Pstat Pstat

Figure 3.3.4. A. STAT signal transduction pathway. Signaling through JAK and STAT is typically activated in response to ligation of cytokine receptors. Once engaged by their cognate ligand, the receptor tyrosine kinases dimerize and autophospho-rylate. This activates JAK and STAT proteins. The activated STATs translocate into the nucleus and initiate gene transcription. The biological outcome of the activation of STATs is often cell proliferation or antiapoptosis.

c-MYC Cyclin D1 proliferation

PIM-1 BCL-X survival

Figure 3.3.4. A. STAT signal transduction pathway. Signaling through JAK and STAT is typically activated in response to ligation of cytokine receptors. Once engaged by their cognate ligand, the receptor tyrosine kinases dimerize and autophospho-rylate. This activates JAK and STAT proteins. The activated STATs translocate into the nucleus and initiate gene transcription. The biological outcome of the activation of STATs is often cell proliferation or antiapoptosis.

transcriptional activity. The heterodimer binds to the E box motif CAC(G,A)TG and interacts with TBP (TATA Binding Protein). cdc25A, cyclin A, and cyclin E, as well as eif-2a and eif-4E are effectors of MYC. A target gene of MYC is cad, which is required for DNA synthesis. c-MYC expression can bypass P16INK4a-mediated growth arrest. MYC can act as a repressor of genes that contain the initiator element in their promoter. MYC represses the expression of the heavy chain of ferritin and stimulates the expression of the iron regulatorypro-tein 2 (irp2). Regulation of the genes controlling intracellular iron concentrations is essential for cell transformation by MYC. Consistently, a reduction in free iron leads to growth arrest and decreased synthesis of the cell cycle regulators P34CDC2 and Cyclin A [Wu et al. 1999]. In nontransformed cells, the expression of the myc proto-oncogene is tightly linked to mitogenic stimuli and is a prerequisite for cell growth. myc is an early response gene, whose expression rises rapidly at the G0 to G1 transition and whose functions are largely linked to G1 and early S progression. Unlike most early response genes, however, myc expression is sustained throughout the phases of the cell cycle. In the absence of mitogens, MYC forms heterodimers with MAD, and recruits mSIN3, NCOR, and Histone Deacetylase. IFN-y and TGF-P lead to the rapid down-regulation of myc and these agents also mediate Gj arrest. CTCF binds to a number of important regulatory regions within the 5' noncod-ing sequence of myc and regulates myc expression. CTCF harbors several autonomous repression domains, including a zinc finger cluster, which silences transcription through binding directly to the corepressor SIN3A and recruiting Histone Deacetylases. Insulator elements, which act as a barrier to prevent neighboring cis-acting elements from regulating a distal gene, mediate their function by CTCF.

Members of the HMG (High Mobility Group) family of non-Histone proteins share a triplicate common DNA-binding domain, the AT hook, that specifically binds to the minor groove of A/T-rich sequences. HMG proteins function as gene transcrip-tional regulatory units. The HMGA (High Mobility Group A) family includes two alternatively spliced forms, HMGA1a and HMGA1b, of HMGA1 (HMGI-Y) and HMGA2. hmgAl is a target gene for MYC. Its promoter contains a MYC/MAX binding site, an E box at nucleotide-1,337. HMGA1 (High

Mobility Group AT-Hook 1) {6p21} is important for the expression of interferon-p. The architectural transcription factor HMGA2 (HMGIC, BABL, LIPO) is expressed almost exclusively in undifferentiated mes-enchymal cells. HMGA2 {12q14.3} plays a critical role in determining body size. The hmgA2 gene spans more than 60 kb and consists of five exons, the first and last of which include long untranslated regions. The hmg genes are abundantly expressed during embryogenesis but not in normal adult tissues.

Negative regulation of STATs operates through tyrosine phosphatases, SOCS (Suppressor of Cytokine Signaling), and PIAS (Protein Inhibitor of Activated STATs). The SOCS family comprises at least eight members, SOCS-1-7 and CIS (Cytokine-Inducible SH2-Containing Protein). SOCSs bind to JAKs and inactivate them. PIAS binds to phosphorylated STAT dimers, preventing DNA recognition.

