Akt Pathway Research Articles

Treatment with onion bulb extract both prevents and reverses allergic inflammation in a murine model of asthma

Context Asthma presents a global health challenge. The main pharmacotherapy is synthetic chemicals and biological-based drugs that are costly, and have significant side effects. In contrast, use of natural products, such as onion (Allium cepa L., Amaryllidaceae) in the treatment of airway diseases has increased world-wide because of their perceived efficacy and little safety concerns. However, their pharmacological actions remain largely uncharacterized. Objective We investigated whether onion bulb extract (OBE) can (1) reverse established asthma phenotype (therapeutic treatment) and/or (2) prevent the development of the asthma phenotype, if given before the immunization process (preventative treatment). Materials and methods Six groups of male Balb/c mice were established for the therapeutic (21 days) and five groups for the preventative (19 days) treatment protocols; including PBS and house dust mite (HDM)-challenged mice treated with vehicle or OBE (30, 60, and 100 mg/kg/i.p.). Airways inflammation was determined using cytology, histology, immunofluorescence, Western blot, and serum IgE. Results Therapeutic (60 mg/kg/i.p.) and preventative (100 mg/kg/i.p.) OBE treatment resulted in down-regulation of HDM-induced airway cellular influx, histopathological changes and the increase in expression of pro-inflammatory signaling pathway EGFR, ERK1/2, AKT, pro-inflammatory cytokines and serum IgE. Discussion and Conclusion Our data show that OBE is an effective anti-inflammatory agent with both therapeutic and preventative anti-asthma effects. These findings imply that onion/OBE may be used as an adjunct therapeutic agent in established asthma and/or to prevent development of allergic asthma. However, further studies to identify the active constituents, and demonstrate proof-of-concept in humans are needed.

TFE3/PI3K/Akt/mTOR Axis in Renal Cell Carcinoma Affects Tumor Microenvironment

The role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in renal cell carcinoma (RCC) progression, metastasis, and resistance to therapies has not been investigated thoroughly. Transcription factor E3 (TFE3) expression is related to a poorer prognosis and tumor microenvironment in patients with RCC. This study aimed to determine the relationship between TFE3 and the PI3K/Akt pathway. TFE3 down-regulation was achieved by transient transfection of siRNA and shRNA in UOK146 cells. TFE3 overexpression was induced by transient transfection with pcDNA3.1 encoding the constitutively active form of TFE3. The cells were treated with mammalian target of rapamycin (mTOR) and PI3K inhibitors. Western blot was performed to detect TFE3, programmed death-ligand 1, phospho-Akt, and Akt. Phospho-Akt expression increased significantly upon TFE3 down-regulation, and decreased significantly upon up-regulation. When RCC cells were treated with a PI3K inhibitor (LY294002), TFE3 expression increased and phospho-Akt expression decreased. Data from this study indicate that TFE3 plays a role in the PI3K/Akt pathway in RCC. The results of this study suggest that PI3K/Akt inhibitors may aid in the treatment of patients with RCC by affecting the tumor microenvironment.

