**9. Connexins**

112 Cancer Prevention – From Mechanisms to Translational Benefits

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

**7. The multifunctional protein and its relation to cellular compartments** 

A single protein can have different functions in a cell and these functions concern the compartment where they are located. One of the best documented examples is that transglutaminase 2 (TG2) may act as a transglutaminase, G-protein kinase, protein disulfide isomerase or as an adapter protein. These multiple biochemical activities are involved in a wide variety of cellular processes such as differentiation, cell death, inflammation, cell migration and others. The specific microhabitats and subcellular compartments of location of the plasma membrane, cytoplasm, nucleus, mitochondria, or extracellular space are important in the development of different biochemical activities by the same protein

Thus, in our search for a drug target, e.g., cancer, we must always know the location of a given protein in the cell and be aware of how this cell places these proteins in different micro environments, and that more often than not these different functions may occur simultaneously. So part of the strategy to find the correct pharmacological targets is the previous understanding of the structure and the establishment of subcellular microenvironments inside the cells and the better knowledge of the complex and dynamic

Therefore, immunohistochemistry provides information that cannot be obtained in any other way, which is the relationship between the pathological state and the dimension of the altered compartment (Oliver and Jamur, 2009; Dabbs, 2010), of great relevance to the

Surprisingly, over the past few decades multifunctional proteins provided the basis of the study of some diseases, including cancer. Alterations and aberrations of the multifunctional proteins regarding their distribution and subcellular localization have been used to diagnose the pathological state. I will consider briefly connexins, β-catenin and kaiso as examples of

subcellular compartmentalization that will be further explained.

**8. The multifunctional proteins, compartments and cancer** 

establishment of the cancer diagnosis, as we shall see soon.

these proteins and their role in cancer development.

of disease etiology (Yoshimura et al., 2011).

structure (Park et al., 2010).

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 cell proliferation and tumor progression.

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

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 & Abdelhay, 2007).

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

Kaiso and Prognosis of Cancer in the Current Epigenetic Paradigm 115

shall see later, this classical interpretation may change when additional information on

In clinical practice aberrant changes in the expression of β-catenin in the nucleus have made it possible to suggest the use of this molecule as a complement to the differential diagnosis of various cancers, including cancers of the gastrointestinal tract, lung and tumors of gynecological origin (Montgomery & Folpe, 2005). Also, the absence or loss of nuclear expression of β-catenin expression associated with strong cytoplasmic P-cadherin was associated with melanoma aggressiveness and poor patient survival, establishing an important prognostic value in these types of cancer for β-catenin (Bachmann et al., 2005).

Fig. 1. Canonical and non-canonical Wnt signaling pathway. Crosstalk between Kaiso, Kaiso–p120ctn, β-catenin and Endossomal compartments. a. In the absence of Wnt ligands or in the case of high E-cadherin concentrations, β-catenin and p120ctn associate with E-

macromolecular complex formed by the cytoplasmic protein APC (adenomatous polyposis coli), Axin and disheveled (DSH) stimulate the phosphorylation of β-catenin. Directly responsible for this phosphorylation is the protein casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Β-catenin phosphorylated is destined to a ubiquitin-mediated degradation of β-catenin. c. The activation of canonical pathway leads to stabilization of β catenin. Therefore, Wnt signaling would finally prevent β-catenin degradation, which could then (d.) translocate to the nucleus, and perform its transcriptional activity, associate with lymphoid enhancer-binding protein (LEF)/T-cell factor (TCF). e. If E-cadherin is mutated or

cadherin, promoting intercellular adhesion. b. In the absence of Wnt binding, a

subcellular compartmentalization is gained.

(Kanczuga-koda et al., 2006). The important diagnostic value of IMH is evident in the resolution stage of the disease.

Although the role of connexins in the process of metastasis is controversial because some studies indicate that connexin expression is inversely proportional to metastatic capacity of a primary tumor (Nicolson et al., 1988), other studies reveal that connexins might be involved in metastasis (Carystinos et al., 2001).

At least it is clear that, unlike previously thought, connexins have a tumor suppressor function, but not from a classical point of view, since there are no mutations of this protein associated with carcinogenesis. So, they seem to have different effects on different stages of carcinogenesis, (depending on the connexin isoform or cell type in which it is expressed). Connexins seem to favor cell proliferation when they are downregulated (cytoplasmic localization) and increase the potential for invasion and metastasis when they are overexpressed (initially in the plasma membrane) (for a review of this literature sees Crespin et al., 2008). As we shall see at the end of this chapter, this issue needs to be clarified for a better understanding of subcellular compartmentalization and mechanisms of regulation of intracellular signaling.
