Akt Pathway Research Articles

Revisiting the follicle-stimulation hormone receptor expression and function in human myometrium and adipose tissue

BackgroundExtragonadal follicle-stimulating hormone receptor (FSHR) expression at low levels has been shown in several normal and tumor tissues, including myometrium and adipose tissue. FSH-FSHR signaling in the myometrium has been suggested to regulate uterine contractile activity and the timing of labor. In contrast, FSH-FSHR has been linked to the activation of brown/beige fat thermogenesis in adipose tissue. The issue of extragonadal FSHR expression and its functionality remains contentious within the scientific community, as contradictory findings necessitate further independent and critical analyses. Hereby, we re-investigated the FSHR expression and its functionality in normal non-pregnant (M-NP) and pregnant (N-P) human myometrium, as well as in human visceral (VAT) and subcutaneous (SCAT) adipose tissue (AT).MethodsFSHR expression at mRNA (real-time qPCR, RNAscope in situ hybridization) and protein (immunohistochemical staining) levels in adipose tissue, myometrium, and adipocytes were evaluated. Myometrium and adipocytes were treated with recombinant (rh)FSH to study its effects on functional pathways. Myometrium contractile activity was measured using a force transducer with digital output and the DASYLab software unit. Cyclic adenosine monophosphate (cAMP) production by myometrium explants and adipocytes was measured using a cAMP ELISA Kit. The activation of the AKT pathway in myometrium and adipocytes was analyzed by Western blot analysis.ResultsContrary to previous observations, we found no expression of FSHR at either mRNA or protein levels in M-NP, N-P, VAT, and SCAT. Treatment with recombinant human FSH (rhFSH) showed no effect on cAMP production or phosphorylation of AKT in M-NP, N-P, as well as in VAT and SCAT. rhFSH treatment did not influence contractile activity in M-NP, N-P.ConclusionsThese findings suggest that the FSHR signaling pathway does not regulate myometrial contractility during pregnancy. Additionally, the absence of FSHR expression in both VAT and SCAT implied that FSHR does not play a role in the functional signaling pathways in adipose tissues. In conclusion, our findings contradict earlier data on the involvement of FSH-FSHR signaling in regulating myometrial contractility near term, as well as in adipose tissue function.

Silver Nanoparticles Reduce ACE2 Expression via Changing Mitochondrial Function in Human Fibroblast-Like Lung Cell and Periodontal Ligament Fibroblast Cells

Silver nanoparticles (AgNPs) have demonstrated antibacterial properties and are widely recognized as one of the most prominent types of nanoparticles. Recent studies have highlighted their effectiveness against coronaviruses. However, the detailed molecular mechanisms underlying the action of AgNPs on viruses and their impacts on the human body remain to be fully elucidated. Thus, we attempt to delineate the preventive effects of AgNPs against SARS-CoV-2 infection. Our findings indicate that treatment with AgNPs reduces ACE2 expression, a key receptor for SARS-CoV-2 particularly in normal oral and lung cells. Additionally, we observed a decrease in the binding affinity of the spike protein to the cell after the AgNP treatment. Through western blot analysis, we identified the involvement of the AKT and/or mTOR signaling pathway in this process. Because AKT and mTOR signaling affects mitochondrial function, we investigated the effects of the AgNP treatment on mitochondria. As a result, we observed the localization of AgNPs within mitochondria. Further, an increase in mitochondrial Fe2+ and reactive oxygen species levels was observed, ultimately resulting in mitochondrial dysfunction. Our results show the efficacy of the AgNP treatment in preventing coronavirus infections.

Adipsin improves diabetic hindlimb ischemia through SERPINE1 dependent angiogenesis

BackgroundAdipsin (complement factor D, CFD), as the first described adipokine, is well-known for its regulatory effects in diabetic cardiovascular complications. However, its role in diabetic hind-limb ischemia was not clarified. This study aimed to evaluate the possible therapeutic effect of Adipsin in hind-limb ischemia in type 2 diabetic mice and to elucidate the molecular mechanisms involved.MethodsA high-fat diet and streptozotocin (HFD/STZ)-induced diabetic mouse model, and a transgenic mouse model with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg) were employed. Hindlimb ischemia was established by femoral artery ligation, and blood flow recovery was monitored using Laser Doppler perfusion imaging. Molecular mechanisms underlying Adipsin-potentiated angiogenesis were examined using RNA sequencing and co-immunoprecipitation/mass spectrometry (Co-IP/MS) analyses.ResultsAdipsin expression was upregulated in non-diabetic mice following HLI, while suppressed in diabetic mice, indicating its potential role in ischemic recovery which is impaired in diabetes. Adipsin-Tg mice exhibited significantly improved blood flow recovery, increased capillary density, and enhanced muscle regeneration in comparison with non-transgenic (NTg) diabetic mice. Adipsin facilitated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) under hyperglycemic and hypoxic conditions. Additionally, it enhanced phosphorylation of AKT, ERK, and eNOS pathways both in vivo and in vitro. RNA sequencing and co-immunoprecipitation/mass spectrometry (Co-IP/MS) analyses identified that Adipsin promoted angiogenesis by interacting with SERBP1, which disrupted the binding of SERBP1 to SERPINE1 mRNA, resulting in reduced SERPINE1 expression and the subsequent activation of the VEGFR2 signaling cascade.ConclusionsAdipsin promotes angiogenesis and facilitates blood perfusion recovery in diabetic mice with HLI by downregulating SERPINE1 through interaction with SERBP1. These findings elucidate a novel therapeutic potential for Adipsin in the management of PAD in diabetic patients, highlighting its role in enhancing angiogenesis and tissue repair.

