Véronique Orian-Rousseau

CD44, a therapeutic target for metastasising tumours

Members of the CD44 family of transmembrane glycoproteins, in particular CD44v6 isoforms, were shown to be metastatic determinants of rat pancreatic tumour cells back in the early 1990s. Furthermore, the expression of several CD44 proteins correlates with aggressive stages of various human cancers. Because of the frequent and homogeneous expression of CD44v6 isoforms in squamous cell carcinoma, antibodies recognising these proteins were used in clinical trials for patients suffering from head and neck squamous cell carcinoma (HNSCC). Although the phase I clinical trials looked promising, the studies were abruptly ended after the death of a patient. Despite the termination of the trials, CD44 certainly remains a valid target for anti-cancer therapy. In this review, alternative strategies targeting CD44 functions are presented. These functions include the binding to hyaluronan (HA), the collaboration with osteopontin and the contribution of CD44 isoforms to receptor tyrosine kinase (RTKs) activation. These new attempts led to the development of peptides that interfere for example with HA binding and that might be used to induce apoptosis in mammary carcinoma or to prevent homing of leukaemia stem cells. Other peptides block RTK activation and thereby inhibit tumour angiogenesis and metastatic spread.

CD44 Acts Both as a Growth‐ and Invasiveness‐Promoting Molecule and as a Tumor‐Suppressing Cofactor

Abstract: High‐molecular‐weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors‐for example, Burkitts lymphoma, neuroblastomas, and prostate cancer‐loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high‐affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins‐for example, ezrin or moesin‐to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor‐promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so‐called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet‐unknown conditions.

A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis

A specific splice variant of the CD44 cell- surface protein family, CD44v6, has been shown to act as a coreceptor for the receptor tyrosine kinase c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another receptor tyrosine kinase, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm, signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting, and tubule formation induced by hepatocyte growth factor (HGF) or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents, suggesting that the coreceptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathologic conditions.

CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylorithat was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.

Chapter 4 Adhesion Proteins Meet Receptors: A Common Theme?

Receptors tyrosine kinases (RTKs) and cell adhesion molecules (CAMs) present on the cell surface sense the surrounding environment and influence the fate of cells. For a long time, it was believed that these molecules were working independently and that the sole binding of a ligand was enough to activate the RTK. It is now apparent that there is, in fact, a very tight connection between RTKs and CAMs and that they work in concert. The CAMs influence the activation, the signaling, or the internalization of the RTKs. Some CAMs have similar functions and are therefore interchangeable. CD44 isoforms exemplify the flexibility of these interactions as they can collaborate with several RTKs and can also be substituted by other CAMs with similar functions. In several instances, CAMs not only control the activation of the receptor by presenting the ligand but also regulate the downstream signaling by organizing a signalosome complex. Furthermore, the functions of the CAMs can be controlled by the cellular environment and the binding to their ligands.

A five-amino-acid peptide blocks Met- and Ron-dependent cell migration.

Various human cancers express elevated levels of the receptor tyrosine kinases Met or Ron and v6-containing isoforms of CD44. The activation of Met and Ron requires the presence of such CD44 v6-containing isoforms that act as coreceptors. Three amino acids within the v6 sequence were identified by mutational analysis to be essential for the coreceptor function: EWQ in the rat sequence and RWH in human. Peptides comprising these three amino acids (the smallest containing only five amino acids) efficiently act as competitors and block ligand-dependent activation of Met or Ron and subsequent cell migration.

CD44 acts as a signaling platform controlling tumor progression and metastasis

Members of the CD44 family of transmembrane glycoproteins emerge as major signal transduction control units. CD44 isoforms participate in several signaling pathways ranging from growth factor-induced signaling to Wnt-regulated pathways. The role of the CD44 family members in tumor progression and metastasis is most likely linked to the function of the various isoforms as signaling hubs. Increasing evidence suggests that these proteins are not solely cancer stem cell (CSC) markers but are directly involved in tumor and metastasis initiation. It is foreseeable that a link between the expression of CD44 isoforms in CSCs and their function as signaling regulators will be drawn in a near future.

Opposing effects of high- and low-molecular weight hyaluronan on CXCL12-induced CXCR4 signaling depend on CD44

The tumor microenvironment makes a decisive contribution to the development and dissemination of cancer, for example, through extracellular matrix components such as hyaluronan (HA), and through chemokines that regulate tumor cell behavior and angiogenesis. Here we report a molecular link between HA, its receptor CD44 and the chemokine CXCL12 in the regulation of cell motility and angiogenesis. High-molecular-weight HA (hHA) was found to augment CXCL12-induced CXCR4 signaling in both HepG2iso cells and primary human umbilical vein endothelial cells, as evidenced by enhanced ERK phosphorylation and increased cell motility. The augmentation of CXCR4 signaling translated into increased vessel sprouting and angiogenesis in a variety of assays. Small HA oligosaccharides (sHA) efficiently inhibited these effects. Both siRNA-mediated reduction of CD44 expression and antibodies that block the interaction of CD44 with HA provided evidence that CXCL12-induced CXCR4 signaling depends on the binding of hHA to CD44. Consistently, CD44 and CXCR4 were found to physically interact in the presence of CXCL12, an interaction that could be inhibited by sHA. These findings provide novel insights into how microenvironmental components interact with cell surface receptors in multi-component complexes to regulate key aspects of tumor growth and progression.

Haploinsufficiency of c-Met in cd44−/− Mice Identifies a Collaboration of CD44 and c-Met In Vivo

ABSTRACT Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44−/− background, as the cd44−/−; met+/− (and cd44−/−; hgf+/− and cd44−/−; gab1+/−) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.

Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine‐rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface‐exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB′, a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full‐length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.