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CpG island hypermethylation is a common mechanism for gene silencing of tumor suppressor genes. A specific profile of gene hypermethylation occurs according to the tumor type and CpG island methylation of particular genes have been used for translational purposes. Genes inhibiting cell invasion and dissemination also undergo CpG island hypermethylation-associated inactivation. These metastasis suppressor genes can also become transcriptionally inactive by other epigenetic mechanism such as an aberrant histone code and a compact conformation of chromatin in a highly dynamic manner. The best characterized example is the E-cadherin gene, but the tumor/metastasis suppressor genes with CpG island hypermethylation in cancer include other members of the cadherin family (H-cadherin, R-cadherin, FAT), heparan sulfate genes (EXT1, GPC3, 3-OST-2), tissue inhibitors of proteinases (TIMP2, TIMP3, TFPI-2), axon guidance molecules (SEMA3B, SLIT-1, SLIT-2, SLIT-3), thrombospondins (THBS1, THBS2) and laminin genes (LAMA3, LAMB3, LAMC2). Preliminary data suggest that DNA demethylating drugs, reactivating these dormant methylated metastasis genes, have an effect against the development of metastasis.

Although there is a wide range of accepted models of tumorigenesis involving genetic lesions, the timing and hierarchy of epigenetic alterations associated with tumor progression and metastasis are still poorly understood. In this regard, the best characterized mouse carcinogenesis system, the multistage skin cancer progression model, has recently been used to identify epigenetic alterations during tumor progression and to provide decisive information about how epigenetic lesions precede metastasis. This model reveals a progressive global loss of genomic methylcytosine that is associated with the degree of tumor aggressiveness and that occurs in the context of increasing numbers of hypermethylated CpG islands of tumor-suppressor genes during the most malignant stages of carcinogenesis. DNA microarrays coupled with demethylating drug treatments confirm the progressive establishment of hypermethylation events from the early stages to the most aggressive phenotypes. It is of particular interest that the transition from epithelial to spindle cell morphology with metastatic potential is associated with prominent epigenetic alterations: E-cadherin methylation, demethylation of the Snail promoter, and a profound decrease of global DNA methylation.

Invasion and metastasis are biological hallmarks of malignant tumors, and metastases are the major cause of cancer deaths. Invasion and destruction of BM is the earliest step in the multi-step process of metastases and it is the earliest morphological feature of invasive tumors. Disruption of organization or integrity of the basement membrane (BM) is a key histologic marker of the transition of a tumor from an in situ carcinoma to an invasive carcinoma. A fundamental and important question is what causes in situ cancers to become invasive even though cancer cells at the preinvasive and invasive stages are morphologically similar. One of the well-established mechanisms for invading and destroying BMs is by matrix metalloproteinases (MMPs), which are up regulated during invasion and metastasis. Developing molecular markers that mark the transition of in situ cancers to invasive cancer are very important because they may predict cancer for those who are at highest risk or those with early invasive cancers. It is logical to presume that disruption of all the homotypic and heterotypic cell adhesion junctions occurs in invasive cells, and that the loss of the involved protein components as well as loss of substances that inhibit tissue invasion may mark the transition from in situ to invasive cancers. This chapter reviews the different cell adhesion junctions and candidate invasion genes, which are inactivated by aberrant promoter methylation and their potential use as molecular markers.

Recent studies have implicated the dysregulation or maladaptation of epigenetic mechanisms to be a central feature of prostate carcinogenesis. Hypermethylation of CpG islands (CGI), clusters of CpG dinucleotides frequently found at gene regulatory regions, has been demonstrated to be one of the most frequent somatic genome alterations associated with prostate carcinogenesis. A few recent studies have explored the role of CGI hypermethylation during prostate cancer progression from the early precursor lesions to distant metastases. This chapter will focus on the time course of CGI hypermethylation changes that occur at each step during the development and progression of prostate cancer in an effort to understand how these epigenetic changes contribute to the formation of prostate cancer metastases. We will begin by giving an overview of the epidemiology, natural progression, and pathogenesis of prostate cancer, then detail the CGI hypermethylation changes that occur at each step along the progression, then postulate the molecular mechanisms that may be involved in generating and propagating these changes, and finally, use the pattern and timing of DNA methylation changes during the natural progression of prostate cancer to derive models that describe how prostate cancer metastases may form.