Abstract LB452: Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors

Abstract Melanoma-initiating stem cells are involved in tumorigenesis, invasion, and drug resistance in malignant melanoma, and are characterized by increased expression of the stem cell marker CD133. We previously showed that CD133 knockout (KO) by CRISPR-Cas9 enhanced MEK inhibitor trametinib (T) -induced apoptosis in patient-derived BAKP melanoma cells by reducing anti-apoptotic p-AKT and p-BAD and increasing pro-apoptotic BAX. Dox-induced CD133 expression, in contrast, diminished apoptosis in T-treated cells, and exhibited elevated p-AKT, p-BAD, and decreased activation of BAX and caspases-3 and -9. This is the first time that CD133 is shown to activate a survival pathway wherein (1) CD133 increases AKT phosphorylation and activation, which induces (2) BAD phosphorylation and inactivation, and (3) reduces caspases-3 and -9 activity leading to apoptosis suppression, increased cell survival and drug resistance in melanoma. We investigated the effects of MEK inhibition with T in combination with the pan-AKT inhibitor capivasertib (AZD5363; C) in BAKP melanoma cells, with or without Dox-induced CD133 expression. Kinetic XTT cell viability and Annexin-Sytox Blue apoptosis assays revealed significantly reduced survival of BAKP cells exposed to a combination of T+C, compared to T alone. To determine the relative contribution of each of the three AKT genes to increased drug resistance of CD133-inducible melanoma stem cells, AKT 1, 2, and/or 3 were knocked out using sgRNAs specific to each of the three AKTs in a CRISPR-Cas9 lentiviral vector, followed by puromycin selection. Gene knockout was confirmed by immunoblot analysis using antibodies specific to each of the three AKTs, as well as by DNA sequence analysis. BAKP control or AKT KO cells were then exposed to T or T+C, and subjected to apoptosis assays, as well as immunoblot analysis with antibodies to apoptosis markers. Single KO of AKT 2 or 3, but not AKT 1, sensitized BAKP cells exposed to T alone, partially obviating the requirement for C to inhibit AKT. Triple KO of AKT 1, 2, and 3 rendered cells even more sensitive to T alone. Targeting nodes of the AKT and MAPK survival pathways with trametinib and AZD5363 highlights the potential for combination therapies for melanoma stem cells increasing the arsenal of more effective treatments for patients with high-risk melanoma. Targeting AKT 1, 2, and/or 3 might be effective in eliminating recalcitrant CD133-positive stem-like cells that may be responsible for tumor recurrence. Citation Format: Cynthia M. Simbulan-Rosenthal, Nusrat Islam, Ryyan Alobaidi, Alejandro Moreno, Veerupaxagouda Patil, Amal Alzharani, Peter Sykora, Dean S. Rosenthal. Effects of AKT1, 2, and 3 knock-out by CRISPR-Cas9 in CD133+human melanoma stem cells treated with MEK and AKT inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB452.

Novel PIKfyve/Tubulin Dual-target Inhibitor as a Promising Therapeutic Strategy for B-cell Acute Lymphoblastic Leukemia.

In B-cell acute lymphoblastic leukemia (B-ALL), current intensive chemotherapies for adult patients fail to achieve durable responses in more than 50% of cases, underscoring the urgent need for new therapeutic regimens for this patient population. The present study aimed to determine whether HZX-02-059, a novel dual-target inhibitor targeting both phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) and tubulin, is lethal to B-ALL cells and is a potential therapeutic for B-ALL patients. Cell proliferation, vacuolization, apoptosis, cell cycle, and in-vivo tumor growth were evaluated. In addition, Genome-wide RNA-sequencing studies were conducted to elucidate the mechanisms of action underlying the anti-leukemia activity of HZX-02-059 in B-ALL. HZX-02-059 was found to inhibit cell proliferation, induce vacuolization, promote apoptosis, block the cell cycle, and reduce in-vivo tumor growth. Downregulation of the p53 pathway and suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway and the downstream transcription factors c-Myc and NF-κB were responsible for these observations. Overall, these findings suggest that HZX-02-059 is a promising agent for the treatment of B-ALL patients resistant to conventional therapies.

M2 macrophage-derived paracrine factor TNFSF13 affects the fibrogenic alterations in endothelial cells and cardiac fibroblasts by mediating the NF-κB and Akt pathway.

