Glial Cell Line Derived Neurotropic Factor GDNF

{5p13.1-p12} specifically promotes the survival and differentiation of dopaminergic neurons, and it enhances the high affinity uptake of dopamine in these cells. Neurturin (NRTN) {19p13.3} is a potent neurotrophic factor. NRTN and GDNF form a distinct TGF-P subfamily, referred to as TRNs (TGF-P-Related Neurotrophins).

The growth factors GDNF and Neurturin ligate a receptor heterodimer of the proto-oncogene product RET {10q11.2} and a glycosyl-phosphatidylinositol-anchored GDNF Receptor a {10q26} surface molecule. This leads to RET tyrosine phosphorylation. The ensuing signal transduction comprises GRB2, SHC, Phosphatidylinositol 3-Kinase. Oncogenic RET also activates RAS, RAF, MEKK1, and I-kB Kinase P. It leads to phosphorylation and degradation of I-kB and to activation of NF-kB, which may promote cell expansion through its antiapoptotic effects [Ludwig etal. 2001].

• Germline activation of the ret gene, attributable to specific point mutations, causes medullary thyroid carcinoma, a neoplastic transformation of the Calcitonin-secreting thyroidal C-cells.

• Mutations of ret may also cause multiple endocrine neoplasia type 2 (C634R in MEN2A, M918T in MEN2B).

• Constitutive kinase activity may be conferred to RET by chromosomal translocation that forms the RET-PTC fusion and occurs in 30% of papillary thyroid carcinomata [Grieco et al. 1990].

Colony-Stimulating Factors. Colony-Stimulating Factors (CSFs) are necessary for the survival and proliferation of hematopoietic progenitor cells. They are named by the cells they stimulate. CSF-1 (Colony Stimulating Factor-1, Macrophage Colony-Stimulating Factor, M-CSF) is synthesized by activated monocytes and macrophages, as well as by fibroblasts and other mesenchymal cells. Mature CSF-1 is secreted as a disulfide-bonded, heterodimeric proteoglycan with a molecular weight of 80 kD. The primary transcript {1p21-p13} encodes a precursor of 554 amino acids, which contains a transmembrane domain. The receptor for CSF-1 is a member of the PDGFR family. CSF-2 (GM-CSF, Granulocyte-M-CSF) {5q31.1} is a homodimeric glycoprotein of 45 kD. The csf-3 (granulocyte colony-stimulating factor, g-csf) gene {7q11.2-7q12} has four introns and two distinct polypeptides are synthesized from the same gene by differential splicing of the message in the second intron. The two polypeptides differ by the presence or absence of three amino acids. G-CSF stimulates the proliferation of promyelocytes and myelocytes.

• Children with type-1 neurofibromatosis (NF1) are predisposed to juvenile myelomonocytic leukemia. GM-CSF (CSF-2) is required to drive the excessive proliferation of the affected myelomonocytic cells.

• CREB (cyclic-AMP Response Element-Binding Protein) is a transcription factor that is a downstream component of the GM-CSF signaling pathway. It is overexpressed in blast cells from patients with acute leukemias. CREB contributes to hematopoiesis, cell proliferation, and acute leukemogenesis [Shankar and Sakamoto 2004].

• G-CSF (CSF-3) is expressed in bladder carcinoma and in glioblastoma multiforme.

Interleukins. Interleukins (ILs) are a group of cytokines that are expressed predominantly by white blood cells as a means of communication. The functions of the immune system depend heavily on the effects of Interleukins. These cytokines often act as growth factors for their target cells.

- IL-1 is secreted by macrophages, monocytes, and dendritic cells. It increases the expression of adhesion molecules on endothelial cells to enable the transmigration of leukocytes.

- IL-2 contributes importantly to cellular immunity. It facilitates G1 to S transition in T-lymphocytes by inactivating P27KIP1.

- IL-6 may induce the differentiation of myelomono-cytic leukemic cells into macrophages.