Breast cancer is the most common malignancy in women and comprises 18% of all female cancers. The incidence of breast cancer increases with age and in the western countries the disease is the single most common cause of death among women aged 40–50, accounting for about a fifth of all deaths in this age group. The advent of mammography screening has led to an increased detection of pre-invasive mammary lesions and a better elucidation of the pathological events that precede the development of invasive breast carcinoma. Invasive breast cancer is classified in two main morphological subtypes ductal carcinoma representing about 80% of breast malignancy, and lobular carcinoma that accounts for approximately 10% of breast cancers. Among pre-invasive breast lesions, the hyperplasia of the usual type (HUT) is morphologically and phenotypically heterogeneous, whereas atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) are homogenous in cell type and marker expression. On the basis of epidemiological and clinical data ADH at the present is seen as a risk factor and not as a direct precursor of DCIS or invasive lesions. Thus the only proliferative lesion that can be considered as a true precursor of invasive breast cancer is DCIS. This model of pathological progression is partially corroborated by genetic studies. In recent years progresses were made in defining some of the critical processes involved in breast cancer development and progression, and CpG island hypermethylation is emerging as one of the main mechanisms for inactivation of cancer related genes in breast tumorigenesis. Three types of genes are involved in carcinogenesis: oncogenes, tumor suppressor genes (TSGs) and stability (caretaker) genes. They encode for proteins involved in a series of pathways that control the basic functions of the cell: proliferation, communication with neighboring cells and with extra cellular matrix, senescence and programmed cell death (apoptosis). Epigenetic mechanism can modulate these pathways by acting directly on tumor suppressor genes and stability genes and indirectly on oncogenes through their regulators. Studies on several tumor types indicate changes in the number of methylated genes as well as an increase in methylation density during tumor progression, but only few studies have investigated changes in promoter hypermethylation during breast cancer progression. This is mainly due to the intrinsic difficulties to collect lesions that might be representative of all stages of the diseases. An increase in promoter hypermethylation was demonstrated for CCND2, ESR1, CDH1, RASSF1A, AP2α, Twist and maspin from DCIS to invasive tumor. In distant metastases from bone, brain and lung the frequency of methylation for CCND2, RASSF1A, Twist, RARβ2 and HIN1 was statistically significant different as compared with the primary tumor. The analysis of six cases of paired primary tumors and lymph node metastasis showed same methylation patterns for all but one case. The identification of changes in methylation distribution during breast cancer progression is fundamental not only for a better comprehension of the mechanisms involved in breast carcinogenesis, but because such alterations may represent potential markers for early cancer detection and for a better definition of the prognosis.

The goal of this chapter is to promote the value of studying maspin regulation as a paradigm for loss of transcriptional control during cancer progression and to highlight the importance of this endeavor in developing a comprehensive picture of the epigenetics of the malignant phenotype. We will attempt to do this through a discussion of the structure and functions of the serpin superfamily of proteins, with an emphasis on maspin, its discovery as a tumor suppressor, and its functional role in cancer. The control of maspin expression in normal tissue by epigenetic mechanisms will be described and how this underlying mechanism is compromised in cancer leading to the inappropriate silencing of maspin in cancers derived from maspin-positive cell types, as well as the activation of maspin in cancers derived from normally maspin-negative cell types. Finally, we will close with speculation that maspin may represent an inaugural member of a class of cell-type restricted genes involved in cancer cause and progression that are controlled by epigenetic mechanisms. During transformation, epigenetic instability and mischief results in a loss of control in the expression of these genes. We propose that these genes, through metastable epigenetic switching mechanisms, can be turned off and on in response to environmental stresses and cues in the cancer cell, thereby allowing tumor cells a phenotypic plasticity that appears necessary for the challenges a tumor cell and its progeny must undertake to migrate from primary tumor site to distant metastatic site. It is proposed that this epigenetic switch can be targeted by therapeutics designed to transcriptional reprogram tumor cells and flip the switch back to non-malignant behavior.

