**12. Kaiso and tumorigenesis**

Almost all the members of the POZ-ZF family were found to be involved in cancer development. BCL-6 and PLZF are oncoproteins linked to non-Hodgkin's lymphoma and acute promyelocytic leukemia, respectively (Chen et al., 1994; Onizuka et al., 1995). FAZF and ZBTB7 are related to Fanconi's anemia and several other human cancers (Hoatlin et al., 1999; Maeda et al., 2005b; Dai et al., 2002; Pessler et al., 1997). On the other hand, ICH-6 and APM1 are candidate tumor suppressors in various human cancers (Schneider et al., 1997; Albagli, 2003).

Like other members of the subfamily POZ-ZF, the Kaiso protein has been implicated in cancer and the first indirect evidence emerged because the target genes of the Kaiso protein

Kaiso and Prognosis of Cancer in the Current Epigenetic Paradigm 119

However, 2009 data has shown for the first time that the subcellular localization of Kaiso in the cytoplasm of a cell is directly associated with the poor prognosis of patients with lung cancer (non-small cell), and around 85 to 95% of lung cancers are non-small cell (Dai et al., 2009). Such data shows a direct relationship between the clinical profile of patients with pathological expression of Kaiso. Therefore, evidence of changes in subcellular localization seems to be relevant to the diagnosis and prognosis of various types of human tumors.

**14. Analysis of the subcellular location of Kaiso in Mielode Chronic Leukemia** 

Data obtained by our group in collaboration with the laboratory of stem cells of the Brazilian National Cancer Institute (INCA of Rio de Janeiro), headed by Dr. Eliana Abdelhay, shows the subcellular distribution of Kaiso by immunofluorescence on cell lines K562, used as a model of CML in the blastic phase. As it can be seen in Figure 2a, the expression of Kaiso is clearly cytoplasmic (Cofre, J., personal communications). As expected, cytoplasmic expression is significantly reduced when using the duplex for inhibition of Kaiso (Rnai) 48 hours after transfection (Figure 2b). As a control, we used the β-tubulin marker and demonstrated that the duplex Kaiso does not modify the expression of this

Fig. 2. Immunofluorescence analysis of kaiso expression. A.Kaiso was expressed in the cytoplasm of K562 cells (a human erythroleukemic line). B. siRNA-Kaiso efficiently downregulates cytoplasm expression after 48 hours transfection. As a control, we used the marker beta-tubulin and showed that siRNA-Kaiso does not modify the expression of this marker

marker after transfection for 48 hours (Figure 2 c and d) .

after 48 hours transfection.

**(CML)** 

(MTA2, MMP2, and siamois CiclinD1) are linked to cell proliferation or metastasis tumor, providing a good indication of the importance, though indirect, of Kaiso in tumorigenesis processes.

More consistent and direct data about the participation of Kaiso in the cancer development process have been recently obtained, when it has been found that Kaiso inhibits activation mediated by β-catenin of the Mmp7 gene (also known as matrilysin), which is well known for metastatic spread (Spring et al., 2005). Recently another study suggests that Kaiso can regulate TCF/LEF1-activity, via modulating HDAC1 and beta-catenin-complex formation (Iioka et al., 2009) (Figure 1f). This shows that Kaiso can directly regulate the signaling pathway of canonical Wnt / β-catenin widely known for its involvement in human tumors. Other evidence also showed that Kaiso rescues the dorsalization of the mesoderm produced by β-catenin and siamois in Xenopus laevis (Park et al., 2005). Siamois is a high mobility group (HMG)-box transcription factor that promotes the dorsalization of the mesoderm of amphibians and is a well-known target of the canonical Wnt pathway involving TCF/LEF. The Kaiso overexpression decreases the ability of TCF/LEF to interact with β-catenin, which implies that Kaiso and TCF/LEF are associated in the nucleus (Van Roy & McCrea, 2005). Other target genes of canonical Wnt pathway, such as Fos, Myc and CCND1 also appear to be directly regulated by Kaiso. On the other hand, the non-canonical Wnt pathways would also be modulated by Kaiso, at least in Xenopus, where it has been demonstrated that Kaiso depletion directly directly activates the Wnt11 promoter (Kim et al, 2004).

However, there is still controversy regarding the Kaiso's oncogenic or tumor suppressor role. As aforementioned, it is known that matrilysin and Wnt11 are repressed by Kaiso, and, thus, it is believed that it might act as a tumor suppressor (Dai et al., 2009). Nevertheless, Kaiso could also act as a methylation-dependent oncogene, repressing the tumor suppressor gene CDKN2A and providing increased survival of colon cancer cells (Lopes et al., 2008). The epigenetic silencing role of kaiso was approached to produce a depletion of Kaiso (by RNA interference) and an increased expression of the tumor suppressor gene CDKN2 was found, which did not affect the DNA methylation levels. As a result, the colon cancer cells were more susceptible to cell death mediated by chemotherapy (Lopes et al., 2008). It is then possible to assess the importance of kaiso as a possible therapeutical target to improve the efficiency of the current cancer treatments.
