Intracellular AKT Signaling Research Articles

Tumor necrosis factor superfamily 14 (TNFSF14/ LIGHT) attenuates neovascularization by reducing circulating endothelial progenitor cells and function

Abstract Background Tumor necrosis factor superfamily 14 (TNFSF14) is also known as LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) can bind to herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT signaling strengthens the synthesis of nitric oxide, reactive oxygen species, and cytokines and affects organ function. LIGHT also stimulates angiogenesis in tumors and induces synthesis of high endothelial venules, degrades extracellular matrix in thoracic aortic dissection, induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed this effect in the current study. Methods C57BL/6 mice underwent hind limb ischemia surgery for model construction. Doppler ultrasound, immunohistochemical staining, and western blotting were employed to analyze the effects of LIGHT on angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. Results In the mice, LIGHT injection inhibited angiogenesis in ischemic limbs. In vitro, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the normal operation of intracellular Akt signaling pathway and endothelial nitrite oxide synthase (eNOS) function. Conclusions LIGHT inhibits angiogenesis after tissue ischemia. LIGHT inhibition of intracellular Akt-eNOS signaling axis and mitochondrial respiration in EPCs impair cell function. These events are the possible causes of impaired angiogenesis.LIGHT decreases the recovery of ischemiaSenescence and dysfunction of EPC

Tumor Necrosis Factor Superfamily 14 (LIGHT) Restricts Neovascularization by Decreasing Circulating Endothelial Progenitor Cells and Function.

Tumor necrosis factor superfamily 14 (TNFSF14) is also known as the LT-related inducible ligand (LIGHT). It can bind to the herpesvirus invasion mediator and lymphotoxin-β receptor to perform its biological activity. LIGHT has multiple physiological functions, including strengthening the synthesis of nitric oxide, reactive oxygen species, and cytokines. LIGHT also stimulates angiogenesis in tumors and induces the synthesis of high endothelial venules; degrades the extracellular matrix in thoracic aortic dissection, and induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed these effects in the current study. In this study, the animal model of hind limb ischemia surgery in C57BL/6 mice was performed. Doppler ultrasound, immunohistochemical staining, and Western blotting were employed to analyze the situation of angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. The results in the animal study showed that LIGHT injection inhibited angiogenesis in ischemic limbs. For the in vitro studies, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the proper functioning of the intracellular Akt signaling pathway, endothelial nitrite oxide synthase (eNOS), and mitochondrial respiration. In conclusion, LIGHT inhibits angiogenesis after tissue ischemia. This may be related to the clamped EPC function.

A Chemical Approach for Profiling Intracellular AKT Signaling Dynamics from Single Cells.

We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.

Abstract B100: Mutant KRAS-induced macrophage migration inhibitory factor (MIF) secretion promotes resistance to cetuximab in colorectal cancer

Abstract The tumor microenvironment is recognized as developing crosstalk between different cells by secretion of growth factors and cytokines, thus providing oncogenic signals enhancing tumor progression and drug resistance. Here, we hypothesized that tumor cells carrying KRAS mutation-induced cytokines may have a critical role in intercellular communication between microenvironment and tumor cells. We first investigated biologic evidence of secreted cytokines in KRAS mutant type (KRAS MT) cell lines. We explored the roles of cytokine that cell morphology, colony-formation ability, and wound-healing ability were enhanced when KRAS wild-type (KRAS WT) cell lines were exposed to conditioned media (CM) from KRAS MT cells, and we found macrophage migration inhibitory factor (MIF) was highly expressed in KRAS MT cells by analysis of proteomics. We also observed secreted MIF level between KRAS WT and MT cell lines, and it was highly secreted in KRAS MT cell lines. In addition, compared with patients whose KRAS mutation was not harbored, MIF expression was higher in patients who have KRAS mutation. The predictive marker of KRAS mutation in colorectal cancer patients is associated with resistance to antiepidermal growth factor receptor (EGFR) antibody cetuximab. Following the hypothesis, we investigated that secreted cytokines have an effect on the cetuximab resistance to surrounding cells including KRAS WT cells. Treatment of CM from KRAS MT cells led to cetuximab resistance in KRAS WT cells and MIF blockade prevented cetuximab resistance. Also, CM from knockout in KRAS MT cells by using CRISPR/Cas9 system resulted in sensitizing to cetuximab resistance compared with CM from KRAS MT cells. In conclusion, we demonstrated for the first time that MIF promotes the cetuximab resistance of KRAS WT colorectal cancer cells, and this effect is mediated by paracrine and autocrine signaling-induced activation of the intracellular AKT signaling pathways and regulated by NF-kb transcription factor through oncogenic KRAS mutation and MIF overexpression, respectively. These findings suggest that MIF may be a promising predictive biomarker for the cetuximab resistance of KRAS wild-type colorectal cancer patients. Citation Format: Jee Eun Jang, Hwang-Phill Kim, Sae Won Han, Tae You Kim. Mutant KRAS-induced macrophage migration inhibitory factor (MIF) secretion promotes resistance to cetuximab in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B100.

Vascular E-Selectin Mediates Chemo-Resistance in Acute Myeloid Leukemia Initiating Cells Via Canonical Receptors PSGL-1 (CD162) and Hcell (CD44) and AKT Signaling

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance.Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44-/-PSGL1-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ.When transplanted into recipient mice CD44-/-PSGL1-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44-/-PSGL1-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling.To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT+ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser473 and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance.To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44-/-PSGL1-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKTSer473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy .In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy. DisclosuresMagnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application.

Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators.