**7.2 Role of STAT family**

The Stat (Stands for signal transducer and activator of transcription) family of proteins are latent cytoplasmic transcription factors which are involved in cytokines signaling pathways. They are necessary for normal cell growth, survival, differentiation, and motility. STAT proteins need activation through tyrosine phosphorylation, which leads to dimerization via conserved structural features phosphotyrosine-SH2 (Src homology domain 2) of two Stat molecules. Fallowing activatin, Stats transport to the nucleus, where they bind to the

enhanced cell proliferation and their ability of anchorage-independent growth of breast cancer cells. These proliferative effects were significantly related to attenuation of two key cell-cycle inhibitors, p27Kip1 and p21Cip1, via Akt/ FOXO1 signaling pathway. FOXO1 is a transcription factor belonging to the Forkhead box-containing class O (FOXO) subfamily. Many biological functions have been shown to be related with FOXO1 including cell-cycle control, differentiation, stress response and apoptosis (37). FOXO proteins could act as tumor suppressors through induction of CDK inhibitors, including p21 Cip1, p27Kip1and p57 (38). Overexpression of *AEG*-1 increases migration and invasion of human glioma cells because of the presence of a lung-homing domain which facilitates breast tumor metastasis to lungs. Recent observations indicate that *AEG*-1 play this role by activating NF-κB pathway. Our recent observations indicate that, AEG-1 facilitates IκBa degradation, resulting in an increase in NF- κB DNA binding activity and NF- κB promoter activity in reporter assays These valuable findings are strengthen the idea which recommend *AEG*-1 as

Another considerable role attributed to AEG-1 is mediating a broad-spectrum chemoresistance. In vitro and in vivo studies showed that knocking down AEG-1 makes several different breast cancer cell lines more sensetive to paclitaxel, doxorubicin, cisplatin, 4-hydroxy cylco phosphamide, hydrogen peroxide, and UV radiation mediated by the prosurvival pathways such as PI3K and NFκ B, or through other downstream genes of MTDH/AEG-1 that directly regulate chemoresistance. AEG-1 has also resulted in chemoresistance neuroblastoma and prostate cancer. In fact, MTDH/AEG-1does not affect

it enhances chemoresistance by increasing cell survival after chemotherapy. Data gathered from Microarray analysis of breast cancer cells showed reduction of expression of chemoresistance genes ALDH3A1, MET, HSP90, and HMOX1, and increased expression of pro-apoptotic genes BNIP3 and TRAIL after MTDH/AEG-1knocking down. Among these genes, ALDH3A1 and MET were established to partially be associated with the chemoresistance role of MTDH/AEG-1 in MDA-MB-231 breast cancer cells. Some other genes also contribute to chemoresistance including drug-metabolizing enzymes for different chemotherapeutic agents, such as dihydropyrimidine dehydrogenase (DPYD), cytochrome P4502B6 (CYP2B6), dihydrodiol dehydrogenase (AKR1C2), and the ATP-binding cassette transporter ABCC11 for drug efflux (40). Roles of MTDH/AEG-1 have been simplified in

There are some studies which suggest that Activated protein C (APC), an anticoagulant serine protease, is related to cell survival, cell migration, angiogenesis and breast cancer invasion. APC recruits EPCR, PAR-1, and EGFR in extracellular matrix in order to increase the invasive properties of MDA-MB-231 cells. Other mechanisms include activation of matrix metalloprotease (MMP) -2 and/or -9 and activation of ERK, Akt, and NF-κB (but not the JNK) pathways. APC does not employ the endogenous plasminogen activation system

The Stat (Stands for signal transducer and activator of transcription) family of proteins are latent cytoplasmic transcription factors which are involved in cytokines signaling pathways. They are necessary for normal cell growth, survival, differentiation, and motility. STAT proteins need activation through tyrosine phosphorylation, which leads to dimerization via conserved structural features phosphotyrosine-SH2 (Src homology domain 2) of two Stat molecules. Fallowing activatin, Stats transport to the nucleus, where they bind to the

a crucial regulator of tumor progression and metastasis (39).

the uptake or retention of chemotherapy a. Instead,

figure 4.

to increase invasion (41).

