elicit differential signaling within the context of a single receptor heterodimer.

The aberrant activation of the ErbB2 receptor tyrosine kinase activity appears to play an active role in the progression of some mammary tumors to malignancy. It is therefore of value to identity compounds that efficiently and specifically inhibit ErbB2 kinase activation. Using cyclic peptide library technology we have developed a method for screening up to 17 billion related compounds in a single experiment for the ability to bind with high affinity to the ErbB2 receptor extracellular domain. Libraries were designed based on the Loop3 structure of the EGF-like growth factors because preliminary observations indicated that a modified form of this subdomain could act as an antagonist of ligand-stimulated ErbB2 activity. Cyclic peptide libraries were synthesized containing ten to 15 residues where up to eight positions were each 19-fold degenerate in the natural ammo acids (excluding cysteine), and at least five positions were fixed with non-degenerate orienting amino acids. Libraries were then incubated at 100-fold molar excess with purified ErbB2 extracellular domain, so that those peptides that bind to ErbB2 with highest affinity were selected from the mixture. Receptor-bound peptides were then isolated and sequenced by Edman degradation to determine the optimal receptor binding motif provided by that library. While specific binding to the receptor extracellular domain was observed, these did not yield any significant high affinity binding motifs.

Sialomucin complex (SMC) is a large, heterodimeric glycoprotein complex composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits and expressed abundantly on the cell surface of ascites 13762 rat mammary adenocarcinoma cells. We have isolated recombinant cDNAs containing different numbers of ASGP-1 mucin repeats, which can be expressed as protein products with variable lengths. To study the anti-adhesive effect of SMC, these cDNAs were transfected into human cancer cell lines. Using a tetracycline-responsive, inducible expression system, we demonstrated that the overexpression of SMC induces morphology changes, cell detachment, and cell-cell dissociation of transfected A375 human melanoma cells in culture. The transition between the adherent and suspension states of the cells is fully reversible and dependent on the SMC expression level. The anti-adhesion effect of SMC was further analyzed kinetically by measuring the cell adhesion of transfected A375 melanoma and MCF-7 breast cancer cell lines to fibronectin, laminin, and collagen IV, demonstrating that SMC disrupts integrin-mediated cell adhesion to extracellular matrix proteins. The degree of this anti-adhesion effect was dependent on the number of mucin repeats in the SMC molecule as well as the level of cell surface expression.

Most of the studies cited in earlier chapters deal with molecular mechanisms of basic cellular processes of relatively homogeneous cell populations. Much of what is significant in biomedical science is concerned with complex, organized cell mixtures: tissues and organs. Thus, it is important to see how the principles we have previously defined apply to such systems. To address some of the questions involved in more complex systems, we have chosen to briefly discuss two topics, early development and neoplasia. In our view these represent two important aspects of multicellularity: 1) the initial and presumably simplest form of the organization of cells; and 2) the failure or reversal of the normal organization.

Development of multicellular organisms can be viewed as an expression of infor-mation encoded in the nuclear genome to provide instructions for the synthe-sis of cell-and tissue-specific proteins. These proteins assemble into structures that allow the differentiated cell to perform its specialized functions. Specific cellular morphologies are as varied as their functions. The span of morphologies expressed by animal cells is astounding, ranging from the simple, spherical shape of the unperturbed lymphocytes to the spinal cord motor neuron which may extend meters in length. The morphology of differentiated cells is not necessarily static, since many cells are capable of radically altering their morphology according to changing conditions in their environment that require them to carry out different functions at different times. For example, neutrophils that circulate as spherical cells in the blood can, in response to signals produced by local tissue inflammation, adopt an “ameboid” migratory configuration to crawl between the lining cells of blood vessels, and home in on the site of inflammation. There they can reorganize their structure yet again to become phagocytic cells (1). The role and dynamics of morphology are not limited to cells of multicellular organisms. The budding yeast, Saccharomyces cerevisiae, responds to mating pheromone by extending a mating projection and by a rearrangement of its cytoskeleton and secretory apparatus (2). Such examples clearly demonstrate that cellular behavior is dependent on cell morphology, and that morphology is both dynamic and responsive to extracellular signals. These morphological changes are not limited to specialized cell functions. Many cells in the organism retain the ability to undergo cell division, often under the influence of signals from their environment or from other cells. This division involves massive internal morphological rearrangements.

One of the defining characteristics of living organisms and cells is movement. At the cellu-lar level motility can be defined as directed shape changes. As described in chapter 1, cell shape depends on two factors, adhesion and the cytoskeleton. Thus, cells move by a cycle which is initiated with a cytoskeleton-dependent protrusive activity (1). This protrusion defines the direction of movement, sometimes in response to some extracellular force such as an attractant. Directionality is set through formation of adhesions by the protrusive element (Fig. 4.1) (2). Translo cation of the cell then occurs through contractile and detachment phases, and the cycle is set to repeat. One of the important lessons in studying cell motility, illustrated in Figure 4.2, is that there are wide variations in motility among different types of cells. However, the fundamental molecular mechanisms appear similar in most cases, so the differences may reside in the organization and control of those mechanisms.