유방암세포주에서 세포성장과 자가포식을 조절하는 Akt의 역할

Abstract

Breast cancer is the most frequently diagnosed life-threatening cancer in women and the most important cause of cancer-related deaths among women. Although significant progress in breast cancer diagnosis and therapy has been made in the last two decades, this disease remains one of the leading causes of death in women. Basic research studies are now in progress to illuminate the molecular pathways and mechanisms that are involved in breast tumorigenesis. This has spurred research on new and more efficient treatments that overcome the limitations of conventional chemotherapy. Targeted therapies come in three types. Hormone receptor antagonists, monoclonal antibodies and inhibitors of catalytic kinase domains. The first targeted therapy in oncology was the use of anti-hormonal compounds in oestrogen and progesterone positive breast cancer. Monoclonal antibodies bind with high specificity to their target antigen on the tumour cell and induce cell death. Kinase inhibitors bind to the ATP-binding pocket of the enzyme and thus inhibit its catalytic reaction. Recent therapies are progressed with combination of three drugs and showed increased effect on breast cancer. But there are still problems to overcome the low efficiency and relapse. Among various kinases involved in breast oncogenesis, Akt was used as a target molecule in this research. Akt pathway is a critical regulator of many essential cellular processes. In addition to playing an important role in normal cellular physiology, activation of PI3K-Akt signaling is one of the most frequent events in cancer. There is growing evidence that the PI3K-Akt pathway is frequently activated in melanomas and plays a functionally important role in this disease. Akt is serine threonine kinase that can phosphorylates multiple downstream factors inducing its activities. Akt has three isoforms reported and isoform specific roles in cancers need to be studied before treatment. To dissect the isoform-specific roles of Akt in breast cancer cells, constitutively active Akt isoforms were introduced into MDA-MB-231 cells. Both Akt1 and Akt2 efficiently inhibited the growth of MDA-MB-231 cells. Over-expression of Akt1 down-regulated ERK activity inhibiting Ser 259 phosphorylation of c-Raf and subsequent downstream signaling. Akt2 over-expression up-regulated the cell cycle inhibitor p27. Cycloheximide decay assays showed that Akt2 increased the stability and nuclear localization of p27, thus inhibiting the cyclin E/CDK2 complex. These results suggest that the inhibition of cell proliferation by Akt1 and Akt2 is mediated by isoform-specific mechanisms. In the second part of this study, the role of Akt in autophagic process was investigated. Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cells own lysosomal apparatus. Autophagy may result in either cell death or survival in cancer cells. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, Akt, which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG in both 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative (DN) Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1overexpression in MDA-MB-231 breast cancer cells down-regulated the mRNA level of UVRAG. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation. In the third part of this study, the effect of S100A4 on Akt-mediated signaling was conducted. S100A4 also known as mts1 is reported as invasive and metastatic marker for many types of cancers. S100A4 interacts with various target genes that affect tumor cell metastasis. But there was not much known information on the other cellular signaling pathway elicited by S100A4. Here, we showed inhibitory effect of S100A4 on β-catenin signaling in breast cancer cells. By overexpressing S100A4 in MCF-7, MDA-MB-231 and MDA-MB-453 breast cancer cells we could observe down-regulated β-catenin expression and β-catenin-dependent TCF/LEF transcriptional activity. The expression of GSK3β which phosphorylates β-catenin and induces proteosomal degradation of β-catenin, was increased in S100A4 overexpressing cell lines. We also found increased GSK3β activity was due to decreased Akt activity by egr-1 induced phosphatase and tensin homolog (PTEN) expression. S100A4 induced egr-1 nuclear localization by binding directly to egr-1, facilitating egr-1 import into the nucleus. This effect was reduced in S100A4 mutants that harbors defect in nuclear localization signals. Collectively we verified here that S100A4 act as a tumor suppressor in breast cancers by down-regulating Akt signaling axis for tumor cell survival.