WNT Receptor


Figure 3.3.7.A. WNT signaling. WNT factors bind to the seven transmembrane spanning serpentine Frizzled Receptors that activate G-Proteins. Receptor ligation induces the activity of the cytoplasmic phosphoprotein Dishevelled (DSH homolog), which inhibits the serine/threonine kinase Glycogen Synthase Kinase-3P (GSK-3P). When GSK-3P is inhibited, P-Catenin becomes hypophosphorylated, the hypophosphorylated form of P-Catenin migrates to the nucleus and interacts with transcription factors, in particular with LEF-1 and TCF-4, thereby inducing gene expression. WNT-dependent signal transduction also antagonizes APC and Axin, which allows the accumulation of y-Catenin (Plakoglobin) and P-Catenin in the nucleus and drives cell proliferation. Furthermore, WNT signaling activates Casein Kinase 1e, which can stabilize P-Catenin.

Enhancer-Binding Factor-1, TCF-1a, T-Cell Factor 1a), thereby inducing gene expression.

- WNT-dependent signal transduction antagonizes the APC/Axin effect and allows the accumulation of y-Catenin (Plakoglobin) and P-Catenin in the nucleus, which then drives cell proliferation.

- WNT signaling activates Casein Kinase 1e, which can stabilize P-Catenin.

WNT signaling proceeds through inhibition of GSK3P and activation of Protein Phosphatase 2A (PP2A), which leads to the dephosphorylation of Axin. The regulatory subunit of PP2A (B56) binds to APC and targets P-Catenin for degradation in the proteasome. PP2A is an intracellular serine/threo-nine protein phosphatase that is expressed as a het-erotrimeric protein-containing conserved catalytic (C) and structural (A) subunits, and a variable regulatory (B) subunit. PP2A is inhibitory to the WNT signaling process.

GSK3 (Glycogen Synthase Kinase3) is a cytoplas-mic serine/threonine kinase that is involved in Insulin signaling and metabolic regulation, as well as in WNT signaling and the specification of cell fates during embryonic development. GSK3 appears in two highly homologous and ubiquitously expressed forms, GSK3a {19q13.1-q13.2} and GSK3P {3q13.3}. The Insulin and WNT signaling pathways differentially regulate GSK3, resulting in distinct downstream events. In the WNT pathway, GSK3 is essential for normal development of the embryo and for regulation of cell proliferation in the adult. WNT signaling inhibits GSK3, resulting in the dephospho-rylation of P-Catenin, which then translocates to the nucleus and activates transcription. A multiprotein complex of P-Catenin, Axin, and APC regulates the phosphorylation of P-Catenin by GSK3 and may prevent cross-talk between the Insulin and WNT signaling pathways. Axin binding to GSK3 inhibits GSK3 phosphorylation on serine 9, thus inactivating downstream events of the Insulin pathway [Weston and Davis 2001]. Many of the GSK3 substrates, including Glycogen Synthetase, must be phosphory-lated before they can dock with the kinase through a phosphate-binding site on arginine 96. Substrates that are part of the WNT signaling pathway, including P-Catenin and Axin, do not require prephosphorylation.

The apc (adenomatouspolyposis coli) gene {5q21} [Kinzler et al. 1991; Nishisho et al. 1991; Groden et al. 1991; Joslyn et al. 1991] contains 15 exons, spanning approximately 125 kb of DNA and encoding an 8.5 kb coding region in the 10 kb mRNA. An alternative form (9A) splices into the interior of exon 9, removing 101 amino acids from the full-length APC polypeptide. The protein coding region of the apc gene is large, encompassing 2,844 amino acids (312 kD). The NH2-terminal domain conatins a series of repeat sequences (amino acids 6-57), which form a-helical structures capable of homodimerization. The central region (amino acids 453-767) contains binding sites for PP2A and ASEF (Figure 3.3.7.B). Axin-2 (Conductin) binds to to the SAMP repeat motif (serine-alanine-methionine-proline) in APC via its RGS (regulator of G-Protein signaling) domain. APC and Axin are substrates for GSK3P and their ability to bind Catenin is enhanced by phosphorylation. Axin-2 forms a complex with P-Catenin, APC, and GSK3P [Behrens et al. 1998].

Figure 3.3.7.B. APC structure. Conserved regions, such as the Armadillo repeats, and regions that interact with other proteins, including Tubulin, the microtubule-associated protein EB1, DLG (Discs Large), P-Catenin and Axin/Conductin, are shown. APC also contains several consensus sites for phosphorylation by P34CDC2, five nuclear export signals (E) and two nuclear import signals (I). Most somatic mutations occur in the mutation cluster region. Most of these mutations lead to truncated proteins. [Reproduced from Fodde et al. 2001. With permission from Macmillan.]

