**5.2 Mutations in tumor suppressor genes**

Transforming growth factor-ß **(TGF- ß)** is a secreted cytokine which induces s growth arrest in normal epithelium. It interacts with the TGF- ß type II receptor (T ß RII) which followed by recruitment and phosphorylation of TGF- ß type I receptor (Tß RI) and activation of downstream signaling cascade. The cytostatic effect of TGF- ß is also seen on early tumor progression and is mediated through the regulation of both apoptosis and cell proliferation. However, TGF-ß signaling increases lung metastasis in some transgenic mouse models. Breast carcinomas are well known for overexpressed TG F- ß. Induct ion of TGF- ß 1 after tumor initiation do not exert much effect on proliferation of tumor, but remarkably increase the lung metastasis. These data support the hypothesis that that TGF -ß 1 may no longer perform an inhibitory role in established tumors (24).

Another important tumor suppressor associated with mammary tumor development is p53. p53 is well-known for its involvement in a variety of cancer types. P53 gene is one of the most altered tumor suppressor genes in human breast cancer, such that around 50% of all breast cancers include mutated form of p53 gene (25).

It has been reported that Insulin-like Growth Factor (IGF) may have effect in breast cancer progression. It has been showed that Retinoic Acid (RA) mediate their inhibitory effects on cell growth of cancerous human breast cancer cells "MCF7" via selective reduction of Insulin Receptor Subtype-1 (IRS-1) and its activity which results in the selective downregulation of IP3-kinase/AKT. High levels of Irs-1 in human breast tumors correlate with elevated incidence of disease recurrence. Although the insulin receptor substrates (IRS) were primarily identified, as the name implied, as a substrate for the insulin receptor (IR), Nowadays it has been known that these adapter proteins, are involved in activation of downstream pathways of several growth factor receptors such as insulin-like growth factor-1 receptor (IGF-1R), vascular endothelial growth factor receptor (VEGF-R), cytokine

Breast Cancer from Molecular Point of View: Pathogenesis and Biomarkers 111

receptors, and some members of the integrin family. Interestingly, loss of either IRS -1 or IRS -2 did not show similar consequence on developing lung metastasis. Metastasis will increase in IRS -1-deficient tumors, IRS-2-deficient tumors shows decreased lung metastasis. It is thought that a compensatory mechanism which upregulate IRS-2 expression is involved in the increased metastasis seen in IRS-1-deficient tumors. These results are very similar to the ones seen with Akt1 and Akt2, where Akt1 was shown to inhibit invasion and metastasis while Akt2 perform in an opposite way. RA influence occurs at post-translational level by increase in ubiquitination and serine phosphorylation of IRS-1. The latter is protein-kinase C (PKC)-dependent, since PKC inhibitors block the process. Activation of PKC-δ by RA has also been reported. Activation of PI3K/PDK/Akt cascade also decreases sensitivity of MCF7 cells to anticancer drugs. Induction of Bcl-2 may contribute to this resistance (26,27). Figure 2 offers a comprehensive diagram which shows the role of PI3/Akt cascade in cellular functions. As it is seen, this pathway plays a vital role in cell proliferation and cell survival. Therefore, logically it is predictable that any signal disregulation in this cascade will be a

In one of the recently-performed experiments, the increasing influence of estradiol (E2) on expression level of iNOS in breast cancer cell line T47D were identified as a result for resistance to tamoxifen. In these cells, administration of oligomycin-2 deoxy glucose (2DG) enhanced tamoxifen antiproliferative effects, which may be due to exacerbated ATP depletion following tamoxifen and oligomycin-2DG co-administration. Oligomycin-2DG neither changed iNOS expression nor affected its attenuated expression due to tamoxifen exposure, suggesting that ATP depletion-mediated sensitivity to tamoxifen is apart from iNOS (28).

Recently, cancer stem cells (CSCs) have attracted a lot of attentions and some roles have been determined for estrogen and progesterone by affecting these cells. It has become clear that the normal and malignant breast contains stem cells (SCs) that play an essential role in the normal development o f the breast and are likely to play a significant role in the genesis and growth of human breast cancer. The CSC hypothesis introduced tissuespecific Stem Cells (SCs) and/or their early progenitors as the main causes of the malignant behavior of cancer. These cells are undifferentiated and, as a result, have the ability to divide into two daughter cells. But, division is asymmetrical and will cause an identical clone of the mother cell and another cell which can divide and fully differentiate into new cell line. This latter daughter cell is named a Progenitor. Physiological functions of breast SCs include producing the early milk ducts and the surrounding stroma at puberty and repair of damaged tissue and renovation the lost ductal and stromal cells

general map from the role of AKT and the signaling crosstalk which discussed above. In addition, Akt has been shown to regulate proteins involved in neuronal function including GABA receptor, ataxin-1, and huntingtin proteins. Akt has been demonstrated to interact with Smad molecules to regulate TGFβ signaling. Finally, lamin A phosphorylation by Akt could play a role in the structural organization of nuclear proteins. These findings make Akt/PKB an important therapeutic target for the treatment of cancer, diabetes, laminopathies, stroke and neurodegenerative disease. [Source: Pathway diagram reproduced courtesy of Cell Signaling

Technology, Inc. (www.cellsignal.com).]

