Shh Pathway Function In Breast Cancer

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Dhh Signaing Testis

Figure 3.3.5.A. SHH signaling. Once activated, SMO transduces signals that activate the transcription factors GLI-1, GLI-2, and GLI-3. They activate genes that support cell proliferation. Somewhat unique is the two-step negative regulation at the level of the receptors. SHH inhibits PTC-1, which inhibits SMO. Hence, the presence of the ligand SHH relieves the PTC-dependent suppression of SMO signaling and allows the activation of GLI.

{2q14} and GLI3 {7p13} can also be regulated by FGF in the embryonic mesoderm, indicating that GLI may integrate several signaling pathways.

The putative tumor suppressors EXT-1 (Exostosin-1) {8q24}, EXT-2 {11p12-p11}, and EXT-3 {19p} have roles in regulating Hedgehog signaling. EXT-1 is a type-II transmembrane glyco-protein resident in the endoplasmic reticulum,whose expression results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). A rate-limiting step in the synthesis of heparan sulfate is the polymerization of alternating GlcA and GlcNAc residues, which is catalyzed by the EXT family of enzymes. EXT-1 and EXT-2 form a het-erooligomeric complex that leads to the accumulation of both proteins in the Golgi apparatus. The Golgi localized EXT-1/EXT-2 complex possesses substantially higher Glycosyl Transferase activity than EXT-1 or EXT-2 alone.

The HH pathway is associated with several forms of cancer.

• GLI1 and GLI2 are implicated in tumorigenesis. GLI amplification may be involved in the development of glioma [Kinzler et al. 1987].

• The SHH^GLI pathway is often abnormally activated in medulloblastomata [Oro et al. 1997]. Ligand-independent activation of the Hedgehog pathway in medulloblastoma occurs either through mutations that render SMO independent of PTC or through mutational inactivation of PTC.

• The SHH^GLI pathway is often abnormally activated in basal cell carcinomata [Oro et al. 1997]. Gain-of-function mutations in smo occur in sporadic basal cell carcinomata. Mutant SMO, unlike its wild-type counterpart, can cooperate with ade-novirus E1A to transform cells. The point mutations W535L in the seventh transmembrane domain of SMO or R562Q in the COOH-terminal cytoplasmic tail of SMO are implicated [Xie et al. 1998]. Genetic mutations leading to a truncated or unstable PTC protein are associated with familial or sporadic basal cell carcinoma. The mutation H133Y constitutes a factor for susceptibility.

• Mutations in the patched gene occur in patients with the basal cell nevus syndrome, a hereditary disease characterized by multiple basal cell carci-nomata and developmental abnormalities. In this condition, the inhibition of SMO signaling is relieved by the mutational inactivation of PTC [Johnson et al. 1996; Hahn et al. 1996a,b,c].

• Rhabdomyosarcoma is associated with mutations that activate the proto-oncogene smo or that inactivate the tumor suppressor ptc. This leads to excessive activation of GLI-1.

• Loss of heterozygosity for markers linked to ext1 and ext2 arises in chondrosarcomata originating in individuals with multiple exostoses as well as in sporadic chondrosarcomata [Hecht et al. 1997].

3.3.6 The Notch pathway

Genes of the notch family [Wharton et al. 1985; Kidd et al. 1986] encode transmembrane receptors that are involved in cell fate decisions during development and postnatal life. There are four notch genes, notch-1 (tan-1) {9q34.3}, notch-2 {1p13-p11}, notch-3 {19p13.2-p13.1}, notch-4 (int-3) {6p21.3}, that are expressed in overlapping but distinct patterns (Figure 3.3.6.A). The Notch-1 and Notch-2 receptors contain 36 EGF repeats in their ectodomains, whereas the Notch-3 receptor harbors 34 repeats and the Notch-4 receptor harbors 29 repeats. In addition to the EGF-like repeats in the extracellular region of Notch, motifs in the intracel-lular region of Notch include two nuclear localization signals, a RAM motif, 6 Ankyrin/CDC10 repeats, PEST sequences, and a glutamine rich domain. No transactivation domain is present in Notch-3 or Notch-4. Notch is a receptor with one transmembrane domain. Although synthesized as a single precursor protein, Notch is cleaved during its transport to the cell surface. Proteolytic processing is part of the maturation and activation of Notch-1 [Chan and Jan 1998]. It is mediated by a Furin-like Convertase within the secretory pathway. Cleavage occurs at an extracellular site, called site 1 (S1), downstream of the recognition sequence RQRR. As a consequence, Notch-1 exists as a heterodimeric receptor. The activation of Notch-1 involves its cleavage between G1743 and V1744 (termed site 3 or S3). Site 3 cleavage occurs in response to ligand binding and serves to release NICD from the membrane. Presenilin is required for the release of the intracellular domain of Notch from the plasma membrane. Ligand binding also facilitates the cleavage at another site, named site 2 (S2), within the extracellular juxtamembrane region. This serves to release the ectodomain repression of NICD production. Site 2 cleavage occurs between A1710