- IL-10 enhances the humoral (antibody-dependent) arm of the immune response.

- IL-13 is expressed in activated T-lymphocytes. It inhibits inflammatory cytokine production induced by lipopolysaccharide in peripheral blood monocytes.

The biological activities of Interleukins are mediated by specific membrane receptors which can be expressed on a wide range of cell types. In most cases, their expression is inducible and subject to several regulatory mechanisms. Many receptors are multi-subunit structures with ligand-binding domains and domains that function as signal transducers due to their intrinsic tyrosine kinase activity. Interleukin Receptors often share common signal-transducing receptor components in the same family, and are associated with specific signal transduction pathways in the interior of the cell. Several cytokine receptors can be converted into soluble binding proteins that regulate ligand access to the cell by specific proteolytic cleavage of receptor ectodomains.

• Polymorphisms in the il-1 gene cluster are associated with a predisposition to gastric cancer. A transition of the normal mucosa to gastritis, which eventually leads to adenocarcinoma, is strongly associated with infection by Helicobacter pylori. Individuals with the il-1ft polymorphisms-31 C/C or -511 T/T, or with the il-1ft receptor penta-allelic 86 bp tandem repeat in intron 2 are at elevated risk. These alleles cause high expression levels of IL-1 and increase the probability of hypochlorhydria, gastric atrophy, and consecutive distal gastric adenocarcinoma.

• A polymorphic site in intron 2 of the il-1ra (IL-1 receptor antagonist) gene influences the IL-1RA plasma level. Elevated IL-1P levels are associated with homozygosity for il-1ra allele 2 (IL-1RN*2). Individuals heterozygous for il-1ra have an increased risk of cervical cancer [Sehouli et al. 2002].

• Pancreas cancer cells express receptors for IL-2 at high density.

• IL-2, IL-6, and IL-7 act as proliferative factors for malignant lymphocytes and plasma cells.

• IL-10 is a vital factor for the differentiation and survival of germinal center B-lymphocytes and is also a negative prognostic factor in non-Hodgkin lymphoma. TIMP-1 regulates the IL-10 expression levels in B-cells and mediates specific B-cell differentiation steps. TIMP-1 expression in B-cell non-Hodgkin lymphoma correlates closely and positively with IL-10 expression and with high histologic grade. Through the inhibition of apoptosis, TIMP-1 may be responsible for the negative prognosis associated with IL-10 expression in these tumors. [Guedez et al. 2001].

• IL-13 and IL-13Ra1 are frequently expressed in Reed-Sternberg cells that exist in Hodgkin lymphoma tissues. IL-13 is important for proliferation and antiapoptosis in Hodgkin lymphoma cells. STAT6 is an important mediator of IL-13 function, which is often activated in Reed-Sternberg cells [Skinnider et al. 2002]. In contrast, IL-13 expression in non-Hodgkin lymphoma is uncommon.

Transforming Growth Factor-b Family. Transforming growth factors (TGFs) are secreted polypep-tides that may reversibly confer a transformed phenotype on cells. TGF-P is the prototypic member of this family of about 30 polypeptide regulatory molecules that also includes Activins (Inhibins), Bone Morphogenic Proteins, Nodal, and the Mullerian Inhibitory Substance. TGF-P exists in three forms (TGF-P1, TGF-P2, TGF-P3), encoded by distinct genes, that act as modulators of tumor growth. The TGF-P protein is released as a latent complex comprising the TGF-P dimer in association with two prosegments. Plasmin activates latent TGF-P. TGF-P is also released by MMP-2 or MMP-9 from its inactive extracellular complex.

