Combined experimental and theoretical tools that include biochemical and molecular biology approaches, genome, transcriptome, and proteome analyses, and comparative methods, have advanced our understanding of signaling pathways that shape development, homeostasis, and disease.
As multiple lines of evidence reveal, signaling pathways originally thought to be primarily involved in activating transcriptional programs and specifying cell fates during development may assume central roles in disease pathogenesis later in life.
The Sonic hedgehog, a critical embryonic signaling pathway conserved from fruit flies to humans, which is involved in cell differentiation, proliferation, and anterior-posterior patterning during development, provides an illustrative example. Signaling in this pathway decreases, but remains active in the adult, where it is involved in stem cell maintenance, homeostasis, and tissue repair.
But its aberrant or uncontrolled activation was reported in a growing number of human malignant tumors. Small molecules that target this, and other signaling pathways involved in tumorigenesis, emerge as attractive therapeutic options.
“We developed an approach to look at Hedgehog signaling in pediatric neuroblastoma,” says Mike Hubank, Ph.D., senior lecturer in molecular hematology and cancer biology at University College London.
In a collaborative research initiative, investigators from Dr. Hubank’s laboratory and those from the lab of his colleague Jonathan Ham, M.D., compared the genomic profiles of samples from pediatric medulloblastoma patients with those from normal cerebellar tissues by using Affymetrix Exon arrays. They reported that different splicing patterns exist between the two groups and between different molecular subgroups among the medulloblastoma patients.
“By examining exon variants with sequencing-based approaches and microarrays, we can analyze splicing and design new experiments to better understand heterogeneities in cell populations,” says Dr. Hubank.
The analysis of 1,262 genes that were found to have at least one exon differentially included between samples from the two groups revealed 174 alternative splicing events, and 11 exons were confirmed by RT-PCR to be differentially spliced.
Similar Splicing Patterns
In addition, the investigators found, in a subgroup of medulloblastoma patients, splicing patterns that were similar to the ones encountered in undifferentiated cerebellar cells, indicating that a disruption in alternative splicing, and the lack of differentiation of these cells, could provide the link between defective or aberrant signaling and the activation of oncogenic pathways.
“It would have been impossible to perform this type of analysis five or six years ago, and the technologies have really helped pull apart the system in such intricate detail,” reports Dr. Hubank.
An important cell-cell communication system is established when ephrin (Eph) receptors, the largest subfamily of receptor tyrosine kinases, interact with their ligands, known as ephrins (Eph receptor interacting proteins). Six GPI membrane-anchored ephrin-A ligands (A1-A6) that preferentially bind ephrin-A receptors (A1-A10), and three transmembrane ephrin-B ligands (B1-B3) that preferentially bind ephrin-B receptors (B1-B6) were described, although some crosstalk is possible.
Ephrin receptors and ligands are expressed on almost all embryonic cell types, and distinct expression patterns are established in adult tissues. The bidirectional signal transduction events that they mediate shape intercellular communication during key events from development, tissue homeostasis, and disease. Ephrin ligand-receptor engagement initiates signal transduction, which is referred to as “forward signaling” when it occurs in the cell bearing the receptor, or “reverse signaling” when it occurs in the cell harboring the ligand.
“We are interested in understanding how ephrin B-type ligands send cellular signals,” says Ira O. Daar, Ph.D. senior investigator and head of the developmental signal transduction section of the laboratory of cell and developmental signaling at the National Cancer Institute. Investigators in Dr. Daar’s lab use Xenopus laevis, a manipulable organism for which a detailed fate map exists for the 32-cell stage, providing an ideal system to dissect cellular and molecular events during development.
Dr. Daar and colleagues revealed that ephrin B1 interacts with a protein called Dishevelled. “We found that, as a result of this interaction, ephrin B1 activates a non-canonical Wnt signaling pathway that is also known as the planar cell polarity pathway and allows retinal progenitor cells to move into the developing eye field,” he explains.
In addition to shedding light on the involvement of ephrin B1 in cell migration, investigators in Dr. Daar’s lab recently unveiled another role of this protein, related to the establishment of cell and tissue boundaries.
“We have shown that ephrin B1 regulates tight junctions by its ability to bind Par6 and prevent Cdc42 from binding,” says Dr. Daar.
Par6, a scaffolding protein of the Par polarity complex that is involved in organizing tight junctions at the apical-lateral border of the polarized epithelial cells, is constitutively bound to the atypical protein kinase C (aPKC). Upon binding the GTP-bound form of Cdc42, it undergoes a conformational change that results in aPKC activation.
Ephrin B1 competes with the active, GTP-bound form of Cdc42 for association with Par6. As a consequence of this competition, aPKC is no longer activated, leading to the disintegration of the tight junctions.
An additional layer of complexity is provided by the presence, in the intracellular domain of ephrin B1, of six conserved tyrosine residues that can be phosphorylated. Phosphorylated ephrin recruits Grb4, a small adaptor protein that binds other cytoskeletal signaling molecules.
Ephrin B1 phosphorylation may occur by multiple mechanisms, and phosphorylated ephrin B1 is impaired in its ability to interact with Par6. As a result, Par6 becomes more available for Cdc42 binding, the power polarity complex can reform, and the aPKC pathway is activated, allowing tight junctions to reform.
One of the clinical implications of these findings is related to the involvement of ephrins in the intestinal epithelium homeostasis and in colorectal cancer progression. Ephrin B signaling blocks the acquisition of malignancy characteristics in colorectal cancer by tumor cell compartmentalization, through an E-cadherin-dependent mechanism, and loss of ephrin B receptor expression is a key step in the transition from adenoma to adenocarcinoma.
Downregulation of the ephrin B1 receptor, which results in lower ephrin B1 phosphorylation levels, could increase its ability to interact with Par6, and result in reduced aPKC signaling and the disruption of tight junctions.
“This is one potential implication that forward and reverse signaling through the ephrin receptor has for understanding tumor metastasis,” explains Dr. Daar.