Increased Akt Activity Research Articles

AKT Regulation of ORAI1-Mediated Calcium Influx in Breast Cancer Cells.

Simple SummaryA remodeling in calcium homeostasis and the protein kinase AKT signaling pathway often promotes tumorigenic traits in cancer cells. Changes in calcium signaling can be mediated through altered expression or activity of calcium channels and pumps, which constitute a class of targetable therapeutic targets. Currently, the interplay between the two signaling pathways in breast cancer cells is unclear. A better understanding of the association between calcium and AKT signaling, and the molecular players involved may identify novel therapeutic strategies for breast cancers with abnormal AKT signaling. Using fluorescence calcium imaging and gene silencing/knockout techniques, we showed that increased AKT activation results in increased calcium entry, and that this is mediated through ORAI1 calcium channels. Future studies exploring therapeutic strategies to target PTEN-deficient or hyperactivated AKT cancers should consider this novel correlation between AKT activation and ORAI1-mediated calcium influx.Although breast cancer cells often exhibit both abnormal AKT signaling and calcium signaling, the association between these two pathways is unclear. Using a combination of pharmacological tools, siRNA and CRISPR/Cas9 gene silencing techniques, we investigated the association between PTEN, AKT phosphorylation and calcium signaling in a basal breast cancer cell line. We found that siRNA-mediated PTEN silencing promotes AKT phosphorylation and calcium influx in MDA-MB-231 cells. This increase in AKT phosphorylation and calcium influx was phenocopied by the pharmacological AKT activator, SC79. The increased calcium influx associated with SC79 is inhibited by silencing AKT2, but not AKT1. This increase in calcium influx is suppressed when the store-operated calcium channel, ORAI1 is silenced. The results from this study open a novel avenue for therapeutic targeting of cancer cells with increased AKT activation. Given the association between ORAI1 and breast cancer, ORAI1 is a possible therapeutic target in cancers with abnormal AKT signaling.

Abstract A016: Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas

Abstract Soft-tissue sarcomas (STS) are a group of lethal mesenchymal tumors that are a major cause of cancer related morbidity for those under the age of 20.PI3K inhibitors have been found to be effective against subset of STS but are associated with significant toxicities limiting their use.We have thought to test a selective fibroblast activation protein (FAP) targeted delivery of PI3K/AKT/mTOR inhibitor omipalisib to STS in pre-clinical setting as FAP is overwhelmingly expressed agressive soft tissue malignancies. Experimental Design: Patient derived fibrosarcomas (aSMA-,FAP+,pAKT+) were isolated by flow cytometry.FAP(-)HT1080 fibrosarcoma cell lines were used as a negative control whereas methylcoanthrene induced mouse fibrosarcoma transfected with human FAP cDNA were used as positive control.Cell expression of FAP,AKT,mTOR was compared using SDS Page,IHC,and flow cytometry.FAP ligand coupled to omipalisib(FAPL-PI3Ki)was then evaluated in-vitro and in small animal models. Results: Cell lines with known FAP expression demonstrated significant uptake of FAPL conjugated NIR fluorochrome.Wild type cells(hFAP-) showed no ligand binding both in flow cytometry and fluorescent microscopy(p<0.05).Escalating doses of FAP-PI3Ki suppressed AKT/mTOR activity in HT-1080 hFAP cells with 10 nM being the smallest effective dose.In hFAP+cell lines 10 nM of FAP-PI3Ki inhibited the AKT/mTOR signaling but inhibition was not observed in hFAP- cell lines demonstrating the need for FAP presence for FAPL-PI3Ki activity(p<0.05).Pan non-targeted PI3Ki inhibited the AKT/mTOR pathway in both hFAP+ and hFAP-cell lines.Densitometric analysis demonstrated that in hFAP+ cells,FAP-PI3Ki at above 10 nM suppresses the AKT/mTOR activity with ratio of pAKT/AKT and pmTOR/mTOR decreasing 11 and 4-fold respectively(p<0.05).FAP-PI3Ki activity significantly reduced when hFAP+ cells were competitively inhibited with free FAPL(4.6x increase in AKT activity,p<0.05).hFAP+ cells treated with FAP-PI3Ki had significant decrease in cell migration.In small animal models,SCID Mice injected with 25 umol/kg of either FAP-PI3Ki and non-targeted PI3Ki showed marked elevation of serum insulin levels in the non-targeted PI3Ki group (0.91 ng/mL vs 0.58 ng/mL,p<0.05).Xenograft tumor digest demonstrated mice treated with FAP-PI3Ki had Akt/mTOR pathway downregulated only in hFAP+ cell lines and this was evident in mice which received 10 nM or higher dosing(pAkt/Akt ratio of 1.1 WT mice vs 0.55 in hFAP+ mice,p<0.05).There was near complete suppression of the mTOR expression in the hFAP+ treatment group (pmTOR/mTOR 1.05 vs 0.087,p<0.05).There was no treatment related mortality in FAPL-PI3Ki group as compared to non-targeted PI3Ki delivery (p<0.05). Conclusion: Our study demonstrated that FAP conjugated modified omipalisib binds FAP expressing soft-tissue sarcomas both in-vitro and in xenograft models in concordance with FAP receptor density.FAP-PI3Ki induces PI3K/AkT/mTOR pathway inhibition similar to non-targeted PI3K inhibitors without significant systemic toxicities observed in pan nontargeted PI3K inhibitors. Citation Format: Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Azra Din, Isvita Marfatia, Steven Albelda, Philip Low, Madduri Srinivasarao, Ramesh Mukkamala, Neil T. Sullivan, Evgeniy Eruslanov, Sunil Singhal. Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A016.

