Pathways

As in adult tissues, signaling through MAPK and other pathways is involved in the regulation of cell proliferation, cell differentiation and apoptosis during ontogenetic development. However, the development of an embryo is, of course, neither a homeostatic replacement nor a simple expansion, but involves many decisions on the developmental fates of individual cells and of cell populations.

From the pluripotent cells in the epiblast, some are developed into primordial germ cells, while others form the germ layers which interact further to form tissues and organs. This requires again cell fate decisions as well as an excellent coordination between the expansion of more or less committed precursor populations by cell proliferation and their differentiation into temporally quiescent or terminally differentiated cells.

In many developing tissues, cells are set aside to form stem cell or precursor populations that allow a continuous supply of differentiated cells exerting specific tissue functions or at least a replenishment of the differentiated cell compartments after tissue damage. Many growth factors and signaling pathways discussed in the preceding part of the chapter are also involved in these processes, but several additional pathways function specifically in cell fate decisions and in the establishment of tissue stem cells and their regulation in adult tissues. Three of them have also become notorious in the context of human cancers. They are the WNT (Figure 6.12), the Hedgehog/SHH (Figure 6.13) and the NOTCH (Figure 6.14) pathways.

Figure 6.12 The WNT/fi-Cateninpathway The inactive state (in the absence of WNT factor binding) is shown on the left and the active state on the right. This simplified scheme does not incorporate cross-talk with other pathways (see e.g. Figure 6.3) and pathway modulators discussed in chapters 13.2 and 16.2.

Figure 6.12 The WNT/fi-Cateninpathway The inactive state (in the absence of WNT factor binding) is shown on the left and the active state on the right. This simplified scheme does not incorporate cross-talk with other pathways (see e.g. Figure 6.3) and pathway modulators discussed in chapters 13.2 and 16.2.

Figure 6.13 The Sonic Hedgehog (SHH) pathway SHH binding to the PTCH1 membrane receptor alleviates inhibition of SMO. By unknown mechanisms GLI1 is released from a protein complex at the microtubules containing SUFU and likely a FU-like protein. GLI1 migrates to the nucleus activating transcription of target genes, which include GLI factors itself as well as PTCH1 and another feedback inhibitor, HIP1. The pathway is also inhibited by active PKA. Note that the GLI factor cascade may differ between cell types; the version shown here is the most likely one in keratinocytes.

Figure 6.13 The Sonic Hedgehog (SHH) pathway SHH binding to the PTCH1 membrane receptor alleviates inhibition of SMO. By unknown mechanisms GLI1 is released from a protein complex at the microtubules containing SUFU and likely a FU-like protein. GLI1 migrates to the nucleus activating transcription of target genes, which include GLI factors itself as well as PTCH1 and another feedback inhibitor, HIP1. The pathway is also inhibited by active PKA. Note that the GLI factor cascade may differ between cell types; the version shown here is the most likely one in keratinocytes.

More than 20 factors belong to the WNT growth factor family in humans. They are «40 kDa proteins with >30% homology towards each other. The proteins are secreted after being being glycosylated and covalent linkage of a lipophilic moiety. Accordingly, the factors bind well to the extracellular matrix and stick to cell membranes, which restricts their diffusion. Therefore, they are limited to acting as paracrine or autocrine factors. During development, WNT proteins drive the expansion and morphogenesis of many different tissues, in particular of tubular and ductular epithelial structures in the kidney and the gut. Of course, WNT factors and pathways interact with others, e.g. with Hedgehog-dependent pathways in the development of the limbs. Individual WNT factors remain involved in the homeostasis of adult tissue and specifically seem to control stem cell compartments, e.g. in the gastrointestinal tract.

Figure 6.14 The NOTCH pathway See text for explanation.

WNT factors are normally recognized by Frizzled receptor proteins (FZD) at the cell surface. There are again several of these that respond to different WNT factors. They are supported by specific LRP proteins (LRP5 and LRP6) which seem to recognize the lipid part of the WNTs and are counteracted by secreted Frizzled related proteins (SFRP). The FZD receptors may not only differ with respect to which WNT they bind, but also with respect to which intracellular pathways they stimulate. At least three different pathways are known, but only one is really well characterized. The decisive step in this pathway is the accumulation and activation of P-Catenin which is prevented by the coordinated action of several proteins including APC, Axin and GSK30 (Figure 6.12, ^13.2). This particular pathway is therefore called the WNT/p-Catenin or 'canonical' WNT pathway. Its constitutive activation is crucial in the development of colon cancer (^13.2). It is also important in other cancers, e.g. in hepatocellular carcinoma (^16.2). Its deregulation is alternatively brought about by loss of function of negative regulators, such as APC

or Axin or by oncogenic mutations activating P-Catenin. APC and CTNNB1 (encoding P-Catenin) therefore behave as tumor suppressor and oncogene, respectively (Table 6.1).