**6. Breast cancer stem cells** 

during adult life.

risk factor for uncontrolled cell proliferation and malignancy.

Fig. 2. PI3K / Akt Signaling.The Akt cascade is activated by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G protein coupled receptors and other stimuli that induce the production of phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) by phosphoinositide 3-kinase (PI3K). These lipids serve as plasma membrane docking sites for proteins that harbor pleckstrin-homology (PH) domains, including Akt and its upstream activator PDK1. There are three highly related isoforms of Akt (Akt1, Akt2, and Akt3) and these represent the major signaling arm of PI3K. For example, Akt is important for insulin signaling and glucose metabolism, with genetic studies in mice revealing a central role for Akt2 in these processes. Akt regulates cell growth through its effects on the mTOR and p70 S6 kinase pathways, as well as cell cycle and cell proliferation through its direct action on the CDK inhibitors p21 and p27, and its indirect effect on the levels of cyclin D1 and p53. Akt is a major mediator of cell survival through direct inhibition of pro-apoptotic signals such as Bad and the Forkhead family of transcription factors. T lymphocyte trafficking to lymphoid tissues is controlled by the expression of adhesion factors downstream of Akt. Figure 2 presents a

Fig. 2. PI3K / Akt Signaling.The Akt cascade is activated by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G protein coupled receptors and other stimuli that induce the production of phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) by phosphoinositide 3-kinase (PI3K). These lipids serve as plasma membrane docking sites for proteins that harbor pleckstrin-homology (PH) domains, including Akt and its upstream activator PDK1. There are three highly related isoforms of Akt (Akt1, Akt2, and Akt3) and these represent the major signaling arm of PI3K. For example, Akt is important for insulin signaling and glucose metabolism, with genetic studies in mice revealing a central role for Akt2 in these processes. Akt regulates cell growth through its effects on the mTOR and p70 S6 kinase pathways, as well as cell cycle and cell proliferation through its direct action on the CDK inhibitors p21 and p27, and its indirect effect on the levels of cyclin D1 and p53. Akt is a major mediator of cell survival through direct inhibition of pro-apoptotic signals such as Bad and the Forkhead family of transcription factors. T lymphocyte trafficking to lymphoid tissues is controlled by the expression of adhesion factors downstream of Akt. Figure 2 presents a

general map from the role of AKT and the signaling crosstalk which discussed above. In addition, Akt has been shown to regulate proteins involved in neuronal function including GABA receptor, ataxin-1, and huntingtin proteins. Akt has been demonstrated to interact with Smad molecules to regulate TGFβ signaling. Finally, lamin A phosphorylation by Akt could play a role in the structural organization of nuclear proteins. These findings make Akt/PKB an important therapeutic target for the treatment of cancer, diabetes, laminopathies, stroke and neurodegenerative disease. [Source: Pathway diagram reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).]

receptors, and some members of the integrin family. Interestingly, loss of either IRS -1 or IRS -2 did not show similar consequence on developing lung metastasis. Metastasis will increase in IRS -1-deficient tumors, IRS-2-deficient tumors shows decreased lung metastasis. It is thought that a compensatory mechanism which upregulate IRS-2 expression is involved in the increased metastasis seen in IRS-1-deficient tumors. These results are very similar to the ones seen with Akt1 and Akt2, where Akt1 was shown to inhibit invasion and metastasis while Akt2 perform in an opposite way. RA influence occurs at post-translational level by increase in ubiquitination and serine phosphorylation of IRS-1. The latter is protein-kinase C (PKC)-dependent, since PKC inhibitors block the process. Activation of PKC-δ by RA has also been reported. Activation of PI3K/PDK/Akt cascade also decreases sensitivity of MCF7 cells to anticancer drugs. Induction of Bcl-2 may contribute to this resistance (26,27). Figure 2 offers a comprehensive diagram which shows the role of PI3/Akt cascade in cellular functions. As it is seen, this pathway plays a vital role in cell proliferation and cell survival. Therefore, logically it is predictable that any signal disregulation in this cascade will be a risk factor for uncontrolled cell proliferation and malignancy.

In one of the recently-performed experiments, the increasing influence of estradiol (E2) on expression level of iNOS in breast cancer cell line T47D were identified as a result for resistance to tamoxifen. In these cells, administration of oligomycin-2 deoxy glucose (2DG) enhanced tamoxifen antiproliferative effects, which may be due to exacerbated ATP depletion following tamoxifen and oligomycin-2DG co-administration. Oligomycin-2DG neither changed iNOS expression nor affected its attenuated expression due to tamoxifen exposure, suggesting that ATP depletion-mediated sensitivity to tamoxifen is apart from iNOS (28).