Figure 3.3.7.B. APC structure. Conserved regions, such as the Armadillo repeats, and regions that interact with other proteins, including Tubulin, the microtubule-associated protein EB1, DLG (Discs Large), P-Catenin and Axin/Conductin, are shown. APC also contains several consensus sites for phosphorylation by P34CDC2, five nuclear export signals (E) and two nuclear import signals (I). Most somatic mutations occur in the mutation cluster region. Most of these mutations lead to truncated proteins. [Reproduced from Fodde et al. 2001. With permission from Macmillan.]

This multiprotein complex (destruction complex) directs P-Catenin to degradation. After P-Catenin has been phosphorylated on four serine/threonine residues in the NH2-terminus by the kinase GSK3P in the complex, it is transferred to the SCF complex (SKP/Cullin/F-Box complex), binds to the F-box protein PTrCP, is ubiquitinated and degraded in the proteasome. APC inhibits RB phosphorylation and reduces the levels of Cyclin D1. This inhibits G1/S progression [Heinen et al. 2002].

The Catenins are a family of proteins that interact with the cytoplasmic portion of the Cadherin family of cell-cell adhesion proteins, thus linking the Cadherins to the Actin cytoskeleton. Catenins are important in the signaling cascade initiated by the WNT family of proteins. The fate of P-Catenin is a critical determinant for cell proliferation with location of P-Catenin in the nucleus mediating the activation of transcription factors and leading to proliferation, whereas degradation of free P-Catenin in the Ubiquitin pathway prevents cell cycle progression. The intracellular localization of P-Catenin can be influenced by sphingolipids. Cytoplasmic P-Catenin is in equilibrium with P-Catenin in adherens junctions. The fraction of P-Catenin in adherens junctions provides a link between E-Cadherin and a-Catenin, which binds to the Actin cytoskeleton. The growth suppressing activity of E-Cadherin is due, at least in part, to the sequestration of P-Catenin and the resulting inhibition of the P-Catenin^TCF-4 pathway. APC binds to microtubule-associated proteins through a domain in its extreme COOH-terminus. Through these associations, APC and P-Catenin can play a role in mitosis.

The LEF/TCF transcription factors include LEF-1 (Lymphoid Enhancer Binding Factor-1, TCF-1a, T-Cell-Specific Transcription Factor-1a), TCF-1, TCF-3, and TCF-4 (T-Cell Factor-4, ITF-2, SEF-2, E2-2). LEF-1 {4q23-q25} is a 48 kD high mobility group transcription factor. LEF-1 belongs to a family of regulatory proteins that share homology with HMG-1 Lymphoid Enhancer Binding Factor-1. Catenin does not bind to DNA, but it does bind to TCF-4, which itself lacks transactivation activity. Together, they induce the expression of genes that support multiple growth pathways.

- Catenin and TCF-4 induce the expression of cyclin D1 and c-myc. Once expressed, MYC binds to SMAD-2 and SMAD-3 and represses the transcription of the p15INK4B gene, thus rendering cells unresponsive to TGF-P-mediated inhibition of cell cyle progression [Feng et al. 2002].

-APCDD1 (Downregulated by APC-1) is a 514 amino acid protein with a molecular mass of about 59 kD that promotes cell growth. Its 2.6 kb transcript is expressed ubiquitously, with abundant levels in the heart, pancreas, prostate, and ovaries. P-Catenin and TCF-4 directly bind to the promoter of apcdd1 {18p11} and induce its transcription. The expression of apcdd1 is inhibited by APC and by Axin [Takahashi et al. 2002].

- TCF-4 induces the expression of ectodermal-neural cortex 1 (end), in colon epithelial cells. ENC-1 increases the growth rate of colon epithelial cells and prevents their differentiation.

- The growth-promoting gene af17 is a likely target for transcription by the P-Catenin/TCF/LEF complex.

- pml is a target gene of P-Catenin and y-Catenin (Plakoglobin) independently of TCF (LEF).

PML, P300, and P-Catenin coactivate the transcription of arf and siamois, but not cyclin D1.