Exendin-4, a glucagon-like peptide-1 receptor agonist, regulates ductus arteriosus by vasodilation and anti-remodeling through the PKA pathway

The mechanisms of ductus arteriosus (DA) closure involve vasoconstriction and vascular remodeling. Previous findings indicate that the glucagon-like peptide-1 receptor agonist (GLP-1RA) exhibits antihypertensive and anti-remodeling effects in the pulmonary circulation. However, its role in the DA remains unknown. This study aimed to investigate whether exendin-4 (Ex-4), a GLP-1RA, can regulate DA patency and elucidate its mechanisms. After confirming the presence of GLP-1R in neonatal rat DA tissue in vivo, the effects of Ex-4 on DA patency in neonatal rats were sequentially examined. Two hours after birth, we observed spontaneous closure of the DA in control rats. In contrast, Ex-4 prevented the closure of DA, accompanied by reduced intimal thickening. Ex-4 attenuated oxygen-induced vasoconstriction in isolated DA rings ex vivo. This effect was diminished in the presence of H89, a PKA inhibitor. In vitro, Ex-4 inhibited platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of DA smooth muscle cells. Additionally, Ex-4 inhibited PDGF-BB-induced reactive oxygen species (ROS) production, calcium mobilization, and signal transduction of MAPK and Akt pathways. Furthermore, Ex-4 preserved the nuclear expression of Nrf2 attenuated by PDGF-BB. Similarly, all these in vitro effects of Ex-4 were blunted by H89. In conclusion, Ex-4 maintains postnatal DA patency through vasodilatation and anti-remodeling via the PKA pathway. The GLP-1R/PKA pathway emerges as a promising target of DA patency in clinical management.

Almonertinib and alflutinib show novel inhibition on rare EGFR S768I mutant cells.

EGFR classical mutations respond well to EGFR tyrosine kinase inhibitors. However, it is uncertain whether currently available EGFR-TKIs are effective against rare EGFR mutations and compound mutations. Herein, the effectiveness of almonertinib and alflutinib, the third-generation tyrosine kinase inhibitors developed in China, on rare EGFR S768I mutations and compound mutations is identified. In this study, using CRISPR method, four EGFR S768I mutation cell lines were constructed, and the sensitivity of EGFR to almonertinib and alflutinib was tested, with positive controls being the 1st (gefitinib), 2nd (afatinib), and 3rd (osimertinib) generation drugs. The present results indicate that almonertinib and alflutinib can effectively inhibit cell viability and proliferation in rare EGFR S768I mutations through the ERK or AKT pathways in a time-dependent manner, by blocking the cell cycle and inhibiting apoptosis. These findings suggest that almonertinib and alflutinib may be potential therapeutic options for non-small cell lung cancer patients with the EGFR S768I mutation.

Transcutaneous auricular vagus nerve stimulation attenuates stroke-heart syndrome: The role of parasympathetic activity

Stroke induces cardiac dysfunction, which increases poststroke mortality and morbidity. An imbalance in the autonomic nervous system resulting from brain injury may serve as the underlying mechanism. The present study investigated whether transcutaneous auricular vagus nerve stimulation (taVNS) attenuates poststroke cardiac dysfunction by activating the parasympathetic nervous system. Adult male mice were subjected to transient middle cerebral artery occlusion (MCAO) and reperfusion surgery. The mice in the treatment group received repeated taVNS starting 60 min after the onset of cerebral ischemia. To assess whether the effects of taVNS were associated with parasympathetic activity, the MCAO mice in the atropine group received intraperitoneal injections of atropine to inhibit parasympathetic activity prior to taVNS. taVNS significantly increased the left ventricular ejection fraction (EF), attenuated myocardial apoptosis, reduced myocardial hypertrophy, and reduced fibrosis following stroke. The beneficial effects of taVNS on cardiac dysfunction were abolished by atropine administration. Heart rate variability (HRV) analysis and western blot analysis revealed that taVNS increased parasympathetic activity but decreased sympathetic dominance in mice with MCAO. Furthermore, the cardioprotective effects of taVNS were associated with muscarinic acetylcholine receptor activation, PI3K–Akt pathway modulation, and eNOS regulation in the heart. Therefore, taVNS alleviates cardiac dysfunction after stroke and is associated with activation of the parasympathetic nervous system.

