James B. McCarthy

Neurite extension by peripheral and central nervous system neurons in response to substratum-bound fibronectin and laminin☆

Dissociated neurons from embryonic chick dorsal root and sympathetic ganglia (peripheral neurons) and from spinal cord and retina (central nervous system neurons) were cultured on plastic substrata treated with purified fibronectin and laminin. Both central and peripheral neurons attached to and extended neurites on laminin. In contrast, only peripheral neurons initiated neurites on fibronectin; central neurons cultured under identical conditions aggregated into clusters and did not extend neurites. Neurite length, number of neurites initiated, and extent of neurite branching on fibronectin- and laminin-treated substrata were evaluated and compared with similar measurements of neuronal response to poly-L-lysine-treated plastic. Poly-L-lysine provides an adhesive surface for neurite elongation, but fibronectin and laminin appear to promote more rapid neurite elongation. Our observations suggest that neuronal interaction with these glycoproteins may involve neuron-specific cell surface components. These responses to laminin and fibronectin in vitro may be related to the presence or absence of these glycoproteins in specific extracellular environments during specific developmental stages.

Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. Decreased adhesion to stroma and fibronectin but increased adhesion to the basement membrane components laminin and collagen type IV.

We studied the adhesion of primitive and committed progenitors from chronic myelogenous leukemia (CML) and normal bone marrow to stroma and to several extracellular matrix components. In contrast to benign primitive progenitors from CML or normal bone marrow, Ph1-positive primitive progenitors from CML bone marrow fail to adhere to normal stromal layers and to fibronectin and its proteolytic fragments, but do adhere to collagen type IV, an extracellular matrix component of basement membranes. Similarly, multilineage colony-forming unit (CFU-MIX) progenitors from CML bone marrow do not adhere to fibronectin or its adhesion promoting fragments but adhere to collagen type IV. Unlike committed progenitors from normal bone marrow, CML single-lineage burst-forming units-erythroid and granulocyte/macrophage colony-forming units fail to adhere to fibronectin or its components but do adhere to both collagen type IV and laminin. Evaluation of adhesion receptor expression demonstrates that fibronectin receptors (alpha 4, alpha 5, and beta 1) are equally present on progenitors from normal and CML bone marrow. However, a fraction of CML progenitors express alpha 2 and alpha 6 receptors, associated with laminin and collagens, whereas these receptors are absent from normal progenitors. These observations indicate that the premature release of malignant Ph1-positive progenitors into the circulation may be caused by loss of adhesive interactions with stroma and/or fibronectin and acquisition of adhesive interactions with basement membrane components. Further study of the altered function of cell-surface adhesion receptors characteristic of the malignant clone in CML may lead to a better understanding of the mechanisms underlying both abnormal expansion and abnormal circulation of malignant progenitors in CML.

The role of cell adhesion proteins—laminin and fibronectin—in the movement of malignant and metastatic cells

SummaryMetastasizing tumor cells must traverse diverse extracellular matrices during dissemination. Extracellular matrices consist of two basic types, interstitial stroma and basement membranes. Extracellular matrices are chemically complex structures that interact with cell surfaces by a number of mechanisms. There has been a great deal of effort in recent years to understand the molecular nature of extracellular matrices, especially as it relates to the adhesion of normal and malignant cell types. Adhesive noncollagenous glycoproteins, such as laminin and fibronectin, serve pivotal roles in basement membrane and stromal matrices, respectively. These proteins participate in establishing the architecture of extracellular matrices as well as in attaching to the surface of cells and affecting cellular phenotype. This phenotypic effect ranges from adhesion and motility to growth and differentiation. Changes in adhesive characteristics and motility of cells have long been suspected to play a role in mediating the spread of malignant neoplasms. This article is designed to review extracellular matrix constituents that are currently known that can mediate the adhesion and motility of malignant neoplasms. The adhesion of normal and malignant cells to matrices is a complex process mediated by several distinct mechanisms which are initially manifested by changes in cytoskeletal architecture. The topic of normal and malignant cell adhesion to matrices will also be discussed in this regard, since any explanation of tumor cell migration must account for the complex dynamic interactions of the cell surface with the substratum as well as with the cytoskeleton. Finally, current efforts designed to understant the molecular nature of tumor cell:matrix interactions that contribute to metastatic behavior will also be discussed. The rationale behind these studies is that selective inhibition of specific tumor:extracellular matrix interactions can provide an avenue for therapeutic intervention of metastatic cancer.

Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation.