The WNT/Ca2+ pathway involves activation of classical PKCs and other Ca2+-dependent protein kinases. Its function is largely undefined, but it may promote cellular differentiation to a greater extent than the 'canonical' pathway. The WNT/polarity pathway involves the small GTP-binding protein RHO and acts on the polarization of the cytoskeleton. It also leads to phosphorylation of JNK1 kinase and thus to cross-talk with this MAPK pathway. These two other pathways are little studied so far in the context of human cancers. Likewise, the significance of altered expression of WNT factors, of FZD receptors and of SFRP1 in several different types of human cancers is still under study. However, at least one instance of WNT factor activation by a retroviral insertion (^4.2) has been documented.

Somewhat comfortingly, there are only three Hedgehog proteins in man. They are Sonic Hedgehog (SHH), Desert hedgehog (DHH), and Indian Hedgehog (IHH). Like WNT factors, they act in a paracrine fashion; and as for WNTs, their diffusion is limited by a lipophilic modification, in this case covalent attachement of cholesterol. Hedgehog factors are perhaps best studied for their role in limb development, but they are certainly also crucial for the development of many other organs, as diverse as the brain and the prostate. Also like WNTs, they contribute to tissue homeostasis in fully grown humans and, in fact, are also implicated in the maintenance of stem cell populations. The most important factor in adult tissues is SHH. Hedgehog factors bind to smoothened receptors such as SMO1 and initiate their own intracellular signaling pathway which ultimately leads to the activation of GLI transcriptional activators. Activation of SMO1 is prevented by PTCH1 (Figure 6.13, ^12.3). Perhaps unsurpringly, there is accumulating evidence of cross-talk between the SHH and WNT pathways inside the cell.

Constitutive activation of SHH signaling appears to lie at the core of several cancers with a precursor cell phenotype such as small cell lung cancer and basal cell carcinoma of the skin (^12.3). This activation can alternatively be brought about by mutations in activating components, such as point mutations in SMO1 or - perhaps -overexpression of GLI transcription factors, or by inactivation of a negative regulatory component, usually PTCH1. So, these have to be regarded as oncogenes and tumor suppressors, respectively (Table 6.1).

Like the WNT and SHH pathways, the NOTCH pathway was first encountered in Drosophila and has been more extensively studied for its function in development in model organisms than in humans. It is now firmly established as a cancer pathway in man, and is also considered to factor in the neurological Alzheimer degenerative diseases. The emerging consensus is that both disruption and overactivity of the NOTCH pathway can promote cancer development. This ambiguity is not entirely unprecedented for cancer pathways, since the TGFp and STAT pathways, e.g., act differently in different cell types. In the case of NOTCH signaling, its ambivalence is related to its pronounced dosage sensitivity which derives from its biological function.

Like the WNT and SHH pathways, the NOTCH pathway also controls stem and precursor cell compartments. More precisely, its characteristic function is the regulation of binary cell fate decisions. This includes the decision of whether a cell remains a tissue precursor or goes on to differentiate, such as in the basal layer of the epidermis. NOTCH signaling is also involved in decisions like whether a differentiating intestinal cell becomes an enterocyte or a secretory Paneth or goblet cell or whether a cell in the lymphocyte lineage enters the T-cell or B-cell sub-lineage. In system theory, this kind of decision is called a bifurcation and its control requires a metastable equilibrium which develops towards either of two opposite states upon slight perturbations, like a coin standing on its edge. This comparison describes the kind of function provided by the NOTCH signaling system.

Four different NOTCH receptors, NOTCH1-4, are known, and are expressed on the cell surface. They are activated by ligands expressed on the cell surface of neighboring cells. Two different kinds of ligands are known. In humans, they comprise the three 'delta-like' DLL1, DLL3, and DLL4 and the 'jagged-like' JAG1 and JAG2 ligands. These ligands differ somewhat in the response they elicit and more substantially in their sensitivity towards modification of NOTCH receptors by FRINGE glycosylases. These elongate glycosyl chains on NOTCH receptors that prevent binding of JAG, but not of DLL proteins.

Importantly, expression of NOTCH receptors and their ligands is each self-reinforcing and cross-inhibitory and therefore tends to become mutually exclusive. Thus within an organized tissue, different types of cells express either receptors or specific ligands. A precursor cell population, e.g., may express a receptor, while cells that have taken a step towards differentiation express a ligand, or the other way round. The latter situation is found, e.g., in human epidermis, where basal cells express NOTCH ligands and cells in upper, differentiated layers express NOTCH1.

NOTCH receptors are heterodimers formed by proteolytic cleavage from a single precursor (Figure 6.14). The protease involved is the y-secretase presenilin whose dysfunction is one cause of Alzheimer disease. The extracellular domain of NOTCH binding the ligands on neighboring cells contains EGF-like repeats and a cystein-rich domain termed LN. The second subunit remains bound to it by a small extracellular domain, continues through the membrane into its larger intracellular segment. This segment contains ankyrin repeats, which mediate protein-protein interactions, a nuclear localization signal, a transcriptional transactivation domain, and a PEST sequence likely responsible for regulated proteolytic degradation.

