Nuclear Akt: target for breast cancer therapy?

Most tumors consist of heterogeneous cells differing in phenotype and function. Up to date the majority of established anti-cancer therapeutics as radiation and chemotherapeutic interventions target specifically fast growing differentiated cells but exclude undifferentiated and quiescent cells. This more undifferentiated subpopulation of cells called cancer stem cells (CSCs) or tumor initiating cells (TICs) is distinguished by a stem cell-related phenotype with the ability of selfrenewing, symmetric and asymmetric cell division and some potential to differentiate. As TICs are attributed to tumor initiation, recurrence and metastasis it is of prime importance to investigate new therapies targeting TICs (2). The first CSCs reported in solid tumors are breast cancer CSCs with ALDHC/high, CD44C/high and CD24¡/low phenotype. One of the most relevant pathways involved in tumor specific features like proliferation, growth, suppressing apoptosis and enhanced survival is the activated PI3K/Akt pathway (2). Akt regulates the cell cycle by phosphorylation of the cyclin dependent kinase (CDK) inhibitors p21 and p27 resulting in their cytoplasmic localization and in that way in transition from G0 to S phase. Though the role of the PI3K/Akt pathway is well studied in human cancer cells, the role of the 3 different isoforms of Akt and their intracellular localization in the context of tumor development remains still unclear. Nuclear Akt is thought to support proliferation or cell death on cell cycle level. Previously it was reported that CD44C/high/CD24¡/low cells are sensitive to blocked PI3K activity and in that way Akt1 is important for the maintenance of the stem cell-like phenotype. In this issue, Jain et al. studied the effects of Akt intracellular localization on the maintenance of CSCs. The group showed that Akt1 localized to the nucleus (Akt-NLS) increases the CSC-like (ALDHC/high/CD44C/ /CD24) population in breast cancer cell lines SKBR3 and MDAMB468. In addition, blocking Akt activity by the Akt inhibitor Triciribine in SKBR3-mammospheres overexpressing Akt-WT or Akt-NLS resulted in a drastical reduction of CSC phenotype. These results are confirmed by 3D soft agar colony formation assay showing that Akt-NLS overexpressing mammospheres produce significantly more colonies than controls and Akt-WT transfected cells. These data point to the role of (nuclear) Akt1 in maintenance of pluripotency and stemness potential. The work of Jain et al. fit very well to the data of Gargini et al. congruently showing a reduced number of CD44C/high/CD24/low colonies after Akt knockdown. Interestingly Akt-NLS as Akt-WT transfected cells exhibit enhanced expression of the stem cell maintenance related factors Oct3/4, cMyc and Nanog on protein levels compared to controls. In contrast wild type Akt upregulates mRNA levels of Oct4, Sox2, cMyc and Nanog whereas Akt-NLS only induces expression of cMyc mRNA. The result indicates Akt1 increases transcriptional levels and a localization dependent stabilization of these pluripotency related factors. Additionally, Jain et al. could show that Akt-NLS overexpression increases cell proliferation and cell survival in breast cancer cells through enhanced phosphorylation of the cell cycle inhibitory protein p21. Indeed, Akt-NLS overexpressing cells contain higher protein levels of p21 than cells transfected with Akt-WT. Moreover, the group could show an opposite regulation of p27 based on subcellular Akt localization. These results indicate functional differences between cytoplasmic and nuclear Akt. Based on these findings, the group studied the functional effect on cell cycle regulatory proteins and could verify that Akt-NLS overexpression leads to a shorthened G0/G1 phase, an increased cell number in G2 phase, an increased cell survival and higher proliferating rates similar to Akt1 in SKBR3 mammospheres. Interestingly mRNA expression profiles differs within Akt-NLS (increase in cyclin D1 and E1 and decrease in A1) and Akt-WT (increase in cyclin A1 and E1) expressing cells indicating different stabilization of cyclin proteins dependant on Akt1 localization. Indeed these data nicely correlate to previous data showing Akt/FoxO3/Bim-dependent apoptosis regulation in mammospheres. In summary, the study indicates that nuclear Akt1 leads to higher proliferation and maintenance of stemness in CSCs of breast cancer cells and recommends further investigations in the development of selective targets in CSCs in cancer therapies focusing on nuclear Akt1 inhibition. In this context it is of prime importance to identify novel nuclear Akt interaction partners, the mode of nuclear entry of Akt and the role of the Akt isoforms in maintenance of CSC pluripotency.