Notch Ligand Drosophila Structure

Figure 3.3.6.A. Notch structure. Left panel: Structure of Notch proteins and their ligands. Drosophila has one Notch receptor (dNotch) and vertebrates have four (Notch 1-4), which are presented on the cell surface as heterodimers. The ectodomain of Notch receptors contains EGF-like repeats and a cysteine-rich Notch/Lin12 domain (LN), which is followed by a transmembrane domain, the RAM domain and six Ankyrin repeats (ANK, CDC10 repeats), two nuclear localization signals (NLS), followed by the transactivation domain (TAD) and a PEST sequence. Notch-1 contains a strong and Notch-2 a weak transactivation domain in the cytoplasmic part of the receptor. Right panel: There are two transmembrane-bound ligands for Notch in Drosophila, delta (Dl), and serrate (Ser). The vertebrates possess three delta homologs, delta-like (DLL)-1, DLL-3, and DLL-4, and two serrate homologs, jagged-1 (JAG-1) and JAG-2. The ligands harbor an NH2-terminal structure called DSL (Delta, Serrate, and LAG-2), which is common to all family members, followed by EGF-like repeats. Serrate, JAG-1 and JAG-2 harbor a cysteine-rich domain (CR) following the EGF-like repeats. [Reproduced from Radtke and Raj 2003. With permission from Macmillan.]

Figure 3.3.6.A. Notch structure. Left panel: Structure of Notch proteins and their ligands. Drosophila has one Notch receptor (dNotch) and vertebrates have four (Notch 1-4), which are presented on the cell surface as heterodimers. The ectodomain of Notch receptors contains EGF-like repeats and a cysteine-rich Notch/Lin12 domain (LN), which is followed by a transmembrane domain, the RAM domain and six Ankyrin repeats (ANK, CDC10 repeats), two nuclear localization signals (NLS), followed by the transactivation domain (TAD) and a PEST sequence. Notch-1 contains a strong and Notch-2 a weak transactivation domain in the cytoplasmic part of the receptor. Right panel: There are two transmembrane-bound ligands for Notch in Drosophila, delta (Dl), and serrate (Ser). The vertebrates possess three delta homologs, delta-like (DLL)-1, DLL-3, and DLL-4, and two serrate homologs, jagged-1 (JAG-1) and JAG-2. The ligands harbor an NH2-terminal structure called DSL (Delta, Serrate, and LAG-2), which is common to all family members, followed by EGF-like repeats. Serrate, JAG-1 and JAG-2 harbor a cysteine-rich domain (CR) following the EGF-like repeats. [Reproduced from Radtke and Raj 2003. With permission from Macmillan.]

and V1711, 12 amino acids outside the transmembrane domain, and generates a transient intermediate peptide termed NEXT (Notch Extracellular Truncation). NEXT accumulates when NICD production is blocked by point mutations or y-Secretase inhibition or by loss of Presenilin-1, and inhibition of NEXT eliminates NICD production. Site 2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch-1 activation [Mumm et al. 2000]. The y-Secretase activity that accounts for the site 2 cleavage is dependent on prior cleavage by the Metalloproteinase TACE (Tumor Necrosis Factor-Converting Enzyme, ADAM17), which plays a prominent role in the activation of the Notch pathway [Brou et al. 2000].

