Epigenetics Of Tissue Homeostasis

stably inherited phenotypes can not only be achieved by mechanisms acting within one cell, but also by interactions between similar or distinct cell types. In many tissues, such interactions occur between the mesenchymal and the epithelial component. They exchange paracrine factors in the steady-state of the tissue. Upon wounding or infection, the steady-state is disturbed and the exchange intensifies leading to wound healing and immune responses.

In the skin, paracrine factors are exchanged between keratinocytes in the epidermis and fibroblasts in the dermis (Figure 8.9). Epithelial keratinocytes in the epidermis produce, a.o., the cytokine interleukin-1 (IL1), of which normally only a fraction reaches the underlying dermal tissue. Mesenchymal cells produce and secrete low amounts of the fibroblast growth factor 7 (FGF7), which is also called keratinocyte growth factor (KGF), because it stimulates the proliferation of

Figure 8.9 Paracrine regulation of wound healing in the skin GF: growth factor. See text for futher explanation.

keratinocytes and other epithelial cells. They likewise produce low amounts of GM-CSF, a factor stimulating the proliferation and maturation of myeloid cells. It acts on keratinocytes, too, promoting their proliferation, but more strongly their differentiation. Upon damage to the epidermis, IL1 is released and binds to its receptor on fibroblasts. This activates the JNK and p38 MAPK pathways (^6.2) and leads to an increased activity of JUN transcriptional activators at the promoters of the FGF7 and GMCSF genes. Increased secretion of FGF7 and GM-CSF stimulates proliferation and differentiation of the keratinocyte compartment, until it is healed and the IL1 concentration returns to normal levels. This is, of course, a simplified description, as many more factors are involved. Moreover, during wound healing a substantial reorganization of the extracellular matrix takes place which is performed, e.g., by proteases secreted from fibroblasts and invading immune cells in response to IL1 and GM-CSF (^9.3). Immune cells are attracted by cytokines, chemokines and other factors from the activated fibroblasts. Another level of regulation is required to limit the ensuing inflammation.

The crucial argument in this example is that normal tissues use mutual paracrine interactions to achieve tissue homeostasis and to react appropriately to its disturbances. This could certainly be considered an epigenetic mechanism. Importantly, this type of interaction also takes place in cancers between tumor cells and the tumor stroma. However, in cancers, these interactions are grossly disturbed and do not lead back to a steady-state, but to a continued expansion of the tumor mass (^9.6). Disturbances of paracrine interactions are also crucially involved in the co-carcinogenic effect of HIV (Box 8.1).

A case in point is angiogenesis (^9.4), an essential process in many cancers,

MIL mutation

Figure 8.10 The vicious cycle of angiogenesis in cancer See text for further explanation.

MIL mutation

Figure 8.10 The vicious cycle of angiogenesis in cancer See text for further explanation.

that can be activated by genetic or epigenetic mechanisms (Figure 8.10). Some cancers carry mutations which lead to the constitutive production of pro-angiogenic growth factors such as VEGF or bFGF (FGF2) that stimulate branching of capillaries and proliferation of endothelial cells. In benign tumors and malignant renal cell carcinomas arising in the Von-Hippel-Lindau syndrome (^15.4), this constitutive production is due to mutations in a regulator of the cellular response to hypoxia. As a consequence, HIF (hypoxia-induced factor) transcription factors are overactive and enhance the production of pro-angiogenic growth factors. So, in this case, a specific genetic change is responsible for increased angiogenesis.

Other cancers do not carry according genetic defects. Instead, when the tumor mass has exceeded the size that allows sufficient supply of oxygen by diffusion, tumor cells become hypoxic. This elicits a normal physiological response, viz. induction of HIF transcription factors leading to the production of angiogenic growth factors. These stimulate angiogenesis. Accordingly, the supply of oxygen (and other nutrients) improves allowing further expansion of the tumor to the point where oxygen becomes limiting again leading to further induction of HIF, angiogenic growth factors and continued angiogenesis. This epigenetic vicious circle is often exacerbated by genetic defects in the cancer cell, e.g. TP53 mutations that diminish the production of anti-angiogenic factors.