Multi-omic network analysis identifies dysregulated neurobiological pathways in opioid addiction

BackgroundOpioid addiction is a worldwide public health crisis. In the United States, for example, opioids cause more drug overdose deaths than any other substance. Yet, opioid addiction treatments have limited efficacy, meaning that additional treatments are needed. MethodsTo help address this problem, we used network-based machine learning techniques to integrate results from genome-wide association studies (GWAS) of opioid use disorder (OUD) and problematic prescription opioid misuse with transcriptomic, proteomic, and epigenetic data from the dorsolateral prefrontal cortex (dlPFC) of opioid overdose victims and controls. ResultsWe identified 211 highly interrelated genes identified by GWAS or dysregulation in the dlPFC of opioid overdose victims that implicated the Akt, BDNF, and ERK pathways, identifying 414 drugs targeting 48 of these opioid addiction-associated genes. Some of the identified drugs are approved to treat other substance use disorders (SUDs) or depression. ConclusionsOur synthesis of multi-omics using a systems biology approach revealed key gene targets that could contribute to drug repurposing, genetics-informed addiction treatment, and future discovery.

Successful Treatment of Abdominal Wall Advanced Endometriosis-Associated Clear Cell Carcinoma with AKT Pathway Inhibitor: Case Report

The emergence of endometriosis-associated clear cell carcinoma (CCC) within the abdominal wall is a notably rare phenomenon. This condition predominantly impacts females who have previously undergone surgical interventions, including hysterectomy or caesarean section (C-section), with the malignant transformation of endometriosis within the post-surgical abdominal scar posited as a likely mechanism. Herein, we delineate a distinctive case of endometriosis-associated CCC emanating from the abdominal wall. The therapeutic approach for the patient encompassed surgical resection, complemented by a regimen of adjuvant chemotherapy, radiotherapy, immunotherapy, and targeted therapy. Despite these measures, the patient experienced disease progression, manifested by bilateral inguinal lymph node involvement and metastasis to the left femoral bone. Advanced molecular diagnostics, specifically next-generation sequencing (NGS) of the resected specimen, identified a targetable PIK3CA E726K mutation. Subsequent treatment with alpelisib and everolimus was initiated, culminating in a sustained complete response.

Investigation of TSRP reverses imatinib resistance through the PI3K / Akt pathway in chronic myeloid leukemia.

Chronic myelogenous leukemia (CML) is a malignant tumor of the blood system, so far there is no effective cure. Imatinib (IM), as the first-line drug for the clinical targeted treatment of CML, has some limiting factors such as drug resistance and relapse, and drug resistance has also emerged in combination with other drugs. At present, traditional Chinese medicine combined with targeted drugs in the treatment of tumor is a research hotspot. The total saponin of L. (TSRP) has an effective anti-tumor activity. Our previous in vitro experiments showed that TSRP can effectively inhibit the proliferation and promote apoptosis of CML cells K562, suggesting that TSRP can effectively reverse the drug resistance of IM, but the mechanism of drug resistance remains unclear. Studies have shown that the PI3K/AKT pathway is the main activation pathway of IM secondary resistance, and is considered to be an innovative therapeutic strategy for targeted cancer treatment, which may be an important mechanism of IM resistance. This project aims to reveal the possible mechanism of TSRP reversing IM resistance through PI3K/AKT signaling pathway through both in vitro and in vivo experiments, providing experimental basis for TSRP combined with IM treatment of CML.

Breast cancer cell resistance to hormonal and targeted therapeutics is correlated with the inactivation of the NR6A1 axis.