Heart failure remains a global threaten to public health, cardiac fibrosis being a crucial event during the development and progression of heart failure. Reportedly, M2 macrophages might affect endothelial cell (ECs) and fibroblast proliferation and functions through paracrine signaling, participating in myocardial fibrosis. In this study, differentially expressed paracrine factors between M0/1 and M2 macrophages were analyzed and the expression of TNFSF13 was most significant in M2 macrophages. Culture medium (CM) of M2 (M2 CM) coculture to ECs and cardiac fibroblasts (CFbs) significantly promoted the cell proliferation of ECs and CFbs, respectively, and elevatedα-smooth muscle actin (α-SMA), collagen I, and vimentin levels within both cell lines; moreover, M2 CM-induced changes in ECs and CFbs were partially abolished by TNFSF13 knockdown in M2 macrophages. Lastly, the NF-κB and Akt signaling pathways were proved to participate in TNFSF13-mediated M2 CM effects on ECs and CFbs. In conclusion, TNFSF13, a paracrine factor upregulated in M2 macrophages, could mediate the promotive effects of M2 CM on EC and CFbproliferation and fibrogenic alterations.

A Meta-analysis of Transcriptome Data to Investigate the Effect of Soy Isoflavones on Breast Cancer Cell.

Breast cancer ranks as the second highest cause of cancer-linked deaths in women, with varying rates between Western and Asian countries. The consumption of phytoestrogens can influence breast cancer occurrence. To comprehend how soy isoflavones impact breast cancer cells, we conducted a meta-analysis, combining gene expression data from multiple studies. This approach aimed to identify crucial transcriptional characteristics driving breast cancer cell response to soy phytoestrogens. The gene expression profiles obtained from the Gene Expression Omnibus and Array Express and were grouped into control and isoflavones exposure conditions. We performed a meta-analysis based on the effect size combination method to identify the differentially expressed genes (DEGs). In addition, we performed Gene Ontology (GO) enrichment analysis, pathway analysis, weighted gene co-expression network analysis (WGCNA) and recursive support vector machine (R-SVM) algorithm. Based on this meta-analysis, we identified 3,890 DEGs, of which 2,173 were up-regulated and 1,717 were down-regulated. For example, SGCG, PLK2, and TBC1D9 were the most highly down-regulated genes and EGR3, WISP2, and FKBP4 were the most highly expressed genes in the isoflavones exposure condition. The functional enrichment and pathway analysis were revealed "cell division" and "cell cycle" among the most enriched terms. Among the identified DEGs, 269 transcription factor (TF) genes belonged to 42 TF families, where the C2H2 ZF, bZIP, and bHLH were the most prominent families. We also employed the R-SVM for detecting the most important genes to classify samples into isoflavones exposure and control conditions. It identified a subset of 100 DEGs related to regulation of cell growth, response to estradiol, and intermediate ribonucleoside monophosphate in the purine (IMP) metabolic process. Moreover, the WGCNA separated the DEGs into five discrete modules strongly enriched for genes involved in cell division, DNA replication, embryonic digit morphogenesis, and cell-cell adhesion. Our analysis provides evidence suggesting that isoflavone affects various mechanisms in cells, including pathways associated with NF-κB, Akt, MAPK, Wnt, Notch, p53, and AR pathways, which can lead to the induction of apoptosis, the alteration of the cell cycle, the inhibition of angiogenesis, and interference in the redox state of cells. These findings can shed light on the molecular mechanisms that underlie the response of breast cancer cells to isoflavones.

Novel artemisinin derivative P31 inhibits VEGF-induced corneal neovascularization through AKT and ERK1/2 pathways

Corneal neovascularization (CoNV)is a major cause of blindness in many ocular diseases. Substantial evidence indicates that vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of corneal neovascularization. Previous evidence showed that artemisinin may inhibit angiogenesis through down regulation of the VEGF receptors. We designed and synthesized artemisinin derivatives, and validated their inhibitory effect on neovascularization in cell and animal models, and explored the mechanisms by which they exert an inhibitory effect on CoNV. Among these derivatives, P31 demonstrated significant anti-angiogenic effects in vivo and in vitro. Besides, P31 inhibited VEGF-induced HUVECs angiogenesis and neovascularization in rabbit model via AKT and ERK pathways. Moreover, P31 alleviated angiogenic and inflammatory responses in suture rabbit cornea. In conclusion, as a novel artemisinin derivative, P31 attenuates corneal neovascularization and has a promising application in ocular diseases.