In long-term bone marrow cultures, stroma-adherent progenitors proliferate significantly less than nonadherent progenitors. Thus, close progenitor-stroma interactions may serve to regulate or restrict rather than promote hematopoietic progenitor proliferation. We hypothesized that signaling through adhesion receptors on hematopoietic cells may contribute to the inhibition of proliferation observed when progenitors are in contact with stroma. We demonstrate that progenitors cultured physically separated from stroma in a transwell proliferate significantly more than progenitors adherent to stroma. Furthermore, proliferation of colony forming cells (CFC) is reduced after specific adhesion to stroma, metabolically inactivated glutaraldehyde-fixed stroma, stromal-extracellular matrix, or the COOH-terminal heparin-binding domain of fibronectin. Nonspecific adhesion to poly-L-lysine fails to inhibit CFC proliferation. That the VLA-4 integrin is one of the receptors that transfers proliferation inhibitory signals was shown using blocking anti-alpha 4 monomeric F(ab) fragments. Furthermore, when synthetic peptides representing specific cell attachment sites within the heparin-binding domain of fibronectin were added to Dexter-type marrow cultures, significantly increased recovery and proliferation of CFC was observed, suggesting that these peptides disrupt adhesion-mediated proliferation inhibitory events. Thus, negative regulation of hematopoiesis may not only depend on the action of growth inhibitory cytokines but also on growth inhibitory signals resulting from direct adhesive interactions between progenitors and marrow stroma.

The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells.

CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a nonintegral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture. Here we describe an autocrine mechanism utilizing cell surface Rhamm-CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines that requires endogenous hyaluronan synthesis and the formation of Rhamm-CD44-ERK1,2 complexes. Motile/invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit more elevated basal activation of ERK1,2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm, and ERK1,2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Combinations of anti-CD44, anti-Rhamm antibodies, and a MEK1 inhibitor (PD098059) had less-than-additive blocking effects, suggesting the action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent autocrine mechanism to coordinate sustained signaling through ERK1,2, leading to high basal motility of invasive breast cancer cells. Therefore, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, such as cell surface Rhamm.

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α4β1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.

Hyaluronan (HA), a secreted glycosaminoglycan component of extracellular matrices, is critical for cellular proliferation and motility during development. However, elevated circulating and cell-associated levels correlate with various types of cancer, including prostate. We have previously shown that aggressive PC3M-LN4 prostate tumor cells synthesize excessive HA relative to less aggressive cells, and express correspondingly higher levels of the HA biosynthetic enzymes HAS2 and HAS3. Inhibition of these enzymes by stable transfection of PC3M-LN4 cells with anti-sense HAS2 or HAS3 expression constructs diminishes HA synthesis and surface retention. In this report, we used these HA-deficient cell lines to examine the role of HA in tumorigenicity. Subcutaneous injection of SCID mice with hyaluronan synthase (HAS) antisense-transfected cells produced tumors threefold to fourfold smaller than control transfectants. Tumors from HAS antisense transfectants were histologically HA-deficient relative to controls. HA deficiency corresponded to threefold reduced cell numbers per tumor, but comparable numbers of apoptotic and proliferative cells. Percentages of apoptotic cells in cultured transfectants were identical to those of control cells, but antisense inhibition of HA synthesis effected slower growth rate of cells in culture. Quantification of blood vessel density within tumor sections revealed 70 to 80% diminished vascularity of HAS antisense tumors. Collectively, the results suggest HAS overexpression by prostate tumor cells may facilitate their growth and proliferation in a complex environment by enhancing intrinsic cell growth rates and promoting angiogenesis. Furthermore, this is the first report of a role for inhibition of HA synthesis in reducing tumor growth kinetics.

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function.

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.

Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions?

Tumor cells use a wide variety of post-translational mechanisms to modify the functional repertoire of their transcriptome. One emerging but still understudied mechanism involves the export of cytoplasmic proteins that then partner with cell-surface receptors and modify both the surface-display kinetics and signaling properties of these receptors. Recent investigations demonstrate moonlighting roles for the proteins epimorphin, FGF1, FGF2, PLK1 and Ku80, to name a few, during oncogenesis and inflammation. Here, we review the molecular mechanisms of unconventional cytoplasmic-protein export by focusing on the mitotic-spindle/hyaluronan-binding protein RHAMM, which is hyper-expressed in many human tumors. Intracellular RHAMM associates with BRCA1 and BARD1; this association attenuates the mitotic-spindle-promoting activity of RHAMM that might contribute to tumor progression by promoting genomic instability. Extracellular RHAMM-CD44 partnering sustains CD44 surface display and enhances CD44-mediated signaling through ERK1 and ERK2 (ERK1/2); it might also contribute to tumor progression by enhancing and/or activating the latent tumor-promoting properties of CD44. The unconventional export of proteins such as RHAMM is a novel process that modifies the roles of tumor suppressors and promoters, such as BRCA1 and CD44, and might provide new targets for therapeutic intervention.

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.