Aim: Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy. Epigenetic alterations, including changes mediated by DNA methyltransferases, play a key role in this process. Previously, we identified that resistance to tamoxifen and rapamycin is associated with the suppression of DNMT3A. This study aims to further explore the mechanisms underlying this suppression, with a focus on identifying NR6A1 as a novel regulatory factor. Methods: Acquisition of resistant breast cancer cell sublines, MTT-test, immunoblotting, transient transfection and reporter analysis, lentiviral infection, qRT-PCR, and analysis of methylation using bisulfite pyrosequencing. Results: Our findings indicate that the development of cross-resistance in breast cancer cells to hormonal and targeted therapies involves a shift in cell signaling to alternative AKT pathways, marked by a localized suppression of the NR6A1/DNMT3A axis and associated DNA methylation changes. We demonstrated the critical role of NR6A1 downregulation in resistance development. Additionally, we observed activation of Snail - a key regulator in the epithelial-mesenchymal transition - as a mediator of the effects of NR6A1 depletion, establishing a direct link between Snail expression and resistance formation. Conclusion: The coordinated suppression of NR6A1 and DNMT3A may contribute to sustaining the resistant phenotype in breast cancer cells. This pathway could serve as a predictive marker, helping guide the selection of optimal therapeutic strategies for breast cancer treatment in the future.

Effect of stem extract of Schisandra Chinensis on improving spermatogenesis disorder induced by Cisplatin in Vivo and in Vitro

BackgroundThe primary adverse effect of chemotherapy drugs is the induction of spermatogenic disorders. Schisandra chinensis (Turcz) Baill. have been preliminarily shown to have a significant ameliorative in spermatogenic disorders. Nevertheless, there are relatively few studies on non-medicinal parts such as stems of S. chinensis, which have good therapeutic potential for side effects caused by cisplatin. Up to now, a total of 238 compounds have been isolated and identified from the vine stem of S. chinensi. The main components are lignans, polysaccharides, volatile oils, and organic acids. Therefore, the development of S. chinensis stem as a source of a novel chemotherapy drug protector is of great significance. MethodsIn the present study, we explored the protective effect of the stem extract of S. chinensis (SCE) against cisplatin-induced spermatogenic disorders and its possible molecular mechanisms in vitro and in vivo. The network pharmacology was used to predict the targets that may act on spermatogenic disorders. Mice were injected intraperitoneally with cisplatin at 2 mg/kg for 7 days to induce spermatogenic disorders. SCE (75, 150, and 300 mg/kg) was administered to mice by gavage for 21 days. TM3 cells were treated with Schisandrol B (Sol B) (0.5, 1, and 2 µM) in the presence or absence of cisplatin (40 µM), and then cell viability, oxidative stress, apoptosis, and mitochondrial function were evaluated. Results16 constituents and target proteins were predicted to have possible effects on spermatogenic disorders by network pharmacology. Both in vivo and in vitro experiments showed a favorable protective effect of SCE against cisplatin-induced spermatogenic disorders. Sol B might as the major chemical component is responsibility for the protective effect. The mechanism may involve the regulation of Nrf2\\HO-1 protein, PI3K\\Akt, apoptosis, and MAPK signaling pathway to modulate 17βHSD, cycp450, Star and other crucial proteins in the testosterone synthesis pathway. Ultimately, this regulatory process aims to ameliorate the disruption of sex hormone secretion by cisplatin. ConclusionThese results indicated that the lignans in SCE could effectively improve the spermatogenesis barrier caused by cisplatin. These findings provided a theoretical basis for the development of non-medicinal parts of S. chinensis, and laid a foundation for improving spermatogenesis disorders caused by chemotherapy drugs.

Micro‐Doses of DNP Preserve Motor and Muscle Function with a Period of Functional Recovery in Amyotrophic Lateral Sclerosis Mice

ObjectiveMitochondrial dysfunction is one of the earliest pathological events observed in amyotrophic lateral sclerosis (ALS). The aim of this study is to evaluate the therapeutic efficacy of 2,4‐dinitrophenol (DNP), a mild mitochondrial uncoupler, in an ALS mouse model to provide preclinical proof‐of‐concept evidence of using DNP as a potential therapeutic drug for ALS.MethodshSOD1G93A mice were treated with 0.5–1.0 mg/kg DNP through daily oral gavage from presymptomatic stage or disease onset until 18 weeks old. Longitudinal behavioral studies were performed weekly or biweekly from 6 to 18 weeks old. In situ muscle contraction measurements in extensor digitorum longus muscles were conducted to evaluate the preservation of contractile force and motor unit numbers in hSOD1G93A mice following DNP treatment. Muscle innervation and inflammatory markers were assessed using immunostaining. Extent of protein oxidation and activation of Akt pathway were also examined.ResultsDNP delayed disease onset; improved motor coordination and muscle performance in vivo; preserved muscle contractile function, neuromuscular junction morphology, and muscle innervation; and reduced inflammation and protein oxidation at 18 weeks old in hSOD1G93A mice. Strikingly, symptomatic hSOD1G93A mice exhibited a period of recovery in running ability at 20 cm/s several weeks after 2,4‐dinitrophenol treatment started at disease onset, offering the first observation in disease phenotype reversal using a small molecule.InterpretationOur results strongly support that micro‐dose DNP may be used as a potential novel treatment for ALS patients, with a possibility for recovery, when used at optimal doses and time of intervention. ANN NEUROL 2024