Alpha-Mangostin Reduces Pericellular Fibronectin on Suspended Tumor Cells and Therapeutically, but Not Prophylactically, Suppresses Distant Metastasis.

Major cancer deaths can be ascribed to distant metastasis to which the assembly of pericellular fibronectin (periFN) on suspended tumor cells (STCs) in the bloodstream that facilitate endothelial attachment can lead. Even though mangosteen pericarps (MP) extracts and the major component α-mangostin (α-MG) exhibit potent cancer chemopreventive properties, whether they can prophylactically and therapeutically be used as dietary nutraceuticals to prevent distant metastasis by suppressing periFN assembly on STCs within the circulation remains obscure. Immunofluorescence staining, MTT assays, flow cytometric assays, immunoblotting, and experimental metastasis mouse models were used to detect the effects of MP extracts or α-MG on periFN on STCs, tumor cell proliferation and apoptosis, the AKT activity, and tumor lung metastasis. The periFN assembly on STCs was significantly diminished upon treatments of STCs with either α-MG or MP extracts in a dose-dependent manner without inhibiting cell proliferation and viability due to increased AKT activity. Pretreatment of STCs with α-MG appeared to suppress tumor lung metastasis and prolong mouse survival rates. Oral gavage with MP extracts could therapeutically, but not prophylactically, prevent lung metastasis of STCs. We concluded that MP extracts or the major component α-MG may therapeutically serve as a potent anti-metastatic nutraceutical.

Abstract P1077: Nicotine Compromises Metabolic Adaptation Of The Heart To Injury By Altering PHLPP Isoforms

Rationale: Cardiovascular (CV) disease remains the leading cause of death and disability in the US and worldwide and tobacco use is a major risk factor for increasing the incidence of disease. Consequently, there is a need for understanding the cardiovascular impact of systemic nicotine on cardiac physiology and heart failure progression following injury. We have demonstrated that removal of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) increases Akt activity and protects the heart from oxidative damage, however downregulation of PHLPP2 causes damage. Preliminary data suggest that exposure to nicotine increases PHLPP1 and decreases PHLPP2 levels in the heart. Therefore, linking the direct pathogenic effects of nicotine on these cell-survival intracellular pathways such as those that utilize PHLPP1 activity needs to be rigorously examined for therapeutic efficacy and intervention. Hypothesis: We hypothesize that exposure to high levels of nicotine will compromise the heart and increase susceptibility to ischemic injury by altering PHLPP isoform expression. Methods: Using in vivo systems we investigated the effect of nicotine exposure on PHLPP expression and cardiomyocyte function and injury. Male and female wild-type WT (n=4) and PHLPP1 KO (n=4) mice were implanted with osmotic minipumps containing nicotine (5mg/kg/day) or saline for 7days and changes in protein and gene expression were examined. A second cohort of WT animals was exposed to nicotine (7 days) and subjected to 1 hour ischemia and 24 hours reperfusion (n=6). Results: Following 7 days of nicotine exposure, we found PHLPP1 protein expression was significantly increased (1.7 ± 0.25 fold) and PHLPP2 significantly decreased (0.8 ± 0.04 fold) in the hearts of WT mice. However, in PHLPP1 KO mice, nicotine exposure significant increased PHLPP2 levels in the heart (1.5 ± 0.044fold). Also, we found that exposure to nicotine suppressed genes important for mitochondrial biogenesis and decreased antioxidant protein expression in the hearts of WT mice while antioxidant signaling was significantly increased in KO hearts. Following 7 days of nicotine exposure WT mice have increased infarct injury (39% ± 3.2, n=6) following 24-hour reperfusion compared to saline treated WT (31% ± 0.55, n=4) with the same area at risk. Preliminary studies found that PHLPP1 KO significantly protected the heart from ischemic injury (26% ± 0.38, n=3), however the effect in the presence of nicotine is under investigation. Conclusions: Our data suggests that nicotine exposure has detrimental effects on the heart by altering PHLPP isoform expression which compromises oxidative signaling and increases injury. Further studies will investigate the mechanism of nicotine induced PHLPP isoform expression both in vitro and in vivo and determine whether targeting PHLPP1 could significantly impact cardiac outcome associated with nicotine use.