The receptors for this family of growth factors are typically composed of a pair of subunits, type I of 53 kD and type II of 70 kD, each of which has a cyto-plasmic portion that functions as a serine-threonine kinase. There are seven distinct type-I receptors, each of which can associate with five distinct type-II receptors to mediate the signals activated by the TGF-P family of ligands. After binding of TGF-P, the kinase TGF-PRII phosphorylates TGF-PRI in the GS sequence, which is located upstream of the kinase domain. This activates the TGF-PRI kinase and allows autophosphorylation as well as the phosphory-lation of downstream targets. Signal transduction from three of the TGF-P type-I Receptors proceeds via the receptor associated SMADs -2 and -3, consecutive oligomerization with SMAD-4, and translocation into the nucleus. Signaling from four of the TGF-P type-I Receptors proceeds through the R-SMADS SMAD-1, -5, or -8. TGF-P receptor engagement primarily inhibits epithelial cell proliferation, typically causing cell cycle arrest in G1

- Through inhibition of c-myc expression. This is required for the induction of p15INK4b (cdkn2B) and p21CIP1/WAF1 (cdkn1A) expression. In proliferating cells, c-MYC is tethered to the proximal promoter regions of the cdkn2B and cdkn1A genes by the zinc finger protein MIZ-1, causing inhibition of transcription. c-MYC down-regulation in response to TGF-P relieves this inhibition.

- Through complex formation of SMADs with

FOXO proteins to activate the transcription of p21 CIP1/WAF1

- Through activation of the transcription factor SP1, which plays a pivotal role in inducing the p15INK4b and p21CIP1/WAF1 genes by TGF-P. In response to TGF-P,p15INK4b is transcribed and its accumulation on CDK4 displaces P27KIP1, which then inhibits the Cyclin E/CDK2 complex.

- Through the down-regulation the CDK2 activator CDC25A. TGF-P increases CDK tyrosine phos-phorylation by repressing the CDK-activating tyrosine phosphatase CDC25A.

- Through repression of the growth-promoting transcription factors id-1, id-2, and id-3. ID

proteins function as negative regulators of basic helix-loop-helix (bHLH) transcription factors, that are crucial for cell differentiation.

- Through phosphorylation of RB. Increased binding of HDAC-1 to RB-1 and to P130RB2 forms a complex that inhibits the gene expression of cdc25A.

- Through activation of serine/threonine kinases. Negative control of proliferation can be exerted via serine/threonine kinase pathways.

In certain cell types, TGF-P-dependent signaling may induce apoptosis. This can be accomplished through down-regulation of BCL-XL or through activation of Caspase-3 and Caspase-8. During development, TGF-P family signals mediate key decisions that specify stem cell maintenance and germ layer differentiation.

Thrombospondin is an extracellular matrix protein with multiple functional domains and pleiomorphic roles in tumorigenesis. At the boundary between the first and second type-1 repeats, Thrombospondin contains a sequence motif, RFK, which binds and activates TGF-p. The interaction between Thrombospondin and TGF-P inhibits tumor growth.

The Activins and Inhibins constitute dimers of the subunits a, encoded by inhibin a{2q33-q36}, PA, encoded by inhibin flA (inhibin b1, activin A, frp, edf {7p15-p13}, PB, encoded by inhibin ftB (inhibin b2, activin B) {2cen-q13}, or PC, encoded by inhibin fiC (activin C) {12q13.1}. Activins are homodimers or heterodimers of related P subunits, while inhibins are heterodimers composed of one a subunit and one P subunit. They have roles in reproduction and development. Activins regulate pituitary function, hormone production in gonadal tissues, and differentiation of erythroid and neural cells. Activins interact with the receptors ACVR-I (ALK-2), ACVR-IB (ALK-4), ACVR-IC (ALK-7), and ACVR-II or ACVR-IIB.

Bone Morphogenic Proteins (BMPs, Osteogenic Proteins, Growth and Differentiation Factors, GDFs) are multifunctional growth factors Their expression is often widespread and dynamic as development proceeds. BMPs are frequently localized to areas of epithelial-mesenchymal interactions, including extraskeletal sites. They have importance in cellular functions and in the embryonic development of heart, nervous system, cartilage, and bone. The BMP family has around 30 members. BMPs are synthesized as large precursor proteins. Upon dimerization, they are proteolytically cleaved at a consensus RX2R site to yield COOH-terminal mature dimers.

