**6. The epigenetics and proteomics walking together toward the diagnosis and prognosis of cancer, in the current epigenetic context**

A major challenge faced by cancer therapy is to be able to predict the early stage of the disease in order to provide an appropriate treatment for the patient (Ludwig and Weinstein, 2005). In this regard, the molecular biomarkers have been useful for distinguishing different subtypes of patients with different clinical profiles and at all stages of disease, expanding our prognostic ability (Seligson et al., 2005).

Over the past decades high-throughput technologies including genomics, epigenome, transcriptome and proteomics have been applied to improve our understanding of cancer pathogenesis in order to develop strategies aimed to improve cancer treatment (Seligson, 2005; Ocak et al., 2009). The ultimate goal of these technologies is to help develop noninvasive methods for specific and sensitive diagnosis and facilitate prediction of the response of a patient to a given therapy, as well as help identify potential therapeutic targets (Ueda et al., 2011).

The most important technologies used in the study of cancer are proteomics and epigenomics that help understand that cancer cell phenotype is primarily determined by proteins, and, thus, a genomic or transcriptome approach of the disease are extremely limited. This can be said because it is known that i. levels and protein expression have a low correlation with mRNA levels, ii. proteins undergo post-translational modifications that may alter its function, iii. in the same cell can express different proteins using a mechanism of differential splicing from the same mRNA and very important as we shall see iv. the same protein may have a different function depending on the cellular compartment where it is located. Therefore, the protein detection techniques, including immunohistochemistry (IMH), in this new context, are of vital importance for understanding cellular processes and disease emergence.

In order to better understand the cancer cell and the development of cancer, proteomic information projects have been created based on epigenetics in which proteins and their interactions with the epigenome inside the cell become the key aspect in the understanding of how cancer cells work (Stefanska et al., 2011; Jerónimo et al., 2011). Therefore, knowledge of machinery and all the protein interactions established by them may be important for the prognosis of the tumor and the development of a proper drug to fight cancer and to determine the mechanisms of the disease.

Kaiso and Prognosis of Cancer in the Current Epigenetic Paradigm 113

Connexins can be channels of intercellular communication or proteins that trigger processes of proliferation or apoptosis, depending on the cellular context (Goodenough & Paul, 2003). Traditionally, it was believed that the role of connexins in cancer development was related to its role in intercellular communication channel or gap junctional intercellular communication activity (GJIC). In fact, cancer was the first disease to be associated with connexin disorders (Loewenstein, 1979). The evidence was mainly related to the use of tumor-promoting agents (non-mutagenic carcinogens) and mitogens that decreased the activity of connexin-mediated intercellular communication (Budunova & Williams, 1994) and, on the other hand, antineoplastic agents or chemicals promoting cell coupling through these proteins (King & Bertram, 2005). It was also shown that tumor-derived cells were deficient in expression of connexins (Lee et al., 1992; Laird et al., 1999) and that studies of overexpression of these proteins showed decrease in cellular proliferation (Yamasaki & Naus, 1996). This created a favorable scenario that could lead one to believe that the decrease in expression of connexins and, thus, intercellular communication, was related to

The most important work on the change in concepts regarding these channels of communication was the transfection of connexin43 that makes it possible reversing the neoplastic phenotype of a strain of human glioblastoma cells and that showed that phenotypic reversion was associated with a cytoplasmic localization of connexins without increasing the ability to establish intercellular communication between cells (Huang et al, 1998). Therefore, a new concept has arisen, according to which connexins could have two different functions not necessarily connected: (i) intercellular communication in the plasma

Concerning the connexin role in regulating cell proliferation, two hypotheses have been developed: 1) a downregulation of connexins from the plasma membrane is an indirect result of the activation of MAPK (mitogen-activated protein kinase) and Akt (phosphoinositide-activated kinase) and 2) they act as negative regulators of intercellular junctions (Kojima et al., 2004). However, other lines of evidence indicated that the connexins were directly involved in the regulation of cell growth and that its downregulation would contribute to (and not be a consequence of) the loss of cell cycle control (Vinken et al., 2006). A detailed study supporting this latter idea used transfection of connexin 43 in human osteosarcoma cells, which inhibited cell proliferation without restoring intercellular communication (Zhang et al., 2001). In this model direct connexin 43 changes the expression of p27/Kip1 (the cyclin-dependent kinase inhibitor). Importantly, Cx43, or at least the carboxy terminal tail of this protein has been localized within the nucleus with the use of immunohistochemistry, confirming an intracellular regulatory role (Dang et al., 2003; Cofre

In primary breast tumors, immunohistochemistry can clearly detect the cytoplasmic expression of connexins 43 and 26, being a commonly used diagnostic test for this stage of the disease (Kanczuga-koda et al., 2006). However, immunohistochemistry shows that in the same metastatic tumor cells taken from the lymph node, the expression of connexin43 and 26 changes and has now expanded, though in the plasma membrane. This increased expression in the cell membrane is considered the earliest event in the process of metastasis

membrane and (ii) direct modulation of cell growth control in cell cytoplasm.

**9. Connexins** 

cell proliferation and tumor progression.

& Abdelhay, 2007).

A good example is the study of the proteins of the methyl-CpG-binding domain (MBD) that "read" and interpret the signals in DNA methylation and are critical mediators of various epigenetic processes. We currently know that the family of MBD proteins is formed by five MBD1 and MeCP2 -4 members. There is also a member of non-classical MBD protein called Kaiso that uses a "zinc finger" domain to bind to methylated DNA and mediate transcriptional repression. The factor Kaiso and its partner p120ctn are considered similar to the β-catenin-TCF/LEF (T-cell factor / Lymphoid Enhancing factor) pair that regulate genes of canonical Wnt pathways, with the peculiarity that Kaiso (the difference in TCF/LEF) can interact with the epigenome in cancer development. As usually, hypermethylation is a recognized gene silencing mechanism in processes of tumorigenesis and drug resistance. Obviously, the MBD protein and Kaiso could be important modulators of tumorigenesis and excellent therapeutic targets for developing anti-cancer therapies (Sansom et el., 2007). Therefore, the role of protein detection methods in the diagnosis, prognosis and even in the development of therapies against cancer is unquestionable in the current epigenetic scenery of disease etiology (Yoshimura et al., 2011).