Adipose-derived stromal cells reverse insulin resistance through inhibition of M1 expression in a type 2 diabetes mellitus mouse model

BackgroundAdipose tissue inflammation is considered as one of the major mechanisms underlying the pathogenesis of insulin resistance and complications in diabetes. Here, we aimed to study the effects of adipose-derived stromal cells on diabetes-induced insulin resistance and M1 cytokine expression.MethodsStromal vascular fractions (SVFs) purified from the inguinal adipose tissue of diabetic mice were treated with plasma from either nondiabetic (Lepr+/+) or diabetic (Leprdb/db) mice and injected into the inguinal white adipose tissue of Leprdb/db mice.ResultsWe found that diabetic plasma treatment induced, whereas nondiabetic plasma suppressed TNF-α, IL-1β, and dipeptidyl peptidase 4 (DPP4) mRNA expression in SVFs in vitro. Importantly, the injection of nondiabetic plasma-treated SVFs significantly decreased TNF-α, IL-6, IL-1β, CCL2, and IL-33 and induced IL-10 mRNA expression in adipose tissue of Leprdb/db mice in vivo. Furthermore, we observed that nondiabetic plasma-treated SVFs increased mRNA expression of Foxp3 in adipose tissue macrophages and Foxp3 in adipose CD4+ T cells, decreased CD11b+CD11c+ cells in adipose tissue, and suppressed mRNA expression of ICAM-1, FCM3, IL-6, IL-1β, iNOS, TNF-α, and DPP4 as well as protein expression of DPP4 and phosphorylated JNK and NF-κB in the liver of Leprdb/db mice. Moreover, we found that nondiabetic plasma-treated SVFs increased Akt activation following insulin administration and attenuated glucose intolerance in Leprdb/db mice.ConclusionsOur results demonstrate that nondiabetic plasma inhibits M1 but increases M2 cytokine expression in adipose tissue of diabetic mice. Most importantly, our findings reveal that nondiabetic plasma-treated SVFs are capable of mitigating diabetes-induced plasma DPP4 activity, liver inflammation, and insulin resistance and that may be mediated through suppressing M1 cytokines but increasing IL-10 and Tregs in adipose tissue. Altogether, our findings suggest that adipose stromal cell-based therapy could potentially be developed as an efficient therapeutic strategy for the treatment of diabetes.

Progerin modulates the IGF-1R/Akt signaling involved in aging.

Progerin, a product of LMNA mutation, leads to multiple nuclear abnormalities in patients with Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disorder. Progerin also accumulates during physiological aging. Here, we demonstrate that impaired insulin-like growth factor 1 receptor (IGF-1R)/Akt signaling pathway results in severe growth retardation and premature aging in Zmpste24−/− mice, a mouse model of progeria. Mechanistically, progerin mislocalizes outside of the nucleus, interacts with the IGF-1R, and down-regulates its expression, leading to inhibited mitochondrial respiration, retarded cell growth, and accelerated cellular senescence. Pharmacological treatment with the PTEN (phosphatase and tensin homolog deleted on chromosome 10) inhibitor bpV (HOpic) increases Akt activity and improves multiple abnormalities in Zmpste24-deficient mice. These findings provide previously unidentified insights into the role of progerin in regulating the IGF-1R/Akt signaling in HGPS and might be useful for treating LMNA-associated progeroid disorders.