BMPs signal through serine/threonine kinase receptors, composed of type-I and type-II subunits. There are three type-I receptors, BMPR-IA (ALK-3), BMPR-IB (ALK-6), and ACVR-IA (ALK-2), and there are three type-II receptors, BMPR-II, ACVR-II, and ACVR-IIB. These receptors are expressed differentially in various tissues. In response to engagement by ligands, the BMP Receptors form a heterotetrameric complex of two type-I and two type-II chains, which initiates signaling. Downstream, SMAD-1, SMAD-5, SMAD-8, and SMAD-9 become phosphorylated. BMP functions are negatively regulated by Chordin and Noggin, which bind BMPs, sequester them in latent complexes, and block their signaling.

The morphogen Nodal {10} is a member of the TGF-P family that is expressed during gastrulation. Nodal has a left-sided expression pattern that plays important roles in left or right axis development. Moreover, Nodal signals from the epiblast pattern the visceral endoderm by activating a SMAD-2-dependent pathway required for the specification of anterior identity in overlying epiblast cells. The transcriptional core-pressor DRAP1 has a very specific role in regulation of Nodal activity during embryogenesis. It interacts with and inhibits DNA binding by the winged-helix transcription factor FOXH1, a critical component of a positive feedback loop for Nodal activity.

The gene for Mullerian-Inhibiting Substance (MIS, Anti-Mullerian Hormone, AMH) {19p13.3-p13.2} has five exons and encodes a 560 amino acid polypeptide. MIS causes regression of the Mullerian duct in the testes during fetal development, thus preventing the development of a uterus and fallopian tubes. It is expressed transiently in the ovaries.

• The T29C polymorphism in the transforming growth factor-b1 gene (leading to L10P) is associated with increased TGF-Pj serum levels and a decreased risk of breast cancer [Ziv et al. 2001].

• In tumor cells, the expression levels of TGF-P Receptors may be reduced by transcriptional silencing of the receptor genes secondary to hyper-methylation of CpG islands or promoter mutations. This confers resistance to the growth inhibitory effects of TGF-P.

• A polymorphic allele (6A) of tgf-prI has a deletion of three alanines from a stretch of nine alanines. Among patients with colon cancer, there is an elevated number of tgf-firI(6A) homozygotes. tgf-brI(6A) acts as a tumor susceptibility allele that contributes to the development of colon cancer by way of reduced TGF-P-mediated growth inhibition [Pasche et al. 1999]. Mutations in tgf-b receptor I arise in chronic lymphocytic leukemia, prostate cancer, gastric cancer, metastatic breast cancer, and glioblastoma. A frameshift mutation in tgf-b receptor l frequently occurs in ovarian cancer. Whereas, retinal cells bear receptors for TGF-P, retinoblas-toma cells lack these receptors. In general, tumors acquire TGF-P resistance at a relatively late stage.

• Inactivating mutations of tgf-brII may be present in 20-25% of colon cancers. Somatic mutations in tgf-b receptor II occur most frequently in the tumors of hereditary nonpolyposis colorectal cancer. A repeat stretch of adenines in the coding sequence is prone to mutations in these patients. This may result in a truncated receptor protein that is incapable of signaling.

• The EWS-FLI oncoprotein, which incorporates a partial coding sequence for the ETS family transcription factor FLI-1, represses the expression of tgf-brII and may account for the decreased responsiveness of Ewing sarcoma cells to TGF-p.

• Cytoplasmic PML is an essential modulator of TGF-P signaling. The PML-RARa oncoprotein of acute promyelocytic leukemia can antagonize cytoplasmic PML function. Consistently, acute promyelocytic leukemia cells have defects in TGF-P signaling.

