AKT Isoforms Interplay in High-Grade Serous Ovarian Cancer Prognosis and Characterization

High-grade serous ovarian cancer (HGSOC) is among the deadliest gynecological malignancies. The acquired resistance to platinum-based therapies and the intrinsic heterogeneity of the disease contribute to the low survival rate. To improve patients’ outcomes, new combinatorial approaches able to target different tumor vulnerabilities and enhance the efficacy of the current therapies are required. AKT inhibitors are promising antineoplastic agents able to act in synergy with PARP inhibitors, but the spectrum of patients who can benefit from this combination is unclear, since the role of the three different isoforms of AKT is still unknown. Here, we study the expression of AKT isoforms on a retrospective cohort of archive tissue by RT-droplet digital PCR (ddPCR) analyzing their association with the clinicopathological features of patients. Based on AKT1/AKT2 and AKT1/AKT3 ratios, we define four AKT classes which were related to patients’ survival, tumor morphology and BRCA1 expression. Moreover, our results show that high AKT3 expression levels were frequently associated with tumors having classic features, a low number of mitoses and the presence of psammoma bodies. Overall, our study obtains new insights on AKT isoforms and their associations with the clinicopathological features of HGSOC patients. These evidences could help to better define the subsets of patients who can benefit from AKT and PARP inhibitors therapy in future clinical trials.

AKT1 is Required for a Complete Palbociclib-induced Senescence Phenotype in BRAF-V600E-Driven Human Melanoma

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by numerous functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation, however induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of the senescence response may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like, non-proliferative state in tumor cells. The AKT kinase family plays an important role in cellular growth and division, and is commonly hyperactive in many cancers including melanoma. AKT activity has also been implicated in regulation of senescence. The three AKT isoforms play both redundant and unique roles in tumorigenesis and cancer progression. To interrogate the role of AKT isoforms in the induction of cellular senescence by Cdk4/6i, we generated isoform specific AKT knockout human BRAF-V600E mutated melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. As a potential mechanism, we evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence. While we showed cGAS-STING function to be dependent on AKT1, pharmacologic inhibition of either cGAS or STING had little effect on senescence. However, we found SASP factors to require NF-kB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1 previously reported in other models. In summary, we provide the first evidence of a novel, isoform specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-kB but independent of cGAS-STING.

Akt Isoforms: A Family Affair in Breast Cancer

Akt, also known as protein kinase B (PKB), belongs to the AGC family of protein kinases. It acts downstream of the phosphatidylinositol 3-kinase (PI3K) and regulates diverse cellular processes, including cell proliferation, cell survival, metabolism, tumor growth and metastasis. The PI3K/Akt signaling pathway is frequently deregulated in breast cancer and plays an important role in the development and progression of breast cancer. There are three closely related members in the Akt family, namely Akt1(PKBα), Akt2(PKBβ) and Akt3(PKBγ). Although Akt isoforms share similar structures, they exhibit redundant, distinct as well as opposite functions. While the Akt signaling pathway is an important target for cancer therapy, an understanding of the isoform-specific function of Akt is critical to effectively target this pathway. However, our perception regarding how Akt isoforms contribute to the genesis and progression of breast cancer changes as we gain new knowledge. The purpose of this review article is to analyze current literatures on distinct functions of Akt isoforms in breast cancer.

Sevoflurane Modulates AKT Isoforms in Triple Negative Breast Cancer Cells. An Experimental Study

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial–mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.

Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox

Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidantN-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression inp53−/−andINK4a−/−/Arf−/−mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 inp47phox−/−MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox. In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important,Akt3alterations correlate with a higher frequency of mutation ofp53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.