Evidence for preserved insulin responsiveness in the aging rat brain

Insulin appears to exert salutary effects in the central nervous system (CNS). Thus, brain insulin resistance has been proposed to play a role in brain aging and dementia but is conceptually complex and unlikely to fit classic definitions established in peripheral tissues. Thus, we sought to characterize brain insulin responsiveness in young (4–5 months) and old (24 months) FBN male rats using a diverse set of assays to determine the extent to which insulin effects in the CNS are impaired with age. When performing hyperinsulinemic-euglycemic clamps in rats, intracerebroventricular (ICV) infusion of insulin in old animals improved peripheral insulin sensitivity by nearly two-fold over old controls and comparable to young rats, suggesting preservation of this insulin-triggered response in aging per se (p < 0.05). We next used an imaging-based approach by comparing ICV vehicle versus insulin and performed resting state functional magnetic resonance imaging (rs-fMRI) to evaluate age- and insulin-related changes in network connectivity within the default mode network. In aging, lower connectivity between the mesial temporal (MT) region and other areas, as well as reduced MT signal complexity, was observed in old rats, which correlated with greater cognitive deficits in old. Despite these stark differences, ICV insulin failed to elicit any significant alteration to the BOLD signal in young rats, while a significant deviation of the BOLD signal was observed in older animals, characterized by augmentation in regions of the septal nucleus and hypothalamus, and reduction in thalamus and nucleus accumbens. In contrast, ex vivo stimulation of hippocampus with 10 nM insulin revealed increased Akt activation in young (p < 0.05), but not old rats. Despite similar circulating levels of insulin and IGF-1, cerebrospinal fluid concentrations of these ligands were reduced with age. Thus, these data highlight the complexity of capturing brain insulin action and demonstrate preserved or heightened brain responses to insulin with age, despite dampened canonical signaling, thereby suggesting impaired CNS input of these ligands may be a feature of reduced brain insulin action, providing further rationale for CNS replacement strategies.

Abstract 156: Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter

Abstract Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer. Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. Here we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. To demonstrate inhibition of Akt, A549 cells stably expressing the reporter were treated with compounds targeting the PI3K/Akt kinase pathway, including allosteric Akt inhibitors MK2206 and API-1, competitive Akt inhibitors AZD5363 and Ipatasertib, and upstream PI3K kinase inhibitors LY294002 and PI-103. Quantification of treatment responses using the Incucyte® Live Cell Analysis System showed concentration-dependent inhibition of Akt activity for all compounds with varying kinetic profiles over 24 hours. To study activation of Akt, HeLa cells were first cultured in the absence of serum to reduce Akt activity. After 4 hours of incubation in serum-free conditions, the cells were treated with either epidermal growth factor (EGF) or recombinant insulin-like growth factor (R3-IGF-1) to activate Akt. An increase in Akt activity was observed for both treatments. While R3-IGF-1-induced activation was sustained over the 12-hour time course, activation by EGF diminished over time. Cell lines with mutations in the tumor suppressor PTEN showed no response to serum starvation or activation with EGF or R3-IGF-1. In addition to monitoring Akt activity over time, the integrated red nuclear restricted protein of the reporter enables concurrent measurements of proliferation. The selective Akt inhibitor MK2206 decreased Akt activity in a similar concentration-dependent manner in both T-47D and MDA-MB-231 cell lines. In contrast, measurements of red object count from the same cells reveal differential effects of MK2206 on proliferation between the two cell lines, with concentration-dependent inhibition of T-47D cell growth but little effect on MDA-MB-231 cells. Overall, these data highlight the utility of the Incucyte® Kinase Akt Lentivirus Reagent to provide valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Citation Format: John N. Rauch, Susan K. Foltin, Libuse Oupicka, Matthew Dilsaver, Grigory S. Filonov, Gillian Lovell, Jasmine Trigg, Cicely L. Schramm. Dynamic live-cell visualization and quantification of Akt activity using a genetically-encoded, fluorescent kinase translocation reporter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 156.

Pro‐inflammatory Cytokines Increase Glycolysis through Upregulation of Uncoupling Protein and Akt Activation in Colorectal Cancer Cells