• The intestinal epithelium is patterned into crypts and villi, with BMP-4 expression occurring exclusively in the intravillus mesenchyme. The TGF-P family member BMP-4 is overexpressed and secreted by human cancer cells with mutant apc (adenomatous polyposis coli) gene. The oncogenic allele of b-catenin is absolutely required for the expression of BMP-4, whose receptor, bmprlA, is mutated in a fraction of the rare inherited gastrointestinal cancer predisposition syndrome juvenile intestinal polyposis [Howe et al. 2001].

• The osteogenic BMP-6 (VGR-1) is expressed in a high proportion of prostate carcinomata, but not in benign prostate tissue. Its levels increase in higher grade tumors. The gene expression is androgen independent. BMP-6 may contribute to the formation of osteoblastic metastases.

• MIS may inhibit the growth of ovarian and endometrial cancers.

Steroid hormones. The growth of some epithelia is regulated by steroid hormones. These types of cells can give rise to tumors that also depend on steroid hormones for growth. Steroid-related neoplasms include breast cancer, prostate cancer, and liver cancer. During tumor progression, these growths may loose their hormone dependence.

The steroid hormones (Figure 3.1.3.I), with the exception of retinoic acid, are derived from cholesterol. All but vitamin D contain the same cyclopen-tanophenanthrene ring as cholesterol. The first reaction in converting C27 cholesterol to C18, C19, and C21 steroids involves the cleavage of a 6-carbon group from cholesterol. It is the principal committing, rate-limiting step in steroid biosynthesis. This cleavage reaction is catalyzed by P450 CYP11A (Desmolase, 450-Linked Side Chain Cleaving Enzyme, P450SCC), which is located in the mitochondria of steroid-producing cells. Various Hydroxylases involved in the synthesis of the individual steroid hormones also are members of the Cytochrome P450 class of enzymes. The adrenal cortex is responsible for the production of glucocor-ticosteroids that regulate carbohydrate metabolism, mineralocorticosteroids that regulate the levels of sodium and potassium, and androgens that are male sex steroids. The adrenal cortex is responsive to ACTH (Adrenocorticotropic Hormone), the levels of which are controlled by the hypothalamus and pituitary gland. The testes and ovaries produce various steroids, the two most abundant ones are testosterone and estradiol. These compounds are under the control of FSH (Follicle-Stimulating Hormone) and LH (Luteinizing Hormone), secreted by the pituitary and GNRH (Gonadotropin-Releasing Hormone), secreted by the hypothalamus.

Androgen. The cells of the normal prostate depend on androgens for growth and survival. In this setting, androgens protect the prostate cells by blocking apoptosis. Androgens may inhibit the Caspases -7, -8, and -9, and may attenuate BAX expression and cleavage. Testosterone is the main circulating androgen, mostly bound to Albumin or SHBG (Sex Hormone-Binding Globulin).

The Androgen Receptor is composed of an NH2-terminal activation domain, a COOH-terminal ligand-binding domain, and a DNA-binding domain containing two zinc fingers in the midre-gion. Like other nuclear receptors, the Androgen

Zona glomerulosa P450ssc

Zona fasciculata P450ssc

Pregnenolone p450c1^^"'---..3ß-DA,H5.isomerase 17-OH pregnenolone


17-OH progesterone P450c21

Zona glomerulosa P450ssc

Pregnenolone 3-ßDH A45-isomerase Progesterone Progesterone

P450c21 11-deoxycorticosterone P450c11


Zona reticularis P450ssc


I P450c17 reg

11-deoxycortisol P450c11

11-deoxycorticosterone P450c11

11-deoxycorticosterone P450c11

Cortisol Corticosterone

Corticosterone aldosterone synthase

17-OH pregnenolone 17,20 lyase Dehydroepiandrosterone (DHEA)


Sulfotransferase ^ DHEA-S

ho h2c ch2oh o ' 2 n c=o aldosterone synthase ho h2c o ' 2 n c=o


Cortisol Corticosterone


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