Correlation between Fatty Acid Transport Proteins and Phosphoinositide 3-Kinase Pathway in Breast Cancer

Abstract Background: Breast cancer (BC) is currently the fifth largest cause of mortality worldwide and has become the most frequent type of cancer. Fatty acid transport proteins (FATPs) assist cancer cells in meeting their higher metabolic needs by increasing fatty acid uptake, which is a significant source of energy for cancer cells. The phosphoinositide 3-kinase/protein kinase B/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway is an important signaling pathway that coordinates the uptake and utilization of various nutrients, including fatty acids. The current study aimed to correlate the FATPs with the PI3K/Akt/mTOR pathway in BC. Methods: Eighty serum samples were collected from BC and control subjects after obtaining an informed consent form. Total ribonucleic acid (RNA) was isolated, and the relative messenger RNA (mRNA) expression of PI3K, Akt, and mTOR was analyzed by a reverse transcriptase-quantitative polymerase chain reaction. Serum FATPs were estimated using commercially available enzyme-linked immunoassay kits. P < 0.05 was indicated as statistically significant. Results: The serum FATPs in subjects with BC differed significantly compared to the control. Relative mRNA expression of PI3K, Akt, and mTOR differed significantly between the groups. Further, on correlating the serum FATPs with PI3K-related signaling molecules showed a significant positive correlation within BC subjects. Conclusion: Significant positive correlation between FATPs and the PI3K, Akt, and mTOR pathway suggests a crucial role of FATPs in promoting BC.

The protective effect of Astaxanthin on scopolamine - induced Alzheimer's model in mice.

To investigate the fundamental mechanisms of the neuroprotective impact of Astaxanthin (AST) in a mouse model of Alzheimer's disease (AD) induced by scopolamine. This research constituted an in vivo animal study encompassing 36 adult male mice, divided into 6 groups: Control, 100mg/kg AST, 2mg/kg scopolamine (AD group), 100mg/kg AST+2mg/kg scopolamine, 3mg/kg galantamine+2mg/kg scopolamine, and 100mg/kg AST+3mg/kg galantamine+2mg/kg scopolamine. After 14 days, the mice's short-term memory, hippocampus tissue, oxidative and inflammatory markers were evaluated. The AST demonstrated a beneficial influence on short-term memory and a reduction in acetylcholinesterase activity in the brain. It exhibited neuroprotective and anti-amyloidogenic properties, significantly decreased pro-inflammatory markers and oxidative stress, and reversed the decline of the Akt-1 and phosphorylated Akt pathway, a crucial regulator of abnormal tau. Furthermore, AST enhanced the effect of galantamine in reducing inflammation and oxidative stress. The findings indicate that AST may offer therapeutic benefits against cognitive dysfunction in AD. This is attributed to its ability to reduce oxidative stress, control neuroinflammation, and enhance Akt-1 and pAkt levels, thereby underscoring its potential in AD treatment strategies.

Small Extracellular Vesicle piR-hsa-30937 Derived from Pancreatic Neuroendocrine Neoplasms Upregulates CD276 in Macrophages to Promote Immune Evasion.

The role of PIWI-interacting RNAs (piRNA) in small extracellular vesicles (sEV) derived from pancreatic neuroendocrine neoplasms (PNEN) in the tumor microenvironment (TME) remains unexplored. We used multiplex IHC to analyze the expression of CD68, CD276 (B7H3), and CD3 on PNEN. CD276+ tumor-associated macrophages (TAM) were more abundant in tumor tissues than nontumor tissues and negatively correlated with T-cell infiltration. Serum sEV piRNA sequencing was performed to identify piRNAs enriched in patients with PNEN. We then investigated the function and mechanism of sEV piR-hsa-30937 in the cross-talk between tumor cells and macrophages in the PNEN TME. PNEN-derived sEV piR-hsa-30937 targeted PTEN to activate the AKT pathway and drive CD276 expression. CD276+ macrophages inhibited T-cell proliferation and IFNγ production. piR-hsa-30937 knockdown and anti-CD276 treatment suppressed progression and metastasis in a preclinical model of PNEN by enhancing T-cell immunity. Thus, our data show that PNEN-derived sEV piR-hsa-30937 promotes CD276 expression in macrophages through the PTEN/AKT pathway and that CD276+ TAMs suppress T-cell antitumor immunity. sEV piR-hsa-30937 and CD276 are potential therapeutic targets for immunotherapy of PNEN.

EGFR Oncogenic Mutations in NSCLC Impair Macrophage Phagocytosis and Mediate Innate Immune Evasion Through Up-Regulation of CD47

IntroductionEGFR-mutated NSCLC is characterized by an immunosuppressive microenvironment that confers limited clinical effectiveness to anti–PD-1 or PD-L1 antibodies. Despite the discouraging outcomes of immunotherapy, novel immune checkpoints are constantly emerging, among which the specific vulnerability for therapeutic intervention in the context of EGFR-mutated NSCLC remains unresolved. MethodsData sets of patient- and cell line-levels were used for screening and mutual validation of association between EGFR mutation and a panel of immune checkpoint-related genes. Regulatory mechanism was elucidated through in vitro manipulation of EGFR signaling pathway and evaluated by immunoblot analysis, quantitative polymerase chain reaction, flow cytometry, immunofluorescence staining, and chromatin immunoprecipitation. In vivo investigation of different therapeutic strategies were conducted using both immunocompetent and immunodeficient mouse models. ResultsAmong all screened immune checkpoints, CD47 emerged as the candidate most relevant to EGFR activation. Mechanistically, EGFR mutation constitutively activated downstream ERK and AKT pathways to respectively up-regulate the transcriptional factors c-Myc and NF-κB, both of which structurally bound to the promotor region of CD47 and actively transcribed this “don’t eat me” signal. Impaired macrophage phagocytosis was observed on introduction of EGFR-sensitizing mutations in NSCLC cell line models, whereas CD47 blockade restored the phagocytic capacity and augmented tumor cell killing in both in vitro and in vivo models. Remarkably, the combination of anti-CD47 antibody with EGFR tyrosine kinase inhibitor revealed an additive antitumor activity compared with monotherapy of either antitumor agent in both immunocompetent and adaptive immunity-deficient mouse models. ConclusionsEGFR-sensitizing mutation facilitates NSCLC’s escape from innate immune attack through up-regulating CD47. Combination therapy incorporating CD47 blockade holds substantial promise for clinical translation in developing more effective therapeutic approaches against EGFR-mutant NSCLC.

Swollen Feet: Considering the Paradoxical Roles of Interleukins in Nephrotic Syndrome.

Interleukins are a family of 40 bioactive peptides that act through cell surface receptors to induce a variety of intracellular responses. While interleukins are most commonly associated with destructive, pro-inflammatory signaling in cells, some also play a role in promoting cellular resilience and survival. This review will highlight recent evidence of the cytoprotective actions of the interleukin 1 receptor (IL-1R)- and common gamma chain receptor (IL-Rγc)-signaling cytokines in nephrotic syndrome (NS). NS results from the injury or loss of glomerular visceral epithelial cells (i.e., podocytes). Although the causes of podocyte dysfunction vary, it is clear that pro-inflammatory cytokines play a significant role in regulating the propagation, duration and severity of disease. Pro-inflammatory cytokines signaling through IL-1R and IL-Rγc have been shown to exert anti-apoptotic effects in podocytes through the phosphoinositol-3-kinase (PI-3K)/AKT pathway, highlighting the potential utility of IL-1R- and IL-Rγc-signaling interleukins for the treatment of podocytopathy in NS. The paradoxical role of interleukins as drivers and mitigators of podocyte injury is complex and ill-defined. Emerging evidence of the cytoprotective role of some interleukins in NS highlights the urgent need for a nuanced understanding of their pro-survival benefits and reveals their potential as podocyte-sparing therapeutics for NS.

Discovery of TK-642 as a highly potent, selective, orally bioavailable pyrazolopyrazine-based allosteric SHP2 inhibitor

Src homology-2-containing protein tyrosine phosphatase 2 (SHP2) is a promising therapeutic target for cancer therapy. In this work, we presented the structure-guided design of 5,6-fused bicyclic allosteric SHP2 inhibitors, leading to the identification of pyrazolopyrazine-based TK-642 as a highly potent, selective, orally bioavailable allosteric SHP2 inhibitor (SHP2WT IC50 = 2.7 nmol/L) with favorable pharmacokinetic profiles (F = 42.5%; t1/2 = 2.47 h). Both dual inhibition biochemical assay and docking analysis indicated that TK-642 likely bound to the “tunnel” allosteric site of SHP2. TK-642 could effectively suppress cell proliferation (KYSE-520 cells IC50 = 5.73 μmol/L) and induce apoptosis in esophageal cancer cells by targeting the SHP2-mediated AKT and ERK signaling pathways. Additionally, oral administration of TK-642 also demonstrated effective anti-tumor effects in the KYSE-520 xenograft mouse model, with a T/C value of 83.69%. Collectively, TK-642 may warrant further investigation as a promising lead compound for the treatment of esophageal cancer.

Akt-driven TGF-β and DKK1 Secretion Impairs F508del CF Airway Epithelium Polarity.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked-down (KD) for CFTR (CFTR KD) were compared to control counterparts, grown at an air-liquid interface (ALI). We show that CFTR mutation in primary HAECs and CFTR KD cells promote the overexpression and over-secretion of TGF-β1 and DKK1 when cultured at ALI. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.

Discovery of cinnamylaldehyde-derived mono-carbonyl curcumin analogs as anti-gastric cancer agents via suppression of STAT3 and AKT pathway

The structural modification of curcumin has always been a hotspot in drug development. In this paper, a class of cinnamylaldehyde-derived mono-carbonyl curcumin analogs (MCAs) with 7-carbon-links were designed and synthesized and their anticancer properties were evaluated. Through screening anti-gastric cancer activity of these compounds, H1 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest in vitro (SGC-7901 and AGS gastric cancer cells). Moreover, the SGC-7901 subcutaneous tumor-bearing mice studies revealed that H1 significantly inhibited the tumor growth of gastric cancer. We explored the possible potential targets of H1 through network pharmacology. Mechanistically, our results demonstrated that H1 showed potential anti-gastric cancer activity through suppression of the STAT3 and AKT signaling pathway in vitro and in vivo, which was validated by molecular docking. Overall, our results indicate the potential of H1 as a potent chemotherapeutic drug against gastric cancer.