Non‐cancerous colonocytes use butyrate, which is a short‐chain fatty acid derived from the fermentation of dietary fiber, as a primary energy source. In contrast, cancerous colonocytes use glucose as their preferred energetic substrate and undergo enhanced glycolysis or the Warburg effect. It has been reported that increased production of pro‐inflammatory cytokines such as interleukin1 beta and tumor necrosis factor alpha (TNFalpha) promote glycolysis. Previously, we have shown that interleukin1 beta increased glycolysis and decreased butyrate oxidation. However, the mechanism underlying how this pro‐inflammatory cytokine increased glycolysis was unclear. Here, we have discovered that Uncoupling Protein 2 (UCP2) appears to mediate the elevation in glycolysis induced by interleukin1 beta. UCP2 is a mitochondrial protein that transports the protons back into the mitochondrial matrix, thus dissipating the proton gradient and reducing ATP production. UCP2 is upregulated in various cancers such as breast, prostate, skin, and colorectal cancers. We hypothesize that interleukin1 beta promotes glycolysis by upregulating UCP2 and activating Akt in colorectal cancer cells. We have found colorectal cancer cells treated with interleukin1 beta showed elevated UCP2 expression and increased Akt activation. Next, we utilized the Seahorse XFe24 analyzer to check whether interleukin1 beta affects proton leak and ATP production in colorectal cancer cells. Preliminary data suggests that interleukin1 beta increases proton leak and decreases ATP production in mitochondria, which is consistent with the upregulation of UCP2 and demonstrates a functional consequence. To study whether UCP2 mediates the interleukin1 beta effect toward increasing glycolysis, we created a stable UCP2 knockdown in colorectal cancer cells and tested whether colorectal cancer cells that had diminished UCP2 from the RNAi knockdown did not show an increase in glycolysis when treated with interleukin1 beta as compared to RNAi mock colorectal cancer cells. Our preliminary data allude to a role for UCP2 in mediating the interleukin1 beta induced increase in glycolysis observed in colorectal cancer cells.

Sera from women with different metabolic and menopause states differentially regulate cell viability and Akt activation in a breast cancer in-vitro model.

Obesity is associated with an increased incidence and aggressiveness of breast cancer and is estimated to increment the development of this tumor by 50 to 86%. These associations are driven, in part, by changes in the serum molecules. Epidemiological studies have reported that Metformin reduces the incidence of obesity-associated cancer, probably by regulating the metabolic state. In this study, we evaluated in a breast cancer in-vitro model the activation of the IR-β/Akt/p70S6K pathway by exposure to human sera with different metabolic and hormonal characteristics. Furthermore, we evaluated the effect of brief Metformin treatment on sera of obese postmenopausal women and its impact on Akt and NF-κB activation. We demonstrated that MCF-7 cells represent a robust cellular model to differentiate Akt pathway activation influenced by the stimulation with sera from obese women, resulting in increased cell viability rates compared to cells stimulated with sera from normal-weight women. In particular, stimulation with sera from postmenopausal obese women showed an increase in the phosphorylation of IR-β and Akt proteins. These effects were reversed after exposure of MCF-7 cells to sera from postmenopausal obese women with insulin resistance with Metformin treatment. Whereas sera from women without insulin resistance affected NF-κB regulation. We further demonstrated that sera from post-Metformin obese women induced an increase in p38 phosphorylation, independent of insulin resistance. Our results suggest a possible mechanism in which obesity-mediated serum molecules could enhance the development of luminal A-breast cancer by increasing Akt activation. Further, we provided evidence that the phenomenon was reversed by Metformin treatment in a subgroup of women.

Increased O-GlcNAcylation promotes IGF-1 receptor/PhosphatidyI Inositol-3 kinase/Akt pathway in cervical cancer cells

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.

Abstract P1-06-03: Promotion of E-cadherin-mediated tumor cell adhesion by COX-2/GSK3β signaling is a targetable mechanism of metastatic breast cancer

