MAPK4 promotes triple negative breast cancer growth and reduces tumor sensitivity to PI3K blockade

About 15–20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade.

Celecoxib-Induced Modulation of Colon Cancer CD133 Expression Occurs through AKT Inhibition and Is Monitored by 89Zr Immuno-PET

We developed an immuno-PET technique that monitors modulation of tumor CD133 expression, which is required for the success of CD133-targeted therapies. Methods. Anti-CD133 antibodies were subjected to sulfhydryl moiety-specific 89Zr conjugation. 89Zr-CD133 IgG was evaluated for specific activity and radiolabel stability. Colon cancer cells underwent binding assays and Western blotting. Biodistribution and PET studies were performed in mice. Results. 89Zr-CD133 IgG showed excellent target specificity with 97.2 ± 0.7 % blocking of HT29 cell binding by an excess antibody. Intravenous 89Zr-CD133 IgG followed biexponential blood clearance and showed CD133-specific uptake in HT29 tumors. 89Zr-CD133 IgG PET/CT and biodistribution studies confirmed high HT29 tumor uptake with lower activities in the blood and normal organs. In HT29 cells, celecoxib dose-dependently decreased CD133 expression and 89Zr-CD133 IgG binding that reached 19.9 ± 2.1 % ( P < 0.005 ) and 50.3 ± 10.9 % ( P < 0.001 ) of baseline levels by 50 μM, respectively. Celecoxib treatment of mice significantly suppressed tumor CD133 expression to 67.5 ± 7.8 % of controls ( P < 0.005 ) and reduced tumor 89Zr-CD133 IgG uptake from 15.5 ± 1.4 % at baseline to 12.3 ± 2.0 % ID / g ( P < 0.01 ). Celecoxib-induced CD133 reduction in HT29 cells and tumors was associated with substantial suppression of AKT activation. There were also reduced HIF-1α accumulation and IκBα/NFκB phosphorylation. Conclusion. 89Zr-CD133 IgG PET provides high-contrast tumor imaging and monitors celecoxib treatment-induced modulation of tumor CD133 expression, which was found to occur through AKT inhibition. This technique may thus be useful for screening drugs that can effectively suppress colon cancer stem cells.

CRISPR/Cas9-mediated Bag-1 knockout increased mesenchymal characteristics of MCF-7 cells via Akt hyperactivation-mediated actin cytoskeleton remodeling

Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma

Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

Hantaan virus replication is promoted via AKT activated mitochondria OXPHOS

Oxidative phosphorylation (OXPHOS) is a vital pathway provides ATP for intracellular activities. Here, we found that Hantaan virus (HTNV) exploited mitochondria OXPHOS to assist its replication in host cells and Protein Kinase B/AKT played a major function in this process. Inhibiting AKT activation by BEZ treatment can inhibit HTNV replication and prevent the increase of OXPHOS level caused by HTNV infection. We also found that HTNV infection can promote AKT translocation to mitochondria, where AKT phosphorylates Polynucleotide phosphorylase (PNPT). Taken together, our research demonstrates that HTNV replication exploits OXPHOS in host cells and it increases OXPHOS function by AKT-PNPT interaction in mitochondria.