Abstract 3787: Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion

Abstract Background: Glutathione peroxidases(GPXs) are member of an antioxidant enzyme family. Their role is to eliminate the reactive oxygen species(ROS). Many available studies confirm that over expression of GPXs can promote cancer cell proliferation and invasion. Among the 8 GPXs, Glutathione peroxidase-1 (GPX1) is the most plentiful. Overexpression of GPX1 has been noted to promote invasion, migration, cisplatin resistance and proliferation in breast, colorectal and lung cancer. NF-kB is a ubiquitous nuclear transcription factor in the human body. that key role in cell proliferation, apoptosis and tumor progression. In reliable studies, inhibition of NF-kB expression has been shown to reduce the malignancy of Esophageal squamous cell carcinoma. Accordingly, the purpose of this study is to identify the GPX1 and NF-kB signaling pathway and their correlation with cancer cell proliferation and invasion in vitro using gastric cancer cell. Methods: In our study, we used cell culture, Northern blotting, cDNA microarray analysis, western blotting, RT-PCR, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, GPX1 knock-down with short hairpin RNS(shRNA), Standard two-chamber invasion assay, Chromatin immunoprecipitation assay. Results: First, we confirmed that the expression level of GPX1 was up-regulated by HGF(hepatocyte growth factor) in gastric cancer cells. The cells were treated with NF-kB inhibitor, Pyrrolidine dithiocarbamate(PDTC), and analyzed by Western blotting to identify the associated pathway of HGF-induced GPX1 with NF-kB. The HGF-induced GPX1 protein level was down regulated by PDTC. The role of GPX1 associated with NF-kB was determined by knock down GPX1 expression using GPX1-sh RNA. The GPX1-sh RNA treated cells showedvdown regulation of NF-kB and urokinase Plasminogen activator(uPA) also. To fine the upper regulator of GPX1 signaling pathway, we treated AKT inhibitor(LY294002) ant then analyzed by Western blotting to identify associated pathway of HGF-induced GPX1 with AKT. When AKT pathway was inhibited, GPX1 and NF-kB were down-regulated in gastric cancer cell. HGF-mediated cell proliferation and invasion in vitro was decreased in GPX1 knock down cell. We identified the putative binding site of GPX1 promotor containing NF-kB binding site and confirmed the function by CHIP assay. Conclusions: This study aimed to investigate relationship between GPX1 and NF-kB in gastric cancer cells. Results showed that HGF induced GPX1 expression through the NF-kB and AKT pathway and GPX1 may play a significant role in cell proliferation and invasion in gastric cancer. Therefore, GPX1 can be a potential therapeutic target of gastric cancer for the treatment. Citation Format: Byeong Il Jang, Ji Yoon Jung, Sung Ae Koh, Kyung Hee Lee. Important role of GPX1 and NF-kb signaling pathway in human gastric cancer including cell proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3787.

Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma

Abstract The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC. Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 630.

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2079.

Abstract 1935: The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance

Abstract Activating mutations in KRAS are found in approximately 30% of non-small cell lung cancer (NSCLC), and among all KRAS mutations, KRAS G12C is the most frequent variant in NSCLC, with a prevalence of approximately 14%. KRAS G12C inhibitors, sotorasib and adagrasib are FDA approved for locally advanced or metastatic KRAS G12C-positive NSCLC. However, clinical evidence suggests that there are various factors limiting the efficacy of KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC, highlighting the urgent need for novel treatment strategies or combinations to improve efficacy. Therefore, we sought to identify effective combinations to enhance the efficacy of KRAS G12C inhibitors using in vitro and in vivo models of KRAS G12C-positive NSCLC. Employing a high throughput in vitro drug screening effort using KRAS mutant NSCLC cell lines, we observed that Focal Adhesion Kinase (FAK) and Steroid Receptor Coactivator (SRC) inhibitors produced a synergistic effect when combined with KRAS G12C inhibitors adagrasib or sotorasib. As determined by reverse phase array and Western blotting, in H358, H23, HCC44, and H2122 cells, KRAS inhibitor treatment alone resulted in an initial suppression of RAS/MAPK and AKT pathway activation which was followed by upregulation of multiple receptor tyrosine kinases and integrins which contributed to the re-activation of the RAS/MAPK and AKT pathways after 24-48 hours. We identified FAK as a key downstream mediator of these bypass pathways. FAK/SRC inhibitors enhanced the efficacy of KRAS G12C inhibitors by preventing this feedback activation. Moreover, we observed increased FAK activation in H358 NSCLC cell lines with acquired resistance to adagrasib or sotorasib as compared to parental cells, and FAK/SRC inhibitors re-sensitized resistant cells to KRAS G12C inhibitors. Using a xenograft model of human KRAS mutant NSCLC, we determined that the combination of repotrectinib, a FAK/SRC inhibitor, and a KRAS G12C inhibitor significantly suppressed tumor growth as compared to KRAS G12C inhibitors alone, and this combination was well tolerated. Overall, our findings indicate that activation of FAK is a key mechanism of adaptive feedback and acquired resistance to KRAS G12C inhibitors in KRAS G12C-positive NSCLC and highlight the therapeutic potential of FAK/SRC inhibitors in combination with KRAS G12C inhibitors. These data support the clinical testing of the combination of FAK/SRC inhibitors and KRAS G12C inhibitors in patients with KRAS G12C-positive NSCLC. Citation Format: Yuji Shibata, Hibiki Udagawa, Monique B. Nilsson, Jacqulyne Robichaux, Ana Galan-Cobo, Marcelo Negrao, David P. Molkentine, Junqin He, Alissa Poteete, Yu Qian, Qian Huang, Ferdinandos Skoulidis, John V. Heymach. The FAK/SRC axis mediates resistance to KRAS G12C inhibitors and its blockade can overcome KRAS inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1935.