Abstract Purpose: Metastatic progression and treatment resistance of breast cancer has been associated with epithelial-mesenchymal transition (EMT), including downregulation of E-cadherin (CDH1) gene expression, which can be initiated by inflammatory signaling such as by COX-2 (PTGS2). However, E-cadherin expression is maintained in many advanced breast cancers, including inflammatory BC (IBC), where it plays an essential role in forming tumor cell emboli within the cancer parenchyma and dermal lymph vasculature and which predict poor outcomes. Thus, the study of IBC offers an opportunity to understand the mechanisms of IBC and other aggressive BCs that lead to E-cadherin-associated cluster-based metastasis, which has recently received heightened recognition. Here, we have investigated the mechanisms that sustain E-cadherin expression in metastatic breast cancer to identify new targeted treatment options. Study Design and Methods: In vitro emboli formation assays and gene/protein expression studies with IBC (SUM149, IBC-3) and non-IBC (SUM159, MCF-7) cell lines along with gene silencing, overexpression, and pharmacological interventions. Analysis of experimental (SUM149) and spontaneous (orthotopic patient-derived xenograft, PDX) lung metastases and of circulating tumor cells (CTCs) in xenograft models treated with celecoxib (Cxb) and/or paclitaxel by imaging cytometry, immunohistochemistry, and/or Western analysis. Results: By analyzing the transcription factor C/EBPδ (CEBPD) and cells derived from inflammatory breast cancer (IBC), an aggressive breast cancer subtype that often presents with E-cadherin-dependent tumor cell emboli, we discovered that COX-2, unexpectedly, sustained E-cadherin protein expression without changing its mRNA levels. Using an in vitro tumor cell emboli culture paradigm (3D), we found that COX-2 or its metabolite PGE2 increased AKT activity and the inhibitory phosphorylation on GSK3β that prevents degradation of p120 catenin (CTNND1), a stabilizer of E-cadherin adhesion complexes. Conversely, the COX-2 inhibitor celecoxib downregulated E-cadherin specifically at the protein level and caused cell death in 3D. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcomes and, along with inhibitory GSK3β phosphorylation, in patient-derived xenografts (PDX) of metastatic triple-negative breast cancers (TNBC). Celecoxib alone decreased E-cadherin protein expression within xenograft primary tumors, reduced circulating tumor cells (CTCs) and clusters, and sensitized lung metastases to paclitaxel treatment. Conclusions: Our study uncovered a novel function of COX-2/PGE2 in promoting E-cadherin protein expression and cell-cell adhesions that are relevant for tumor cell cluster-based metastasis. Indeed, COX-2 inhibition reduced CTC clusters in a xenograft model, and sentized established metastases to chemotherapy. These results suggest that patients with COX-2+/E-cadherin+ metastastic BC, including IBC, may specifically benefit from targeting the PGE2 pathway in cobniation therapy approaches. Citation Format: Kuppusamy Balamurugan, Saadiya Sehareen, Savitri Krishnamurthy, Shikha Sharan, Wei Tang, Naoto Ueno, Stefan Ambs, Dipak Poria, Esta Sterneck. Promotion of E-cadherin-mediated tumor cell adhesion by COX-2/GSK3β signaling is a targetable mechanism of metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-06-03.

MiR-193a-3p increases glycolysis under hypoxia by facilitating Akt phosphorylation and PFKFB3 activation in human macrophages

Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.

DNA-PK promotes activation of the survival kinase AKT in response to DNA damage through an mTORC2-ECT2 pathway.

The kinase AKT (also known as protein kinase B) is a key regulator of cell proliferation, survival, and metabolism. In addition to being activated by growth factors, AKT is activated in response to DNA damage. Here, we found that the DNA damage response kinase DNA-PK sustains cell survival through a phosphorylation event that leads to increased AKT activity. In various cancer and noncancer cells in culture, DNA damage caused by ionizing radiation or topoisomerase inhibitors triggered DNA-PK–dependent phosphorylation of the mTOR complex 2 (mTORC2) subunit Sin1, which enabled its interaction with the guanine nucleotide exchange factor ECT2. Depleting Sin1 or ECT2 or disrupting the protein interaction or catalytic function of ECT2 attenuated DNA damage–induced AKT activation, thereby enhancing cellular sensitivity to DNA-damaging agents. Our findings elucidate a mechanism mediating DNA damage–induced AKT activation and cell survival.

A detrimental role of NLRP6 in host iron metabolism during Salmonella infection

Maintaining host iron homeostasis is an essential component of nutritional immunity responsible for sequestrating iron from pathogens and controlling infection. Nucleotide-oligomerization domain-like receptors (NLRs) contribute to cytoplasmic sensing and antimicrobial response orchestration. However, it remains unknown whether and how NLRs may regulate host iron metabolism, an important component of nutritional immunity. Here, we demonstrated that NLRP6, a member of the NLR family, has an unconventional role in regulating host iron metabolism that perturbs host resistance to bacterial infection. NLRP6 deficiency is advantageous for maintaining cellular iron homeostasis in both macrophages and enterocytes through increasing the unique iron exporter ferroportin-mediated iron efflux in a nuclear factor erythroid-derived 2–related factor 2 (NRF2)-dependent manner. Additional studies uncovered a novel mechanism underlying NRF2 regulation and operating through NLRP6/AKT interaction and that causes a decrease in AKT phosphorylation, which in turn reduces NRF2 nuclear translocation. In the absence of NLRP6, increased AKT activation promotes NRF2/KEAP1 dissociation via increasing mTOR-mediated p62 phosphorylation and downregulates KEAP1 transcription by promoting FOXO3A phosphorylation. Together, our observations provide new insights into the mechanism of nutritional immunity by revealing a novel function of NLRP6 in regulating iron metabolism, and suggest NLRP6 as a therapeutic target for limiting bacterial iron acquisition.