It is important to realize that by such paracrine interactions the tumor cells change the character of the normal cells with which they interact. While these cells need not become genetically altered (although this has been occasionally reported), they are persisently activated, which can alter their properties considerably. Experimentally, it can be demonstrated that stromal cells from malignant tumors acquire an 'epigenetic memory', i.e., their activated state tends to persist even if the actual cancer cells are removed. This is plausible, if one considers the role of epigenetics in cell differentiation (^8.5).

Interactions between tumor cells and neighboring normal stromal cells are particularly important during metastasis. Setting up stable interactions is crucial for the survival and eventual expansion of metastatic cells. This presupposes a selection for those cancer cells which fit into the target tissue and their successful adaptation to the local environment. For instance, metastatic prostate cancer cells adapt so well to the microenvironment in the bone by interacting with local osteoblasts and osteoclasts that they have been termed 'osteomimetic' (^19.4). As in normal tissues, these mutual interactions are to a great deal mediated by exchange of paracrine growth factors, and to some extent by direct cell-to-cell interactions.

A final example of epigenetic mechanisms relevant to both normal tissues and cancer concerns stem cells. Stem cells are defined as cells with unlimited proliferation potential, the ability to generate differentiated derivatives, and the ability to do this by assymetric division generating another stem cell and a more differentiated daughter cell (Figure 8.11). Stem cells which can give yield to any cell-type (in principle) are called pluripotent. In a healthy adult human, two types of stem cells are present: those of the germ-line and tissue stem cells. Tissue stem cells are probably not pluripotent. Rather, they can give rise to a limited number of diverse cell types. They are therefore also labeled as 'tissue precursor' cells and as

Figure 8.11 Properties of stem cells Stem cells are characterized by the abilities to self-renew, divide assymetrically, and give rise to diverse differentiated cell types.

'multi- or oligopotent'. As the DNA of stem cells does not differ from that in (most) somatic cells, they must be defined by epigenetic mechanisms. In fact, both intercellular and intracellular mechanisms are involved.

Intracellular mechanisms include the expression of hTERT and of active telomerase, which allows the escape from replicative senescence (^7.4). The other mechanism inducing replicative senescence upon continued cell proliferation, viz. induction of CDK inhibitors (^7.4) is likewise repressed. Specifically, accumulation of p16INK4A appears to be prevented by a polycomb repressor complex with BMI1 (^8.4) as its crucial component. It is not precisely clear, how pluripotency is maintained. In germ cells, expression of specific transcription factors appears to be involved. For instance, the transcriptional activator OCT3/4, now systematically called POUF5, is expressed in the germ line and in the early embryo. Its expression is lost, when pluripotent cells in the epiblast become committed to specific tissues. It is also strongly expressed in germ cell cancers and is necessary for their continuous proliferation. Germ cells and perhaps tissue precursor cells also have patterns of DNA methylation different from those typical for somatic cells. These are accordingly reflected in germ cell cancers. In germ cells as in germ cell cancers, moreover, expression levels of both RB1 and TP53 may be relatively low.

10 A note of caution: definitions in this field are often very loosely used. So, it is not unusual to find all basal cells in an epithelium summarily denoted as tissue precursor cells or tissue stem cells being called 'pluripotent' on the argument that they can give rise to distinct cell types.

This may prohibit a commitment to specific pathways of differentiation as well as replicative senescence.

In the testes and ovaries, respectively, primordial germ cells reside in an environment that allows their maintenance and organized differentiation towards mature oocytes and spermatozoa. Oogenesis is almost completed after the fetal period, whereas spermatogenesis continues throughout life and the stem cells remain present in the epithelia of the seminiferous tubules of the testes. There, they divide assymetrically to give rise to spermatozoa after several differentiation steps including meisosis. The location within the testicular epithelia is on one hand crucial for the survival of these cells, as they tend to undergo apoptosis outside this environment. On the other hand, primordial stem cells can principally develop into tumors when placed into the wrong environment. This is exemplified by experimental teratocarcinomas in specific mouse strains, which appear to arise by purely epigenetic mechanisms. However, germ cell tumors in humans do show chromosomal aberrations. In contrast, normal primordial germ cells contain the same amount and the same sequence of DNA as somatic cells. Thus, the mechanisms that make them immortal and pluripotent are purely epigenetic. The tubules of the testes are an example ofa stem cell 'niche'. This niche is actively maintained not so much by the germ cells themselves, but by the surrounding testicular tissue. Cells in this tissue provide, e.g., SCF (stem cell factor), a ligand for the receptor tyrosine kinase KIT.