Abstract C031: Targeting colorectal cancer-associated fibroblasts with all-trans retinoic acid to reprogram the tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment (TME) and are known for their role in supporting tumor growth and progression through interactions with cancer cells and other stromal elements. Liver metastatic CAFs (lmCAFs), in particular, play an important role in establishing the secondary niche that facilitates tumor metastasis. Reprogramming the stroma, particularly targeting the pro-tumorigenic functions of lmCAFs, has emerged as a promising therapeutic intervention to disrupt the supportive environment tumors rely on for progression. An initial drug screen of FDA-approved oncology therapies was conducted on patient-derived colorectal cancer (CRC) CAFs and tumor organoids (PDTOs) to identify compounds that may be re-purposed as stroma targeting agents. All-trans retinoic acid (ATRA) was shown to specifically inhibit the proliferation of CAFs without affecting CRC-PDTOs. ATRA is an active metabolite of Vitamin A that exerts its canonical biological effects by binding to nuclear retinoic acid receptors (RAR). The differential RAR expression in multiple CAFs and PDTOs was confirmed with bulk RNA sequencing, with higher expression found in the CAF lines. Subsequent analysis included the qPCR assessment of glycolysis enzyme levels, CAF activation markers, and ATRA signaling in lmCAFs following treatment with ATRA for 5 days. Protein expression of key components of the TGF-b signaling pathway, including SMAD 2/3, Akt, and b-catenin, were also analyzed. Treatment of lmCAFs with ATRA induced expression of RARb and ALDH1A1, biomarkers associated with ATRA signaling pathways. In one patient-derived CAF line, ATRA treatment exhibited a decrease in CAF activation markers (e.g. aSMA) and glycolytic enzymes (e.g. lactate dehydrogenase). On the other hand, another CAF line showed increased fibroblast activation and a shift away from metabolic dependency on glycolysis upon ATRA treatment. Additionally, ATRA targeted TGF-b signaling through the pSMAD 2/3 and Akt pathways, highlighting the multifaceted impact on CAF function and heterogeneity. ATRA effectively reprograms some CAFs by inhibiting their proliferation and altering key signaling pathways, thereby disrupting the pro-tumorigenic environment of the TME. The results of this study suggest that ATRA holds potential as a therapeutic strategy to target and reprogram the CAFs within the permissive stromal components essential for tumor progression. Citation Format: Brandon Choi, Yuyuan Zhou, Seungil Kim, Shannon M. Mumenthaler. Targeting colorectal cancer-associated fibroblasts with all-trans retinoic acid to reprogram the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C031.

Unveiling the role of Pleckstrin-2 in tumor progression and immune modulation: insights from a comprehensive pan-cancer analysis with focus on lung cancer

Cancer remains a leading cause of mortality globally, highlighting the need for novel biomarkers to enhance prognosis and therapeutic strategies. Pleckstrin-2 (PLEK2), a member of the pleckstrin family, has been implicated in processes critical to tumor progression, but its role across cancers remains underexplored. This study systematically examined the expression patterns, prognostic relevance, and functional impact of PLEK2 across multiple cancer types. Using data from The Cancer Genome Atlas (TCGA), Genotype Tissue Expression Project (GTEx), and the Human Protein Atlas, we analyzed PLEK2 expression in both cancerous and normal tissues, revealing significant overexpression of PLEK2 in various cancers at the mRNA and protein levels. Single-cell RNA sequencing further indicated predominant expression of PLEK2 in tumor cells and macrophages within the tumor microenvironment. Survival analysis demonstrated that elevated PLEK2 expression correlated with poor prognosis in specific cancers, though its impact varied across cancer types. Functional assays showed that PLEK2 knockdown inhibited proliferation and migration in human cancer cell lines. In vivo studies using a Lewis lung carcinoma (LLC) model confirmed that PLEK2 knockdown suppressed tumor growth and enhanced the efficacy of PD-1 immunotherapy. Mechanistically, PLEK2 knockdown was associated with reduced AKT pathway activation, diminished tumor-associated macrophage infiltration, and increased CD8 T cell presence. Compounds like Navitoclax were also identified as potential PLEK2 inhibitors. In conclusion, PLEK2 played a multifaceted role in cancer progression and immune response modulation. Targeting PLEK2 might suppress tumor growth and overcome immunotherapy resistance, offering a promising biomarker and therapeutic target to improve cancer treatment outcomes.