Abstract P132: Promotion of E-cadherin-mediated tumor cell adhesion by COX-2/GSK3b signaling is a targetable mechanism of metastatic breast cancer

Abstract Purpose: Metastatic progression and treatment resistance of breast cancer has been associated with epithelial-mesenchymal transition (EMT), including downregulation of E-cadherin (CDH1) gene expression, which can be initiated by inflammatory signaling such as by COX-2 (PTGS2). However, E-cadherin expression is maintained in many advanced breast cancers, including inflammatory BC (IBC), where it plays an essential role in forming tumor cell emboli within the cancer parenchyma and dermal lymph vasculature and which predict poor outcomes. Thus, the study of IBC offers an opportunity to understand the mechanisms of IBC and other aggressive BCs that lead to E-cadherin-associated cluster-based metastasis, which has recently received heightened recognition. Here, we have investigated the mechanisms that sustain E-cadherin expression in metastatic breast cancer to identify new targeted treatment options. Study Design and Methods: In vitro emboli formation assays and gene/protein expression studies with IBC (SUM149, IBC-3) and non-IBC (SUM159, MCF-7) cell lines along with gene silencing, overexpression, and pharmacological interventions. Analysis of experimental (SUM149) and spontaneous (orthotopic patient-derived xenograft, PDX) lung metastases and of circulating tumor cells (CTCs) in xenograft models treated with celecoxib (Cxb) and/or paclitaxel by imaging cytometry, immunohistochemistry, and/or Western analysis. Results: By analyzing the transcription factor C/EBPδ (CEBPD) and cells derived from inflammatory breast cancer (IBC), an aggressive breast cancer subtype that often presents with E-cadherin-dependent tumor cell emboli, we discovered that COX-2, unexpectedly, sustained E-cadherin protein expression without changing its mRNA levels. Using an in vitro tumor cell emboli culture paradigm (3D), we found that COX-2 or its metabolite PGE2 increased AKT activity and the inhibitory phosphorylation on GSK3β that prevents degradation of p120 catenin (CTNND1), a stabilizer of E-cadherin adhesion complexes. Conversely, the COX-2 inhibitor celecoxib downregulated E-cadherin specifically at the protein level and caused cell death in 3D. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcomes and, along with inhibitory GSK3β phosphorylation, in patient-derived xenografts (PDX) of metastatic triple-negative breast cancers (TNBC). Celecoxib alone decreased E-cadherin protein expression within xenograft primary tumors, reduced circulating tumor cells (CTCs) and clusters, and sensitized lung metastases to paclitaxel treatment. Conclusions: Our study uncovered a novel function of COX-2/PGE2 in promoting E-cadherin protein expression and cell-cell adhesions that are relevant for tumor cell cluster-based metastasis. Indeed, COX-2 inhibition reduced CTC clusters in a xenograft model, and sentized established metastases to chemotherapy. These results suggest that patients with COX-2+/E-cadherin+ metastastic BC, including IBC, may specifically benefit from targeting the PGE2 pathway in cobniation therapy approaches. Citation Format: Balamurugan Kuppusamy, Saadiya Sehareen, Savitri Krishnamurthy, Shikha Sharana, Wei Tang, Naoto T. Ueno, Stefan Ambs, Dipak K. Poria, Esta Sternecka. Promotion of E-cadherin-mediated tumor cell adhesion by COX-2/GSK3b signaling is a targetable mechanism of metastatic breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P132.

Changes in Thyroid Hormone Signaling Mediate Cardiac Dysfunction in the Tg197 Mouse Model of Arthritis: Potential Therapeutic Implications.

Background Rheumatoid Arthritis (RA) patients show a higher risk of heart failure. The present study investigated possible causes of cardiac dysfunction related to thyroid hormone (TH) signaling in a RA mouse model. Methods A TNF-driven mouse model of RA[TghuTNF (Tg197)] was used. Cardiac function was evaluated by echocardiography. SERCA2a and phospholamban protein levels in left ventricle (LV) tissue, thyroid hormone levels in serum, TH receptors in LV and TH-related kinase signaling pathways were measured. T3 hormone was administered in female Tg197 mice. Results We show LV and atrial dilatation with systolic dysfunction in Tg197 animals, accompanied by downregulated SERCA2a. We suggest an interaction of pro-inflammatory and thyroid hormone signaling indicated by increased p38 MAPK and downregulation of TRβ1 receptor in Tg197 hearts. Interestingly, female Tg197 mice showed a worse cardiac phenotype related to reduced T3 levels and Akt activation. T3 supplementation increased Akt activation, restored SERCA2a expression and improved cardiac function in female Tg197 mice. Conclusions TNF overexpression of Tg197 mice results in cardiac dysfunction via p38 MAPK activation and downregulation of TRβ1. Gender-specific reduction in T3 levels could cause the worse cardiac phenotype observed in female mice, while T3 administration improves cardiac function and calcium handling via modified Akt activation.