Tissue stem (precursor) cells are less obvious and in humans they are only beginning to become characterized. Like primordial germ cells, however, they do not differ in DNA amount or sequence from their differentiated progeny. Instead, their state appears to be determined by their location in particular niches within a tissue, e.g. near the basis of crypts in the intestine or near the root of hair bulbs in the skin. Maintenance of their state appears to be achieved partly by growth factors produced by the surrounding mesenchyme. WNT factors in the intestine and WNT factors as well as SHH in the skin are thought to be essential (^6.10). Less is known about the intracellular mechanisms which maintain their stem cell character. Very likely, telomerase expression is involved and may be stimulated via MYC through WNT or SHH-dependent pathways.

In contrast to primordial germ cells or cells of the epiblast, tissue-stem cells may not be pluripotent. Rather, they may be committed to a limited spectrum of differentiation fates, which may, however, involve quite different types of cells. For instance, stem cells of the large intestine are precursors of enterocytes, enteroendocrine, Paneth and goblet cells, which exhibit quite different functions. Moreover, if transplanted into a different environment, tissue stem cells may show a great deal of plasticity. Thus, bone marrow stem cells can not only give rise to many different types of blood cells and of the immune system, but even to some kinds of epithelial cells such as hepatocytes or to endothelial cells. Again, these different differentiation potentials must be imposed by epigenetic mechanisms that are at present insufficiently understood.

Clearly, most cancers likewise develop some kind of stem cell phenotype, by different mechanisms (Figure 8.12). In a number of cancers pathways involved in f\ A

SC TA DO

Figure 8.12 Relationship of cancer cells to stem cells Cancer cells can be derived directly from stem cells (SC) retaining their characteristics, but with at least partially blocked differentiation (left), from transient amplifying (TA) cells which do not terminally differentiate and/or secondarily acquire a stem cell phenotype (center), or from differentiated cell types that fail to turn off proliferation and/or revert to a less differentiated precursor stage (right).

SC TA DO

Figure 8.12 Relationship of cancer cells to stem cells Cancer cells can be derived directly from stem cells (SC) retaining their characteristics, but with at least partially blocked differentiation (left), from transient amplifying (TA) cells which do not terminally differentiate and/or secondarily acquire a stem cell phenotype (center), or from differentiated cell types that fail to turn off proliferation and/or revert to a less differentiated precursor stage (right).

the maintenance of tissue stem cells are activated by mutations in components of these pathways or by autocrine mechanisms. This mechanism is likely responsible for the precursor cell phenotype of colorectal cancers (^13.2) and basal cell carcinoma of the skin (^12.3). Chronic myelocytic leukemia (CML) is also clearly a stem cell disease caused by overactivity of 'cancer pathways' (^10.4).

Other cancers also resemble stem cells in possessing apparently unlimited proliferation potential, and most express telomerase. In some cancers, there is even evidence for a subpopulation which gives rise to a larger fraction of more differentiated tumor cells. However, it appears that in many cancers the stem cell properties are acquired secondarily. So, cancers do not necessarily develop directly from stem cells, although some certainly do. Others may develop from cells at a more differentiated stage that resume the phenotype of their precursor cell. Still others may acquire only selected aspects of a stem cell phenotype, such as telomerase expression. While the stem cell properties of cancers are often caused by genetic aberrations, a stem cell phenotype can be established by purely epigenetic mechanisms and this could well important in some cancers.