Endothelial progenitor cell-derived conditioned medium mitigates chronic cerebral ischemic injury through macrophage migration inhibitory factor-activated AKT pathway

BackgroundChronic cerebral ischemia (CCI) is a significant health issue characterized by hypoperfusion due to damage or occlusion of the cerebral or carotid arteries. CCI may lead to progressive cognitive impairment that is considered as a prelude to neurodegenerative diseases, including dementia and Alzheimer's disease (AD). Endothelial progenitor cells (EPCs) have been implicated in vascular repair in ischemic cerebrovascular diseases, primarily by differentiating into endothelial cells (ECs) or through paracrine effects. However, the clinical transplantation of stem cell therapies remains limited. In this study, we investigated the effects of EPC-derived conditioned medium (EPC-CM) on the impaired vasculature and neurological function in a rodent model of CCI and the mechanism involved.MethodsEPC-CM was analyzed by cytokine array to identify key factors involved in angiogenesis and cellular senescence. The effects and mechanism of the candidate factors in the EPC-CM were validated in vitro using oxygen–glucose deprivation (OGD)-injured ECs and EPCs. The therapeutic effects of EPC-CM and the identified key factor were further examined in a rat model of CCI, which was induced by bilateral internal carotid artery ligation (BICAL). EPC-CM was administered via intracisternal injection one week post BICAL. The cerebral microvasculature and neurobehavior of the rats were examined three weeks after BICAL.ResultsMacrophage migration inhibitory factor (MIF) was identified as a key factor in the EPC-CM. Recombinant MIF protein promoted angiogenesis and prevented senescence in the injured EPCs and ECs. The effect was similar to that of the EPC-CM. These therapeutic effects were diminished when the EPC-CM was co-treated with MIF-specific antibody (Ab). Additionally, the vascular, motor, and cognitive improvements observed in the BICAL rats treated with EPC-CM were abolished by co-treated with MIF Ab. Furthermore, we found MIF promoted angiogenesis and anti-senescence via activating the AKT pathway. Inhibition of the AKT pathway diminished the protective effects of MIF in the in vitro study.ConclusionsWe demonstrated that EPC-CM protected the brain from chronic ischemic injury and promoted functional recovery through MIF-mediated AKT pathway. These findings suggest EPC-CM holds potential as a novel cell-free therapeutic approach for treating CCI through the actions of MIF.

Abstract B013: Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression

Abstract Breast cancer (BC) is the most diagnosed cancer and the second leading cancer-related cause of mortality among women in the US. Despite advances in treatment, BC remains a challenge due to its metastatic potential. The most aggressive BC subtype, triple negative (TN), often harbors mutations in the tumor suppressor gene, p53. One of the effects of mutant p53 involves the inhibition of the tumor and metastatic suppressive functions of TAp63, a p53 family member. Loss of TAp63 leads to the formation of metastatic mammary adenocarcinomas in mice, in part through the regulation of a group of nine long non-coding RNAs (lncRNAs), that we identified and named TAp63 regulated oncogenic lncRNAs (TROLLs). We previously characterized two TROLLs, TROLL-2 and TROLL-3, as markers of tumor progression in multiple cancer types via the activation of the AKT pathway. Currently we are investigating the role of another member, TROLL-9. To investigate the biological role of TROLL-9, we performed in vivo experiments using preclinical mouse models. Specifically, MCF-DCIS, MCF-CA1D, and MDA-MB-231 triple negative breast cancer (TNBC) cells constitutively expressing TROLL-9 were generated and utilized for two mouse models: orthotopic xenografts for primary tumor growth and spontaneous metastasis. The former was designed to identify the potential role of TROLL-9 in tumor formation, whereas the latter involved the resection of a primary tumor and monitoring for metastases via bioluminescence imaging. Notably, TROLL-9 overexpression led to increased formation of lung and livers metastases in all these models, suggesting a role for TROLL-9 in the metastatic cascade. In order to elucidate the mechanism of function of TROLL-9, two complementary approaches were used. Firstly, pulldown using MS2-TRAP followed by mass spectrometry (MS) was performed and led to the identification of putative protein interactors. In parallel, Laser Capture Microscopy (LCM) of the lung metastases followed by RNA-sequencing was used to assess for any gene expression changes due to TROLL-9 overexpression. Integration of the two datasets is currently being performed to unveil the network of genes and proteins perturbed by TROLL-9. Furthermore, to understand the relevance in BC patients, TROLL-9 expression levels were analyzed in the TCGA BC dataset. Interestingly, differences in TROLL-9 levels across BC subtypes were observed, thus supporting its putative role in TNBC. Overall, our data suggest a role for TROLL-9 in BC progression. Our findings provide evidence for further characterizing its mechanism of function as a potential therapeutic target for the treatment of invasive BC, particularly TNBC harboring TP53 mutations. Citation Format: Avani A Deshpande, Marco Napoli, John H Lockhart, Christina L Carr, Jaden R Baldwin, Elsa R Flores. Investigation of a novel lncRNA, TROLL-9, in breast cancer formation and progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B013.