**7.2 Role of STAT family** 

Fig. 4. MTDH/AEG-1 promotes tumor progression through the integration of multiple signaling pathways. Oncogenic Ha-Ras increases *MTDH/AEG-1* expression through the activation of the PI3K/Akt pathway, which phosphorylates and inactivates GSK3β, and subsequently enhances the stabilization and binding of c-Myc to the *MTDH/AEG-1* promoter. MTDH/AEG-1 can activate AKT, NFκB, and Wnt/β-catenin pathways to promote proliferation, survival, and invasion. Activation of NFκB signaling is in part mediated by the direct interaction of MTDH/AEG-1 with p65 and CBP, a general transcriptional co-activator. MTDH/AEG-1 activates the Wnt/β-catenin pathway through increasing the activity of MAPK kinases ERK and p38, which phosphorylates GSK3β and stabilized β-catenin. Furthermore, MTDH/AEG-1 increases the expression of LEF-1, a transcriptional cofactor for β-catenin. The prometastasis function of MTDH/AEG-1 is mediated by the interaction of the LHD of MTDH/AEG-1 with an unknown receptor in endothelial cells. The broad spectrum chemoresistance function of MTDH/AEG-1 is mediated by a number of downstream genes that promote the resistance to multiple

Breast Cancer from Molecular Point of View: Pathogenesis and Biomarkers 117

promoter of target genes and activate their transcription. Dimerized status of STATs is transient in normal non-transformed cells. But in transformed cancerous cells, Stat proteins in particular, Stat3 are found in a permanent active dimerized manner. Activated form of STAT3 has been found in more than 50% of primary breast tumors and tumor-derived cell lines. It has been reported that expression of a constitutively active form of Stat3 (Stat3C) is sufficient for promoting cellular transformation and bearing an immortalized breast cell line. Since the IL-6/gp130/Jak signaling pathway has a crucial role in Stat3 activation in human breast cancer, blockade of this pathway may be an important therapeutic plan in

As it is mentioned above, dysregulation protein expression can result in increased metastatic properties of breast cancer. As a fact, reduction in cell adhesion and increased cell motility is necessary for tumor metastasis. Therefore, cell adhesion molecules have roles in promoting and inhibiting metastasis. Specific families of adhesion molecules including selectins, integrins, lectins, and cadherins have been established to be associated with metastasis (43- 47). The cells have to pass the basement membrane to reach the surrounding vessels and spread to other sites. This process involves proteolysis and motility and need proteolytic enzymes to work. Three major categories of proteolytic enzymes including the matrix metalloproteinases (48), serine proteinases, and cathepsins (discussed above) are implicated in metastasis. Cell motility is another factor which cells need to be able to metastasize to other tissues. Several factors are necessary for cellular motility, including the autocrine motility factor, autotaxin, and hepatocyte growth factor (HGF). HGF will result in

Chemoattractants and their corresponding receptors are the other factors affecting metastasis rocess. Osteonectin (a glycoprotein secreted by osteoblasts in bone, initiating mineralization and promoting mineral crystal formation) engages breast and prostate cancer cells to bone. Recently presented data indicate that chemokine receptors CXCR4 and CCR7 express in breast carcinoma cells predisposed for metastasis to lymph nodes and bone (24). Metastasis-associated protein 1 (MTA1) mRNA expression is parallel to metastatic potential. Function of the MTA1 gene product in tumor progression and metastasis is still unknown, although it is thought that MTA1 is found in the chromatin remodeling histone deacetylase

Osteopontin was identied as a metastasis associated gene. Osteopontin appears to be useful for prognosis in that elevated plasma levels and immunohistochemical staining of tumor cells are found in metastatic breast cancer patients. It is important, however, to note that not all studies show correlations. For example, immunohistochemical staining showed

*E-cadherin* (a member of the cadherin superfamily of Ca2+-dependent adhesion cell surface molecules, expressed predominantly in epithelial tissues) has been demonstrated to correlates negatively with the potential of tumor invasion. Reduction and/or loss of Ecadherin expression in carcinomas will result in increased tumor metastasis because of the

breast cancer therapy (42). Role of STAT3 has been shown in figure 5

developing more as well as larger axillary lymph node metastases (24).

no correlation with lymph node involvement or histological grade (24).

reduction in tumor cell adhesiveness and increased cell motility (49)

complex (24).