The engagement by the Notch ligands, Delta-Like-1 (DLL-1), DLL-3, DLL-4, Serrate/Jagged-1 (JAG-1), or JAG-2, presented on adjacent cells, initiates a proteolytic cascade that leads to its cleavage and release of the NICD. NICD then translocates to the nucleus, where it associates with HLH transcription factors of the CSL family, specifically with CBF1 (Suppressor of Hairless, LAG1, RBPSUH)

{9p13-p12}, and functions as a transcriptional activator. In the absence of Notch, CBF-1 binds to the promoters of its target genes and recruits corepres-sors and Histone Deacetylases, which inhibit transcription. When Notch is present, it competes with the inhibitory proteins for binding to CBF-1 and then recruits coactivators. In this setting, CBF-1 activates the gene expression of hes-1 (hairy/enhancer of split-1) {3q28-q29}. It may lead to the regulation of programmed cell death by inhibition of NUR77-mediated apoptosis. The promoters for erbB2 and nf-kB2 also contain CSL binding sites (Figure 3.3.6.B).

The outcome of Notch signaling is highly context dependent [Radtke and Raj 2003]. - In hematopoietic precursor cells, signaling induced by JAG-1 maintains the precursor pool. Further Notch signaling inhibits B-lymphocyte development and drives the progenitor cells into the T-lymphocyte lineage. Maturation of the T-lymphocytes beyond the CD4+CD8+ (double positive) stage then requires the discontinuation of Notch-1 signaling.

Notch Cell Development

Figure3.3.6.B. Notch signaling. Notch proteins are synthesized as precursors that are processed by a Furin-Like Convertase in the Golgi organelle before being transported to the cell surface, where they reside as heterodimers. Interaction of Notch receptors with their ligands, such as Delta-Like or Jagged, leads to a cascade of proteolytic cleavages. The first cleavage is mediated by TACE (Tumor necrosis factor-a-converting enzyme/metalloproteinase), followed by a second cleavage mediated by the y-Secretase activity of Presenilins (PS), which liberates the cytoplasmic domain NIC (Notch Intracellular Domain) of the Notch receptors. The liberated NIC enters the nucleus and binds to the transcription factor CSL, which displaces corepressors (CoR) and recruits coactivators (CoA), leading to transcriptional activation of downstream target genes. This pathway represents the CSL-dependent pathway. Genetic evidence also points to the existence of a CSL-independent pathway. [Reproduced from Radtke and Raj 2003. With permission from Macmillan.]

Figure3.3.6.B. Notch signaling. Notch proteins are synthesized as precursors that are processed by a Furin-Like Convertase in the Golgi organelle before being transported to the cell surface, where they reside as heterodimers. Interaction of Notch receptors with their ligands, such as Delta-Like or Jagged, leads to a cascade of proteolytic cleavages. The first cleavage is mediated by TACE (Tumor necrosis factor-a-converting enzyme/metalloproteinase), followed by a second cleavage mediated by the y-Secretase activity of Presenilins (PS), which liberates the cytoplasmic domain NIC (Notch Intracellular Domain) of the Notch receptors. The liberated NIC enters the nucleus and binds to the transcription factor CSL, which displaces corepressors (CoR) and recruits coactivators (CoA), leading to transcriptional activation of downstream target genes. This pathway represents the CSL-dependent pathway. Genetic evidence also points to the existence of a CSL-independent pathway. [Reproduced from Radtke and Raj 2003. With permission from Macmillan.]

- Notch signaling participates in cell fate decisions. Notch signaling induces neuronal progenitor cells to retain a stem cell character by preventing them from undergoing neurogenesis. Contact-mediated Notch signaling regulates the capacity of neurons to extend neurites. Up-regulation of Notch activity is concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth. In neurons with low Notch activity, which readily extend neurites, up-regulation of Notch activity either inhibits the extension or causes a retraction of neurites. Conversely, in more mature neurons that have ceased their growth after establishing numerous connections and display high Notch activity, the inhibition of Notch signaling promotes neurite extension.