The Anti-Inflammatory and Uric Acid Lowering Effects of Si-Miao-San on Gout

BackgroundSi-Miao-San (SMS) is a well-known traditional Chinese medicine. This study aims to evaluate the anti-inflammatory effects of SMS on gouty arthritis and its potential mechanism of action.MethodsThe effects and mechanism of SMS were evaluated in monosodium urate (MSU)-treated mice or macrophages. The expression of cytokines and PI3K/Akt was analyzed using real-time PCR and Western blotting analyses. Macrophage polarization was assessed with immunofluorescence assays, real-time PCR, and Western blotting. Mass spectrometry was used to screen the active ingredients of SMS.ResultsPretreatment with SMS ameliorated MSU-induced acute gouty arthritis in mice with increased PI3K/Akt activation and M2 macrophage polarization in the joint tissues. In vitro, SMS treatment significantly inhibited MSU-triggered inflammatory response, increased p-Akt and Arg-1 expression in macrophages, and promoted M2 macrophage polarization. These effects of SMS were inhibited when PI3K/Akt activation was blocked by LY294002 in the macrophages. Moreover, SMS significantly reduced serum uric acid levels in the hyperuricemia mice. Using mass spectrometry, the plant hormones ecdysone and estrone were detected as the potentially effective ingredients of SMS.ConclusionSMS ameliorated MSU-induced gouty arthritis and inhibited hyperuricemia. The anti-inflammatory mechanism of SMS may exert anti-inflammatory effects by promoting M2 polarization via PI3K/Akt signaling. Ecdysone and estrone might be the potentially effective ingredients of SMS. This research may provide evidence for the application of SMS in the treatment of gout.

Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase—AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2−/− but not of Bmal1−/− mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2−/− mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2−/− genes.

Oxyresveratrol Inhibits TNF-α-Stimulated Cell Proliferation in Human Immortalized Keratinocytes (HaCaT) by Suppressing AKT Activation

Psoriasis is a complex inflammatory disease characterized by hyperproliferative keratinocyte caused by active PI3K/AKT signaling. TNF-α concentrated in the psoriatic lesions stimulates AKT activation. We previously discovered that oxyresveratrol inhibited inflammation via suppressing AKT phosphorylation, therefore oxyresveratrol may possess a conserved property to block AKT activation and proliferation in keratinocyte in response to TNF-α. Our current study proved that oxyresveratrol exhibited potent anti-proliferative effects against TNF-α. These effects are explained by the findings that oxyresveratrol could potentially inhibit TNF-α-stimulated AKT and GSK3-β activation in a dose-dependent manner, and its inhibitory pattern was comparable to that of a specific PI3K inhibitor. Results from immunofluorescence supported that oxyresveratrol effectively inhibited AKT and GSK3-β activation in individual cells upon TNF-α stimulation. Furthermore, functional assay confirmed that oxyresveratrol repressed the expansion of the HaCaT colony over 3 days, and this was caused by the ability of oxyresveratrol to induce cell cycle arrest at S and G2/M phases and the reduction in the expression of a proliferative marker (Ki-67) and a survival marker (MCL-1). Given the importance of TNF-α and the PI3K/AKT pathway in the psoriatic phenotype, we anticipate that oxyresveratrol, which targets the TNF-α-stimulated PI3K/AKT pathway, would represent a promising psoriasis therapy in the near future.

Aerobic Exercise Training and In Vivo Akt Activation Counteract Cancer Cachexia by Inducing a Hypertrophic Profile through eIF-2α Modulation

Cancer cachexia is a multifactorial and devastating syndrome characterized by severe skeletal muscle mass loss and dysfunction. As cachexia still has neither a cure nor an effective treatment, better understanding of skeletal muscle plasticity in the context of cancer is of great importance. Although aerobic exercise training (AET) has been shown as an important complementary therapy for chronic diseases and associated comorbidities, the impact of AET on skeletal muscle mass maintenance during cancer progression has not been well documented yet. Here, we show that previous AET induced a protective mechanism against tumor-induced muscle wasting by modulating the Akt/mTORC1 signaling and eukaryotic initiation factors, specifically eIF2-α. Thereafter, it was determined whether the in vivo Akt activation would induce a hypertrophic profile in cachectic muscles. As observed for the first time, Akt-induced hypertrophy was able and sufficient to either prevent or revert cancer cachexia by modulating both Akt/mTORC1 pathway and the eIF-2α activation, and induced a better muscle functionality. These findings provide evidence that skeletal muscle tissue still preserves hypertrophic potential to be stimulated by either AET or gene therapy to counteract cancer cachexia.