Further reading

Wolffe A (1999) Chromatin: structure and function. 3rd ed. Academic Press Szyf M (ed.) DNA methylation and cancer therapy. Landes Biosciences, online

Ross SA (2000) Retinoids in embryonic development. Physiol Rev. 80, 1021-1054 Fereira R (2001) The Rb/chromatin connection and epigenetic control: opinion. Oncogene 20, 3128-3133 Ehrlich M (2002) DNA methylation in cancer: too much, but also too little. Oncogene 21, 5400-5413 Geiman TM, Robertson KD (2002) Chromatin remodeling, histone modifications, and DNA methylation:

how does it all fit together. J. Cell. Biochem. 87, 117-125 Jacobs JJL, van Lohuizen M (2002) Polycomb repression: from cellular memory to cellular proliferation and cancer. BBA 1602, 151-161 Johnstone RW (2002) Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat. Rev. Drug Discov. 1, 287-299

Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415-428

Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662-673

Mueller MM, Fusenig NE (2002) Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. Differentiation 70, 486-497 Reik A, Gregory PD, Urnov FD (2002) Biotechnologies and therapeutics: chromatin as a target. Curr.

Opin. Genet. Devel. 12, 233-242 Costa RH et al (2003) Transcription factors in liver development, differentiation, and regeneration.

Hepatology 38, 1331-1347 Esteller M (2003) Cancer epigenetics: DNA methylation and chromatin alterations in human cancer. Adv.

Exp. Med. Biol. 532, 39-49 Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. NEJM 349, 2042-2054

Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 Suppl., 245-254 Jaffe LF (2003) Epigenetic theories of cancer initiation. Adv. Cancer Res. 90, 209-230 Lessard J, Sauvageau G (2003) Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423, 255-260 McLaughlin F, Finn P, La Thangue NB (2003) The cell cycle, chromatin and cancer: mechanism-based therapeutics come of age. Drug Discov Today 8, 793-802 Ohlsson R et al (2003) Epigenetic variability and the evolution of human cancer. Adv. Cancer Res. 88, 145-168

Otte AP, Kwaks THJ (2003) Gene repression by Polycomb group protein complexes: a distinct complex for every occasion? Curr. Opin. Genet. Devel. 13, 448-454 Passague E et al (2003) Normal and leukemic hematopoesis: Are leukemias a stem cell disorder or a reacquisition of stem cell characteristics. PNAS USA 100, Suppl.1, 11842-11849 Sims III RJ, Nishioka K, Reinberg D (2003) Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629-639 Walter J, Paulsen M (2003) Imprinting and disease. Semin. Cell Dev. Biol. 14, 101-110 Egger G et al (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457463

Nakayama M et al (2004) GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer. J. Cell. Biochem. 91, 540-552 Roberts CWM, Orkin SH (2004) The SWI/SNF complex - chromatin and cancer. Nat. Rev. Cancer 4, 133-142

Box 8.1 Carcinogenesis by HIV

Like HTLV-I, HIV1 contributes to human cancer development, and like HTLV-I it expresses a number of accessory proteins in addition to the usual set encoded by the gag, pol and env genes (cf. Fig. 4.1).

HIV1 does not cause tumors itself, but instead facilitates the development of a specific set of cancers through immunosuppression. In many of these, other viruses may be active. They include certain lymphomas (EBV-associated?), squamous carcinomas (HPV-associated?), and specifically, Kaposi sarcoma (KS). In addition to immunosuppression, HIV acts through one or several paracrine mechanisms.

The actual causative virus in KS is a 165 kb herpes virus, HHV8 or KHSV. Kaposi sarcoma consists of a mixture of mesenchymal cell types, which may partially be derived from an undifferentiated precursor cell. It is not quite clear which proteins of the virus are oncogenic. Some suppress apoptosis. Others act as cytokines and still others induce cytokine receptors. In KS, HIV may contribute more directly, beyond immunosuppression. HIV-infected cells release the viral transactivator protein tat, which may promote replication of KHSV and expression of viral proteins in cells harboring that virus. Moreover, HIV tat may induce secretion of the cytokine IL6 in uninfected cells, promoting the proliferation of KHSV-infected and other cells in the tumor.

HIV1 action in carcinogenesis might be schematically illustrated as follows:

Bellan C, De Falco G, Lazzi S, Leoncini L (2003) Pathological aspects of AIDS malignancies. Oncogene 22, 6639-6645

Scadden DT (2003) AIDS-related malignancies. Ann. Rev. Med. 54, 285-303.

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