Following activation by ligands, the intracellular NOTCH domain (sometimes called TAM) becomes free to move into the nucleus, where it replaces repressor proteins from the CBF1 transcription factor (also known by multiple other names such as CSL or RBJ1) to activate its target genes. Activation of NOTCH receptors additionally elicits CSL-independent events which are not as well elucidated. NOTCH target genes appear to be different in different cell types. Thus, in neuronal precursors NOTCH signaling inhibits expression of neuron-specific genes. In keratinocyte precursors, it induces differentiation markers and the p21CIP1 CDK inhibitor causing cell cycle arrest. NOTCH signaling often inhibits WNT/p-Catenin, SHH and AP1 signaling, and, conversely, supports NFKB activation.

As would be expected from its function in normal epidermis, NOTCH receptors appear to function as tumor suppressors in this organ. Down-regulation of NOTCH1 and NOTCH2 is a regular finding in basal cell carcinoma, although the extent to which it contributes to this tumor is debated. As NOTCH signaling inhibits the SHH pathway, its loss may exacerbate the overactivity of this pathway that causes this type of cancer (^12.3). Similarly, loss of NOTCH function may be a prerequisite for formation of small cell lung cancers, which also show activation of the SHH pathway, likely by an autocrine mechanism. In this case, NOTCH activity appears to prevent the precursor cells from adopting the neuroendocrine, 'stem-cell-like' phenotype displayed by these cancers.

In other cancers, NOTCH proteins undoubtedly function as oncogenes. One type of T-cell acute leukemias (T-ALL) is characterized by a translocation between chromosome 7 and chromosome 9, t(7;9) (q34;q34.3), which leads to the overexpression of the cytoplasmic domain of NOTCH1 under the influence of the T-cell receptor P enhancer. Constitutively active NOTCH signaling appears to direct an inappropriately large fraction of lymphocyte precursors towards a T-cell fate where they become malignant by further mutations. In a similar fashion, NOTCH overactivity appears to cooperate with viral oncoproteins such as the SV40 Tantigen in mesothelioma and HPV E6 and E7 in genital cancers. Here, the inhibitory effect of NOTCH signaling on the cell cycle is abrogated by the viral oncoproteins whereas its precursor cell maintenance function remains active and contributes to expansion of the tumor.

Further reading

Krauss G (2003) Biochemistry of signal transduction and regulation 3rd ed. Wiley-VCH Polakis P (2000) Wnt signaling and cancer. Genes Devel. 14, 1837-1851

Taipale J, Beachy PA (2001) The Hedgehog and Wnt signalling pathways in cancer. Nature 411, 349-354 Attisano L, Wrana JL (2002) Signal transduction by the TGFP superfamily. Science 296, 1646-1647 Cantley LC (2002) The Phosphoinositide 3-Kinase pathway. Science 296, 1655-1657 Hood JD, Cheresh DA (2002) Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2, 91100

Martin KH et al (2002) Integrin connections map: to infinity and beyond. Science 296, 1652-1653 Sears RC, Nevins JR (2002) Signaling networks that link cell proliferation and cell fate. JBC 277, 1161711620

Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-kinase/AKT pathway in human cancer. Nat. Rev. Cancer 2, 489-501

Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-P family signalling. Nature 425, 577-584 Heinrich PC et al (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem. J. 374, 1-20

Knowles MA, Hornigold N, Pitt E (2003) Tuberous sclerosis complex (TSC) gene involvement in sporadic tumours. Biochem. Soc. Transact. 31, 597-602 Maillard I, Pear WS (2003) Notch and cancer: best to avoid the ups and downs. Cancer Cell 1, 203-205 Nickoloff BJ, Osborne BA, Miele L (2003) Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents. Oncogene 22, 6598-6608 Parsons JT (2003) Focal adhesion kinase: the first ten years. J. Cell Sci. 116, 1409-1416 Pouyssegur J, Lenormand P (2003) Fidelity and spatio-temporal control in MAP kinase (ERKs)

signalling. Eur. J. Biochem. 270, 3291-3299 Radtke F, Raj K (2003) The role of Notch in tumorigenesis: oncogene or tumour suppressor. Nat. Rev. Cancer 3, 756-767

Siegel PM, Massague J (2003) Cytostatic and apoptotic actions of TGF-P in homeostasis and cancer. Nat Rev Cancer 3: 807-820

Van Es, Barker N, Clevers H (2003) You Wnt some, you lose some: oncogenes in the Wnt signaling pathway. Curr. Opin. Genet. Devel. 13, 28-33 Greten FR, Karin M (2004) The IKK/NF-kappaB activation pathway-a target for prevention and treatment of cancer. Cancer Lett. 206, 193-199 Jin H, Varner J (2004) Integrins: roles in cancer development and as treatment targets. Brit. J. Cancer 90, 561-565

Lum L, Beachy PA (2004) The hedgehog response network: sensors, switches and routers. Science 304, 1755-1759

Slee EA, O'Connor DJ, Lu X (2004) To die or not to die: how does p53 decide? Oncogene 23, 2809-2818 Yu H, Jove R (2004) The STATs of cancer - new molecular targets come of age. Nat. Rev. Cancer 4, 97105

0 0

Post a comment