• Allelic loss and point mutations can occur in the apc (adenomatouspolyposis coli) tumor suppressor gene on chromosome 5q21. More than 120 distinct germline and somatic mutations are documented in the apc gene. Somatic loss-of-function mutations in apc may initiate colorectal cancer development, whereas germline mutations are responsible for familial adenomatous polyposis (FAP). Multiple colonic polyp development characterizes the disease. These polyps arise during the second and third decades of life and become adenomata and malignant carcinomata later in life. The vast majority of these mutations occur in the mutation cluster region (exon 15, codons 1,286-1,513), and lead to COOH-terminal truncations of the APC protein. This results in a lack of Conductin-bind-ing motifs, such as the SAMP repeats, and a lack of the variable number of 20 amino acid repeats that are associated with the down-regulation of intra-cellular P-Catenin. Normally, the APC/Axin-2/ P-Catenin complex stimulates the breakdown of P-Catenin. Therefore, mutations that cause a loss of APC, or a loss of the portion of the APC protein that interacts with P-Catenin, can lead to a constitutive activation of TCF (LEF-1) and unrestricted growth. Cells with mutant apc, often in colorectal tumors, have an abundance of spindle microtubules that fail to connect to kinetochores and are characterized by chromosomal instability. Somatic mutations of apc also occur in cancers of the stomach, pancreas, thyroid, ovary and breast.

• Methylation in the promoter region of apc may lead to inactivation in gastrointestinal tumors. Aberrant methylation occurs early in colorectal carcinogenesis.

• BMP4 is overexpressed and secreted by human cancer cells with mutant adenomatous polyposis coli gene. The oncogenic allele of b-catenin is absolutely required for the expression of the TGF-P family member BMP-4, whose receptor, bmpr1A, is mutated in a fraction of the rare inherited gastrointestinal cancer predisposition syndrome juvenile intestinal poly-posis [Howe et al. 2001]. This indicates the presence of regulatory interactions between the WNT and BMP signaling pathways [Kim et al. 2002].

• In normal colonic epithelium, survivin is preferentially expressed in the lower crypt. The expression of survivin correlates inversely with the expression of APC, because it is down-regulated by APC^P-Catenin^TCF-4 signaling. The gradual transformation of colorectal epithelium to carcinomata is associated with the progressive inhibition of apoptosis. Survivin is highly expressed in the majority of colorectal carcino-mata [Zhang et al. 2001], likely accounting for this phenomenon.

> The expression of apcdd1 is directly regulated by the P-Catenin/TCF-4 complex. Its expression levels are reduced by APC or Axin-1 activity. Elevated expression of APCDD1 promotes the proliferation of colonic epithelial cells and the molecule is frequently overexpressed in colorectal cancer [Takahashi et al. 2002].

The PP2A-dependent decrease in P-Catenin is blocked by certain oncogenic mutations in b-catenin [Seeling et al. 1999]. In colorectal tumors with intact apc gene, gain-of-function mutations of b-catenin, that alter functionally significant phosphorylation sites, are frequent. In colorectal cancers with activating b-catenin mutations, an inappropriately activated high mobility group transcription factor TCF-4 leads to overexpression of the target genes c-myc and tcf-1, which then promote neoplastic growth [Roose et al. 1999]. Oncogenic mutants of P-Catenin that lack GSK3P phosphorylation sites do not bind P-TrCP. This protects P-Catenin from Ubiquitin-mediated degradation. The aberrant accumulation of P-Catenin in tumors is often associated with mutational inactivation of the P53 tumor suppressor. High-level expression of transcriptionally active P53 down-regulates P-Catenin. This inhibitory effect is likely mediated by the Ubiquitin-proteasome system and requires active GSK3P. These processes imply that there may be a selective pressure for the loss of wild-type p53 expression in cancers that are driven by excessive accumulation of P-Catenin [Sadot et al. 2001].

> In medulloblastoma, while mutations of apc are rare, a hot spot region of b-catenin (ctnnb1) mutations occurs in a subset of tumors. Point mutations and deletions in axin-1 may also arise in medul-loblastoma.

»The WNT signaling pathway is often up-regulated in epidermal cancers. The gene for the APC homolog apc-2 (apcl) is located on chromosome 19p13.3, a region that is commonly lost in ovarian cancer. High frequency apc-2 allelic imbalance in ovarian cancers implies that APC-2 may act as a tumor suppressor in this type of malignancy.

3.3.8 The SMAD pathway

SMADs (SMA- and MAD-Related Proteins) mediate signals from members of the TGF-P superfamily of cytokines. There are three classes of SMADs (Figure 3.3.8.A),

-Receptor-regulated SMADs (R-SMADs, comprising SMADs -1, -2, -3, -5, and -8)

- Common mediator SMADs (co-SMADs, comprising SMADs -4 and -10)

- Inhibitory SMADs (I-SMADs, comprising SMADs -6 and -7)

Receptor ligation leads to serine phosphorylation of R-SMADs in the COOH-terminal domain, their dissociation and assembly into complexes with the co-SMADs, SMAD-4 (MADH4, DPC4) and SMAD-10 (SMAD-4P) followed by a translocation of the complexes into the nucleus where the SMADs


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