Abstract 3358: Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells

Abstract Glioblastoma (GBM; WHO grade 4) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. We have recently shown that TRIB1, a Ser/Thr pseudokinase contributes to therapeutic resistance in GBM cells by activating both ERK and Akt pathways. Here we show that, increased expression of TRIB1 contributed to RT/TMZ resistance by activating pro-survival Akt signaling resulting in increased viability of GBM cells. High level of Akt phosphorylation/activation is correlated with poor prognosis of GBM patients. Many pan Akt inhibitors have been developed which are currently in various phases of clinical trials. The three Akt isoforms (1, 2 and 3) are known to play a role in cancer during proliferation, transformation and metastasis. A previous report has shown that TRIB1 interacts with Akt1 in Drosophila and blocks its activation. In this study we show that Akt binds and forms a complex with TRIB1 in GBM cells and this interaction could be isoform and cell line specific. We utilized patient derived xenograft (PDX) GBM cell lines and established GBM cell lines in this study. Stable TRIB1 overexpressing cell lines were generated by antibiotic selection. Co-immunoprecipitation (Co-IP) was performed utilizing Anti-FLAG or anti-c-Myc magnetic agarose beads to evaluate protein-protein interactions. Western blotting was done to detect protein levels. The FLAG tagged TRIB1 deletion mutants were a generous gift from Dr. Takuro Nakamura (Division of Carcinogenesis, The Cancer Institute, Tokyo, Japan). Cells were transfected using Lipofectamine 2000 reagent. Co-IP studies showed that TRIB1 formed a complex with Akt in PDX GBM cell lines. We then transiently transfected respective TRIB1 deletion mutants lacking either N and C terminal residues in HEK 293 cells and performed Co-IP with Anti-FLAG magnetic agarose beads. We observed that all TRIB1 deletion mutants except TRIB1-ΔN2 (lacking first 160 amino acids) were able to pull down Akt. The western blot results showed that each Akt isoform had a variable protein expression across different cell lines. Co-IP studies in the PDX cell lines showed that TRIB1 interacted with all three Akt isoforms in T08-387 cells whereas TRIB1 consistently interacted with only Akt3 but not Akt1/2 in GBM30 cells that lack wild-type p53 expression. Our data suggest that TRIB1 forms a complex with Akt in PDX GBM cell lines. The potential Akt binding site on TRIB1 lies between 90-160 amino acids. TRIB1 can interact with different Akt isoforms in a cell type dependent manner thereby playing a role in their activation and consequent tumorigenic processes. Therefore, targeting TRIB1 mediated Akt activation could be considered a better therapeutic option over targeting Akt directly, which may decrease Akt’s oncogenic activities without risking the toxicity associated with available Akt- specific inhibitors. Citation Format: Karnika Singh, Chunhua Han, Heather Manring, Saikh Jaharul Haque, Arnab Chakravarti. Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3358.