Browsing by Subject "Akt"

Now showing 1 - 3 of 3

Open Access
Functional effects of ATAD2 gene expression in breast cancer
(2016-08) Özel, Buse Nurten
The ATAD2 gene is a newly investigated gene of which the expression levels are associated with the disease prognosis in many types of cancer and especially breast cancer and that is known to be overexpressed usually through gene amplification and E2F/RB pathway activation. ATAD2 (ATPase family, AAA domain-containing 2) can be overexpressed due to amplification or other regulatory mechanisms in many cancers such as lung, breast, prostate and liver. The fact that ATAD2 has an AAA+ ATPase and bromodomain indicates that it may be a good target for anti-cancer therapy. However, it is necessary to clarify the role of the ATAD2 gene in tumorigenesis before strategies that target ATAD2 are developed. We evaluated a regulator that consistently shows high expression in breast cancer in this study. ATAD2 (ATPase family AAA domain-containing protein 2) is a gene that regulates important cellular activities such as cell proliferation and invasion. This study aimed to clarify the mechanism of action of ATAD2 in breast cancer. The ATAD2 expression of the MCF7 and T47D cell lines with high ATAD2 gene expression was silenced with siRNA and the expression levels of all genes were screened. Gene chip expression analyses revealed that the suppression of ATAD2 in breast cancer cells indicated a role in the regulation of microtubule organization, cell growth, cell adhesion and important signal pathways such as EGFR, FGFR, MAPK, and PI3K. Functional studies with breast cancer cells have supported the gene expression analysis results. Our study revealed that silencing of ATAD2 lead to suppression of ER(-) breast cancer cell migration but not ER(+) cancer cell migration. The same experiments causes a marked decrease in the colony formation capacity and proliferation potential of HCC1937 cells while there was no change in SKBR3 and ER(+) cells. ATAD2 silencing also lead to a senescence response in all breast cancer cells. We investigated the molecular mechanisms of action of ATAD2 to determine the factors underlying the biological effect. The MCF7 and HCC1937 cells were used to clarify its action on the main cellular signal pathways. We found that ATAD2 silencing induced an apoptosis response in both cell types. Intrinsic pathways are activated with caspase-9 cleavage in MCF7 cells while high Bcl2 and BclXL expression prevents caspase-9 cleavage in HCC1937 cells. Decreased ATAD2 did not cause a difference in the p53 protein level in either cells but while p21 expression was increased in just MCF7 cells, RB phosphorylation was inhibited in both cell lines. The results indicate that dysregulation of proteins involved in intracellular control pathways triggers the senescence mechanism. ERα gene expression has been shown to be suppressed as a result of siRNA suppression of ATAD2 gene expression in MCF7 cells. This result indicates that ATAD2 has a role in ERα regulation. ATAD2 gene expression has been found to decrease following Gefitinib suppression of EGFR signaling while EGF treatment of serum-starved MCF7 cells caused increased ATAD2 gene expression. These results indicate that EGFR could be a possible upstream activator of ATAD2. This gene expression pattern also points towards a positive feedback mechanism between ATAD2+ERα and EGFR. Although it is known that EGFR is frequently overexpressed in breast cancer and cross-talk with the estrogen receptor, we do not have detailed information on the mechanism of their interactions. ‘Pathway Enrichment’ analysis of microarray studies have revealed EGFR signaling as one of pathways enriched in the genes downregulated with decreased ATAD2 expression. The silencing of ATAD2 and ERα together prevents EGFR expression in MCF7 cells while silencing of ATAD2 by itself in HCC1937 cells does not cause a change in EGFR expression but prevents its phosphorylation in the Tyr1173 region of the receptor. The ATAD2-suppressed EGFR activity in HCC1937 cells did not lead any change in the Akt level or MEK/ERK activity. The down-stream signaling pathway analysis of the EGFR has revealed that Akt protein expression is suppressed when ATAD2 is silenced in MCF7 cells. The increase in the MEK/ERK signaling activity with decreased ERα expression in the same cells was suppressed with decreased ATAD2 expression. In conclusion, the high expression of the ATAD2 gene in breast cancer stimulates growth of cancer cells while its interaction with the EGFR signaling pathway could be one of the causes of the pro-oncogenic effects of the gene. Its suppression together with EGFR could provide an option for new therapeutic applications in breast cancer studies.