The New PI3K/mTOR Inhibitor GNE-477 Inhibits the Malignant Behavior of Human Glioblastoma Cells.

The most common primary central nervous system tumor in adults is glioblastoma multiforme (GBM). The high invasiveness of GBM cells is an important factor leading to inevitable tumor recurrence and a poor prognosis of patients. GNE-477, a novel PI3K/mTOR inhibitor, has been reported to exert antiproliferative effects on other cancer cells. However, researchers have not clearly determined whether GNE-477 produces antitumor effects on GBM. In the present study, GNE-477 significantly inhibited the proliferation, migration and invasion of U87 and U251 cells. In addition, GNE-477 also induced apoptosis of GBM cells, arresting the cell cycle in G0/G1 phase. More importantly, GNE-477 also reduced the levels of AKT and mTOR phosphorylation in the AKT/mTOR signaling pathway in a concentration-dependent manner. An increase in AKT activity induced by SC79 rescued the GNE-477-mediated inhibition of GBM cell proliferation and apoptosis. The antitumor effects of GNE-477 and the regulatory effects on related molecules were further confirmed in vivo using a nude mouse intracranial xenograft model. In conclusion, our study indicated that GNE-477 exerted significant antitumor effects on GBM cells in vitro and in vivo by downregulating the AKT/mTOR pathway.

Ticagrelor and Dapagliflozin Have Additive Effects in Ameliorating Diabetic Nephropathy in Mice with Type-2 Diabetes Mellitus.

Ticagrelor and dapagliflozin can suppress the activation of the NOD-like receptor 3 (NLRP3)-inflammasome and activate AMP-activated protein kinase (AMPK). The anti-inflammatory effects of dapagliflozin has been shown to depend on AMPK activation. Dapagliflozin and ticagrelor have been shown to have additive effects on the progression of diabetic cardiomyopathy in BTBR ob/ob mice with type-2 diabetes. We assessed whether dapagliflozin and ticagrelor have additive effects on the activation of the NLRP3-inflammasome and the progression of diabetic nephropathy in mice with type-2 diabetes. Eight-week-old BTBR received either no-drug, dapagliflozin (1.5mg/kg/d), ticagrelor (100mg/kg/d), or their combination for 12weeks. Blood was assessed weekly for glucose and urine for glucose and albumin. After 12weeks, blood creatinine, cystatin C, inflammasome activation, and insulin were assessed by ELISA. Renal cortex samples were assessed by hematoxylin and eosin and periodic acid-Schiff staining. RT-PCR and immunoblotting were used to evaluate fibrosis and the activation of Akt, AMPK and the inflammasome. Both ticagrelor and dapagliflozin reduced serum creatinine and cystatin C levels and urinary albumin. Both drugs attenuated the increase in glomerular area and mesangial matrix index. Both drugs decreased collagen-1 and collagen-3 expression and the activation of the NLRP3-inflammasome. Both drugs increased P-AMPK levels, but only dapagliflozin increased P-Akt levels. Overall, the protective effects of dapagliflozin and ticagrelor were additive. Dapagliflozin and ticagrelor attenuated the progression of diabetic nephropathy in BTBR ob/ob mice with additive effects of the combination. This was associated with AMPK activation and reduced activation of the NLRP3 inflammasome, whereas only dapagliflozin increased Akt activation.

Inhibition of RPS6K reveals context-dependent Akt activity in luminal breast cancer cells.

Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contribute to the risk and advancement of many cancer types by activating cell survival cascades. Similarities between these pathways have thus far prevented the development of pharmacological interventions that specifically target either Ins or IGF1 signaling. To identify differences in early Ins and IGF1 signaling mechanisms, we developed a dual receptor (IGF1R & InsR) computational response model. The model suggested that ribosomal protein S6 kinase (RPS6K) plays a critical role in regulating MAPK and Akt activation levels in response to Ins and IGF1 stimulation. As predicted, perturbing RPS6K kinase activity led to an increased Akt activation with Ins stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating the emergence of compensation mechanisms without disrupting InsR signaling.