• Constitutively activated STATs exist in a wide variety of cancers, including almost all head and neck cancers. They are activated by tyrosine phosphory-lation through the persistent activity of tyrosine kinases, including SRC, EGF Receptor, JAKs, or BCR-ABL. Such oncogenic tyrosine kinases are often activated as a consequence of permanent lig-and-receptor engagement in autocrine or paracrine cytokine and growth factor signaling or represent constitutively active enzymes as a result of genetic alterations. Persistent signaling of specific STATs, in particular STATs -3 and -5, directly contributes to oncogenesis by stimulating cell proliferation and preventing apoptosis. This is accomplished through the up-regulation of gene expression of apoptosis inhibitors and cell cycle regulators, including bcl-XL, mcl-1, cyclins D1, cyclin D2, and c-myc. The oncogenic potential of STATs (-)3 and (-)5 can be directly activated by point mutations. Hepatocellular carcinoma harbors persistently active STAT3 in association with hypermethylation, and hence suppression, of socs1, which encodes a negative regulator of STAT activity.

• In contrast to STAT-3 or STAT-5, STAT-1 plays important roles in growth arrest and apoptosis, and it is implied as a tumor suppressor.

• ctcfis located in a small region of overlap for common chromosomal deletions in sporadic breast and prostate tumors, suggesting that CTCF acts as a tumor suppressor. Its absence may lead to overexpression of myc.

• Deregulated expression of myc genes is frequent in cancer. c-MYC is glycosylated by 0-linked N-acetylglucosamine on threonine 58, a phosphory-lation site and a mutational hot spot in lymphomata. Three forms of c-MYC are distinguishable according to no modification, phosphorylation, or glycosylation of T58. Growth factor deprivation may increase T58 glycosylation and correspondingly decrease its phosphorylation, while exposure to growth factors has the opposite effect. A kinase responsible for T58 phosphoryla-tion is the GSK3. The T58 phosphorylated form of c-MYC predominantly accumulates in the cytoplasm rather than the nucleus [Kamemura et al. 2002].

• ID proteins coordinate proliferation and differentiation. ID-2 acts as a dominant antagonist of basic helix-loop-helix transcription factors and proteins of the RB family. ID-2 may be an effector of N-MYC in neuroblastomata. ID-2 is recruited by MYC oncoproteins to bypass the tumor suppressor function of RB [Lasorella et al. 2000].

• hmg genes are frequently overexpressed in neo-plasias of the thyroid, prostate, cervix, colorec-tum, and pancreas as well as in pituitary adenomata.

• MYC plays important roles in the pathogenesis of Burkitt lymphoma. MYC induces hmgA gene expression. The expression of HMGA1 protein is increased in Burkitt lymphoma cells.

• In translocations associated with lipoma, the 3' end of the hmgA2 gene is deleted. Most of the breaks occur within the third intron. Chimeric transcripts are formed, in which HMGIC DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivator domain [Ashar et al. 1995]. lhfp is the fusion partner of hmgA2 in lipoma with t(12; 13). The expressed fusion transcript encodes the three DNA-binding domains of HMGA2, followed by 69 amino acids encoded by frameshifted lhfp sequences [Petit et al. 1999]. LPP (Lipoma Preferred Partner) is fused with HMGA2 by a t(3;12) translocation in some forms of benign lipoma. Additional fusions include HMGIC-LHFP, HMGIC-RAD51L1, HMGIC-HEI10, HMGIC-ALDH2, and HMGIC-COX6C.

• Pulmonary chondroid hamartomata are benign tumors of the lungs, characterized by a more or less high degree of mesenchymal metaplasia. In most cases, rearrangements of hmgA2 underlie this condition [Kazmierczak et al 1996].

3.3.5 The SHH pathway

Signaling by the hedgehog family of secreted glyco-proteins is implicated in the determination of embryonic cell fate, in the maintenance of somatic cell fate, in the specification of organ size, and in the patterning of various tissues. They include skin, lung, brain, bone, and blood. There are three known Hedgehog families, Sonic Hedgehog (SHH) {7q36}, Desert Hedgehog (DHH) {12q13.1}, and Indian Hedgehog (IHH) {2q33-q35}. SHH is a receptor that transduces signals, which are instrumental in patterning the early embryo. It is expressed in the Hensen node, the floorplate of the neural tube, the early gut endoderm, the posterior of the limb buds, and throughout the notochord. SHH contributes to the patterning of the ventral neural tube, the anterior-posterior limb axis, and the ventral somites. IHH is expressed in the prehypertrophic chondro-cytes of cartilage elements, where it regulates the rate of hypertrophic differentiation. The distribution of DHH is very restricted, limited primarily to the Sertoli cells of developing testes and to the Schwann cells of peripheral nerves.