Open Access
Inhibition of Akt signaling in hepatoma cells induces apoptotic cell death independent of Akt activation status
(Springer, 2011) Buontempo, F.; Ersahin, T.; Missiroli, S.; Senturk, S.; Etro, D.; Ozturk, M.; Capitani, S.; Cetin Atalay, R.; Neri, M. L.
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is involved in cell survival and anti-apoptotic signaling. Akt has been shown to be constitutively expressed in a variety of human tumors including hepatocellular carcinoma (HCC). In this report we analyzed the status of Akt pathway in three HCC cell lines, and tested cytotoxic effects of Akt pathway inhibitors LY294002, Wortmannin and Inhibitor VIII. In Mahlavu human hepatoma cells Akt was constitutively activated, as demonstrated by its Ser473 phosphorylation, downstream hyperphosphorylation of BAD on Ser136, and by a specific cell-free kinase assay. In contrast, Huh7 and HepG2 did not show hyperactivation when tested by the same criteria. Akt enzyme hyperactivation in Mahlavu was associated with a loss of PTEN protein expression. Akt signaling was inhibited by the upstream kinase inhibitors, LY294002, Wortmannin, as well as by the specific Akt Inhibitor VIII in all three hepatoma cell lines. Cytotoxicity assays with Akt inhibitors in the same cell lines indicated that they were all sensitive, but with different IC50 values as assayed by RT-CES. We also demonstrated that the cytotoxic effect was through apoptotic cell death. Our findings provide evidence for its constitutive activation in one HCC cell line, and that HCC cell lines, independent of their Akt activation status respond to Akt inhibitors by apoptotic cell death. Thus, Akt inhibition may be considered as an attractive therapeutic intervention in liver cancer. © Springer Science+Business Media, LLC 2010.
Open Access
Liver cancer cells are sensitive to Lanatoside C induced cell death independent of their PTEN status
(Elsevier, 2016) Durmaz, I.; Guven, E. B.; Ersahin, T.; Ozturk, M.; Calis, I.; Cetin Atalay, R.
Background Hepatocellular carcinoma is the second deadliest cancer with limited treatment options. Loss of PTEN causes the P13K/Akt pathway to be hyperactive which contributes to cell survival and resistance to therapeutics in various cancers, including the liver cancer. Hence molecules targeting this pathway present good therapeutic strategies for liver cancer. Hypothesis It was previously reported that Cardiac glycosides possessed antitumor activity by inducing apoptosis of multiple cancer cells through oxidative stress. However, whether Cardiac glycoside Lanatoside C can induce oxidative stress in liver cancer cells and induce cell death both in vitro and in vivo remains unknown. Methods Cell viability was measured by SRB assay. Cell death analysis was investigated by propidium iodide staining with flow cytometry and PARP cleavage. DCFH-DA staining and cytometry were used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Antitumor activity was investigated on mice xenografts in vivo. Results In this study, we found that Cardiac glycosides, particularly Lanatoside C from Digitalis ferruginea could significantly inhibit PTEN protein adequate Huh7 and PTEN deficient Mahlavu human liver cancer cell proliferation by the induction of apoptosis and G2/M arrest in the cells. Lanatoside C was further shown to induce oxidative stress and alter ERK and Akt pathways. Consequently, JNK1 activation resulted in extrinsic apoptotic pathway stimulation in both cells while JNK2 activation involved in the inhibition of cell survival only in PTEN deficient cells. Furthermore, nude mice xenografts followed by MRI showed that Lanatoside C caused a significant decrease in the tumor size. In this study apoptosis induction by Lanatoside C was characterized through ROS altered ERK and Akt pathways in both PTEN adequate epithelial and deficient mesenchymal liver cancer cells. Conclusion The results indicated that Lanatoside C could be contemplated in liver cancer therapeutics, particularly in PTEN deficient tumors. This is due to Lanatoside C's stress inducing action on ERK and Akt pathways through differential activation of JNK1 and JNK2 by GSK3β. © 2015 Elsevier GmbH. © 2016 Elsevier GmbH. All rights reserved.