mAbs to receptor tyrosine kinases such as EGF receptor͞ErbB-1 and HER2͞ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Abreceptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.ErbB ͉ growth factor ͉ oncogene ͉ signal transduction ͉ antibody T he four receptor tyrosine kinases of the ErbB family and their multiple ligand molecules form a layered signaling network, which is implicated in human cancer (reviewed in ref.1): overexpression of ErbB-1͞EGF receptor (EGFR) has been implicated as a feature of poor prognosis in various human malignancies. Moreover, deletion mutants of EGFR exist in brain tumors and point mutations have recently been reported in lung cancer (2). By contrast, ErbB-2͞HER2 is rarely mutated in solid tumors. Instead, the erbB-2 gene is frequently amplified in breast, ovarian, and lung cancer (3). Because of their oncogenic potential and accessibility, ErbB proteins have emerged as attractive targets for pharmaceutical interventions. One major strategy involves the use of mAbs. Early studies uncovered the tumor-inhibitory potential of mAbs directed at ErbB-1 and ErbB-2 (4, 5), and later studies indicated that anti-ErbB mAbs are effective when combined with various chemotherapeutic agents (6, 7). Indeed, the clinical benefit of combining mAbs with certain chemotherapeutic agents was notable, which led to the approval of mAbs to ErbB-2 (Herceptin) and EGFR (C225͞ Cetuximab) for the treatment of breast and colorectal cancer, respectively.Two types of mechanisms have been implicated in ErbBdirected immunotherapy. The first involves mAb-mediated recruitment to tumors of natural killer cells through the Fc-␥ activation receptors of these immune effector cells (8). The second type of mechanisms relates to intrinsic mAb activities, which include blockade of ligand binding or receptor heterodimerization (9), inhibition of downstream signaling to Akt (10), and acceleration of receptor internalization (11,12). The latter mechanism is particularly attractive because ligand-induced endocytosis and degradation of active receptor tyrosine kinases (RTKs) is considered a major physiological process underlying attenuation of growth-promoting signals (13).Several studies reported cooperative effects of mAb combinations (9, 12, 14-16), whereas others found that bivalent, Fc-lacking versions of anti-ErbB mAbs inhibit tumorigenic growth in animals (17, 18). These observations ...

During embryogenesis, paraxial mesoderm cells contribute skeletal muscle progenitors, whereas cardiac progenitors originate in the lateral splanchnic mesoderm (SpM). Here we focus on a subset of the SpM that contributes to the anterior or secondary heart field (AHF/SHF), and lies adjacent to the cranial paraxial mesoderm (CPM), the precursors for the head musculature. Molecular analyses in chick embryos delineated the boundaries between the CPM, undifferentiated SpM progenitors of the AHF/SHF, and differentiating cardiac cells. We then revealed the regionalization of branchial arch mesoderm: CPM cells contribute to the proximal region of the myogenic core, which gives rise to the mandibular adductor muscle. SpM cells contribute to the myogenic cells in the distal region of the branchial arch that later form the intermandibular muscle. Gene expression analyses of these branchiomeric muscles in chick uncovered a distinct molecular signature for both CPM-and SpM-derived muscles. Islet1 (Isl1) is expressed in the SpM/AHF and branchial arch in both chick and mouse embryos. Lineage studies using Isl1-Cre mice revealed the significant contribution of Isl1 + cells to ventral/distal branchiomeric (stylohyoid, mylohyoid and digastric) and laryngeal muscles. By contrast, the Isl1 lineage contributes to mastication muscles (masseter, pterygoid and temporalis) to a lesser extent, with virtually no contribution to intrinsic and extrinsic tongue muscles or extraocular muscles. In addition, in vivo activation of the Wnt/␤-catenin pathway in chick embryos resulted in marked inhibition of Isl1, whereas inhibition of this pathway increased Isl1 expression. Our findings demonstrate, for the first time, the contribution of Isl1 + SpM cells to a subset of branchiomeric skeletal muscles.

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.

In the vertebrate head, mesoderm cells fuse together to form a myofiber,which is attached to specific cranial neural crest (CNC)-derived skeletal elements in a highly coordinated manner. Although it has long been recognized that CNC plays a role in the formation of the head musculature, the precise molecular underpinnings of this process remain elusive. In the present study we explored the nature of the crosstalk between CNC and mesoderm cells during head muscle development, employing three models for genetic perturbations of CNC development in mice, as well as experimental ablation of CNC in chick embryos. We demonstrate that although early myogenesis is CNC-independent, the migration, patterning and differentiation of muscle precursors are regulated by CNC. In the absence of CNC cells, accumulated myoblasts are kept in a proliferative state, presumably because of an increase of Fgf8 in adjacent tissues, which leads to abnormalities in both differentiation and subsequent myofiber organization in the head. These results have uncovered a surprising degree of complexity and multiple distinct roles for CNC in the patterning and differentiation of muscles during craniofacial development. We suggest that CNC cells control craniofacial development by regulating positional interactions with mesoderm-derived muscle progenitors that together shape the cranial musculoskeletal architecture in vertebrate embryos.