The products of the shh and patched genes normally promote organ development in the brain and peripheral nervous system. They convey a key inductive signal in patterning of the ventral neural tube. SHH function is required for the induction of motor neurons by both the notochord and midline neural cells. In the cerebellum, Hedgehog signaling delays neuronal differentiation and induces the proliferation of cerebellar granular neuronal precursors (CGNPs). Activation of the Hedgehog pathway normally requires the inactivation of the 12 transmembrane spanning protein PTC by Hedgehog Ligand, thus releasing the seven transmembrane spanning protein SMO for activation of target genes of the cubitus interruptus/gli family of transcription factors, which are associated with gliomata.

Hedgehog (HH) signaling promotes the expression of G1/S Cyclins, including Cyclins D and E, and results in the growth of cells. SHH signaling also opposes epithelial cell cycle arrest by P21CIP1/WAF1. After cleavage of the signal sequence, the Hedgehog protein precursor of approximately 45 kD undergoes autocatalytic internal cleavage. This yields an approximately 20 kD NH2-terminal domain that has signaling activity and a 25 kD COOH-terminal domain that is active in precursor processing. Hedgehog protein autoprocessing includes peptide bond cleavage and the attachment of a lipophilic adduct to the COOH-terminal region. The lipophilic modification is critical for the spatially restricted tissue localization of the Hedgehog signal domain. Cholesterol is the lipophilic moiety cova-lently attached to the NH2-terminal signaling domain during autoprocessing and the COOH-ter-minal domain acts as an intramolecular Cholesteryl Transferase. Hedgehog proteins bind to PTC or to a PTC/SMO complex and thereby induce SMO activity (Figure 3.3.5.A).

The SHH Receptor and tumor suppressor PTC (Patched) {9q22.3} is a 12-transmembrane spanning member of a class of gene products that are important in controlling early epithelial proliferation. In the absence of HH signaling, PTC represses the genes for wnt, tgf-b, and ptc. PTC suppresses the signaling of SMO (Smoothened) {7q31-7q32} by inhibiting its association with P-Arrestin-2. The ligation of PTC by SHH relieves this inhibition. This leads to phospho-rylation of SMO by GRK-2 (G-Protein-Coupled Receptor Kinase-2), interaction with P-Arrestin-2, and endocytosis of SMO in Clathryn-coated pits. PTC induces apoptosis in neuroepithelial cells unless its ligand SHH is present to block the signal. SHH is required for the survival of these cells. The high-affinity binding between PTC and SHH may also provide mitogenic or differentiative signals to basal cells in the skin throughout life. Cell cycle progression following SHH signaling depends on contributions by the PDGF pathway. The transcription of ptc is induced by Hedgehog pathway activity, thus serving as a negative feedback loop.

Ligation of the seven transmembrane spanning receptor SMO by the lipid-anchored cell surface lig-and SHH activates PTC, which prevents the PKA-dependent phosphorylation and consecutive cleavage of the Krüppel family zinc finger protein GLI. In the nucleus, proteolyzed GLI acts as a repressor of hedgehog target genes. In healthy tissues, gli gene products are mainly active in precursor cells. The GLI proteins reside in the nucleus and the cytoplasm. In the cytoplasm, they are components of multiprotein complexes that are tethered to the cytoskeleton. In the absence of SHH, GLI is cleaved by the proteasome and COOH-terminally truncated forms translocate to the nucleus. Because the short forms of GLI retain their DNA-binding domain, but have lost their transactivation domain, they act as transcriptional repressors. Following SHH signaling, GLI cleavage is inhibited. There are three GLI proteins. The transcriptional activity of GLI1 {12q13.2-q13.3} is negatively regulated by SUFU (Suppressor of Fused). The activation of GLI2

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