Abstract B011: RNA-based therapeutics to target the p53 pathway in cancer

Abstract Mutations in the TP53 gene and activation of the PI3K/AKT pathway are the two most frequent genetic alterations across multiple human tumors. We discovered a crucial mechanism of crosstalk between these two events consisting of two oncogenic lncRNAs, TROLL-2 and TROLL-3, whose expression is induced by mutant p53. We demonstrated that these lncRNAs drive tumor and metastasis formation in several orthotopic models, including breast cancer, lung adenocarcinoma, and melanoma. The investigation of these mouse models and a pan-cancer analysis of human cancers showed that these lncRNAs are markers of tumor progression and lead to the phosphorylation and subsequent activation of AKT by promoting the cytoplasmic localization of the scaffold protein WDR26. Notably, we have now proven that TROLL-2 and TROLL-3 are also involved in the response to cancer therapies. Indeed, the downregulation of these lncRNAs via siRNAs bypasses the AKT-driven resistance to chemotherapeutics (e.g. Adriamycin, docetaxel, and paclitaxel) in triple-negative breast cancer cells, to inhibitors of mutant KRAS (e.g. sotorasib) and EGFR (e.g. erlotinib) in lung adenocarcinoma cells, and to inhibitors of PARP (e.g. niraparib) in ovarian cancer cells. To target these lncRNAs in a clinically relevant manner, we designed LNA GapmeRs, also known as antisense oligonucleotides (ASOs), to downregulate these two lncRNAs directly. The efficacy of the ASOs was confirmed in ex vivo specimens of tumors and metastases of lung adenocarcinoma and ovarian cancer patients. These clinical specimens were also analyzed via single-cell RNA sequencing and phospho-proteomics to unveil the common vs. unique downstream effects of targeting each lncRNA. When administered together, the ASOs targeting TROLL-2 and TROLL-3 effectively decreased the levels of these lncRNAs, which in turn caused WDR26 sequestration in the nucleus, thus preventing the activation of the AKT pathway and the proliferation of these cancer specimens. Furthermore, we confirmed that the antitumor activities of these ASOs were also maintained in vivo, as proven by the decreased tumor proliferation observed in breast cancer xenograft models. Finally, to improve the delivery of these ASOs in vivo, the usage of non-viral systems, including lipid-based and metallic-based nanoparticles, is being tested in both ex vivo and in vivo preclinical models. Overall, our results unveiled TROLL-2 and TROLL-3 as suitable targets to counteract tumor growth and promote the efficacy of chemotherapeutics and targeted therapies in ex vivo and in vivo cancer models. Citation Format: Marco Napoli, Ashley K Lu, Avani A Deshpande, Jaden R Baldwin, Christina L Carr, Michael R Dunne, Omar K Farha, Michelle H Teplensky, Elsa R Flores. RNA-based therapeutics to target the p53 pathway in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B011.

Abstract A025: RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant type of ovarian cancer with a high frequency of p53 mutation and hyperactivation of the AKT pathway. Even though roughly 85% of patients achieve remission after initial surgical debulking and chemotherapy, four out of five HGSOC patients will experience disease recurrence, resulting in a 5-year survival rate of 43%. We have identified and characterized two TAp63 Regulated Oncogenic Long non-coding RNAs (TROLL-2 and TROLL-3) whose transcription is induced by mutant p53 and high expression level correlate with a more aggressive ovarian cancer grade and phosphorylation of AKT. In-depth analysis demonstrated that TROLL-2 and TROLL-3 directly activate AKT in tumor cells, thus linking p53 alterations to AKT activity. The use of AKT inhibitors in the clinic is limited because of frequent adverse effects, likely due to the constitutive expression of AKT in all cells. TROLL-2 and TROLL-3 expression is limited to tumor cells and provides a more specific target for decreasing AKT activation, potentially minimizing off-target effects. We have optimized reduction of TROLL-2 and TROLL-3 expression using antisense oligonucleotides (ASOs) in an ex vivo perfusion platform for 3D culture of microtumors from primary patient derived tumors. Samples show maximum reduction of proliferation (EdU) and phosphorylation of AKT when TROLL-2 and TROLL-3 ASOs are used concurrently. We also performed phospho-proteomics and single cell RNAseq to compare individual ASO-mediated knockdown of each lncRNA to combined targeted ASO treatments. We have developed primary patient derived xenograft models using ovarian tumors orthotopically implanted into NSG mice generating a relevant preclinical model to determine the efficacy of ASO treatment. To reduce the rapid degradation and clearance of ASOs in circulation, ASOs were encapsulated into lipid nanoparticles and metal-organic frameworks (MOFs) and we are currently testing these in vivo. Since AKT activation is associated with increased staging and resistance to platinum and targeted therapies in ovarian cancer patients, our data provides preclinical rationale for the implementation of ASOs and the use of new delivery methods as a relevant translational therapy and novel method to exploit the specificity of TROLL-2 and TROLL-3 expression in HGSOC. Citation Format: Ashley Lui, Marco Napoli, Jaden R Baldwin, Christina L Carr, Angie Rivera, Duy Nguyen, W. Gregory Sawyer, Michael R Dunne, Erin George, Omar K Farha, Michelle Teplensky, Elsa R Flores. RNA-Based Therapeutics to target the p53 pathway in high-grade serous ovarian cancer: TAp63-Regulated Oncogenic LncRNAs (TROLLs) as novel therapeutic targets in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A025.

