Jean Schneikert

The canonical Wnt signalling pathway and its APC partner in colon cancer development.

The flat surface of the colon is covered by an epithelium composed of four differentiated cell types (enterocytes, enteroendocrine, goblet and Paneth cells) that invaginates at regular intervals to form crypts (fig 1). The bottom of the crypts is occupied by a few stem cells that give rise to actively dividing precursor cells that populate the bottom two-thirds of the crypts.1 Proliferation occurs under the influence of growth factors from the Wnt family that may be produced by the underlying stromal cells underneath the stem cell compartment or by the epithelial cells themselves. The precursors migrate upward in an ordered fashion, which is also controlled by Wnt factors,2 and they stop proliferation when they reach the top third of the crypt, probably because they are too far from the Wnt source. Meanwhile, they continue their migration movement and colonise the surface of the colon. After about a week, epithelial cells undergo apoptosis and are shed in the lumen of the gut. The Paneth cells constitute an exception, as they migrate downward and occupy the very bottom of the crypt. Thus, the epithelium of the colon is under perpetual renewal. Wnt growth factors activate a cascade of intracellular events, which is known as the canonical Wnt pathway that ultimately leads to the expression of a genetic program controlling the co-ordinated expansion, fate and sorting of the epithelial cell population. In colorectal cancer, epithelial cells initially proliferate inappropriately because they acquired mutations in components of the pathway, thereby mimicking the effect of a permanent Wnt stimulation. Thus, the mutated cell recapitulates a progenitor-like phenotype, independent of its position in the epithelium.3 Canonical Wnt signalling has received considerable attention from cancer researchers over the years, because of its essential role in the homeostasis of the colonic epithelium and its deregulation …

RAP46 Is a Negative Regulator of Glucocorticoid Receptor Action and Hormone-induced Apoptosis

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.

AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.

APC is a multifunctional tumor suppressor protein that negatively controls Wnt signaling, but also regulates cell adhesion and migration by interacting with the plasma membrane and the microtubule cytoskeleton. Although the molecular basis for the microtubule association of APC is well understood, molecular mechanisms that underlie its plasma membrane localization have remained elusive. We show here that APC is recruited to the plasma membrane by binding to APC membrane recruitment 1 (AMER1), a novel membrane-associated protein that interacts with the ARM repeat domain of APC. The N-terminus of AMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2]-binding domains, which mediate its localization to the plasma membrane. Overexpression of AMER1 increases APC levels and redirects APC from microtubule ends to the plasma membrane of epithelial cells. Conversely, siRNA-mediated knockdown of AMER1 reduces the overall levels of APC, promotes its association with microtubule ends in cellular protrusions and disturbs intercellular junctions. These data indicate that AMER1 controls the subcellular distribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.

Amer1/WTX couples Wnt‐induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low‐density lipoprotein receptor‐related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β‐catenin signalling, which requires Wnt‐induced formation of phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P2‐binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt‐induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P2. Amer1 translocates to the plasma membrane in a PtdIns(4,5)P2‐dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P2. Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β‐catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

CAG-repeat expansion in androgen receptor in Kennedy's disease is not a loss of function mutation.

Expansion of CAG trinucleotide repeats in androgen receptor gene is present in patients with a rare X-linked inherited form of motor neuron disorder termed Kennedys disease or spinal and bulbar muscular atrophy (SBMA). This is a late onset progressive disease often associated with mild signs of androgen insensitivity. Defects in androgen receptor (AR) action have been linked to the expansion of the CAG trinucleotide repeats and postulated to be the cause of the disease. We have identified a trinucleotide repeat of 45 in the N-terminus of the AR in two brothers with SBMA and several members in their family (range in the general population is 11-35). Treatment of the patients with androgens failed to improve their clinical symptoms and provided no hint of an anomalous function of the AR. Consistently, functional analysis of the mutant receptor showed hormone binding, transactivation and transrepression potentials identical to that of the wild-type receptor. These results together argue against SBMA being a loss of function mutation of the AR.

β-Catenin degradation mediated by the CID domain of APC provides a model for the selection of APC mutations in colorectal, desmoid and duodenal tumours

Biallelic mutation of the ADENOMATOUS POLYPOSIS COLI (APC) gene is a hallmark of sporadic colorectal cancer and colorectal, duodenal and desmoid tumours that develop in familial adenomatous polyposis (FAP) patients. The mutations affecting both APC alleles are interdependent, the position of the first APC mutation determining where the second hit will occur. This results in a complex pattern of mutation distribution in the APC sequence that translates into the stabilization of beta-catenin that in turn feeds the affected cells with a permanent mitogenic signal. We describe here a new APC domain, the beta-catenin inhibitory domain (CID) of APC located between the second and third 20 amino acid repeats and therefore present in many truncated APC products found in human tumours. In truncated APC, the CID is absolutely necessary to down-regulate the transcriptional activity and the level of beta-catenin, even when an axin/conductin binding site is present. The activity of the CID is dramatically reduced in several colon cancer cell lines and can be inhibited by shorter truncated APC lacking the CID. The CID is a direct target of the selective pressure acting on APC during tumourigenesis. It explains the interdependence of both APC mutations, not only in colorectal but also in duodenal and desmoid tumours.

Hsp70–RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor

Receptor‐associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N‐ and C‐termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N‐terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C‐terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46‐mediated downregulation of glucocorticoid receptor activity.

Truncated APC is required for cell proliferation and DNA replication.

The tumour suppressor APC is truncated in most colon cancers, which leads to the stabilization of β‐catenin and to the constitutive activation of Wnt signalling. However, it is not clear why colon cancer cells retain the truncated APC fragment. Here, we show that a decrease of APC levels achieved by RNA interference impairs cell proliferation and DNA replication, not only in 293 cells that express a wild‐type protein, but also in SW480 colon cancer cells that express exclusively a truncated APC fragment. This correlates with a reduction of the levels of cyclin A, cyclin A‐dependent kinase activity, p27kip1 and the catalytic subunit of DNA polymerase δ. Thus, our data suggest that colon cancer cells retain a truncated APC fragment because it is essential for cell proliferation.

N-terminal sequences of the human androgen receptor in DNA binding and transrepressing functions

Androgen receptor is a ligand binding transcription factor that controls several physiological processes ranging from the development of the male sexual organs to the acquisition of secondary sex characteristics. It is composed of a carboxy-terminal ligand binding domain, a centrally located DNA binding domain and an amino terminal modulator region. Detailed study on the DNA and carboxy-terminal regions have been carried out, but only limited information is available on the activity of the N-terminus. With the use of truncated and chimeric receptor constructs we have demonstrated in transient transfection experiments that the N-terminus of the androgen receptor contributes to DNA binding, transactivation and transrepression functions of the receptor. We have shown that specific sequences at the N-terminus are needed for transactivation but we were unable to identify discrete sequences in this region for the DNA binding and transrepression functions. Sequences from the transcription factor NFI/X3 that bear no homology to the N-terminus of the androgen receptor nevertheless functionally replaced it in enhancing DNA binding, transrepression but not transactivation functions of the receptor. Thus, it appears that the structure rather than sequence specific elements determines the contribution of the N-terminus of the androgen receptor to DNA binding and transrepression functions.