Inactivation or loss of function of E-cadherin, the principal cell adhesion molecule in epithelial cells, is thought to be an important step in tumour progression and metastasis. In recent years, efforts have been made to understand how E-cadherin expression and function is regulated during these processes. Several mechanisms have been shown to be involved in the regulation of E-cadherin expression, including genetic, epigenetic and transcriptional changes. However, the complete picture of how this molecule is regulated still remains to be fully elucidated. As our understanding of how epigenetic mechanisms influence the control of gene expression expands it becomes clear that the epigenetic modification of genes involved in metastasis could influence the acquisition of malignant cell behaviour. In this chapter, we will focus our attention on the epigenetic control of the E-cadherin gene and discuss how this might be integrated with the known transcriptional repressors of E-cadherin. Understanding the epigenetic control of E-cadherin may help to identify new targets for drug design to block the metastatic process, the most aggressive and lethal consequence of tumour progression.

During the development of the nervous system, guidance cues are required to correctly direct the developing axons. These cues are highly conserved in evolution and may have diverse functions and receptors. The Slit proteins are members of these cues and along with their roundabout (robo) receptors, they act as repulsive cues for robo-expressing axons preventing them from crossing or re-crossing the midline. There is increasing evidence that the slit-robo interactions are not limited to axon guidance. The repulsive effect on axons due to slit-robo binding is mirrored in the immune system as well as in breast tumour cells; Slit proteins act as inhibitors of cell migration and invasion. Our group recently demonstrated that both SLIT and ROBO genes are inactivated in human cancers by promoter region CpG island hypermethylation with the subsequent silencing of gene expression. Restoring expression after treatment with a demethylating agent, provided further evidence that promoter hypermethylation was responsible for silencing SLIT-ROBO genes in several human cancers. Whilst Robo1 homozygous mutant mice die at birth due to incomplete lung development, the heterozygous mice show increased predisposition to tumour development concurrent with the inactivation of the remaining wild type Robo1 allele by promoter region CpG island hypermethylation. SEMA3B , another axon guidance molecule, was recently demonstrated to be epigenetically inactivated in human cancers and suppressed tumour growth. Hence, evidence is accumulating for the role of axon guidance molecules in human cancer development. Unlike mutational inactivation, epigenetic inactivation is a reversible event. This presents new and exciting opportunities for clinical management of cancer. In addition, promoter hypermethylation of genes is increasingly being developed as molecular biomarkers for non-invasive screens for early detection of cancer. Furthermore, the SLIT(s) gene products are secretary proteins, which may also lead to the development of novel therapeutic approaches. This chapter summarises the literature on SLIT-ROBO gene families in relation to human diseases.

Localized cancer, before it metastasizes, can be cured by surgery. The high mortality rate associated with most cancers, however, is due to the propensity of tumors to metastasize while the primary tumor is small and undetected. Metastasis, which occurs through a complex series of events, involves various gene products that dictate the progression of a cancer from a precursor lesion, to localized disease, and finally to metastatic disease. The expression of certain genes or alterations in gene structure or gene products may result in the progression of benign tumor cells to an invasive and metastatic state. Thus, the process of cancer metastasis requires, among other steps, changes in signaling pathways, activation of target gene products, enhanced cell survival, and increased epithelial-to-mesenchymal transition. A proper understanding of the progression of tumors to the metastatic stage and of the events that occur in highly malignant cells is important in the development of new therapeutic approaches for the diagnosis, treatment, and prognosis of highly progressive tumors. The molecular mechanisms that cause a cancer to exhibit more malignant behavior are widely believed to involve the deregulation of genetic and epigenetic cascades. We will here highlight the discovery and emerging significance of one family of regulators or chromatin modifiers, namely, the metastasis-associated antigens.

Recent advances in drug discoveries and understanding of epigenetic regulation of gene expression have brought histone deacetylases (HDACs) in spotlight. B reast C ancer M etastasis S uppressor 1 (BRMS1), a gene shown to functionally suppress metastasis of breast cancer and melanoma, is a member of mSin3-HDAC complex. This chapter reviews the emerging understanding of the molecular mechanisms of BRMS1 action and possible players involved in it. New evidence of BRMS1-family of proteins and their possible role in histone code is discussed.