Cell Division Cycle 42 Improves Renal Functions, Fibrosis, Th1/Th17 Infiltration and Inflammation to Some Degree in Diabetic Nephropathy.

Our two previous studies observed that cell division cycle 42 (CDC42) was lower and correlated with improved renal function and inflammation in diabetic nephropathy (DN) patients, and CDC42 inhibited renal tubular epithelial cell fibrosis and inflammation under high glucose condition. Sequentially, this current study aimed to investigate the effect of CDC42 on improving renal function, fibrosis, and inflammation in DN mice, and its interaction with T cell receptor (TCR) related pathways. Mice were treated by streptozotocin to construct early-stage DN model, then transfected with CDC42 overexpression adenovirus, followed by simultaneous treatment of LY294002 (PI3K/AKT inhibitor) and CI-1040 (ERK inhibitor), respectively. CDC42 reduced blood glucose, creatinine, and 24h urine protein in DN mice, but only showed a tendency to decrease blood urea nitrogen without statistical significance. Hematoxylin&eosin staining revealed that CDC42 descended the glomerular volume, basement membrane thickness, and inflammatory cell infiltration in kidney. Meanwhile, CDC42 lowered fibronectin, TGF-β1, and Collagen I expressions in kidney, but not decreased α-SMA significantly. Besides, CDC42 decreased T-helper (Th) 1 and Th17 cells in kidney, and reduced serum IFN-γ, IL-1β, IL-17A, and TNF-α but not IL-6. Regarding TCR-related pathways, CDC42 activated AKT and ERK pathways but not JNK pathway. However, the treatment of LY294002 and CI-1040 had limited effect on attenuating CDC42's functions on renal function and fibrotic markers. CDC42 improves renal functions, fibrosis, Th1/Th17 infiltration and inflammation to some degree in DN mice, these functions may be independent to AKT and ERK pathways.

Identifying the crucial binding domain of histatin-1 to recombinant TMEM97 in activating chemotactic migration in human corneal epithelial cells

TMEM97, also known as the sigma-2 receptor, plays a crucial role as an endoplasmic reticular protein involved in various physiological processes such as wound healing, and cholesterol metabolism. Moreover, TMEM97 has been implicated in multiple human diseases including neurodegenerative disorders and cancers. Histatin peptides are endogenous peptides with diverse biological effects, including antimicrobial, immunomodulatory, and wound healing functions. Recent studies have revealed that histatin-1 (Hst1) acts as an endogenous ligand for TMEM97 and is essential for Hst1-induced corneal epithelial migration. In this study, we sought to establish the crucial Hst1 residues that facilitate binding to TMEM97. The purified full-length (FL)-TMEM97 expressed from Escherichia coli exhibited comparable binding affinity, as indicated by the dissociation equilibrium constant (KD) determined by Surface plasmon resonance (SPR), to commercially sourced TMEM97 expressed in mammalian cells. SPR analysis revealed that TMEM97 bound to FL-Hst1 and selected deletion mutants of Hst1. Truncation experiments pinpointed the central region of Hst1 as crucial for its binding to TMEM97, with the loss of residues 15–19 either significantly weakening or completely abolishing the binding interaction. Furthermore, alanine substitution mutant experiments highlighted residues 9–19 as critical for the interaction between TMEM97 and Hst1. Functional assays including migration and signaling were also compared for Hst1 and mutant Hst1. Collectively, these findings underscore the specific binding of Hst1 to TMEM97 and elucidate the critical regions within Hst1 necessary for this interaction which is critically important for the epithelial migration and signaling changes in the ERK and Akt pathways.