- The epithelium of the skin comprises several layers of keratinocytes representing progressive stages of differentiation. Notch-2 is expressed in the basal cell layer; Notch-3 is expressed in the basal cell and the suprabasal cell layers. Notch signaling enhances the differentiation of ker-atinocytes. NICD directly stimulates the expression of p2lCIP1/WAF1 and represses the expression of c-fos, which causes withdrawal from the cell cycle and facilitates terminal differentiation.

- During lung development, differentiation pathways converge to generate pulmonary epithelial cells or neuroendocrine airway cells. Notch-1 and HES-1 are highly expressed in nonneuroendocrine airway epithelial cells, whereas MASH-1 (Mammalian Achaete Scute Homolog-1, Human Achaete Scute Homolog-1, hASH-1, ASCL1) expression is restricted to neuroendocrine cells. The activation of HES by Notch represses mash-1 {12q22-q23} and determines the differentiation along the pulmonary epithelial cell lineage.

Notch signaling can be modulated on various levels. The elongation of 0-linked fucose residues on certain EGF repeats by Glycosyl Transferases of the Fringe family prevents the activation of Notch signaling by Jagged, but not by Data Like ligands.

In various forms of cancer, Notch signaling is altered.

• Transcripts of the gene notch-1 (tan1, translocation-associated notch homolog 1) are generated in many normal fetal tissues but are most abundant in lym-phoid tissues. The Notch pathway is overactive in some forms of leukemia. Chromosome 7q34-q35, which contains the gene tcrp for the T-Cell Antigen Receptor p chain, is a common site for translocations in T-cell neoplasms. In the translocation t(7;9)(q34;q34.3), which occurs in acute T-cell lym-phoblastic leukemia, the locus on chromosome 9 contains a gene of the notch family. Breakpoints within 100 bp of an intron in tan1 result in the truncation of tan1 transcripts [Ellisen et al. 1991]. In this translocation, only the COOH-terminal portion of Notch-1 is juxtaposed. The expressed truncated Notch-1 protein corresponds to NICD. This maintains the T-lymphocytes in an immature CD4+CD8+

(double-positive) state and predisposes them to full transformation. Because the overexpression of NICD in hematopoietic cells gives rise exclusively to T-cell neoplasms, Notch-mediated transformation depends on a T-lymphocyte-specific signal transduced through the pre-TCR (pre-T-Lymphocyte Antigen Receptor).

• The Notch pathway is overactive in various epithelial tumors, including mammary adenocarcinoma and colon adenocarcinoma. It is by itself insufficient to cause cancer and needs to partner with other oncoproteins. These partners, RAS, MYC, HPV E6, and HPV E7, share the common property of overriding the G1/S cell cycle checkpoint. In epithelial cancers, oncogenic RAS activates Notch. Full transformation by Notch then requires active signals from the ERK (MAP Kinase) and PI-3 Kinase pathways downstream of RAS [Fitzgerald et al. 2000].

• In keeping with the context dependence of Notch signaling, Notch-1 and Notch-2 can have tumor-suppressive effects. In small cell lung cancer cells with neuroendocrine differentiation, the proteolytic cleavage products of Notch-1 or Notch-2 can cause cell cycle arrest, in part by repressing mash-1.

• Basic helix-loop-helix transcription factors of the MASH family are regulators of development in the central nervous system and neural crest. MASH-1 is highly expressed in two neuroendocrine cancers, medullary thyroid cancer and small cell lung cancer [Ball et al. 1993].

3.3.7 The APC pathway

The WNT family of proteins is involved in embryonic patterning and in the development of the nervous system. There are more than 30 WNT proteins, which are secreted factors that interact with cell surface receptors. WNT factors bind to the seven transmembrane spanning serpentine Frizzled Receptors, which cooperate with LRP6 (Low Density Lipoprotein Receptor Related Protein) to activate G-Proteins. Their signaling critically depends on P-Catenin and regulates cell proliferation (Figure 3.3.7.A). - WNT-WNT Receptor interaction induces the activity of the cytoplasmic phosphoprotein Dishevelled. Activated Dishevelled inhibits the ser-ine/threonine kinase 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 (Lymphoid

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