**8.1 E-cadherin** 

**8. Metastasis suppressor genes** 

Tissue Inhibitors of Metalloproteinases

chemotherapeutic agents. Proteins with direct interactions with MTDH/AEG1 are shown in green. Dotted line indicates pathways yet to be fully validated or characterized. [Source: Figure 1 from Ref. 40] With permission

Fig. 5. Role of Stat3 signaling pathway to cancer metastasis. Activatin of STAT3 happens by recruitment to phosphotyrosine motifs within complexes of growth factor receptors (e.g., epidermal growth factor receptor), cytokine receptors (e.g., IL-6 receptor), or non-receptor tyrosine kinases (e.g., Src and BCR-ABL) through their SH2 domain. Stat3 is then phosphorylated on a tyrosine residue by activated tyrosine kinases in receptor complexes. Phosphorylated Stat3 forms homodimers and heterodimers and translocates to the nucleus. In the nucleus, Stat3 dimers bind to specific promoter elements of target genes and regulate gene expression. The Stat3 signaling pathway regulates cancer metastasis by regulating the expression of genes that are critical to cell survival, cell proliferation, invasion, angiogenesis, and tumor immune evasion.

promoter of target genes and activate their transcription. Dimerized status of STATs is transient in normal non-transformed cells. But in transformed cancerous cells, Stat proteins in particular, Stat3 are found in a permanent active dimerized manner. Activated form of STAT3 has been found in more than 50% of primary breast tumors and tumor-derived cell lines. It has been reported that expression of a constitutively active form of Stat3 (Stat3C) is sufficient for promoting cellular transformation and bearing an immortalized breast cell line. Since the IL-6/gp130/Jak signaling pathway has a crucial role in Stat3 activation in human breast cancer, blockade of this pathway may be an important therapeutic plan in breast cancer therapy (42). Role of STAT3 has been shown in figure 5

As it is mentioned above, dysregulation protein expression can result in increased metastatic properties of breast cancer. As a fact, reduction in cell adhesion and increased cell motility is necessary for tumor metastasis. Therefore, cell adhesion molecules have roles in promoting and inhibiting metastasis. Specific families of adhesion molecules including selectins, integrins, lectins, and cadherins have been established to be associated with metastasis (43- 47). The cells have to pass the basement membrane to reach the surrounding vessels and spread to other sites. This process involves proteolysis and motility and need proteolytic enzymes to work. Three major categories of proteolytic enzymes including the matrix metalloproteinases (48), serine proteinases, and cathepsins (discussed above) are implicated in metastasis. Cell motility is another factor which cells need to be able to metastasize to other tissues. Several factors are necessary for cellular motility, including the autocrine motility factor, autotaxin, and hepatocyte growth factor (HGF). HGF will result in developing more as well as larger axillary lymph node metastases (24).

Chemoattractants and their corresponding receptors are the other factors affecting metastasis rocess. Osteonectin (a glycoprotein secreted by osteoblasts in bone, initiating mineralization and promoting mineral crystal formation) engages breast and prostate cancer cells to bone. Recently presented data indicate that chemokine receptors CXCR4 and CCR7 express in breast carcinoma cells predisposed for metastasis to lymph nodes and bone (24).

Metastasis-associated protein 1 (MTA1) mRNA expression is parallel to metastatic potential. Function of the MTA1 gene product in tumor progression and metastasis is still unknown, although it is thought that MTA1 is found in the chromatin remodeling histone deacetylase complex (24).

Osteopontin was identied as a metastasis associated gene. Osteopontin appears to be useful for prognosis in that elevated plasma levels and immunohistochemical staining of tumor cells are found in metastatic breast cancer patients. It is important, however, to note that not all studies show correlations. For example, immunohistochemical staining showed no correlation with lymph node involvement or histological grade (24).
